WO1997017371A1 - ISOLATION DE L'apo(a), COMPOSITIONS ET PROCEDES D'UTILISATION - Google Patents
ISOLATION DE L'apo(a), COMPOSITIONS ET PROCEDES D'UTILISATION Download PDFInfo
- Publication number
- WO1997017371A1 WO1997017371A1 PCT/US1996/018136 US9618136W WO9717371A1 WO 1997017371 A1 WO1997017371 A1 WO 1997017371A1 US 9618136 W US9618136 W US 9618136W WO 9717371 A1 WO9717371 A1 WO 9717371A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- apo
- apolipoprotein
- lipoprotein
- lysine
- ldl
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/775—Apolipopeptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates generally to the field of molecular biology. More particularly, certain embodiments concern methods and compositions comprising purified active apo(a) isoforms, apoBlOO, and reconstituted lipoprotein(a) [Lp(a)]. In certain embodiments, the invention concerns the use of apo(a) in diagnostic kits in the determination and diagnosis of cardiovascular diseases and lipoprotein disorders. Methods are disclosed for purifying and quantitating apo(a) isoforms, as well as quantitating and reconstituting active Lp(a) complexes.
- Insoluble lipids are transported in the plasma as soluble lipid particles that comprises a core of hydrophobic cholesteryl esters and triglycerides surrounded by a surface monolayer of amphipathic phosphoiipids with which free cholesterol and apolipoproteins are associated.
- Apolipoproteins serve as cofactors for enzymes of lipid metabolism and as recognition factors that allow the secretion and uptake of lipoproteins.
- Lipoproteins are characterized by their relative density, which in turn is determined by the relative amounts of lipid and protein (Havel and Kane, 1989).
- VLDL Very low density lipoproteins
- chylomicrons are triglyceride-rich lipoproteins.
- VLDL arise from the liver and chylomicrons are from the gut, as they enter the plasma the triglyceride core is hydrolyzed, leaving triglyceride depleted chylomicrons and VLDL.
- the chylomicrons are taken up by the liver but a large number of the VLDL are further modified in the plasma through the action of lipases and cholesterol ester transferase to yield LDL.
- High density lipoproteins are generated from excess surface components of VLDL and chylomicrons during the hydrolysis ofthe triglycerides.
- Apolipoprotein B100 There are two types of apolipoproteins involved in the assembly of lipoprotein: those that remain an integral part of the lipoprotein (a) and those that are more loosely associated and can exchange between particles.
- ApoB 100 is found in the liver and is an integral protein of lipoprotein, its intestinal counterpart is apoB48.
- the readily exchangeable lipoproteins include the soluble apolipoproteins. These are gradually lost during triglyceride hydrolysis until LDL contains only apoB proteins.
- the soluble apolipoproteins are part of a multigene family.
- Lipoprotein (a) (Lp(a)) is composed of apo (a) associated through a disulfide linkage to apoBlOO. High concentrations of Lp(a) in the plasma are a major determinant of coronary heart disease as discussed later. The predicted structure of apo (a) shows homology with plasminogen. The apo (a) component is highly polymorphic leading to a huge variability in Lp(a) structure. The concentration of Lp(a) in the plasma may vary greatly. One of the determinants for this variability seems to be an inverse relationship between the kringle IV domain, and the amount of Lp(a) concentration. Apo (a) is synthesized in the liver of man and other primates. 2. Lp(a) Assembly
- Lp(a) assembly was first shown to occur in the plasma of mice transgenic for human apo(a) given intravenously human LDL (Chiesa et al. , 1992).
- the inventors have previously performed studies in human primary hepatocyte cultures (Edelstein et al, 1994) which identified both intracellularly and in the cell medium, VLDL-like particles having as their protein moiety apoBlOO covalently linked to apo(a). The abundance of these particles increased after feeding the cells with oleate bound to albumin.
- the issue of Lp(a) assembly is clearly a complex issue, which is clouded by uncertainties and discrepancies. The confounding factors might relate to the different models studied and the difficulty of extrapolating them to human biology. Even in the case of the Lp(a) of rhesus monkeys, a species phylogenetically close to man, Tomlinson et al.
- Lp(a) As a risk factor for cardiovascular disease is well- documented in a number of prospective and retrospective epidemiological studies (Albers and Marcovina, 1994; Scanu, 1993; Scanu et al, 1991 ; Utermann, 1995). Unfortunately, limited knowledge exists of the mechanism(s) responsible for the role of Lp(a) in cardiovascular pathogenicity. On structural considerations, Lp(a) may be atherogenic, thrombogenic or both. Some evidence also has been obtained for a function of Lp(a) as a regulator of smooth muscle cell migration and proliferation (Grainger et al, 1993).
- lysine binding may be related to fibrin binding and that Lp(a), by inhibiting plasmin generation, may contribute to the process of atherosclerosis (Loscalzo et al, 1990). It also has been shown that apo(a) kringle IV- 10 plays a dominant role in lysine binding through the lysine binding site,
- mice transgenic for human apo(a) when fed a high fat diet become more susceptible to atherosclerosis as compared to their non-transgenic littermates.
- Apo(a) in its free form is potentially more reactive than the apo(a) bound to apoB 100-containing lipoproteins.
- apo(a) has prevented the preparation of apo(a)- containing diagnostic reagents for analysis of clinical samples, and thwarted efforts to optimize isolation and purification methods aimed at producing active apo(a).
- the development of methods to obtain a free apo(a) is imperative for functional and metabolic studies in both animal models and in developing human therapeutics. This need is particularly critical, since a dissociation between apo(a) and LDL may occur at tissue sites, despite the fact that Lp(a) is the more prevalent species in circulating plasma.
- Lp(a) is the more prevalent species in circulating plasma.
- apo(a) free or bound to components of the extracellular matrix can be present in atherosclerotic areas of the arterial wall (Rath et al, 1989; Beisiegel, 1991 ).
- the present invention provides novel methods and compositions for the preparation of "native" active apo(a) isoforms dissociated from Lp(a)'s using mild reductive conditions which cause the cleavage of the interchain disulfide between apo(a) and apoBlOO without an effect on the intrachain disulfides ofthe individual kringles.
- the present invention provides methods for the purification of apolipoprotein
- the reducing agents used in the present invention may be one of any reducing agent capable of reducing the disulfide bond of Lp(a) to produce an active apo (a) fraction.
- a reducing agent is homocysteine, N-acetyl cysteine, 2- mercaptoethanol, 3-mercaptopropionate, 2-aminoethanol, dithiothreitol or dithioerythritol.
- the reducing agent is dithioerythritol at a concentration between about 0.5mM and 2.0mM.
- the lysine analogues are used in the present invention in concentrations sufficient to prevent non-covalent lysine mediated interactions between apo (a) and apoBlOO, but insufficient to allow precipitation of said proteins from solution.
- Examples of lysine analogues used in the present invention may include trans 4(arnino-methyl)-cyclohexanecarboxylic acid, N-acetyl-L-lysine, p-benzylamine sulfonic acid, hexylamine, benzamidine, benzylamine, L-proline and ⁇ -aminocaproic acid (EACA).
- EACA ⁇ -aminocaproic acid
- the lysine analogue used was EACA in a final concentration ranging between 50mM and 200mM and most preferably between lOOmM and 150mM final concentration.
- the separation of apo (a) from the rest of the reaction milieu may be achieved by any conventional separation techniques well known to those of skill in the art. Such separation methods include affinity chromatography, ion exchange chromatography, high performance liquid chromatography, ultracentrifugation, and density flotational ultracentrifugation. In the most preferred embodiments of the present invention, the apo (a) is separated using density ultrcentrifugational flotation at 1.21 g/ml for 20 hours. Of course, these are only exemplary centrifugational parameters that may be adapted by any practitioner skilled in the art.
- the purification methods disclosed herein provide means for isolating in large quantity intact, active apo(a) from a variety of sources. Reconstitution of these apo(a)'s with isolated apoBlOO would therefore provide the basis for the preparation of specific Lp(a) formulations of various molecular weights. This represents a significant advancement over the prior art, since it was not previously possible to isolate the various apo(a) isoforms from mature Lp(a) without denaturing and inactivating those apo(a) isoforms.
- the present invention further describes a method for the purification of fragments of apolipoprotein (a) comprising the steps of providing a composition comprising lipoprotein (a) contacting said lipoprotein (a) composition with a reducing agent; further contacting said lipoprotein (a) composition with a lysine analog to produce a reaction mixture containing lipoprotein (a), reducing agent, and lysine analog; incubating said mixture under conditions whereby LDL, unreacted lipoprotein (a) and free apolipoprotein (a) are produced; separating apolipoprotein (a) from said mixture; contacting said apolipoprotein (a) with a concentration of a proteolytic enzyme whereby fragments of apolipoprotein (a) are produced; and separating said fragments.
- the proteolytic enzyme cleaves the bond between Ile3520-Leu3521 of apolipoprotein (a) to produce an Fl fragment and an F2 fragment.
- the preferred proteolytic enzyme to be used in the present invention is elastase.
- the ratio of apo (a): elastase is 25: 1.
- the F 1 fragment produced in the present invention has an apparent molecular weight of about 220kDa and said molecular weight varies with size of the phenotype.
- the F2 fragment produced has an apparent molecular weight of about 170kDa and does not vary appreciably with phenotype.
- the F2 fragment of the present invention may be further characterized as possessing binding sites for fibrinogen and fibronectin.
- the present invention further comprises a method of screening for elastase activity in diseased tissue comprising contacting tissue with a composition comprising apolipoprotein (a); determining the presence of fragments of apolipoprotein a, wherein the presence of fragments is indicative of the presence of elastase activity in the diseased state.
- the fragments indicative of a diseased state comprise either an Fl fragment, an F2 fragment or a mixture thereof.
- Fl fragment have an apparent molecular weight of about 220kDa wherein the molecular weight varies according to the size of the phenotype.
- F2 fragments fragment have an apparent molecular weight of about 170kDa.
- Certain embodiments of the present invention disclose a method of screening for inhibitors of elastase activity comprising the steps of obtaining apolipoprotein (a); contacting said apolipoprotein (a) with elastase and a candidate substance for the inhibition of elastase activity; and comparing the cleavage products of apolipoprotein (a) in the presence of the candidate substance with the cleavage products in the absence of the candidate substance whereby the lack of fragments F 1 and F2 in the presence ofthe candidate substance is indicative of inhibition of elastase activity.
- the present invention discloses an apo (a) formed according to a method comprising the steps of providing a composition comprising lipoprotein (a); contacting said lipoprotein (a) composition with a reducing agent; further contacting said lipoprotein (a) composition with a lysine analog to produce a reaction mixture containing lipoprotein (a), reducing agent, and lysine analog; incubating said reaction mixture under conditions whereby LDL, unreacted lipoprotein (a) and free apolipoprotein (a) are produced.
- the present invention discloses fragments of apolipoprotein (a) formed according to a method comprising the steps of providing a composition comprising lipoprotein (a); contacting said lipoprotein (a) composition with a reducing agent; further contacting said lipoprotein (a) composition with a lysine analog to produce a reaction mixture containing lipoprotein (a), reducing agent, and lysine analog; incubating said reaction mixture under conditions whereby LDL, unreacted lipoprotein (a) and free apolipoprotein (a) are produced; separating apolipoprotein (a) from said mixture; contacting said apolipoprotein (a) with a concentration of a proteolytic enzyme to produce fragments of apolipoprotein (a).
- the present invention also relates to methods of identifying and quantitating apo(a) and Lp(a), Fl fragments, F2 fragments, or variants thereof.
- the present invention contemplates diagnostic kits for screening agents or determination of apo(a) isoforms, Lp(a) or apoBlOO.
- Said kits can contain active polypeptides ofthe invention.
- These kits can contain reagents for detecting an interaction between an agent, or antibody and one of the purified peptides of the present invention, such as apo(a).
- the provided reagent can be radio-, fluorescence- or enzyme-labeled.
- kits can contain a known radiolabeled agent capable of binding or interacting with one ofthe purified peptides ofthe present invention, such as purified apo(a).
- a known radiolabeled agent capable of binding or interacting with one ofthe purified peptides ofthe present invention, such as purified apo(a).
- an antibody capable of detecting an apo(a) or an apoBlOO complex can be provided.
- an antibody capable of detecting a specific apo(a) isoform or an apoB 100-protein/lipoprotein complex can be provided.
- an antibody capable of detecting an Fl or an F2 fragment can be provided.
- Reagents for the detection ofthe lipoprotein complex can be provided. For example, if the compound provided is Lp(a), reagents for detecting the activity of total native or reconstituted Lp(a) (or its particular apo(a) isoform) can be provided.
- the present invention contemplates a diagnostic kit for detecting Lp(a) or specific components of Lp(a) such as apo(a) or apoBlOO.
- the kit can contain a polynucleotide probe or alternatively an antibody immunoreactive with a Lp(a) or one of its components such as apo(a) or apoBlOO.
- the present invention concerns immunodetection methods and associated kits. It is proposed that the apo(a) isoforms or apoBlOO peptides of the present invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect apo(a), apoBlOO or apo(a)/ apoBlOO- related epitope-containing peptides.
- these methods will include first obtaining a sample suspected of containing such a protein, pepjide or antibody, contacting the sample with an antibody or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of an immunocomplex, and then detecting the presence ofthe immunocomplex.
- immunocomplex formation is quite well known in the art and may be achieved through the application of numerous approaches.
- the present invention contemplates the application of ELISA, RIA, immunoblot (e.g., dot blot), indirect immunofluorescence techniques and the like.
- immunocomplex formation will be detected through the use of a label, such as a radiolabel or an enzyme tag (such as alkaline phosphatase, horseradish peroxidase, or the like).
- a label such as a radiolabel or an enzyme tag (such as alkaline phosphatase, horseradish peroxidase, or the like).
- a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.
- any sample suspected of comprising either an apo(a) or apoBlOO peptide or an apo(a)/apoB 100-related peptide or antibody sought to be detected, as the case may be, may be employed. It is contemplated that such embodiments may have application in the titering of antigen or antibody samples, in the selection of hybridomas, and the like.
- compositions comprising isolated and purified active apo(a) isoforms and/or apoBlOO proteins, native and reconstituted Lp(a)s Fl fragments, F2 fragments, as well as native and reconstituted Lp(a) complexes.
- Pharmaceutical compositions prepared in accordance with the present invention find use in a variety of diagnostic and assay kits, as well as providing sources of both active purified apo(a) isoforms and native or reconstituted Lp(a)'s derived from such apo(a) isoforms.
- Such compositions may be used in the production of antibodies to particular apo(a) isoforms and/or apoBlOO.
- Such methods generally involve administering to an animaL a pharmaceutical composition comprising an immunologically effective amount of an apo(a) isoform or apoBlOO composition.
- This composition may include an immunologically-effective amount of either an apo(a) or apoBlOO peptide or an apo(a) or apoBlOO-encoding nucleic acid composition.
- Such compositions may also be used to generate an immune response in an animal.
- kits that may be employed to detect and/or quantitate the presence of apo(a) isoforms or apo(a)-related proteins or peptides and/or antibodies in a sample.
- kits in accordance with the present invention will include a suitable apo(a) protein or peptide or antibody directed against such a protein or peptide, together with an immunodetection reagent and a means for containing the antibody or antigen and reagent.
- the components of the diagnostic kits may be packaged either in aqueous media or in lyophilized form.
- the immunodetection reagent will typically comprise a label associated with the antibody or antigen, or associated with a secondary binding ligand.
- exemplary ligands might include a secondary antibody directed against the first antibody or antigen or a biotin or avidin (or streptavidin) ligand having an associated label.
- a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention.
- the kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user ofthe kit.
- the reagent(s) of the kit can be provided as a liquid solution, attached to a solid support or as a dried powder.
- the liquid solution is an aqueous solution.
- the solid support can be chromatograph media, a test plate having a plurality of wells, or a microscope slide.
- the reagent provided is a dry powder, the powder can be reconstituted by the addition of a suitable solvent, that may be provided.
- the container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antigen or antibody may be placed, and preferably suitably aliquoted. Where a second binding ligand is provided, the kit will also generally contain a second vial or other container into which this ligand or antibody may be placed.
- the kits of the present invention will also typically include a means for containing the antibody, antigen, and reagent containers in close confinement for commercial sale. Such containers may include injection or blow- molded plastic containers into which the desired vials are retained.
- FIG. 1 Far-ultraviolet circular dichroic spectra of apo(a), parent Lp(a) and
- RLp(a) Free apo(a), ( — ); parent Lp(a), ( ) isolated from subject K.B. containing the 289 kDa apo(a) isoform; RLp(a), ( ) containing the 289 kDa apo(a) isoform and
- FIG. 2A, FIG. 2B and FIG. 2C depict Lys-SepharoseTM affinity chromatography of human wild-type and rhesus Lp(a) and their respective apo(a)s isolated from Lp(a) by reduction with 2 mM DTE.
- the dissociated apo(a), 0.3 mg, was isolated by sedimentation at d 1.127 g/mL.
- FIG.2A shows human Lp(a) from subject P.T.
- FIG. 2B depicts Rhesus Lp(a) and
- FIG. 2C shows human and rhesus apo(a) chromatographed separately on the same column but displayed here on the one graph. All samples were dialyzed against Buffer A and loaded onto 2 mL columns. The sample was applied at a flow rate of 7.8 mL/h. After washing with four column volumes of PBS. the bound components were eluted with 200 mM EACA at a flow rate of 15 mL/h.
- FIG. 3A and FIG. 3B illustrate the effect of time of incubation on the reassembly of Lp(a) from apo(a) and LDL.
- FIG. 3B shows apo(a) from subject B.K. incubated with VLDL ( ⁇ - ⁇ ) from a dyslipidemic subject under the same conditions. The results are presented as the percent ofthe total apo(a) mass in the reaction mixture that underwent reassembly.
- FIG. 4A, FIG. 4B and FIG. 4C illustrate size distribution data obtained from electron micrographs of human Lp(a), RLp(a) and LDL. All samples were obtained from subject K.B. with the 289 kDa apo(a) phenotype at a concentration of 50 mg/mL in 10 mM NH HCO 3
- FIG. 4A shows control Lp(a). The electron micrograph was taken at an instrumental magnification of 46,000. The bar graph shows the size distribution of particles.
- FIG. 4B shows RLp(a) isolated by lysine-SepharoseTM chromatography. The electron micrograph was taken at an instrumental magnification of 46,000. The bar graph shows the size distribution of particles.
- FIG. 4C illustrates control LDL. The electron micrograph was taken at an instrumental magnification of 46,000. The bar graph shows the size distribution of particles.
- FIG. 5 Models of Lp(a) assembly for T ⁇ 72 and Arg72 apo(a). Kringles IV- 4 to 10 and kringle V representing the COOH terminal domain of apo(a) are drawn as circles and the protease region (P) as a rectangle. The interchain disulfide between kringle IV-9 and apoB 100 is depicted as present in both the wild-type and mutant apo(a). When the disulfide bridge is intact, lysine binding of Lp(a) occurs via the
- FIG. 6 Fractionation by lysine-SepharoseTM ofthe products from the limited digestion of apo(a) with pancreatic elastase.
- the apo(a) digest was dialyzed against buffer A before loading onto a 7 ml column. The sample was applied at a flow rate of 7.8 ml/h. After washing with three column volumes of PBS, the column was further washed with 500 mM NaCl to elute non-specifically bound material; thereafter, the bound component was eluted with 200 mM EACA at a flow rate of 15 ml/h.
- FIG. 7 Alignment of the partial amino acid sequences of Fl and F2.
- Fl and F2 were purified from the elastase digests of Lp(a) and apo(a) as outlined in the text and prepared for microsequencing as described.
- Apo(a) is composed of repeats of KIV numbered 1-10, one KV and a protease domain (P) according to the nomenclature of Scanu and Edelstein (1995).
- the first 20 amino acids obtained by sequencing Fl (SEQ ID NO:8) were aligned to the amino acid sequence of the mature apo(a) without the signal peptide sequence (SEQ ID NO:7).
- the amino acid sequence of F2 began with leucine at position 3521 (SEE SEQ ID NO:9).
- the elastase cleavage site was determined to be in the linker region between KIV-4 and 5 (SEQ ID NO: 10).
- the dashed lines refer to undetermined amino acids and the dotted lines are continuation of sequences upstream and downstream of the protein sequences. Alignments to the deduced amino acid sequence of apo(a) (McLean et al, 1987) were performed with the ClustalW sequence alignment program.
- FIG. 8 Schematic diagrams ofthe structure of Lp(a), R-miniLp(a), apo(a), Fl and F2. These structures are drawn to emphasize the kringles and are not meant to be actual depictions of Lp(a) or apo(a) structure.
- KIV-9 contains the single sulfhydryl which is in covalent linkage to apoBlOO of LDL.
- FIG. 9A, FIG. 9B and FIG. 9C Bar graphs of the size distribution of particles in electron micrographs of human Lp(a) (FIG. 9A), miniLp(a) (FIG. 9B)and R-miniLp(a) (FIG. 9C). All samples were at a concentration of 50 ⁇ g/ml in 10 mM NH 4 HCO 3 .
- FIG. 10A and FIG. 10B Binding of R-miniLp(a) and apo(a) fragments to fibrinogen and fibronectin.
- the binding to fibrinogen (FIG. 10A) was expressed as a specific lysine-mediated binding obtained by subtracting the binding in the presence of 200 mM EACA from the total binding.
- the binding to fibronectin (FIG. 10B) is represented by the total binding due to the absence of an inhibitory effect of EACA.
- the data are the means of two determinations for a representative study.
- FIG. 11 Clearance of Lp(a) and derivatives from mouse plasma: Mice were injected with 25-250 ⁇ g of R-miniLp(a) ( ⁇ , closed squares), Lp(a) (D, open squares), F2 ( ⁇ , open triangles), apo(a) (•, closed circles), unfractionated apo(a) digest (o, open circles) and Fl ( ⁇ , closed inverted triangles) in sterile PBS. Blood samples (100 ⁇ l) were collected from the orbital vein in heparinized haematocrit tubes and plasma apo(a) immunoreactive components were measured by ELISA as described in the Examples.
- the pathogenicity of Lp(a) has been documented in a number of studies. It is unknown whether the pathogenicity arises from the Lp(a) molecule er se and/or from apo (a) in its free from. A major drawback in answering these questions is the lack of readily available, purified apo (a) of known size.
- the present invention provides methods and compositions for the preparation of "native" active apo(a) isoforms dissociated from Lp(a)'s using mild reductive conditions which cause the cleavage of the interchain disulfide between apo(a) and apoBlOO without an effect on the intrachain disulfides of the individual kringles. The present invention further provides convenient methods for the screening of inhibitors of elastase activity.
- the purification of apo (a) involves obtaining a composition of Lp(a) and subjecting the composition to reductive cleavage in a manner that allows the formation of cleavage products apo (a) and apoBlOO. These products are then separated to yield purified apo (a).
- Lipoprotein(a),Lp(a) refers to a class of lipoprotein particles in which apoBlOO is covalently linked to apolipoprotein(a),apo(a), by a disulfide bond (Koschinsky et al, 1993; Brunner et al, 1993; Van Der Hoek et al, 1994; Scanu and Fless, 1990).
- Apo(a) is a large glycoprotein, polymo ⁇ hic in size (250-800 kDa), containing kringles highly homologous to kringles 4 and 5 of plasminogen (Scanu and Edelstein, 1995).
- LBS functional lysine binding site
- kringle IV- 10 comprising two anionic, Asp55 and Asp57, two cationic, Arg35 and Arg71, and three non-polar, T ⁇ 62, T ⁇ 72 and Phe64 amino acids (Guevara et al, 1992, 1993).
- LBS is also present in plasminogen kringle 4 except that Lys35 has been replaced by Arg.
- Lp(a) has been assayed using a variety of immunological methods, with these studies indicating an elevated Lp(a) level was associated with an increased risk of cardiovascular and cerebrovascular disease (Albers and Marcovina, 1994).
- Lp(a) The assembly of Lp(a) was also shown to occur in mice transgenic for human apoB 100 and a 17 kringle apo(a) construct (Linton et al, 1993; Callow et al, 1994). In all cases, the reassembled lipoprotein was reported to have an apo(a) disulfide linked to apoB 100. In fact, the requirement for this disulfide has been documented by Brunner et al, ( ⁇ 993).
- Lp(a) is known to be made in the liver of primates. The inventors' present study also indicates that Lp(a) may be present in other species.
- the LDL and VLDL in the plasma represents the primary source for the purification of Lp(a).
- Plasma may be collected from any primate source for the pu ⁇ oses of the invention, or indeed any other source suspected of possessing Lp(a).
- the Lp(a) component of the plasma can then be separated from other components of the plasma using ultracentrifugational flotation at a density of 1.21 g/mL for 20 hours at 50, OOO ⁇ rn followed by affinity chromatography using lysine-SepharoseTM.
- the ultra centrifugational procedure is only exemplary and those of skill in the art will be able to vary them according to the particular equipment and study need without undue experimentation.
- the plasma may be supplemented with various inhibitors, for example, EACA and proline to prevent the Lp(a) from interacting with LDL components ofthe plasma.
- the Lp(a) sample is purified using affinity chromatography lysine-SepharoseTM chromatography.
- columns are packed in a ratio of, for example 5: 1 of lysine-SepharoseTM :Lp(a) protein.
- the columns are equilibrated with an appropriate buffer containing for example, 0.02%NaN 3 .
- NaN 3 is a bacteriostatic agent and may be substituted with any other suitable agent known to those of skill in the art.
- Lp(a) fractions eluted from the lysine-SepharoseTM columns are dialyzed to remove excess salts in a suitable buffer containing 0.02% NaN3 at a pH of 7.5.
- the product purity can be assessed by for example, mobility on, 1% agarose gels, Western blots of SDS PAGE, utilizing anti-Lp(a) antibodies.
- the present invention also discloses methods for enhanced production of Lp(a) by recombinant methodologies in a suitable eukaryotic or bacterial hosts, employing DNA constructs to transform cell lines, yeast cells, or Gram-positive or Gram-negative bacterial cells can also be used.
- Escherichia coli expression systems are well known to those of skill in the art, as is the use of other bacterial species such as Bacillus subtilis or Streptococcus sanguis.
- Further aspects of the invention include high level expression vectors inco ⁇ orating DNA encoding the novel genes encoding components of Lp(a) and particular variants thereof. It is contemplated that vectors providing enhanced expression of Lp(a) components in other systems such as S. mutans will also be obtainable. Where it is desirable, modifications of the physical properties of Lp(a) components may be sought to increase its solubility or expression in liquid culture. The genes may be placed under control of a high expression promoter or the components of the expression system altered to enhance expression. 3. Isolation of Apo (a) from Lp (a)
- the novel methods of the present invention are employed to generate dissociated active apo (a) from the Lp(a) complex.
- the purified intact Lp(a) protein is subjected to mild reductive cleavage wherein a known volume of Lp(a) of a suitable concentration for example, 1 mg/ml in buffer of pH 7.5 is incubated with a reductant such as DTE at a final concentration of 1.5-2mM.
- EACA ⁇ -aminocaproic acid
- EACA may be substituted by other lysine analogues, for example, compounds such as trans 4(amino-methyl)-cyclohexanecarboxylic acid, N-acetyl-L-lysine, p-benzylamine sulfonic acid, hexylamine, benzamidine, benzylamine, L-proline.
- lysine analogues for example, compounds such as trans 4(amino-methyl)-cyclohexanecarboxylic acid, N-acetyl-L-lysine, p-benzylamine sulfonic acid, hexylamine, benzamidine, benzylamine, L-proline.
- the reaction mixture is stirred slowly in an anoxic environment and is kept protected from light.
- the incubation period is for a time sufficient to allow the dissociation of apo (a) and apoB 100 from Lp(a), in a preferred embodiment this period is 1 hour, however, it is understood that this time may vary with each experimental procedure.
- the dissociated Lp(a) components are then dialyzed in a suitable buffer of pH containing 0.02% NaN3 and lOOmM EACA. After dialysis the density of the mixture is adjusted with 60% sucrose in the same buffer. The final solution is ultracentrifuged at 2-10°C for a suitable period of time to allow density separation of free apo (a) from LDL and unreacted Lp(a).
- the purified apo (a) is in the bottom layer of the supernatant.
- the conditions for the separation of apo (a) from the reaction mixture using sucrose density ultracentrifugation is only exemplary, and other methods commonly used by those of skill in the art may be used.
- the yield of apo (a) from the above process is between 80-100 percent. Of course, this yield may vary according to the particular conditions and reagents used by the practitioner.
- the methodology described may facilitate the isolation of a variety of various apo(a) isoforms in large quantity. These isoforms can then be separated using chromatographic and other separation techniques to yield highly pure isoforms of active apo (a).
- These purified apo (a) isoforms can be used in the reassembly of Lp(a) to generate known Lp(a)s of specific size and characteristics to facilitate the study of Lp(a) metabolism and to monitor its pathogenic properties.
- Lp(a) may be treated with a reducing agent in the presence of a lysine analogue.
- the lysine analog is supplied to prevent the interaction of apo (a) with apoBlOO.
- the reducing agent is supplied to break the disulfide bond of Lp (a).
- Lysine analogs for this invention include but are not limited to compounds such as EACA, trans 4(amino-methyl)-cyclohexanecarboxylic acid, N-acetyl-L-lysine, p- benzylamine sulfonic acid, hexylamine, benzamidine, benzylamine, L-proline or any other lysine analogue known to the artisan skilled in the art may be used.
- Example of reducing agents that may be used in this invention include but are not limited to homocysteine, N-acetyl cysteine, 2-mercaptoethanol, 3-mercaptopropionate, 2- aminoethanol, dithiothreitol, and DTE.
- the mixture of Lp (a), l-2mM DTE as a reducing agent and 50mM EACA as a lysine analog is incubated for a suitable period of time in a suitable buffer of pH 7.4.
- a heparin-SepharoseTM column is equilibrated with a suitable buffer containing 50mM EACA and ImM DTE.
- the mixture is applied to the equilibrated column, the column is washed with the same buffer and Ihe first eluate is collected.
- the first eluate from the column contains the apo (a) dissociated from Lp (a).
- the "free" apo (a) is dialyzed against an appropriate buffer, the dialysis product is pure apo (a) that may be freeze dried and stored at -20°C or used immediately.
- the column is further washed with the buffer for a total of three column volumes followed by 3 volumes of 2M NaCl in the buffer.
- the high salt concentration serves to dissociate the remaining unreacted Lp(a) and LDL free of apo (a).
- heparin-SepharoseTM chromatography An alternative to heparin-SepharoseTM chromatography is lysine chromatography.
- Lp(a) is treated with a suitable reducing agent for example 1 -2mM DTE for a suitable period of time and then applied to a lysine SepharoseTM column that has been equilibrated with a suitable buffer of pH 7.4 containing for example ImM DTE (elution buffer C). the column is washed with the same buffer and the first volume of elute is collected.
- This fraction contains LDL dissociated from apo (a).
- Lp(a) and apo (a) as purified in the steps above can be subjected to proteolytic cleavage to yield fragments of a known size. These fragments have been characterized as the Fl and F2 fragments from apo (a).
- Apo (a) is subjected to proteolytic cleavage using elastase an apo (a): elastase ratio of 5: 1 to 100: 1 can be used for the generation of the proteolytic fragments. In some embodiments a ratio of 50: 1 was used in other a ratio of 10: 1 was used. In preferred embodiments a ratio of 25: 1 apo protein: elastase protein was used.
- the apo (a) and elastase enzyme were incubated in a suitable buffer containing KI for time period of 1-24 hours at about 18-22°C.
- the time period was between 2 hours and 20 hours in other embodiments of the invention the time period of for incubation varied between 4 hours and 15 hours in other embodiments ofthe invention the time for incubation was between 5 hours and 10 hours.
- the incubation time was 2 hours. Of course it is understood that this time could be greater or less depending on the demands ofthe particular assay being performed.
- this second incubation time is 20 minutes. Of course this time can vary from one study to another.
- Lp(a) can also be used to generate fragments through elastase mediated cleavage.
- the Fl and F2 fragments may then be separated according to conventional protein separation methodology well known to those of skill in the art. These methods include but are not limited to HPLC, affinity chromatography, centrifugation, and electrophoresis among others. Ion exchange and affinity chromatography would be useful in the isolation of purified Fl and F2 fragments.
- the F2 fragment binds to LDL, lysine-Sepharose, heparin-Sepharose, fibrinogen, and fibronectin.
- columns of lysine-Sepharose, heparin-Sepharose or of immobilized LDL fibrinogen. and fibronectin are utilized to bind F2 from a mixture of Fl and F2 generated by elastolytic activity.
- the Fl fraction is recovered in the buffered flow-through volume from these columns.
- the F2 fragments can then be preferentially eluted from the columns by adjusting the buffer to a high ionic strength, for example 500mM Nacl. and containing a high concentration of lysine analog for example 200mM EACA.
- the purified Fl and F2 fragments can then be lyophilized and stored at -20°C.
- the proteolytic fragments generated in the digestion are then subjected to Western blot analysis and probed with anti apo (a) antibodies.
- apo(a) the cleavage by pancreatic elastase at the Ile3520-Leu3521 bond generated two discrete fragments which was called Fl and F2.
- Fl comprised kringles IV- 1, IV-2 repeats, IV-3 and IV-4 and corresponded to the N-terminal domain of apo(a). Consistent with the chemical data was the observation that by electrophoretic criteria the size of Fl varied according to apo(a) isoform size which is dependent on the number of kringle IV-2 repeats (Lackner et al, 1991; McLean et al, 1987).
- F2 comprised kringles IV-5 to IV-10, kringle V and the protease region and represented the C-terminal domain of apo(a).
- elastase cleavage of Lp(a) produced Fl and F2.
- F2 was linked to LDL in the form of an LDL/F2 complex which was called miniLp(a) because it was smaller than the parent Lp(a) by having only one apo(a) fragment and also by electrophoretic criteria.
- Huby et al (1995) reported the generation of a miniLp(a) particle by subjecting Lp(a) to limited digestion by thermolysin. This enzyme also cleaved apo(a) in the linker between kringles IV-4 and IV-5 but at the Ala3513-Phe3514 bond which is seven amino acids upstream of the elastase cleavage site.
- the miniLp(a) generated by the thermolysin digestion method contains a truncated apo(a) which is 7 amino acids longer than their F2.
- thermolysin- nor elastase-generated miniLp(a) particles are ideal end-products because both enzymes cause a partial proteolysis of apoBlOO with retention of the fragments on the lipoprotein particle.
- a better way to produce miniLp(a) is by the reassembly approach exploiting the capacity of F2 to covalently associate with LDL.
- R-miniLp(a) resembles elastase-derived miniLp(a) but it has the important advantage of containing intact apoBlOO. For these reasons, R-miniLp(a)) was preferentially used in the inventors' work.
- F2 is the fragment which has potentially relevant biological functions.
- animal studies unveiled further differences in the F 1 and F2 fragments.
- Fl injected into mice had a short residence time in the plasma and was also rapidly excreted in the urine in the form of several fragments. After injection into the mouse the Fl fraction appears as distinct 4-5 electrophoretic bands in the size range of 220-135 kDa and as markedly smaller ones in the urine (size range 100-33 kDa). These fragments show that elastase activity in the plasma acts on apo (a).
- F2 has different retention characteristics to Fl in that there is a longer retention time and relatively small amounts are excreted.
- fragments of apo(a) are spontaneously present in their plasma. These fragments represent about 5% of the total plasma Lp(a) protein and are significantly larger than those in the urine. Moreover, the fragments in the urine have th,e size and band pattern of those seen in the urine of injected mice.
- Elastase-dependent apo(a) fragmentation generating Fl and F2 fragments could be occurring under pathological conditions, for instance at sites of inflammation involving an active recruitment of polymo ⁇ honuclear cells and macrophages.
- apo(a) fragments has been reported in human atherosclerotic lesions (Hoff et al. , 1994). The present studies show that these fragments would be of the F2-type and thus unaffected by the Fl -dependent size polymo ⁇ hism of apo(a). As a corollary to this, F2 would be more pathogenic than Fl from the cardiovascular standpoint.
- the present invention describes a novel method for the disassembly and reassembly of both human and rhesus Lp(a). This technology has been applied to the analysis of some of the determinants of Lp(a) assembly taking advantage of the Lys + and Lys " Lp(a) models.
- Elastase is serine protease whose normal role is in the phagocytosis and defense against microbial infection (Bode et al, 1989). Elastases are also capable of destroying many connective tissues and elastase activity has been found in numerous diseased states including pulmonary emphysema and rheumatoid arthritis (Tetley, 1993). Natural inhibitors of elastase activity include bovine pancreatic tryptin inhibitor, ⁇ t proteinase inhibitor, and macroglobulin.
- the present invention seeks to overcome drawbacks inherent in the prior art by providing compositions and kits for screening for inhibitors of elastase activity.
- the invention provides methods for screening for inhibitors of elastase activity by testing for the presence of Fl and F2 fragments in the absence of the candidate substance and comparing such results to the assay performed in the presence of candidate inhibitors of elastase.
- the inventors have discovered that the use of elastase on apo (a) and on Lp(a) characteristically yields two fragments of known size and functional characteristics.
- the present invention concerns a method for identifying further inhibitors. It is contemplated that this screening technique will prove useful in the general identification of any compound that will inhibit elastase activity in cells.
- the active compounds may include fragments or parts of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive. However, prior to testing of such compounds in humans or animal models, it will possibly be necessary to test a variety of candidates to determine which have potential.
- the present invention is directed to a method for determining the ability of a candidate substance to inhibit the activity of elastase the method including generally the steps of: (a) obtaining apo (a);
- a candidate substance As being capable of inhibiting elastase activity, one would measure or determine activity in the absence of the added candidate substance by adding apo (a) or Lp(a) and monitoring the generation of Fl and F2 fragments arising as a result of elastase activity. One would then add the candidate substance to the cell and re-determine the levels ofthe Fl and F2 fragments that arise upon addition of apo (a) or Lp(a) in the presence of the candidate substance.
- a candidate substance which reduces the elastase activity, and thereby reduces the levels of Fl and F2 generated, relative to the activity in its absence is indicative of a candidate substance with inhibitory capability.
- the candidate screening assay is quite simple to set up and perform, and is related in many ways to the assay discussed above for determining elastase activity.
- a candidate substance with the enzyme, under conditions which would allow the formation of Fl and F2 fragments but for inclusion of an inhibitor substance.
- Elastase may be supplied in a pure form or in an endogenous from cellular extracts. "Effective amounts" in certain circumstances are those amounts effective to reproducibly reduce elastase activity, or to reduce the Fl and F2 fragments, in comparison to their normal levels. Compounds that achieve significant appropriate changes in activity will be used.
- New inhibitors of elastase activity may be used in vivo for the treatment of various diseased states. Natural inhibitors of elastase activity, such as Al proteinase inhibitor and macroglobulin are inactivated in numerous diseased states, thereby leading to uncontrolled elastolytic activity. Inhibitors of elastase activity as isolated according to the present invention may be used inhibit the activity of elastase in such diseased states hence limiting the amount of breakdown ofthe extracellular matrix. Pharmaceutical compositions comprising such inhibitors can be formulated for the treatment of inflammation or to inhibit the elastolytic breakdown of cellular tissue that occurs as a result of unchecked elastase activity. Methods for in vitro and in situ delivery protocols of such formulations are well within the grasp of those skilled in the art. E. PHARMACEUTICAL COMPOSITIONS
- compositions disclosed herein may be used in the preparation of pharmaceutical preparations for administration to an animal. Such administration may be desirable for the induction of an immune response or for diagnosis of a specific disease or disorder. Such administration may be oral, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be inco ⁇ orated directly with the food of the diet.
- the compounds may be inco ⁇ orated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
- Such compositions and preparations should contain at least 0.1% of compound.
- the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
- the tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
- a binder as gum tragacanth, acacia, cornstarch, or gelatin
- excipients such as dicalcium phosphate
- a disintegrating agent such as corn starch, potato starch, alginic acid and the like
- a lubricant such as magnesium stearate
- a sweetening agent such as sucrose, lactose or saccharin may be added or a flavor
- any material may be present as coatings or to otherwise modify the physical form of the dosage unit.
- tablets, pills, or capsules may be coated with shellac, sugar or both.
- a syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
- any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
- the active compounds may be inco ⁇ orated into sustained-release preparation and formulations.
- the active compounds may also be administered parenterally or intraperitoneally.
- Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
- Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
- the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
- the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
- the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
- a coating such as lecithin
- surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium stearate, and gelatin.
- Sterile injectable solutions are prepared by inco ⁇ orating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by inco ⁇ orating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like.
- the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be inco ⁇ orated into the compositions.
- the polypeptides of the present invention may be inco ⁇ orated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
- a mouthwash may be prepared inco ⁇ orating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
- the active ingredient may be inco ⁇ orated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
- the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
- the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
- compositions that do not produce an allergic or similar untoward reaction when administered to a human.
- aqueous composition that contains a protein (a)s an active ingredient is well understood in the art.
- such compositions are prepared as injectables, either as liquid solutions or ⁇ uspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
- the preparation can also be emulsified.
- compositions of the present invention may be formulated in a neutral or salt form.
- Pharmaceutically-acceptablesalts include the acid addition salts (formed with the free amino groups ofthe protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
- solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
- the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
- the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
- aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
- sterile aqueous media which can be employed will be known to those of skill in the art in light ofthe present disclosure.
- one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example,
- the present invention contemplates an antibody that is immunoreactive with apo (a), Lp(a), Fl, F2 or any other polypeptide of the invention.
- An antibody can be a polyclonal or a monoclonal antibody. In a preferred embodiment, an antibody is a monoclonal antibody. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Howell and Lane, 1988).
- a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal.
- an immunogen comprising a polypeptide of the present invention
- a wide range of animal species can be used for the production of antisera.
- an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
- Antibodies both polyclonal and monoclonal, specific for isoforms of antigen may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art.
- a composition containing antigenic epitopes of the compounds ofthe present invention can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the compounds of the present invention.
- Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
- monoclonal antibodies To obtain monoclonal antibodies, one would also immunize an experimental animal, often preferably a mouse, with an antigenic composition. One would then, after a period of time sufficient to allow antibody generation, obtain a population of spleen or lymph cells from the animal. The spleen or lymph cells can then be fused with cell lines, such as human or mouse myeloma strains, to produce antibody-secreting hybridomas. These hybridomas may be isolated to obtain individual clones which can then be screened for production of antibody to the desired peptide .
- spleen cells are removed and fused, using a standard fusion protocol with plasmacytoma cells to produce hybridomas secreting monoclonal antibodies against apo(a) related antigen, as used herein apo (a) related antigen or peptide refers to Lp(a), apo (a), Fl fragments of apo (a), F2 fragments of apo (a), or variants thereof.
- Hybridomas which produce monoclonal antibodies to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods. Hybridoma clones can then be cultured in liquid media and the culture supematants purified to provide the antigen-specific monoclonal antibodies.
- the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Westem blot methods, as well as other procedures which may utilize antibody specific to apo(a) related antigen epitopes.
- monoclonal antibodies specific to the particular apo(a) isoforms may be utilized in other useful applications.
- their use in immunoabsorbent protocols may be useful in purifying native or recombinant apo(a) isoforms or variants thereof.
- both poly- and monoclonal antibodies against apo (a) related antigens may be used in a variety of embodiments. For example, they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding apo(a) or related proteins. They may also be used in inhibition studies to analyze the effects of apo(a) related peptides in cells or animals.
- Anti-apo(a) related antigen antibodies will also be useful in immunolocalization studies to analyze the distribution of apo(a) related peptides during various cellular events, for example, to determine the cellular or tissue- specific distribution of the apo(a) related peptide under different physiological conditions.
- a particularly useful application of such antibodies is in purifying native or recombinant apo(a) related peptide, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light ofthe present disclosure.
- a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal.
- an immunogenic composition in accordance with the present invention
- a wide range of animal species can be used for the production of antisera.
- the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because ofthe relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
- a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
- exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine semm albumin (BSA). Other albumins such as ovalbumin, mouse semm albumin or rabbit semm albumin can also be used as carriers.
- KLH keyhole limpet hemocyanin
- BSA bovine semm albumin
- Other albumins such as ovalbumin, mouse semm albumin or rabbit semm albumin can also be used as carriers.
- Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
- the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
- adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
- the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization.
- a variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal).
- the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved.
- the immunized animal can be bled and the semm isolated and stored, and/or the animal can be used to generate mAbs.
- mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, inco ⁇ orated herein by reference.
- this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified apo(a) isoform protein, polypeptide or peptide.
- the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
- Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep frog cells is also possible.
- the use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the B ALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
- somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol.
- B-cells B-lymphocytes
- These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
- a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
- the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
- Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
- any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984).
- the immunized animal is a mouse
- rats one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
- NS-1 myeloma cell line also termed
- P3-NS-l-Ag4-l which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573.
- Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
- Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a
- 2: 1 ratio though the ratio may vary from about 20:1 to about 1 :1, respectively, in the presence of an agent or agents (chemical or electrical) that promote, the fusion of cell membranes.
- Fusion methods using Sendai vims have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al, (1977).
- PEG polyethylene glycol
- the use of electrically induced fusion methods is also appropriate (Goding, 1986).
- Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10 " to 1 x 10 " .
- the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
- Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
- the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
- HAT medium a source of nucleotides
- azaserine the media is supplemented with hypoxanthine.
- the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium.
- the myeloma cells are defective in key enzymes ofthe salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.
- HPRT hypoxanthine phosphoribosyl transferase
- the B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
- This culturing provides a population of hybridomas from which specific hybridomas are selected.
- selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supematants (after about two to three weeks) for the desired reactivity.
- the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobindirig assays, and the like.
- the selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs.
- the cell lines may be exploited for mAb production in two basic ways.
- a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
- the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
- the body fluids ofthe animal such as semm or ascites fluid, can then be tapped to provide mAbs in high concentration.
- the individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
- mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
- ELISAs may also be used in conjunction with the invention.
- the novel active dissociated apo(a) isoforms may be used in the preparation of specific apo(a) antibodies and the Fl and F2. It is known that Lp(a) is involved in the various cardiovascular diseases.
- the present invention provides for the preparation of monoclonal antibodies to apo (a), Fl and F2 and also for Lp(a). These antibodies can be used in characterizing the Lp(a) status of healthy and diseased tissues, through techniques such as ELISAs and Westem blotting.
- anti-Lp(a), anti-apo(a), anti-F 1 and anti-F2 antibodies or antibodies to variants thereof, in an ELISA assay is contemplated.
- Apo(a), Fl and F2 proteins or antigenic sequences derived therefrom are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells, of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine semm albumin (BSA), casein or solutions of powdered milk. This allows for blocking of nonspecific adso ⁇ tion sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
- BSA bovine semm albumin
- the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation.
- Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween®. These added agents also tend to assist in the reduction of nonspecific background.
- the layered antisera is then allowed to incubate for from about 2 to about 4 hr, at temperatures preferably on the order of about 25° to about 27°C. Following incubation, the antisera- contacted surface is washed so as to remove non-immunocomplexed material.
- a preferred washing procedure includes washing with a solution such as PBS/Tween®, or borate buffer.
- the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first.
- the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
- a urease or peroxidase- conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hr at room temperature in a PBS-containing solution such as PBS/Tween®).
- the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol pu ⁇ le or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonicacid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectmm spectrophotometer.
- a chromogenic substrate such as urea and bromocresol pu ⁇ le or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonicacid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label.
- Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectmm spectrophotometer.
- the antibodies ofthe present invention are particularly useful for the isolation of antigens by immunoprecipitation.
- Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein.
- For the isolation of membrane proteins cells must be solubilized into detergent micelles.
- Nonionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.
- the antibodies of the present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, e.g., enzyme-substratepairs.
- compositions of the present invention will find great use in immunoblot or
- the anti-apo(a) related antibodies may be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof.
- a solid support matrix such as nitrocellulose, nylon or combinations thereof.
- these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background. This is especially useful when the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal.
- Immunologically-based detection methods for use in conjunction with Westem blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard.
- Lp(a-), Lp(a) devoid of apo(a);
- VLDL very low density lipoprotein
- LDL low density lipoprotein
- TG triglycerides
- PAGE polyacrylamide gel electrophoresis
- GGE native gradient gel electrophoresis
- PMSF phenylmethylsulfonylfluoride
- BHT ⁇ -hydroxytoluene
- EDTA ethylenediaminetetraaceticacid
- ⁇ -ME ⁇ -mercaptoethanol
- LBS lysine-bindingsite
- Lys " deficient lysine-binding
- Lys + positive lysine binding
- BD binding domain
- human fibrinogen and human plasma fibronectin from Sigma Chemical Co. (St. Louis, MO); Kallikrein inactivator (KI) from Calbiochem, San Diego, CA; molecular weight standards for native gradient gels from Pharmacia-LKB (Alameda, CA); lambda DNA size standards and polyacrylamide from Bio * Rad (Richmond, CA); Immobilon-P® membranes from Millipore (Bedford. MA) and an enhanced chemiluminescent kit (ECL Westem Blotting Detection kit) from Amersham (Arlington Heights, IL). All other chemicals were of reagent grade.
- Antisera to purified preparations of Lp(a) and LDL were raised in the rabbit and affinity-purified antibodies to apo(a), Lp(a) and LDL were prepared as previously described (Fless et al, 1989).
- Anti-Lp(a) were shown to be devoid of immunoreactivity to LDL and plasminogen, and anti-LDL were unreactive to Lp(a) and apo(a).
- Monoclonal antibodies to apo(a) KV were prepared in the inventors' laboratory.
- Buffer A was 10 mM phosphate containing 1 mM EDTA, 0.02% NaN 3 , pH 7.5.
- Buffer B was 10 mM phosphate containing 1 mM EDTA, 0.02% NaN 3 and 100 mM EACA, pH 7.5. All other buffers were prepared as described in the text.
- the four wild-type subjects were two males (one Afro-American, one Caucasian) and two females (Caucasians), all healthy with Lp(a) protein levels in the range of 15-43 mg/dL (Table 2). All of them were heterozygous for apo(a) size isoforms based on protein and genomic analyses.
- One healthy subject male, Caucasian
- One healthy subject with the T ⁇ 72- Arg mutation in apo(a) kringle IV- 10 had plasma Lp(a) protein levels of 0.16 mg/dL (Scanu et al. , 1994), (Table 2). and had a single allele and a single apo(a) isoform.
- the plasma from all subjects was obtained by plasmapheresis performed in the Blood Bank ofthe University of Chicago. TABLE 2 Lp(a) Levels, Phenotypes and Genotypes of Wild-Type and Mutant Subjects
- the steps for Lp(a) and LDL isolation were carried out immediately after blood drawing.
- the plasma samples used for the isolation of VLDL were obtained from either normolipidemic healthy human donors or dyslipidemic subjects (type IV) before receiving treatment at the University of Chicago Lipid Clinic and had plasma levels of Lp(a) protein below 1 mg/dL.
- the inventors used an additional 10 subjects for studying the apo(a) fragments in their plasma and urine. These were also healthy subjects with a known phenotype and genotype.
- Their plasma Lp(a) protein levels varied between 0.1 and 10 mg/dl and their size isoforms varied between 300 and 600 kDa.
- Apo(a) phenotyping was performed on either reduced plasma, isolated apo(a) or Lp(a) samples by SDS-PAGE followed by immunoblotting using anti-Lp(a) (Edelstein et al. , 1995). The mobility of the individual apo(a) bands was compared with isolated apo(a) isoforms of known molecular weights (Fless et al, 1994).
- DNA plugs were prepared from blood mononuclear cells, subsequently fractionated by pulsed field electrophoresis and the blots probed with an apo(a) specific probe essentially as described earlier (Lackner et al. , 1991 ).
- the plasma obtained by plasmapheresis was adjusted with 0.15% EDTA, 0.01% NaN 3 10,000 U/L KI and 1 mM PMSF.
- Wild-type Lp(a) were isolated by sequential ultracentrifugation and lysine-SepharoseTM chromatography as previously described (Fless et al, 1994).
- the purified Lp(a) were filter sterilized and stored at 4°C. An aliquot of both blood and plasma from each subject was utilized for apo(a) genotyping and phenotyping, respectively. In three of the subjects, D.G. B.K. and P.T. the two apo(a) phenotypes were close in size (Table 2); thus Lp(a) species containing a single phenotype could not be separated by the procedure used. On the other hand, in the case of K.B., the high differential in apo(a) size and thus Lp(a) density permitted the separation of two Lp(a)s, one containing the 289 kDa and the other the 488 kDa phenotype. The technique used was an adaptation of that described by Fless et al, (1994).
- the four rhesus monkeys studied were from the same pedigree previously described (Scanu et al, 1993). All had high plasma Lp(a) protein levels varying between 20 and 40 mg/dL, exhibited a single band phenotype and were housed at the Southwest Foundation for Biomedical Research in San Antonio, TX. The monkeys were fasted ovemight before collecting 20 mL of venous blood in tubes containing 0.01% EDTA. The isolation procedure was essentially as described previously (Scanu et al, 1993). Since rhesus apo(a) is Lys " , the inventors could not utilize lysine- SepharoseTM chromatography for the isolation of Lp(a).
- the top layer containing mainly Lp(a) was removed, dialyzed against 10 mM Tris-HCL, 0.01% EDTA, 0.01% NaN 3 and subjected to FPLC ion-exchange chromatography as fully described by Scanu et al, (1993).
- the isolated Lp(a) were filter sterilized and stored refrigerated in 33 M phosphate buffer containing 2 mM PMSF, 0.15% EDTA and 0.0 l%NaN 3 pH 7.5 under nitrogen.
- EACA EACA to a final concentration of 100 mM was then added in small increments and the reaction mixture was protected from light with aluminum foil and rotated slowly (7 ⁇ m) in a general pu ⁇ ose rotator at room temperature for 1 h under nitrogen gas. Subsequently, the incubated mixture was dialyzed for 2 h at room temperature against 2 changes of 4 liters each of Buffer B purged with nitrogen gas.
- the yield of free apo(a) was 90-100%.
- the resulting apo(a) behaved in a similar way but the yields, based on the ELISA of the Lp(a) remaining in the floating fraction, were significantly lower than those obtained with 2 mM DTE; about 55 and 70% with 0.5 and 1 mM DTE, respectively vs. 90% with 2 mM DTE.
- the disassembly process was the same whether starting from Lp(a) preparations containing either one or two phenotypes.
- the apo(a) was incubated with 100 mM EACA for 60 min at 37°C before the addition of LDL and the mixture then incubated for the desired time.
- an aliquot (125 ⁇ L) of the reaction mixture was diluted with an equal volume of 60% sucrose in buffer A containing 200 mM EACA and spun in a TLA 100 rotor (tube capacity, 250 ⁇ L) at 412,160 x g at 15°C for 18 h.
- the top fraction (105 ⁇ L) was removed and quantitated by ELISA designed to measure the apoB100:apo(a) complex (Fless et al, 1989).
- the bottom 100 ⁇ l containing free apo(a) was also quantitated by a sandwich ELISA specific for apo(a) using anti-Lp(a) for coating and alkaline phosphatase-conjugated anti-Lp(a) for detection.
- the reassembly reaction mixture after dialysis against Buffer B, was subjected to density gradient ultracentrifugation (Nilsson et al, 1981 ) which effects an efficient separation of LDL from Lp(a) and apo(a).
- the fractions containing Lp(a) were dialyzed against Buffer A and further purified to homogeneity by affinity chromatography with lysine-SepharoseTM or FPLC.
- the procedure was the same as that described for the reassembly with LDL.
- the molar ratio of apoB 100 to apo(a) was based on the concentration of apoB 100 in VLDL as measured by ELISA.
- apo(a) was incubated in Buffer A containing 50 mM DTE, 6 M guanidine HCl for 2 hrs at room temperature. The mixture was made 150 mM with respect to iodoacetamide and the incubation continued for an additional hour. Before use, the reduced and alkylated apo(a) was dialyzed against buffer A. 12. Limited proteolysis of Lp(a) and apo(a)
- Lp(a) and apo(a), containing KI were incubated at 20°C with porcine pancreatic elastase at various molar ratios of Lp(a) or apo(a) protein to enzyme (5, 10, 25, 50, 100 to 1) for time periods of 1 to 24 h.
- the digestion mixtures were then examined on Westem blots of SDS-PAGE probed with anti-apo(a).
- the inventors' end point for limited proteolysis was the production f two major bands of 220 kDa and 170 kDa. Minor amounts ( ⁇ 10%) of smaller molecular weight bands were also observed.
- CNBr-activated SepharoseTM 4B was coupled to the ⁇ -amino group of lysine essentially according to the instructions supplied by Pharmacia-LKB.
- the amount of lysine crosslinked to the beads was assessed as described (Wilkie and Landry, 1988) and ranged between 16 and 21 ⁇ moles of lysine per mL bead suspension.
- Chromatography was performed at room temperature on a Bio-Rad Econo® Chromatography system. Columns were packed with lysine-SepharoseTM at a ratio of 5 mL of packing material to 1 mg of Lp(a) protein and equilibrated with PBS containing 1 mM EDTA, and 0.02% NaN 3 . After loading, the column was washed with at least 3 column volumes of equilibrating buffer at the same flow rate. Fractions containing ap ⁇ (a) or Lp(a) were pooled and dialyzed against Buffer A.
- TBS buffer 50 mM Tris-HCl, pH 7.5, 150 mM NaCl
- Nonspecific binding sites were blocked with 2% BSA in TBS for 2 h at 22°C.
- TBST buffer TBS supplemented with 0.1% BSA and 0.02% Tween-20
- various concentrations of Lp(a) and the derived fragments were added to the wells in TBS buffer with or without 200 mM EACA and incubated for 2 h at 22°C.
- [Fn 0 ] represents the total number of fibrinogen or fibronectin binding sites
- [Fn] the number of moles of Lp(a) or apo(a) adsorbed on fibrinogen or fibronectin
- [X] refers to Lp(a), apo(a), mini-Lp(a) or apo(a) fragments and K the association constant.
- SDS-PAGE (3.5% separating gel, 2.75% stacking gel), was performed on a Novex system (Novex, San Diego, CA) for 1.5 hr at constant voltage (120 V) at room temperature.
- the samples were prepared by heating at 95°C for 5 min in sample buffer which consisted of 94 mM Phosphate buffer, pH 7.0, 1% SDS and 2 M urea with or without 3% ⁇ -ME.
- sample buffer which consisted of 94 mM Phosphate buffer, pH 7.0, 1% SDS and 2 M urea with or without 3% ⁇ -ME.
- the gels were placed onto Immobilon-P® sheets which were previously wetted with a buffer containing 48 mM Tris, 39 mM glycine, pH 8.9.
- the Immobilon-P® blots were blocked in PBS containing 5% non-fat dry powdered milk and 0.3% Tween® 20 followed by incubation with anti- Lp(a) or anti-apoB antibody. In specified cases, monoclonal antibodies directed against kringle V of apo(a) were used. The blots were washed and incubated with anti-rabbit horseradish peroxidase-labeledlgG. Subsequently, the blots were developed with the ECL Westem Detection Reagent according to the manufacturer's instmctions. 18. Amino Acid Analyses
- Apo(a) fragments (10-30 ⁇ g) were eiectrophoresed under reducing conditions as outlined above. After electrophoresis, the gels were electroblotted onto Immobilon PSQ sequence grade membranes (Millipore Co ⁇ ., Bedford, MA) as described above in the immunoblotting section. The blots were rinsed in distilled water, stained with Coomassie Blue R250 (0.025% in 40% methanol) and destained with 50% methanol. The stained bands were cut from the membrane, further washed with 40% methanol and allowed to air dry. Reduction with DTT and alkylation with iodoacetamide was performed directly on the PSQ membrane which was then subjected to automated Edman degradation on an Applied Biosystems 477A unit using procedures recommended by the manufacturer.
- each grid was coated with one drop of 1 % phosphotungstic acid. The excess phosphotungstic acid was removed and the grids were air dried and examined in a Philips CM 10 electron microscope at an accelerating voltage of 100 kV. 20. Circular Dichroic Measurements
- CD spectra were measured on a Jasco J-600 spectropolarimeter (Jasco, Japan) and analyzed using the J-700 software after conversion ofthe data using the Softsec File conversion program (Softwood Co., CT). Spectra were recorded at protein concentrations ranging from about 0.1 mg/mL to 2 mg/mL in cuvettes of 0.01 to 0.1 cm path length. Mean residue ellipticities were calculated using the following mean residue weights: 1 12.8 for apo(a) and 1 13.3 for Lp(a) and RLp(a).
- the secondary stmcture content was calculated by two methods, using the program V ARSLC 1 starting with a set of 33 reference proteins (Manavalan and Johnson, 1987), and the program CONTIN (Provencher and Glockner, 1981). All samples were previously dialyzed against Buffer A but without PMSF since this reagent interfered with CD abso ⁇ tion in the far ultraviolet region ofthe spectrum.
- Triglycerides were determined by a test kit from Sigma (TG INT #336) and phosphoiipids as inorganic phosphorous using the Fiske and Subbarow reagent (Fisher, USA) following the method of Bartlett, (1959) and using the factor 25 to convert inorganic phosphate to phospholipid mass.
- Lp(a) and LDL protein were quantitated by a sandwich ELISA essentially as previously described (Fless et al, 1989) except that anti-Lp(a) IgG was used as the capture antibody and anti-apoB IgG conjugated to alkaline phosphatase as the detection antibody.
- anti-Lp(a) IgG was used as the capture antibody
- anti-apoB IgG conjugated to alkaline phosphatase as the detection antibody.
- anti-apo(a) IgG conjugated to alkaline phosphatase was used 22 as the detection antibody.
- mice Balb/c female mice (10-12 weeks) from Jackson Laboratories (Bar Harbor, ME) were used. All mice were housed in individual cages under normal light. The evening before the study, the mice were given a 10% sucrose solution to drink ad libitum in place of water. The following morning the mice were anesthetized with Metafane and 25-250 ⁇ g of either Lp(a), apo(a), or the fragments obtained from the elastase digestion in a volume of 200 ⁇ L were injected into the tail vein. The mice were then placed in metabolic cages, given access to standard lab chow and the 10% sucrose solution. Blood samples were withdrawn from the orbital vein into heparinized hematocrit tubes at the specified time points and immediately iced.
- Urine was collected at 0-3, 3-5 and 5-24 h.
- ELISA quantitation sensitive to ⁇ 0.0015 mg/dl of apo(a), was performed on the urine samples to determine the levels of apo(a) reacting material. These results indicated to what degree the sample was to be concentrated for electrophoretic detection which was estimated to be > 0.03 mg/dl.
- the urine was concentrated in Amicon Centriprep filters and the extent of concentration for each representative sample is stated in the legends to the Figures.
- Mouse plasma and urine were analyzed for the levels of apo(a), Lp(a) and kringle V by a sandwich ELISA using monospecific antibodies as described above. The decay of Lp(a), apo(a) or fragments in plasma was expressed as:
- percent of injected dose (mg/dl), / (mg/dl), x 100
- Modeling was performed on a molecular graphics workstation from Silicon Graphics Inc. using the modeling system Insight II v.95.0 and the programs Buider, Biopolymer and Discover (Biosym/MSI, San Diego, CA). Since crystallographic coordinates are not available for the linker regions the inventors used the amino acid sequence deduced from the cDNA sequence (McLean et al, 1987) and built each amino acid sequentially in a linear fashion. The secondary stmcture was then constmcted based on the algorithms of Chou-Fasman (1978) and Gamier/Robeson (1978) and the model was subjected to energy minimization.
- the Lp(a) from subjects containing two apo(a) phenotypes (488 and 289 kDa) was separated into two Lp(a) species each containing one of the two phenotypes and then incubated with 2 mM DTE in the presence of 100 mM EACA.
- the Westem blots of the products dissociated from Lp(a) by the action of DTE showed that only free apo(a) was present in the sedimenting fraction but not in the floating fraction which contained only apoBlOO and barely detectable quantities of unreacted Lp(a).
- the theoretical number of fully reduced cysteines was calculated to be 106.
- the isolated apo(a) examined by far-ultraviolet CD spectroscopy gave a spectmm (FIG. 1 ) characterized by a strong negative band at 203 nm and a positive band at 222-232 nm.
- the CD deconvolution programs are mainly suited to globular proteins, these values agreed with those predicted for apo(a) by Guevara et al, (1992).
- Apo(a) from subject D.G. was incubated with LDL (subject D.G.) at an apoB100:apo(a) molar ratio of 25:1 for 24 h at 37°C and the reassembled Lp(a) separated by lysine-SepharoseTM chromatography and analyzed on Westem blots of 3.5% polyacrylamide slab SDS-PAGE gels with antibodies to Lp(a) and apoBlOO.
- Lanes 1-3 anti-Lp(a) blots of unreduced gels of parent Lp(a), free apo(a) and RLp(a) respectively; lanes 4-6, anti-apoBlOO blots of unreduced gels of parent Lp(a), RLp(a) and control LDL respectively; lanes 7-9, blots of reduced samples as in lanes 1-3; lanes 10-12, blots of reduced samples as in lanes 4-6. Markers for Lp(a) and apo(a) are shown on the left ofthe unreduced gels and apo(a) and apoBlOO on the right ofthe reduced gels.
- Lysine and proline have been previously reported to inhibit the assembly of Lp(a) (Chiesa et al, 1992; Phillips et al, 1993; Trieu et al, 1991).
- incubation of apo(a) with either of the two reagents prior to incubation with LDL significantly inhibited Lp(a) reassembly.
- a maximum inhibition of 90% was attained.
- EACA appeared to be a relatively more potent inhibitor than proline (Table 3).
- EACA or proline 100 mM was preincubated with apo(a) for 1 h at 37°C before incubation with LDL.
- EACA or proline 100 mM was added after the complex was formed and incubated for 1 h.
- Both EACA and proline, each at 100 mM. were preincubated with apo(a) for 1 h at 37°C before interaction with LDL.
- Lp(a) was isolated from plasma and utilized for the preparation of apo(a). The latter and LDL were used to prepare RLp(a). Apo(a), Lp(a) and LDL were obtained from subject K.B. with the 289 kDa phenotype. The percent was calculated from four studies;
- apo(a) linked to apoBlOO can be found in triglyceride- rich particles isolated from hyperlipidemic plasma (Scanu et al, 1992).
- the in vitro interaction of apo(a) isolated from an Lp(a) having a single apo(a) isoform (289 kDa) was examined with preparations of VLDL isolated from the plasma of two hypertriglyceridemic subjects (R.W. and R.Z.) with type IV dyslipoproteinemia and very low plasma levels of Lp(a) protein, i.e., 0.1 and 0.3 mg/dL. respectively.
- Rhesus apo(a), at an apoBlOO.apo(a) molar ratio of 50: 1 also bound equally to either autologous LDL or rhesus Lp(a-) or human wild-type LDL.
- the reaction between rhesus apo(a) and human LDL followed a time course of reassembly (FIG. 3 A) similar to that of the wild-type human apo(a), reaching a maximum of about 60% in 5 h.
- Westem blot analyses ofthe reassembled fraction which was contained in the d 1.127 g/mL floating fraction, showed that an apoB 100:apo(a) complex was formed and only dissociated in the presence of ⁇ -ME.
- EACA and proline inhibited the reassembly, although inhibition by either of these reagents was slightly more efficient than in the human model (Table 3).
- a novel method for isolating wild-type and mutant apo(a) phenotypes in a free form has been developed by subjecting each parent Lp(a) to mild reductive conditions using 2 mM DTE and 100 mM of the lysine analogue, EACA. This procedure caused the cleavage of the interchain disulfide between one of the unpaired cysteines (Cys- 3734,4190,4300)of apoBlOO and the unpaired Cys-4057 in apo(a) kringle IV-9 without an apparent disruption of the intrachain disulfides in the kringles of apo(a).
- apo(a) The isolation of a water-soluble, functionally competent apo(a) is an important development particularly when one considers that essentially all apo(a) in the plasma is covalently linked to apoB 100 and that much of the current information on apo(a) has been derived from results on recombinant products which may not necessarily reflect the properties of "native" apo(a).
- This development was dependent on various factors: 1) the use of very low concentrations of DTE; 2) the presence in the reaction mixture of EACA forthe pu ⁇ oseof inhibiting the reassociation between apo(a) and LDL, 3) at the end ofthe reaction, the use of sucrose in order to achieve the necessary medium density for separating apo(a) from the LDL moiety by ultracentrifugal flotation.
- sucrose had to be used because in its absence, apo(a) came out of solution at the NaCl concentrations required to float LDL and any unreacted Lp(a). Moreover, sucrose proved to be a good stabilizing factor in storing free apo(a) at -80°C.
- the ability to prepare relatively large amounts of free apo(a) of a defined phenotype, provides a powerful tool for gaining a broader knowledge on the stmctural properties of this unique glycoprotein and for defining, on a physiological level, its role in the process of Lp(a) assembly.
- the lys/pro binding domain plays a dominant role in Lp(a) assembly (see FIG. 5). This would explain why human subjects and rhesus monkeys with a functionally defective LBS in kringle IV- 10 are competent to form Lp(a).
- lys/pro domain spans the apo(a) region between kringle IV-4 and kringle IV-9 including the interkringle linkers. This would be in keeping with the finding that the kringle IV-2 repeats have no affinity for lysine (Li et al, 1992) and are unable to sustain Lp(a) assembly (Ernst et al, 1995; Frank et al, 1994a, b).
- VLDL is competent to form a covalent complex with apo(a) and corroborates the results of previous studies demonstrating the occurrence in human plasma of apo(a) linked to triglyceride-rich lipoprotein particles (Bersot t al, 1986; Scanu et al, 1992; Selinger et al, 1993).
- Lp(a) represents a broad class of lipoprotein particles, both cholesteryl ester and triglyceride-rich having as a protein moiety apoB 100 linked to apo(a).
- One aspect of the present invention was to determine whether a reassembly defect could account for the very low plasma levels of Lp(a) present in the human mutant with kringle IV- 10 Arg72 in contrast to the normal levels exhibited by the rhesus monkeys with the same substitution.
- Behind this hypothesis was the assumption that free apo(a) incompetent to affiliate with the apoBlOO of LDL. would be cleared from the plasma at a comparatively higher rate than Lp(a).
- this hypothesis was not supported by the experimental findings.
- the apo(a) present in the plasma of the human mutant was in the form of Lp(a).
- the free apo(a) was not supported by the experimental findings.
- Lp(a) obtained by the mild reduction of Lp(a) from either the human mutant or the rhesus monkey, when mixed with either human or rhesus LDL formed an Lp(a) complex with comparable kinetics.
- the LDL obtained by the reduction of either the human mutant or rhesus Lp(a) was able to restore an Lp(a) complex when mixed with wild-type free apo(a).
- apo(a) can be readily dissociated from L ⁇ (a) in vitro and that the resulting native apo(a) has the capacity to covalently link again with apoBlOO to reconstitute either CE-rich or TG-rich lipoproteins.
- the domain in apo(a) involved in the reassembly process is spatially removed from kringle IV- 10 which is responsible for the binding of Lp(a) to lysine-SepharoseTM.
- Another aspect of the invention is the defining of the hydrodynamic and conformational properties of free apo(a).
- This example illustrates the properties of free apo(a). obtained by the novel methods of Example 1.
- hydrodynamic studies use a Beckman Optima XLA analytical ultracentrifuge with the analyses being conducted on apo(a) in the absence and in the presence of EACA and proline.
- the rationale for using these two reagents is that both interfere with the Lp(a) reassembly process and it was necessary to determine whether this would directly affect the stmctural properties of apo(a).
- Common hydrodynamic parameters are measured, i.e., sedimentation and diffusion coefficients, viscosity and Stokes radius.
- the molecular weight of apo(a) is determined in phosphate buffer at pH 7.2 as a function of apo(a) concentration in order to assess whether apo(a) is monomeric or self-associated in solution.
- the Stokes radius of the monomer is determined either from the diffusion coefficient or by combining the sedimentation equilibrium with the sedimentation velocity data.
- viscosity measurements may be carried out in a Cannon-Manning Semi-micro ELC 50 viscometer. Based on viscosity values, it is possible to assess the overall conformation of apo(a), i.e.. whether the protein is flexible and extended (high intrinsic viscosity between 10 and 30 cm 3 /g) or is in a compact and rigid stmcture similar to globular proteins (intrinsic viscosity between 3.3 and 4.0 cm 3 /g). The effect on this conformation by increased concentrations of EACA and L- proline may also be determined.
- the secondary stmcture analyses of apo(a) in the presence and absence of EACA and proline is carried out on a Jasco J-600 spectropolarimeter(Jasco, Japan) and analyzed using the Jasco-700 software after converting the data by the Softsec File conversion program (Softwood Co., CT). Spectra are recorded at protein concentrations ranging from about 0.1 mg/ml to 2 mg/ml in cuvettes of 0.01 to 0.1 cm path length.
- Mean residue ellipticities are calculated using a mean residue weight of 112.8 for apo(a).
- the secondary stmcture content is calculated by two methods, one using the program VARSLC 1 starting with a set of 33 reference proteins (Manavalan and Johnson, 1987), and the other, the program CONTIN (Provencher and Glockner, 1981 ). All samples are dialyzed against Buffer A with or without EACA or proline but without PMSF (antiproteolytic agent) since this reagent interferes with CD abso ⁇ tion in the far ultraviolet region of the spectrum. The effect of size polymo ⁇ hism on the hydrodynamic and conformational properties of apo(a) may also be determined.
- VARSLC 1 The secondary stmcture content is calculated by two methods, one using the program VARSLC 1 starting with a set of 33 reference proteins (Manavalan and Johnson, 1987), and the other, the program CONTIN (Provencher and Glockner, 1981 ). All samples are dialyzed against Buffer A with or without EACA or proline but without PMSF (antiproteolytic agent) since this reagent interferes with CD abs
- This example illustrates the effects of elastase enzymes on apo (a) and further relates to the stmctural and functional properties of fragments from proteolytic cleavage.
- porcine pancreatic elastase Studies with porcine pancreatic elastase.
- porcine elastase The choice of porcine elastase, is based on previous studies showing that this enzyme cleaves plasminogen into two lysine binding fragments and a domain called mini plasminogen, acting at the level of the small neutral amino acids located in the interkringle regions (Sottrup- Jensen et al, 1978). Studies have shown that when apo(a) is digested by porcine elastase it generates three fractions, one of which is able to bind to lysine-SepharoseTM.
- This example illustrates the analysis of apo(a) and Lp(a) by comparing the fragments obtained from the elastase digestion of Lp(a) and apo(a) and identifying the specific apo(a) region which interacts with apoB 100 and fibrin.
- apo(a) is incubated with porcine pancreatic elastase (EC 3.4.21.36) at a molar ratio of 25: 1 (apo(a):elastase) in 50 mM Tris-HCl. 100 mM NaCl, pH 7.5 for 2 hr at room temperature. The reaction is terminated by the addition of 5 mM DFP and the product immediately applied to a lysine-SepharoseTM column equilibrated with 10 mM PBS, pH 7.5. The column is then washed with 3 column volumes each of PBS. 0.5 M NaCl and 200 mM of the lysine analogue, EACA.
- Each eluted fraction is analyzed on Westem blots of reduced and non-reduced SDS-PAGE (4-20% polyacrylamide gradient, Novex precast gels) and probed with monospecific antibodies directed against whole apo(a) and kringle V.
- gels are stained with Coomassie Blue to identify fragments that would not be recognized by the antibodies.
- those fractions exhibiting size heterogeneity are purified by molecular sieving chromatography using gel media of appropriate pore size (SephadexTM, SephacrylTM or SuperoseTM; Pharmacia).
- Lp(a) is digested with porcine elastase as outlined for apo(a).
- the digest is centrifuged at d 1.21 g/ml in order to separate the floating fractions containing lipids from those which are lipid-free protein (sedimenting fraction).
- the floating and sedimenting fractions are dialyzed in membranes of 10,000 kDa and 3,500 kDa cutoff, respectively, against PBS.
- each fraction is applied to a lysine-SepharoseTM column and the homogeneity of the eluted fractions determined by SDS-PAGE in 4-20% and 3.5% gels (the 3.5% gel is used since some of the fractions will contain an apoBlOO: apo(a) complex too large to resolve on the 4-20% gradient gel).
- the fractions are further purified following the procedure described above for apo(a) except that, in addition, the gels are probed with antibodies directed against apoB 100.
- thermolysin In parallel with elastase studies, studies are also performed with thermolysin, which also cleaves at the level ofthe neutral amino acids. Studies by Huby et al. (1994; 1995) have previously shown that digestion of Lp(a) by this enzyme releases fragments identified as the N-and C- terminal portions of the apo(a) in Lp(a).
- Lp(a) or apo(a) are digested with thermolysin (from Bacillus thermoproteolyticus, 40 units/mg of lyophilisate; Sigma) at 37°C for 30 min in 125 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl 2 , 0.01% EDTA and 0.01% NaN 3 , pH 8.0 at a thermolysin:apo(a) mass ratio of 1 :500.
- the reaction is stopped with 10 mM EDTA, and the purification of the resulting fragments is performed in the same manner as described for the elastase digested apo(a) and Lp(a). 2. Structural and functional characterization of the purified enzymatic digests.
- the F2-reassembled particle migrated electrophoretically as a single band which was immunostainable by both anti-apo(a) and anti-apoBlOO.
- anti-apo(a) staining revealed a band corresponding to fragment F2; in turn, immunostaining with anti-apoBlOO showed one band which migrated in the position of apoBlOO.
- MiniLp(a) obtained from the elastase digestion of Lp(a), exhibited fragments of apoBlOO. Therefore, in functional studies the inventors utilized R-miniLp(a) particles where the fragmentation of apoBlOO was absent.
- R-miniLp(a) appeared somewhat more homogeneous in size than native Lp(a) or miniLp(a).
- a EACA-inhibitable, or lysine-mediated binding obtained by subtracting the binding in the presence of 0.2 M EACA from the total binding.
- b Total binding since in the presence of 0.2 M EACA the binding to fibronectin was not significantly affected.
- c Kd and Bmax were calculated as described in Material and Methods. The values are expressed as mean ⁇ SD from at least three independent studies in duplicate.
- Fibronectin binding F2 and R-miniLp(a) bound to immobilized fibronectin in a saturable, concentration-dependent manner (FIG. 10B) and exhibited apparent Kds comparable to those obtained with apo(a) and Lp(a) (Table 7). In contrast, Fl failed to bind to fibronectin. of note, EACA did not affect the binding. As in the case of fibrinogen. the B max was significantly decreased when F2 and apo(a) were complexed to LDL (Table 7). Based on these results, the inventors concluded that F2 contains binding sites for both fibrinogen and fibronectin and that these sites are partially hidden when this fragment associates with LDL.
- fragments are incubated with 100 mM of EACA or proline for 60 min at 37°C before the addition of LDL and the incubation continued for an additional 6 h.
- an aliquot (125 ⁇ l) of the reaction mixture is diluted with an equal volume of 60% sucrose in buffer A containing 200 mM EACA and spun in a TLA100 rotor (tube capacity, 250 ⁇ l) at 100,000 ⁇ m at 15°C for 18 h using a TL 100 ultracentrifuge (Beckman Instmments, C A).
- the top fraction ( 105 ⁇ l) is removed and quantitated by ELISA designed to measure the apoB 100:apo(a) complex.
- the bottom 100 ⁇ l containing free apo(a) is quantitated by a sandwich ELISA specific for apo(a) using affinity-purified polyclonal anti-apo(a) for coating and alkaline phosphatase-conjugated anti-apo(a) for detection.
- the results are expressed as the percent ofthe total apo(a) that reassembled to an apoB 100:apo(a) complex in 6 h.
- Fibrin binding The binding assays are performed in triplicate according to the procedure of Ha ⁇ el et al. ( 1989) with some modifications. 96-well plates are incubated with 1 mg/well fibrinogen (Sigma) in Tris buffered saline (TBS) for 2 h at 37°C. After the wells are emptied, 2% BSA in TBS buffer is added to the plates for 1.5 h at 22°C. The wells are washed three times with TBST buffer (TBS buffer complemented with 0.02% TweenTM-20) and further treated with 3 ng/well of plasmin (Enzyme Research Laboratories) for 40 min at 37°C.
- TBS Tris buffered saline
- Plasmin is inactivated by incubation of the wells with TBST containing the protease inhibitor /?-nitrophenyl/?'-guanidinobenzoate(Sigma) at a final concentration of 0.1 mmol/L for 20 min at 22°C. After two additional washes with TBST, various concentrations of purified fragments from digested apo(a) and Lp(a) diluted with TBS with or without 200 mM of EACA or L-proline are added and incubated ovemight at 22°C. Thereafter, the wells are washed three times with TBST. rabbit anti-apo(a) semm (1 :500) added and incubated for 1 h at 22°C.
- constmcts are prepared where amino acids are either deleted or mutated and then expressed in CHO cells and human embryonic kidney 293 cells (HEK).
- the choice of CHO cells is based on the work by Frank et al (1994; 1994) who expressed in these cells constmcts of apo(a) varying in size from 3 to 18 kringle IV repeats. These cells have already been used in the expression of wild-type and mutated (T ⁇ 72 ⁇ Arg) human kringle IV- 10.
- the HEK 293 cells were utilized by Van der Hoek et al.
- PCRTM amplification using primers derived from the published apo(a) cDNA sequence are used for cloning into the CMV-promoter driven mammalian expression vector pcDNA3 (Invitrogen).
- the expression plasmids are transfected into CHO or HEK293 cells and selected for neomycin resistance at 400 mg/ml.
- the expression level of the secreted products is determined by ELISA assays of the conditioned medium using monospecific anti-apo(a) antibodies.
- the expressed wild ⁇ type and mutated fragments are purified by a combination of molecular sieve chromatography and affinity chromatography using an anti-apo(a) SepharoseTM column.
- the expressed apo(a) fragments are tested for their capacity to bind to either apoB 100 or fibrin using the techniques already outlined.
- the inventors' studies have shown that limited proteolysis by pancreatic elastase cleaves human apo(a) at the Lle3520-Leu3521 bond located in the linker between kringles IV-4 and IV-5. Since the same cleavage pattern was obtained with both apo(a) and Lp(a), it is apparent that the elastase-sensitive site is not hindered by the linkage of apo(a) to LDL, suggesting that enzyme site accessibility may depend on the intrinsic properties of the linker between kringles IV-4 and IV-5. It is interesting to note that the cleavage by elastase occurs at the end of the ⁇ -structure region where the neutral amino acid He appears more exposed and available. The Ile-Leu cutting site is consonant with the known specificity for neutral amino acids by the enzymes of the elastase family (Barrett and McDonald, 1980).
- mice The choice ofthe mouse as the animal model is based on the fact that this a null species in terms of apo(a) and it is easy to manipulate experimentally. Moreover the mouse has been used productively in transgenic studies.
- mice are anesthetized with Metafane and injected into the tail vein with 25 mg of either pure apo(a) or Lp(a). Each mouse is under anesthesia at each bleed time point. Seven bleedings, three from the right and three from the left orbital vein and one from the heart are carried out. Bleeds are performed after 1, 15, 30, 120, 180, 300 and 420 minutes. The amount of blood at each interval is 50 ⁇ l collected in heparinized hematocrit tubes. The plasma is separated by centrifugation and used for ELISA specific for human apo(a). The inventors carried out ultracentrifugal and gel electrophoretic analyses in order to define whether the injected products are present in the mouse plasma as free or LDL-bound apo(a). The kinetic behavior of the products injected is determined by standard techniques.
- FIG. 1 1 shows a semi-log plot of the percent of injected dose as a function of time. The slopes of the curves between 3 and 7 h could be separated into three groups: R-miniLp(a) with the slowest clearance, Lp(a) and F2 intermediate and apo(a), the unfractionated apo(a) digest and Fl the fastest.
- Lipoproteins, apolipoproteins, or the fragments purified from digested apo(a) were prepared as outlined above and injected into the tail vein of normal female mice. Plasma decays were followed for 24 h by quantitative ELISA. dumber of mice. Values are means ⁇ SEM.
- c Apo(a) was digested with elastase under limited conditions as outlined above. The whole unfractionated digest was dialyzed against 10 mM phosphate buffer, pH 7.2, filter sterilized and injected into the tail vein of mice.
- the inventors also examined on anti-apo(a) immunostained blots of reduced 4% gels, the plasma samples taken one hour after injection into the mouse of Lp(a) and derivatives. Compared to the electrophoretic patterns before injection of Lp(a) and apo(a). those after injection contained new bands in addition to the major apo(a) component, suggesting that a degradation process had occurred in vivo. It should be noted that the extent of Lp(a) and apo(a) degradation in plasma was less than 5%, and that the majority of the injected material remained intact. The banding pattern of Fl before and after injection was comparable in banding pattern but ncreased in band intensity. On the other hand, after injection, F2 exhibited an additional faster migrating band designated by an arrow.
- the inventors next examined the urine of mice 0-5 h after injection of Lp(a) and derivatives by anti-apo(a) immunostained blots of reduced 4-12% gradient gels.
- the urinary patterns of Lp(a), apo(a) and Fl showed a broad spectrum of bands, three of which were comparable, migrating at 85, 57 and 33 kDa, respectively.
- the urinary pattern after injection of F2 exhibited two major bands migrating in a more narrow size range (70 to 62 kDa).
- KV specific ELISA none ofthe urine samples contained KV.
- the Fl pattern was characterized by several bands which were observed as early as one hour after injection, and by ELISA, represented less than 0.5% of that injected.
- the F2 pattern was characterized by fewer bands which appears later (5 h) and represented less than 0.5% of the injected material. 2.
- apo(a) is labeled with I and Lp(a-) with I according to procedures established in that laboratory. Aliquots of these two radiolabeled products are then injected intravenously into normal volunteers in order to obtain data on the kinetics of these two Lp(a) components.
- I apo(a) and I Lp(a-) is first mixed to reconstitute a double-labeled Lp(a) which is then injected intravenously into normal volunteers.
- These kinetic studies allow the inventors to determine whether Lp(a) is catabolized as a lipoprotein particle or whether apo(a) dissociates and is metabolized independent of the LDL component of Lp(a).
- Example 3 above described the functional divergence between the F2 and Fl fragments generated by elastase cleavage of apo (a).
- An important difference between Fl and F2 was also unveiled by the studies in which mice were injected intravenously with these two fragments. Based on ELISA and gel analyses, the injected F12 had a short residence time in the plasma and was also rapidly excreted in the urine in the form of several fragments. It was interesting to note that the relatively homogeneous Fl after injection into the tail vein of the mouse, appeared in the plasma as distinct 4-5 electrophoretic bands in the size range of 220-135 kDa and as markedly smaller ones in the urine (size range 100-33 kDa).
- injected F2 had a comparatively longer plasma residence time and was excreted in rather minute amounts in the form of fragments which could only be detected in 15-30 fold urine concentrates.
- the metabolic divergence between Fl and F2 was also documented by the studies in which the whole unfractionated elastase digest of apo(a) was injected into the mouse showing again that most of the rapidly excreted apo(a) urinary fragments were of Fl derivation.
- apo(a) fragments of the Fl type were also detected in 3-10 fold urine concentrates of mice injected intravenously with preparations of Lp(a) or apo(a) which had not been previously digested with elastase. Contrary to the relative homogeneity of these materials prior to injection, several bands were also present in the plasma of the injected mice. Taken together, these data show the normal mouse may have an active elastase-like system capable of cleaving Lp(a) or apo(a). This inte ⁇ retation may appear at variance with the results of the studies by Mooser et al.
- mice provide a useful basis for inte ⁇ reting the results of their studies in normal human subjects.
- fragments of apo(a) are spontaneously present in their plasma.
- ELISA they_ represented about 5% of the total plasma Lp(a) protein and were significantly larger than those in the urine.
- the fragments in the urine had the size and band pattern of those seen in the urine of injected mice.
- the results invite the speculation that in both animal species these fragments might have derived from the action of elastase-like enzymes probably from the formed elements of the blood. Accordingly, both mice and man would have an enzymatic make-up able to digest a small portion of the total apo(a) mass.
- mice Even though the mouse is not an apo(a) animal, it produces plasminogen, a five kringle zymogen, which has a high degree of homology with apo(a) (McLean et al, 1987).
- elastase is known to cleave plasminogen in vitro in a predictable manner (Sottrup- Jensen et al, 1978; Castellino et al, 1990); moreover, recent studies have shown that plasminogen fragments are present in mouse urine (O'Reilly et al, 1994). The latter studies are of interest in that those fragments were found to have angiostatic action.
- apo(a) proteolysis would be more likely to occur at the level of cell membranes of polymo ⁇ honuclear cells, platelets, or macrophages. Accordingly, elastase-dependent apo(a) fragmentation would be expected to occur under pathological conditions, for instance at sites of inflammation involving an active recruitment of polymo ⁇ honuclear cells and macrophages.
- apo(a) fragments has been reported in human atherosclerotic lesions (Hoff et al, 1994). The present studies show that these fragments would be of the F2 type and thus unaffected by the Fl -dependent size polymo ⁇ hism of apo(a). As a corollary to this, F2 would be more pathogenic than Fl from the cardiovascular standpoint.
- the Lp(a) in plasma is known to be polymo ⁇ hic in size and density. This polymo ⁇ hism may be contributed by both the size/density of the apoB 100-containing lipoprotein particles and by the size of apo(a). In order to determine the relative contribution by each of these two components to Lp(a) assembly, the following studies are performed.
- LDL in Lp(a), or Lp(a-) may differ from that of autologous LDL in that Lp(a-) has a higher molecular weight (about 12% higher)), a lower buoyant density and a significant difference in lipid composition (mol/mol), in particular, a higher triglyceride content (Fless et al, 1986).
- Lp(a-) has a higher molecular weight (about 12% higher)), a lower buoyant density and a significant difference in lipid composition (mol/mol), in particular, a higher triglyceride content
- apoBlOO in Lp(a) derives from a pool that is metabolically different than that of apoBlOO in autologous LDL.
- LDL dissociated from Lp(a) and autologous LDL both isolated from the same subject, for their ability to interact with apo(a) and form R-Lp(a).
- Lp(a-) is prepared from the disassembly of Lp(a) by mild reduction with DTE and further purified from unreduced Lp(a) by lysine-SepharoseTM chromatography. The Lp(a-) will elute in the unbound fraction.
- LDL is isolated from the autologous plasma by sequential flotation at d 1.030 - 1.050 g/ml. The purity of the lipoproteins is ascertained by Westem blot analyses of SDS-PAGE using mono-specific antibodies against apo(a) and LDL and their chemical composition determined. Comparisons are made on the percent of the total apo(a) that reassembled with apoB 100 after a 6 h incubation and the effect of EACA and proline.
- LDL of d 1.019-1.063 g/ml is dialyzed to d 1.04 g/ml in NaBr at 4°C with four solution changes over 24 h and further fractionated as described by Tribble et al ( 1992).
- Dialyzed LDL (3.8 ml) is layered over a NaBr solution of d 1.054 g/ml (4.7 ml) in a
- Plasma from normolipidemic subjects containing Trasylol 10,000 KlU/ml
- EDTA (1 mM), NaN 3 (0.01%) and PMSF (1 mM) is centrifuged at d 1.006 g/ml for 20 h at 15°C at 45,000 ⁇ m.
- the top floating fraction is further fractionated into three subclasses of S f 100-400, S f 60-100 and S f 20-60 by Lindgren's method (Lindgren et al, 1972) and the chemical composition of each subfraction determined. From the studies of Gianturco and Bradley (1986) it is known that surface composition and reactivity of VLDL from hyperlipidemic subjects may differ from that of normolipidemic VLDL.
- LpL are isolated from bovine raw milk and purified by heparin-SepharoseTM chromatography by methods previously established (Chung and Scanu, 1977). The activity of the stock LpL are assayed according to Iverius and Lindquist-Ostland(1986) using a radiolabeled triolein emulsion as substrate.
- VLDL incubations are carried out in 10 mM Tris-HCl buffer with 150 mM NaCl, pH 7.5 containing 6% fatty acid-poor albumin in covered polypropylene tubes.
- Lipolysis of VLDL is initiated by adding LpL and the mixture incubated in a shaking thermostatically controlled 37°C water bath for various time periods (0-120 min). The reaction is terminated by inactivating the enzyme with 2 mM diethyl-/ nitrophenyl phosphate (E600) (Williams et al, 1992). The extent of lipolysis is monitored by measuring the levels of free fatty acids before and after incubation using the NEFA C test kit from Wako Chemicals, Inc. Dallas, TX.
- This enzymatic test is insensitive to glycerol, is highly reproducible, rapid (1 h) and detects as little as 0.25 mEq fatty acid/L.
- the lipolyzed VLDL is then separated from any unreacted VLDL and albumin by flotation as described above and the composition determined.
- the inventors initiated a study of the hydrodynamics of Lp(a). To this end the inventors measured the sedimentation coefficient, partial specific volume, molecular weight and in some instances the diffusion coefficient of homogenous Lp(a) species with defined apo(a) mass is thought to contribute to Lp(a) density, which in turn has a direct effect on the sedimentation rate of Lp(a) in a centrifugal field, the inventors measured the partial specific volume (which is the reciprocal of the Lp(a) particle buoyant density).
- Lp(a) mass does not correlate with apo(a) mass or kringle number.
- Frictional ratios i.e., f/fo values were calculated for each Lp(a) phenotype assuming a hydration of 0.37 g H 2 O / g Lp(a).
- f/fo values did not correlate with apo(a) mass or kringle number.
- the mean f/fo of six different Lp(a) phenotypes was calculated to be 1.23 ⁇ 0.05.
- the mean f/fo of two LDL preparations that were assumed to be hydrated to the extent of 0.34 g H 2 O / g LDL was 1.01.
- the reduction in the magnitude of the sedimentation coefficient is proportional to the concentration of EACA. Assuming that n EACA molecules bind to Lp(a) at equivalent binding sites the inventors were able to fit plots of sedimentation rate vs. EACA concentration and derive the dissociation constant and number of binding sites. The mean dissociation constant was 3.7 x 10 " and the mean number of EACA molecules bound was 4.8. The magnitude of the change in the sedimentation rate was directly proportional to the mass of apo(a) or number of kringle 4 domains. For Lp(a) phenotypes with large apo(a) polymo ⁇ hs, this change represented almost a 50% decrease in the sedimentation rate.
- the inventors also calculated the frictional ratio of Lp(a) from the minimum sedimentation rates achieved at maximal EACA concentrations. These ratios were directly proportional to apo(a) kringle 4 number and had a regression coefficient of 0.996. This was in contrast to the native or compact form of Lp(a) which did not exhibit a similar correlation. Assuming a hydration of 0.37 g H 2 O / g Lp(a), frictional ratios of Lp(a) increased in magnitude linearly from 1.4 to 2.1 for particles with the smallest to the largest apo(a) polymo ⁇ h.
- SDS-gel electrophoresis is used routinely in the characterization of apo(a) polymo ⁇ hs, and apo(a) molecular weights are frequently obtained with the use of apoB-100 in addition to high molecular weight standards from either BioRad or Pharmacia.
- Using apoBlOO as a standard is also problematic, as its mobility on polyacrylamide gels is anomalously fast, leading to apo(a) molecular weights that are exaggerated.
- the inventors also investigated 4% acrylamide gels using the Laemmli buffer system because of their wide use in the field, although the inventors were aware that the crosslinked phosphorylase B standards were not compatible with this system. The inventors found that the gels had to be overloaded with phosphorylase B for band visualization. These gels also overestimated the molecular weight of apo(a) by 20-25%, and underestimated that of apoB by 40%.
- the inventors focused their studies on the Lp(a) isolated from the plasma of subject KB exhibiting two apo(a) alleles (70 and 90 kb), determined by pulse field gel electrophoresis and two apo(a) isoforms (289 and 488 kDa).
- the Lp(a) species were isolated by lysine-SepharoseTM and then repurified by density gradient ultracentrifugation. In each case the inventors observed a broad density profile spanning from d 1.030 to 1.090 g/ml with three main components of average peak density of 1.032, 1.052 and 1.078 g/ml. The first two fractions contained the 289 kDa isoform and the third, more dense, the 488 kDa isoform. Each of Lp(a) species were subjected to the action of LpL.
- the products were separated by ultracentrifugation in a performed density gradient, 0- 12% NaBr, the pertinent fractions combined and examined for their chemical composition and also by SDS-PAGE followed by Westem blotting using a mono ⁇ specific Lp(a) antibody.
- d l .055 g/ml.
- Table 5 lists the chemical composition of this Lp(a) species before and after lipolysis. Most significantly, the TG content was reduced by 30% while the remaining lipids were unaffected.
- This Lp(a) particle had the 488 kDa isoform and was the most dense of the particles that the inventors examined. Upon sample lipolysis the density gradient ultracentrifugal profile showed a peak shift to a higher density without changes in peak width.
- Lp(a) heterogeneity is any partially controlled by the apo(a) gene and that the lipids, and in particular the core TG, are important contributors to lipoprotein density.
- the inventors' studies also indicate that LpL is a main modulator of the TG-rich Lp(a) as is the case for apoB 100-containing lipoproteins (VLDL, IDL and light LDL) suggesting that at least some of the LpL species in the plasma are generated via the lipolytic conversion of TG-rich Lp(a) to CE-Lp(a). The functional properties of these various Lp(a) species and clarification.
- the inventors have been able to prepare in vitro a functionally competent apo(a) starting from human Lp(a) preparations of a defined size, density, composition and apo(a) phenotype.
- the disulfide between apoBlOO and apo(a) is more accessible to cleavage that the three intrachain disulfides that stabilize each apo(a) kringle
- the inventors subjected Lp(a) purified from plasma to reduction with 2 mM dithiothreitol (DTE), in the presence of 100 mM of the lysine analog ⁇ -aminocaproic acid (EACA).
- the dissociated apo(a) isolated by sedimentation in a sucrose solution of d 1.127 g/ml, retained its binding capacity for lysine-SepharoseTM and also interacted with cholesteryl ester (CE)-rich, low density lipoproteins (LDL) to restore a complex, CE-Lp(a), which was indistinguishable from the parent Lp(a) in chemical conformation (circular dichroism) and size (electron microscopy).
- CE cholesteryl ester
- LDL low density lipoproteins
- the theoretical number of fully reduced cysteines was calculated to be 139.
- the isolated apo(a) examined by far-ultraviolet CD spectroscopy gave a spectrum characterized by a strong negative band at 203 nm and a positive band at 222-232 nm.
- the analysis of the spect m by the CONTIN and VARSLCI methods indicated 0-2% ⁇ -helix, 66% ⁇ -structure and the remainder 32% in mainly random conformation.
- apo(a) of a defined phenotype was incubated with an homologous preparation of LDL at 37°C.
- the amount of Lp(a) formed was quantitated by ELISA designed to measure the apoB100:apo(a) complex. Westem blots probed with antibodies directed against apo(a) and apoBlOO, showed that the band corresponding to the reassembled product contained both apoB 100 and apo(a), even though the sample had been boiled in SDS prior to gel electrophoresis, suggesting a covalent association between apoBlOO and apo(a).
- the reassembly of Lp(a) was hampered by the presence of high molarity (100 mM) solutions of either EACA or proline.
- the inhibitory effect was dose dependent until reaching the 500 mM concentration, when there was an almost 90% level of inhibition.
- EACA appeared to be a relatively more potent inhibitor than proline.
- the inventors incubated apo(a) with 100 mM each of EACA and proline prior to incubation with LDL. Under these conditions the inventors obtained a two-fold inhibition of the Lp(a) reassembly, as compared to the incubation with a single inhibitor.
- the inventors have previously shown that an apo(a) linked to apoBlOO can be found in triglyceride-rich particles isolated from hyperlipidemic plasma.
- the inventors examined the in vitro interaction of apo(a) with a single apo(a) isoform (289 kDa) with preparation of VLDL isolated from the plasma of two hypertriglyceridemic subjects with type IV dyslipoproteinemia and very low levels of plasma Lp(a) protein, i.e., 0.1 and 0.3 mg/dl and TG levels of 300 and 277 mg/dl, respectively.
- the experimental conditions for the reassembly were as described previously using a 100:1 VLDL apoB100:apo(a) weight ratio.
- rLp(a) had the same mobility as control Lp(a) indicating that both had a similar size or Stoke " s radius.
- the conformation of rLp(a) closely resembled that of native Lp(a).
- the inventors have previously identified human mutants having a Lys " Lp(a) and Arg72 of T ⁇ 72 (Lys + , wild-type) in the LBS of kringle IV- 10.
- the inventors have compared the behavior of Lp(a) and apo(a) (obtained by mild reduction as described above) from human wild-type (WT) and mutant (M) subjects with respect to their binding to lysine-SepharoseTM and PM-fibrinogen. Lysine-SepharoseTM affinity chromatography showed that contrary to WT, the Lp(a) from the M subject failed to bind.
- Lp(a) and apo(a) were digested with pancreatic elastase under conditions of limited proteolysis as described. Lp(a) and apo(a) containing a single phenotype were incubated with porcine pancreatic elastase in 50 mM Tris-HCl containing 100 mM NaCl, pH 8.0. The apo(a):elastase ratio was 50: 1 (mol/mol). The digestion was allowed to proceed for 5 h at room temperature with gentle stirring. The reaction was terminated by the addition of diisopropylfluorophosphate(DF) to 5 mM and incubated for an additional 30 min at room temperature.
- DF diisopropylfluorophosphate
- the mixture was applied to a lysine-SepharoseTM column previously equilibrated with 10 mM phosphate buffer containing 100 mM NaCl, pH 7.5. The column was then washed with 3 column volumes of equilibration buffer followed by three column volumes of phosphate buffer containing 500 mM NaCl and finally with 200 mM EACA.
- the Lp(a) and apo (a) digests were subjected to 4% SDS-PAGE immunoblot analysis using a rabbit anti- apo(a) polyclonal antibody.
- Immunostained gels were n under unreduced (left) and reduced (right) conditions.
- digested Lp(a) and apo(a) exhibited a common migrating band, designated as Fl .
- the banding patterns of digested Lp(a) and apo(a) were identical.
- Fl had an apparent mass of 220 kDa and was associated with a second band with an apparent mass of 170 kDa, designated F2, and a set of faint ones differing in size by approximately 20 kDa. which is the apparent size of a single kringle.
- Digested Lp(a) was diluted 1 : 1 (v/v) with 60% sucrose in buffer A containing 200 mM EACA to a final density of 1.127 g/ml and the mixture centrifuged in a TL 100 tabletop ultracentrifuge at 15°C, ovemight. Two fractions were collected; a top, (0.5ml), and a sedimenting one (1 ml). In the anti-apo(a) immunostained unreduced gels, the top fraction contained a band representing undigested Lp(a) and a second one which migrated faster than Lp(a). Both bands were also detected with an anti- apoBlOO antibody.
- the d 1.127 g/ml top was treated with 1.5 mM DTE in the presence of 100 mM EACA and centrifuged in 30% sucrose according to the method utilized by the inventors for the isolation of free apo(a) (Edelstein et al, 1995).
- the sedimenting lipid-free fraction contained a small amount of undigested apo(a) and F2.
- the latter was purified by molecular sieving on Superose 6.
- the isolated F2 exhibited a band with an apparent mass of 170 kDa, as detected by an anti-apo(a) antibody. Based on specific ELISAs.
- F2 contained KV but apoBlOO.
- the limited proteolysis of Lp(a) by elastase produced two fractions: one of them, referred to as Fl, was lipid-free, sedimented at d 1.127 g/ml, and reacted against anti-apo(a) but not against anti-KV or anti-aopBlOO; the second one, was lipid-rich, floated at d 1.127 g/ml and reacted against anti-apoB 100, anti-apo(a) and anti-KV. Mild reduction of this fraction generated a 170 kDa band, F2, reactive against anti-apo(a) and anti-KV and non reactive against anti-apoBlOO.
- the inventors inte ⁇ reted the above results to indicate that the d 1.127 g/ml floating fraction is a lipoprotein particle containing apoBlOO covalently linked to F2.
- the inventors called this lipoprotein, miniLp(a). This nomenclature was recently proposed by Huby et al. (1995) to designate a lipoprotein particle which they obtained by digesting Lp(a) with thermolysin. In those studies thermolysin caused hydrolysis of apoBlOO. In the current studies elastase also caused a partial cleavage of apoBlOO in Lp(a) and the resulting fragments remained associated with the lipoprotein particle (see Section on Functional studies).
- limited elastase digestion cleaves Lp(a) into a lipid-free fraction, Fl and a miniLp(a) particle in which F2 is linked covalently to LDL containing a partially hydrolyzed apoBlOO.
- Free apo(a) digested with pancreatic elastase was applied to a lysine- SepharoseTM affinity column which was then washed with three column volumes of PBS, 500 mM NaCl and 200 mM EACA (FIG. 6). Two major peaks were observed, one eluting with PBS and one with EACA. Electrophoretic analyses on reduced gels (4-12%) probed with anti-apo(a), showed that the unbound fraction eluting with PBS, represented Fl migrating in the 220 kDa position and the fraction, eluting with EACA represented F2, migrating in the 170 kDa position . Of the two fractions, only F2 reacted against anti-KV.
- FIG. 7 shows the partial NH 2 -terminal sequences of Fl and F2 obtained from the elastase digestion of Lp(a) and apo(a).
- the inventors located the cleavage site at the Ile3520-Leu3521 bond in the linker region between kringles IV-4 and IV-5. From these data the inventors concluded that the apo(a) fragment which eluted from the lysine-SepharoseTM column with PBS (FIG.
- mice were injected in the tail vein with 25 ⁇ g of apo(a) or Lp(a) in a total volume of 200 ⁇ l and bled at timed intervals from the orbital vein.
- Mouse plasma was analyzed for the levels of apo(a) or Lp(a) by a sandwich ELISA using mono ⁇ specific antibodies directed against apo(a), Lp(a) and human apoBlOO. Between 120 and 420 min after the initial injection the slope of the decay curve was linear. The T 1 2 value for this interval was significantly higher ( 10.5 h) for Lp(a) than for apo(a) ( 1.65 h).
- This mutation was found in a subject whose Lp(a) exhibited a lack of lysine binding.
- the mutation was localized to the lysine-binding pocket of the kringle.
- the lysine-binding pocket consists of a hydrophobic V-shaped trough which has the form of an elongated, open shallow depression located at the kringle surface.
- the depression is lined by aromatic rings Phe-64, Trp-62 and T ⁇ -72, the latter two oriented in an antiparallel manner to each other.
- zwitterionic ligands such as w-amino acids interact with the anionic and cationic sites at each end of the depression while the aliphatic backbone of each ligand is tubulized by its interaction with the hydrophobic trough.
- Modeling of the lysine-binding pocket in the mutant and wild-type kringle clearly showed that the Arg72 substitution in the mutant kringle prevented the docking of lysine in the binding pocket.
- This mutation is not associated with a lysine-binding and is present in about 45% of the subjects. Modeling of the wild-type and mutant kringle showed that these amino acids are at the kringle surface, remote from the lysine-binding site and thus should not affect lysine binding, in keeping with the results ofthe clinical studies.
- the inventors have successfully transfected both HepG2 and McA cells with apo(a) expression vectors encoding an apo(a) with 6, 10 or 17 repeats of the kringle 4 like domain. Each of these constmcts also encodes kringle 5 and the protease domain. Single clones have been selected and apo(a) secretion determined quantitatively by ELISA.
- apo(a) is synthesized as a precursor which undergoes extensive posttransiational processing, resulting in the mature form ofthe protein which can be identified beginning at 60 minutes of chase.
- Treatment with brefeldin-A completely blocked the processing of apo(a) into the mature form, consistent with its previously demonstrated effects in disrupting normal endoplasmic reticulum to Golgi transport of secretory proteins.
- cell lysates were prepared from either control or brefeldin-A treated cells and aliquots incubated with human LDL, followed by immunoprecipitation of apo(a). Lysates prepared from brefeldin-A treated cells were found to support the assembly of a B-(a) complex, indicating that the immature form of apo(a) is competent to associate with apoB 100.
- the apo(a) expression plasmid was transfected into several different CHO cell lines with well characterized defects in glycosylation.
- the apo(a) expression plasmid was transfected into the parental (Pro 5) and four defective CHO cell lines, which were subsequently radiolabeled.
- the radiolabeled media was mixed with LDL apoBlOO, followed by immunoprecipitation with anti-apo(a) antisera. All lines secreted apo(a). However, apo(a) secreted from the mutant CHO cells demonstrated altered electrophoretic mobility, as predicted from the differing degrees of glycosylation defect.
- Lp(a) has a defective in lysine and fibrin binding and is present in the plasma in very low levels ( ⁇ 1.0 mg/ml).
- the DNA isolated from peripheral blood and amplified by PCRTM demonstrated a T ⁇ 72 ⁇ Arg mutation in kringle IV- 10.
- the Met66 of kringle IV- 10 was replaced by Thr.
- the Thr— Met substitution caused no significant changes in the binding of Lp(a) to lysine/fibrin.
- Human wild-type and mutant (T ⁇ 72 ⁇ Arg) kringle IV- 10 has been expressed in both E. coli (non-glycosylatedform) and CHO cells (glycosylated form).
- the Arg72 mutant was prepared by introducing the T— > A mutation on apo(a) kringle IV- 10 amplified from human liver mRNA by the reverse transcriptase PCRTM technique. The yield of kringles from E. coli was significantly higher than that from CHO cells.
- the Met66— ⁇ Thr mutant has also been expressed on E. coli.
- the introns between the regions of kringle V and protease domain were amplified.
- the amplification primers were selected by assuming that the position of the three introns was the same as in the plasminogen gene.
- the primer set #1 amplified a 700 bp length fragment encompassing the area of kringle IV-37 exon b, intron 1 and part ofthe KV exon a.
- the primer set #2 amplified a 2.2 kb fragment encompassing part of KV exon a, intron 2 and part of the kringle V exon b.
- Primer set #3 amplified a 2.9 kb fragment encompassing part of the kringle V exon b, intron 3 and a part ofthe protease region exon.
- Each fragment was amplified by PCRTM by carefully selected conditions, then gel purified using a QI AEX® kit (Qiagen) and directly cloned into a TA vector (Invitrogen) using the "original" TA cloning kit.
- the positive clones were identified by blue/white color selection, digested with EcoRI and the digests sequenced by using the "fmol® DNA sequence system” (Promega) using as sequencing primers the same ones employed for PCRTM amplification.
- the sequences obtained for the kringle V and the protease regions corresponded to those previously reported from cDNA sequencing.
- the intronic regions were homologous but not identical to those on the plasminogen gene.
- the knowledge of the sequence of three introns allowed the generation of primers for the amplification ofthe exons coding for the KV and protease domains.
- Lp(a) The interaction of Lp(a) with these sites was time-dependent, specific, saturable divalent ion independent and temperature-sensitive, characteristics of plasminogen binding to these same sites.
- the affinity of plasminogen and Lp(a) for these sites also was similar (K d equal to 1-3 ⁇ M), but Lp(a) bound to fewer sites (10-fold less). This difference in the number of binding sites may reflect steric limitations in Lp(a) binding or the recognition of subpopulations of plasminogen binding sites.
- Lp(a) An additional and previously undescribed interaction of Lp(a) with cells involving a low affinity interaction with non-selective (recognizing both LDL and HDL) lipoprotein binding sites was also demonstrated. Binding of Lp(a) to these sites, as well as to plasminogen receptors, was inhibited by gangliosides but not the constituents of gangliosides. Thus, Lp(a) can interact with plasminogen binding sites on cells via its LBS, providing support for the hypothesis.
- Lp(a) is captured from plasma with a monoclonal antibody specific for apo(a). The immunocaptured Lp(a) is then reacted with anti-LBS. Binding of the anti-LBS antibody is quantitated with an alkaline phosphatase conjugated disclosing antibody. Using this assay, the LBS activity of an unknown Lp(a) sample can be quantitated relative to a reference Lp(a) standard (fully retained on lysine-SepharoseTM).
- LBS-Lp(a) values in plasma samples ranged from 0 - 100% of total Lp(a) levels.
- the LBS-Lp(a) activity correlated poorly with total Lp(a) concentrations in the plasma.
- the LBS-Lp(a) assay also has been used to assess whether metabolic modification alters LBS function. Lp(a) was preincubated with lipoprotein lipase, sphingomyelinase, phospholipase C, phospholipase A2 or thrombin was assessed for alterations of LBS activity. The most marked change in LBS activity was induced by phospholipase A2. which resulted in a 56% increase in the LBS activity.
- a two-compartment system with a monolayer of bovine aortic endothelial cells forming a barrier, was used to compare the transport and retention of Lp(a) and LDL into the subendothelial matrix. Baseline values for transport and retention of Lp(a) and LDL were not significantly different. Incubation with sphingomyelinase or lipoprotein lipase caused modest and similar increases (1.2-2.0-fold, respectively) in transport and retention of the two lipoproteins. In contrast, incubation with phospholipase A2 (PLA2), caused a 4-fold increase in retention of Lp(a) on the subendothelial matrix, but only a 2-fold increase in LDL retention.
- PDA2 phospholipase A2
- PLA2 did not have a differential effect on the binding of Lp(a) and LDL to the cells, suggesting that PLA2 increased retention of Lp(a) in the subendothelial matrix.
- PLA2 did not have a differential effect on the binding of Lp(a) and LDL to the cells, suggesting that PLA2 increased retention of Lp(a) in the subendothelial matrix.
- retention of Lp(a) on membranes (without cells) coated with extracellular proteins (fibronectin, laminin, or collagen 1 ) was 4-10 times higher than LDL.
- the increase in binding to the subendothelial matrix may be related to an enhanced LBS function, arising from the modification of Lp(a) by PLA2.
- compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept ofthe invention as defined by the appended claims.
- Huby et al "Characterization of the N-terminal and C-terminal Domains of Human apolipoprotein(a): Relevance to Fibrin Binding," Biochemistry, 34:7385-7393, 1995. Huby et al, "Stmctural Domains of apolipoprotein(a) and Its Interaction with apolipoprotein B-100 in the Lipoprotein(a) Particle," Biochemistry, 33:3335- 3341 , 1994.
- Macrophagesand Fibroblasts irter/o.sc/er. Thromb., 14:770-779, 1994.
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Cited By (5)
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US6210906B1 (en) | 1998-01-20 | 2001-04-03 | Abbott Laboratories | Specific antibodies to kringle 5 of apo(a) and methods of use therefor |
US7723508B2 (en) * | 2003-06-02 | 2010-05-25 | Isis Pharmaceuticals, Inc. | Modulation of apolipoprotein (A) expression |
US9574193B2 (en) | 2012-05-17 | 2017-02-21 | Ionis Pharmaceuticals, Inc. | Methods and compositions for modulating apolipoprotein (a) expression |
CN110850107A (zh) * | 2019-11-22 | 2020-02-28 | 武汉市长立生物技术有限责任公司 | 一种检测脂蛋白(a)分子浓度的试剂盒及其应用 |
US11634711B2 (en) | 2012-05-17 | 2023-04-25 | Ionis Pharmaceuticals, Inc. | Methods and compositions for modulating apolipoprotein (a) expression |
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
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US6210906B1 (en) | 1998-01-20 | 2001-04-03 | Abbott Laboratories | Specific antibodies to kringle 5 of apo(a) and methods of use therefor |
US7741305B2 (en) | 2001-08-07 | 2010-06-22 | Isis Pharmaceuticals, Inc. | Modulation of apolipoprotein (a) expression |
US8138328B2 (en) | 2001-08-07 | 2012-03-20 | Isis Pharmaceuticals, Inc. | Modulation of apolipoprotein (A) expression |
US7723508B2 (en) * | 2003-06-02 | 2010-05-25 | Isis Pharmaceuticals, Inc. | Modulation of apolipoprotein (A) expression |
US8673632B2 (en) | 2003-06-02 | 2014-03-18 | Isis Pharmaceuticals, Inc. | Modulation of apolipoprotein (a) expression |
US9574193B2 (en) | 2012-05-17 | 2017-02-21 | Ionis Pharmaceuticals, Inc. | Methods and compositions for modulating apolipoprotein (a) expression |
US11634711B2 (en) | 2012-05-17 | 2023-04-25 | Ionis Pharmaceuticals, Inc. | Methods and compositions for modulating apolipoprotein (a) expression |
US11859180B2 (en) | 2012-05-17 | 2024-01-02 | Ionis Pharmaceuticals, Inc. | Antisense oligonucleotide compositions |
CN110850107A (zh) * | 2019-11-22 | 2020-02-28 | 武汉市长立生物技术有限责任公司 | 一种检测脂蛋白(a)分子浓度的试剂盒及其应用 |
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