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WO1996002566A9 - Vitronectin binding protein - Google Patents

Vitronectin binding protein

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Publication number
WO1996002566A9
WO1996002566A9 PCT/SE1995/000861 SE9500861W WO9602566A9 WO 1996002566 A9 WO1996002566 A9 WO 1996002566A9 SE 9500861 W SE9500861 W SE 9500861W WO 9602566 A9 WO9602566 A9 WO 9602566A9
Authority
WO
WIPO (PCT)
Prior art keywords
protein
vitronectin
binding protein
quinquedecapeptide
binding
Prior art date
Application number
PCT/SE1995/000861
Other languages
French (fr)
Other versions
WO1996002566A1 (en
Filing date
Publication date
Priority claimed from SE9402490A external-priority patent/SE9402490D0/en
Application filed filed Critical
Priority to EP95926088A priority Critical patent/EP0776336A1/en
Priority to JP8504958A priority patent/JPH10505581A/en
Publication of WO1996002566A1 publication Critical patent/WO1996002566A1/en
Publication of WO1996002566A9 publication Critical patent/WO1996002566A9/en

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Definitions

  • the present invention relates to a vitronectin binding protein, a method for preparation thereof, a quinquedecapeptide that is useful for preparing the protein, an antibody against the protein, pharmaceutical compositions and a diagnostic or analytical kit comprising said protein, use of the protein for the preparation of a pharmaceutical composition, a diagnostic or analytical kit comprising an antibody against said protein and a method for purifying vitronectin.
  • Vitronectin is a plasma protein that occurs in concentrations around 300 mg/1.
  • the major part of the plasma form of vitronectin does not bind to heparin or Staphylococcus aureus .
  • a denatured or immobilized form of vitronectin supports binding of heparin and S . aureus .
  • S . aureus can bind to vitronectin when it is included as a stationary matrix protein. It is highly likely that blocking of S. aureus binding to vitronectin in matrix, severly hampers the infectious process.
  • Extracellular matrix is composed of a complex mixture of macromolecules that serve as a substrate for cellular adhesion.
  • Many potentially pathogenic staphylococcal strains have the ability to bind to ECM proteins such as fibronectin [1, 2], collagen [3, 4, 5], laminin [6], fibrinogen [7] and vitronectin (Vn) [8, 9], as well as heparan sulphate glycosaminoglycans [10] presented in ECM and on eukaryotic cell surface.
  • Bacteria possess specific binding molecules which interact with distinct sites of the proteins in the ECM of the host [11]. It has been shown earlier that this adherence is of vital importance for the infectious process. Thus these binding functions are suitable targets for antimicrobial therapy and prophylactic treatment.
  • Another procedure for its purification is based on an FPLC Mono-Q column to separate the staphylococcal cell surface proteins, and to obtain the 60 kDa bacterial protein retaining Vn-binding ability.
  • a 60 kDa protein that bind to vitronectin is already known [9]. However the protein has neither been purified nor characterized.
  • bacteria colonise body sites by sequentially engaging their surface-bound adhesins with specific substances availabe on epithelial cells, endothelial cells, leukocytes, or the ECM. It is generally accepted that this recognition process is required to establish bacteria at a given site. Aside from the ability to recognise a host component, these binding proteins play a substantial role in determining the outcome of the host-parasite interacion. They may initiate invasion by the pathogen, either themselves or by engaging a cascade of secondary molecules. Undoubtedly many bacterial cell surface binding proteins have not yet been discovered, identified, isolated or characterized.
  • the present invention provides both a new protein that binds to vitronectin and a new quinquedecapeptide that is derived from the heparin binding domain of vitronectin.
  • the quinquedecapeptide constitutes the vitronectin region that is responsible for binding the new protein.
  • a first aspect of the invention consists of a vitronectin binding protein having an apparent molecular weight of 60 kDa and having affinity to the quinquedecapeptide AKKQRFRHR RKGYR and having the N-terminal sequence MNKTDLINAVAEVADLVGKV, or variants, subfragments, multiples or mixtures thereof.
  • a part of the vitronectin binding region of the protein has been found to be encoded by the nucleotide sequence 5'- CCCTCAACTGTTTCAAACAATATAATTGATGAACTTAAACAAGTTGG TGAATACAATCAAATTTTCACAACTGAAGTTGACGGTACAGTCATAAACAATTTATGT AAATNCAAAATGCCAAGAAGACATTGGATTACTTCCATTGCTTCTTGGCATTTTTTCA GGG -3'. With the reading frame begining at the first nucleotide this corresponds to the amino acid sequence
  • nucleotide sequence is an unidentified nucleotide, corresponding to X in the amino acid sequence, which is an unidentified amino acid.
  • the four possible nucleotides give the four possible amino acids: phenylalanine, serine, tyrosine or cysteine. A clone was obtained and the nucleotide sequence was determined according to the general method described in K. Jacobsson and L. Frykberg, 1995, BioTechniques, Cloning of Ligand-Binding Domains of Bacterial Receptors by Phage Display, Vol. 18. No. 5, pages 878-885.
  • subfragment in the description and claims meant a part- fragment of the given domains or fragments which include parts from the various domains having mutually the same binding properties.
  • variant in the desciption and claims meant proteins or peptides in which the original amino acid sequence has been modified or changed by insertion, addition, substitution, inversion or exclusion of one or more amino acids, although while retaining or improving the binding properties, also including functional derivatives.
  • the invention also relates to those proteins which contain several arrays (multiples) of the binding domains or mixtures of the binding domains with retained binding properties.
  • the invention also relates to mixtures of the various domains of amino acid sequences having mutually the same binding properties.
  • a second aspect of the invention consists of a method for preparing a vitronectin binding protein according to the first aspect. This can be done by a method comprising culturing a staphylococcal bacterial strain, preferably Staphylococcus aureus, strain V8, preparing a cell surface protein extract of the bacteria and either applying said extract to an affinity purification step using the peptide AKKQRFRHRNRKGYR or applying said extract to an ion-exchange purification step. From a culture of 100 ml 0.2 mg protein extract of cell wall proteins was obtained and therefrom 2-3 ⁇ q of the protein according to the invention was obtained, as in the Examples described below.
  • bacteria than S. aureus can be used for the production of the protein, such as other bacteria from the genus Staphylococcus.
  • Various media can be used, such as Brain-Heart Infusion, Luria Broth or Todd-Hewitt Broth (THB) .
  • a preferred medium is especially THB.
  • the medium used should preferably be a rich and complex medium.
  • the bacteria can be grown at temperatures between 33 and 42°C, preferably between 35 and 39°C and most preferably at 37°C.
  • the pH is suitably ranging from 6.7 to 7.8, preferably from 7.0 to 7.5 and most preferably the culture is performed at the physiological pH, which is 7.3.
  • enzymatic digestion can be used.
  • the protein according to the invention may also be produced by cultivation of a genetically engineered host, e. g. E. coli. that has been provided with the gene for the protein of the invention.
  • the protein may be provided as an extracellular or intracellular protein.
  • An alternative to removing the protein from the cell surface is to disintegrate the cells and then clarifying the disintegrate by for example centrifugation, filtration or extraction.
  • the disintegration can be performed by chemical methods using for example lysozymes, lysostaphin, alkalis, solvents or surfactants, physical methods such as osmotic shock, drying or freeze-thawing, mechanical methods such as high pressure, bead milling or ultrasonication.
  • lysostaphin In a preferred method for disintegration lysostaphin is used.
  • the method described in Example 2 below can be altered by substituting one or more steps or adding one or more steps by one or more of the following steps: precipitation, extraction of different kinds, adsorption, isoelectric focusing and evaporation.
  • precipitation As further alternatives to the chromatography mehods described in Example 3 and 4 below can be mentioned gel filtration, reverse phase HPLC, hydrofobic chromatography etc.
  • a third aspect of the invention consists of the quinquedecapeptide AKKQRFRHRNRKGYR or a variant thereof, a pharmaceutical composition comprising an effective amount of said quinquedecapeptide or a variant thereof and pharmaceutically acceptable excipients, diluents and carriers.
  • the excipients, diluents and carriers may include all known agents suitable for use as such in lotions, pastes, creams, ointments, rinsing fluids, e.g. for use in the eyes or in the nose, and other compositions for topical use.
  • the composition may also be administered rectally or on a plaster.
  • additives and administration forms are not to be understood as limiting, but merely illustrative.
  • a purpose with the third aspect is to inhibit bacterial adhesion to stationary vitronectin in connection with infections.
  • the aspect defines a peptide that can be used for such a purpose.
  • a fourth aspect of the invention consists of an antibody, such as a monoclonal or polyclonal antibody, against a vitronectin binding protein according to the first aspect.
  • the antibodies could be produced by immunizing an animal, e. g. as in Example 8 a rat, by administration of the protein of interest. It is well known how to immunize an animal with an antigen, in this case the protein according to the invention, collect the blood, isolate the serum and use the antibodies that react with the protein.
  • monoclonal antibodies could be produced by a) immunizing mice with the protein according to the invention, and further by a method known per se b) isolating a lymphocyte mixture from the mice, c) fusing B-lymphocytes with B-myeloma mouse cells to produce a hybridoma, d) transferring the mixture of produced hybridomas and non- hybridized B-myeloma cells to a culture medium for the produced hybridomas, and cultivating and selecting the produced hybridomas over the non-hybridized B-myeloma cells, e) subcloning the hybridoma that produces the antibody that is directed specifically against the protein according to the invention, and at will f) isolating the antibody.
  • the antibody according to the invention can be used in a diagnostic or analytical kit as described below. They can also be used as a prophylactic or therapeutic agent for e.g. intravenous, intramuscular or subcutaneous administration. Administered in this way, the antibodies according to the invention can provide passive immunization to the organism being treated.
  • the third and fourth aspects concern providing means to inhibit the in vivo invasion of the human or animal body, of particularly S . aureus , although other microbial infections where the microbe binds to vitronectin are included in the invention.
  • Many micoorganisms do bind to vitronectin when initiating invasion of an organism.
  • Those microbes may interact with the quinquedecapeptide according to the third aspect and the interaction of such microbes with the substances according to the invention is therefore included in the invention. All such microorganisms could also cross-react with the antibodies according to the fourth aspect of the invention.
  • these aspects concern all pathogens that interact with vitronectin.
  • a fifth aspect of the invention consists of a pharmaceutical composition comprising an effecive amount of a vitronectin binding protein according to the first aspect and pharmaceutically acceptable excipients, diluents or carriers, and a vaccine composition comprising an effecitive amount of a vitronectin binding protein according to the first aspect, and possibly pharmaceutically acceptable adjuvants and excipients.
  • the excipients, diluents and carriers for the pharmaceutical composition may include all known agents suitable for use as such in lotions, pastes, creams, ointments, rinsing fluids, e.g. for use in the eyes or in the nose, and other compositions for topical use.
  • the composition may also be administered rectally or on a plaster.
  • Suitable adjuvants are those conventionally used in the field.
  • suitable excipients are mannitol, lactose, starch, cellulose, glucose etc., only to mention a few. The examples given are not to be regarded as limiting the invention.
  • a purpose with the fifth aspect is to competitively inhibit the adhesion of bacteria.
  • the pharmaceutical composition can be applied locally or adminstered intravenously, intramuscularly, subcutaneously, etc. to inhibit bacterial invasion.
  • the vaccine can be used to immunize a mammal including the human body, or other vertebrates.
  • a sixth aspect of the invention consists of a diagnostic or analytical kit comprising a vitronectin binding protein according to the first aspect or the quinquedecapeptide according to the third aspect, or a diagnostic or analytical kit comprising an antibody.
  • the kit may for instance be used for assaying antibodies against the VnBP according to the first aspect. In this way it could be indicated that the organism has been infected with the bacteria of interest, or that the organism has sufficient immunological protection against future infections. For example it is possible to determine if a pregnant woman has antibodies enough to protect her baby from such an infection. Various samples could also be assayed for the presence of the VnBP according to the first aspect. Antibodies or the quinquedecapeptide can be used for this purpose. In the kit serum or solutions containing the protein according to the first aspect may be present for use as a standard.
  • a seventh aspect of the invention consists of a method for purifying vitronectin by using immobilized vitronectin binding protein in an affinity column.
  • Vitronectin is unstable and difficult to purify, e. g. because of its high molecular weight and the fact that it tends to aggregate. With this aspect of the invention vitronectin could be produced more easily and cheaper.
  • Fig. 1 represents the isolation of a bacterial protein recognising the quinquedecapeptide.
  • a 60 kD staphylococcal protein was isolated using a peptide affinity column.
  • Left lane shows molecular weight markers.
  • Fig. 2 illustrates the separation of staphylococcal cell surface proteins using FPLC on a Mono-Q column.
  • the putative vitronectin- binding protein was indicated as VnBP.
  • Fig. 3 represents the results of SDS-PAGE and western blot analysis of the putative VnBP.
  • Lane a molecular weight markers
  • Lane b silver staining of the fraction having the ability to bind immobilised Vn, revealing a single band having a molecular mass of 60 kDa.
  • the protein transblotted onto a nitrocellular membrane was able to bind soluble Vn, which was subsequently detected using rabbit anti-Vn polyclonal antibodies (lane c) , or monoclonal antibody against Vn (lane d) .
  • Lanes e and f were respective controls when Vn was ommitted.
  • Fig. 4 represents the detection of the putative VnBP from the staphylococcal cell surface protein extract.
  • Lane a the LiCl- extract profile was stained with Coomassie Blue on a nitrocellular membrane after SDS-PAGE separation;
  • Lane b the separated proteins were probed with Vn and was subsequently detected using rabbit anti-Vn polyclonal antibodies;
  • Lane c control experiment of the rabbit antibodies when Vn was ommitted;
  • Lane d high concentration of normal rabbit serum (1:200) was used to detect staphylococcal protein A (SpA) from the strain V8.
  • SpA staphylococcal protein A
  • Fig. 5 illustrates the effects of synthetic peptides on binding of - ⁇ -labelled S . aureus cells to immobilised Vn, fibronectin or fibrninogen on microtiter plates. The results of the peptide competition are expressed as relative inhibition compared with a PBS control. 15-mer: quinquedecapeptide; 13-mer: tridecapeptide.
  • Fig. 6 illustrates the detection of the putative VnBP using a microtiterplate assay. Captured bacterial protein by immobilised Vn on the microtiterplates was detected using rat antibodies against the LiCl-protein extract, which was then probed with secondary antibodies against rat Igs.
  • Staphylococcus aureus prototype strain V8 a well-known protease producer, was obtained from Dr. Staffan Arvidsson, Karolinska Institute, Sweden. The bacteria were grown in Todd- Hewitt Broth (THB) (Difco, Detroit, MI, USA) with 100 ml inoculum in 2 liter flasks at 37°C for 20 h on a gyratory shaker (New
  • Bacterial cells were harvested at the late stationary phase by centrifugation (5,000 X g for 30 min at 4°C) and washed twice with 0.1 M phosphate buffer saline (PBS, pH 7.0). All common chemicals were of analytical grade, purchased from KEBO (Spanga, Sweden) .
  • LiCl-extract of staphylococcal cell surface protein was prepared as described elsewhere [10]. Briefly, every 1 g of bacteria was resuspended in 5 ml 1 M LiCl and the mixture was incubated at 37°C for 2 h on a gyratory shaker (New Brunswick, USA) with vigorous agitation. After centrifugation (5000 x g for 15 min at 4°C) sedimenting the bacteria, the supernatant was dialysed (membrane tubing MWCO: 12- 14 000, Spectrum Medical Industries Inc., Los Angeles, CA, USA) against 0,02 M Tris-HCl buffer pH 9.0 over night to remove LiCl.
  • the sample was centrifuged (5000 x g for 15 min at 4 °C) , and the protein concentration in the clear supernatant was determined using Bio-Rad protein assay. The extract was then stored at -20°C as 2 ml aliquots.
  • VnBP was purified on a HiTrap NHS column (Pharmacia) coupled to the quinquedecapeptide (AKKQRFRHRNRKGYR) comprising the heparin binding consensus sequence of human vitronectin.
  • the VnBP purified this way is shown in Figure 1.
  • Bacterial protein recognising the synthetic peptide (Ala 347 -Arg-*" 1 ) was isolated by the following procedure.
  • HiTrapTM N-hydroxysuccinimide (NHS)-activated affinity columns were purchased from Pharmacia, Uppsala, Sweden. Five milligrams of the synthetic peptide was used to couple to a 1 ml column followed by washing and deactivation according to the manufacturer's instruction. Coupling efficiency was generally higher than 85%. About 20 ml LiCl-extract containing approximately 2 mg protein (Bio-Rad protein assay) was applied to the peptide-column through a P-3 peristaltic pump (Pharmacia) , followed by equilibration with PBS at 4°C.
  • Staphylococcal cell surface proteins were separated using FPLC on
  • the chromatography procedure was as follwed: 5 min buffer A equilibration, 4 min sample loading onto the Mono-Q column, again 5 min buffer A returning to base-line, 30 min gradient 0-70 % buffer B, 10 min gradient 70-77.5 % buffer B, 13 min gradient 77.5-100 % buffer B, 5 min 100 % buffer B, and 2 min 100 % buffer A.
  • a 10-ml super-loading loop (Pharmacia) was used with 20 min sample loading time, and the protein separation pattern was proved to be reproducible.
  • FIG. 2 A separation profile of an LiCl-extract of S . aureus is shown in Fig. 2.
  • the chromatography achieved optimal resolution of a number of bacterial proteins from staphylococcal cell surface. Occasionally some peaks drifted earlier or later with a margin of 1-2 min (0,5-2 ml). In both our pilot experiments and later scale-up runs the chromatogram was proved to be easily reproducible, and was therefore complementary to the SDS-PAGE profile of the staphylococcal cell surface proteins published previously [10]. As such there has been developed a convenient, inexpensive and reliable method to release and analyze bacterial cell surface proteins, many of which are yet to be identified and characterized.
  • Human Vn was purified as described by Yatohgo et al [16]. The purified preparation was homogeneous as judged by SDS-PAGE (two bands corresponding to 65 and 75 kDa) and western blot analysis with mouse anti-human Vn monoclonal antibody (Boehringer Mannheim GmbH, Germany) and did not cross-react with antibodies against fibronectin.
  • the LiCl-protein extract was separated using the Mini-PROTEAN II cell (Bio-Rad) with 4% stacking gel and 7.5% separating gel. In western blot experiments, the separated protein was tranferred to nitrocellulose membranes (Schleicher Sc Schuell) at 65°C for 30 min following SDS-PAGE.
  • the membranes were blocked with 3% BSA in PBS for aproximatly 3h at room temperature, and then incubated with 5 ⁇ g/ml Vn in PBST for 1 h at room temperature.
  • the membranes were then incubated with 5 ⁇ g/ml rabbit anti-Vn polyclonal antibodies (Calbiochem) or monoclonal antibody aganist Vn (Boehringer Mannheim Biochemica) in PBS for 1 h at room temperature with gentle agitation. Secondary antibodies against rabbit or mouse Igs conjugated with alkaline phosphatase in 1:1000 dilution were added to the membranes.
  • the membranes were developed with bromochloroindolyl phosphate/nitro blue tetrazolium (Bio-Rad) according to the manufacturers' recomendation. Between each step the membranes were washed twice with 5 ml PBST for 5 min. As shown in Fig. 3, a single protein band having a molecular mass of 60 kDa was revealed after silver staining (lane b) , and in the western blot experiments.
  • This protein transblotted onto a nitrocellular membrane, had the ability to bind soluble Vn at a concentration of 5 ⁇ g/ml in PBS, which was subsequently detected using either rabbit anti-Vn polyclonal antibodies or monoclonal antibody directed against Vn (lane c and e) .
  • Control experiments with both anti-Vn mentioned above, omitting Vn were performed (lane d and f) .
  • an LiCl-extract transblotted onto a nitrocellular membrane was stained with Coomassie blue (Fig. 4, lane a).
  • aureus strain V8 the strain employed in the present study, has a novel structure lacking an IgG-binding domain (58 amino acid) and two octapeptide repetitions located in the membrane attaching region.
  • IgG-binding domain 58 amino acid
  • two octapeptide repetitions located in the membrane attaching region.
  • staphylococcal protein A from the strain V8 having a molecular mass slightly above 45 kDa (lane d) , which coincided with published data [13].
  • lane d staphylococcal protein A from the strain V8 having a molecular mass slightly above 45 kDa
  • Amino acid analysis was performed as follows. About 50 ⁇ g of the purified 60 kDa Vn-binding protein was hydrolyzed with 6 M HCl containing 2 mg/ml phenol for 24 h at ll ⁇ °C. The amino acid composition was determined by the Protein and Peptide Unit at the Deptartment of Medical Biochemistry and Biophysics, Karolinska Institute, Sweden. Approximately 1.5 nmol purified protein was used for N-terminal sequence determination after methanolic HCl de-blocking treatment [18]. Briefly, the protein sample was dissolved in 1 N HCl in anhydrous methanol and the mixture was incubated at 37°C for 5 h followed by dialysis against deionized water overnight at 4°C and lyophilization. The freeze-dried sample was dissolved in 30 % Acetonitrile and sequencing was performed by the Biomolecular Resource Facility at Lund University, Lund, Sweden.
  • S . aureus has the ability to bind to heparin and heparan sulphate [10].
  • a heparin binding protein was purified from an S . aureus extract by passing it through a heparin column. The purified protein was subjected to N-terminal amino acid analysis according to Example 6, and as shown in Table II the heparan sulphate binding protein and the VnBP seem to be the same protein.
  • Antibodies aganist the LiCl-extract were produced in Wistar rats according to standard methods. Briefly, each of four rats was immunized with 100 ⁇ g staphylococcal protein extract subcutaneously at three occasions with a three-week span in between. The antigens were given with Freund's complete adjuvant (Sigma) at the first and with incomplete adjuvant (Sigma) at the following two injections. The antibody titer aganist the LiCl- extract was monitered using ELISA on icrotiterplates coated with the bacterial protein extract and detected using anti-rat Igs conjugated with horseradish peroxidase (The Binding Sites, Birmingham, UK) as secondary antibodies. The rat sera were pooled and stored at -20°C as 0.5 ml aliquots.
  • a 15 amino acid long peptide (Ala 47 -Arg 61 ) was synthesised using t-butoxycarbonyl-protected amino acids with an automated peptide synthesiser (model 430A, Applied Biosystems, Inc.), and was subsequently purified on FPLC.
  • the amino acid sequence of the synthetic peptide was determined according to the manufacturer's instructions from Applied Biosystems.
  • a 13 amino acid long peptide (Asn 371 -Leu 3 ° 3 ) was kindly provided by Dr. W. St ⁇ ber (Behringwerke. Marburg, Germany) .
  • the J H-labelled bacteria were washed extensively with PBS and resuspended to a final density of 10° cells per ml (specific activity: approximately 200 CFU/cpm) and used promptly for microtiter plate adhesion assays.
  • Microtiter plates (96-well) (Costar, Cambridge, MA, USA) were coated with 0.2 ⁇ g/well of Vn 1 preparation, fibronectin (Sigma Chemical Co) of fibrinogen (Kabi, Sweden) in lOO ⁇ l PBS overnight at 4°C and additional binding sites were blocked using 3% BSA in PBS containing 0.05% Tween-20 for 2 h at 37°C, similar to a published procedure [17].
  • VnBP vitronectin binding protein
  • the plates were developed with 1.2- phenylendiamine (DAKO, Glostrup, Denmark) in 0.1 M citric acid- phosphate buffer pH 5.0 and approximately 0.01% hydrogen peroxide (KEBO) as recomended by the manufacturer.
  • the plates were read at 490 nm with a Bio-Rad ELISA reader. Several repetitions of duplicate determinations were made and averaged in all cases.
  • NAME Torkel Wadstrom
  • NAME Olin Liang, c/o Bovin
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • FRAGMENT TYPE N-terminal
  • ORGANISM Staphylococcus aureus
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Staphylococcus aureus
  • ORGANISM Staphylococcus aureus

Abstract

The present invention concerns a new vitronectin binding protein from Streptococcus aureus. The protein is involved in the infectious process of the microorganism. The invention also concerns a method for preparation thereof and a quinquedecapeptide with the amino acid sequence AKKQRFRHRNRKGYR, that is useful for preparing the protein and blocking the protein binding site to vitronectin. Further the invention relates to an antibody against the protein, pharmaceutical compositions and a diagnostic or analytical kit comprising said protein, peptide or antibody and a vaccine. Finally, the invention concerns a method for purifying vitronectin by using the vitronectin binding protein in an affinity column.

Description

VITRONECTIN BINDING PROTEIN
Technical Field of the Invention
The present invention relates to a vitronectin binding protein, a method for preparation thereof, a quinquedecapeptide that is useful for preparing the protein, an antibody against the protein, pharmaceutical compositions and a diagnostic or analytical kit comprising said protein, use of the protein for the preparation of a pharmaceutical composition, a diagnostic or analytical kit comprising an antibody against said protein and a method for purifying vitronectin.
Description of Related Art
Vitronectin is a plasma protein that occurs in concentrations around 300 mg/1. The major part of the plasma form of vitronectin does not bind to heparin or Staphylococcus aureus . However, a denatured or immobilized form of vitronectin supports binding of heparin and S . aureus . Also, S . aureus can bind to vitronectin when it is included as a stationary matrix protein. It is highly likely that blocking of S. aureus binding to vitronectin in matrix, severly hampers the infectious process.
Extracellular matrix (ECM) is composed of a complex mixture of macromolecules that serve as a substrate for cellular adhesion. Many potentially pathogenic staphylococcal strains have the ability to bind to ECM proteins such as fibronectin [1, 2], collagen [3, 4, 5], laminin [6], fibrinogen [7] and vitronectin (Vn) [8, 9], as well as heparan sulphate glycosaminoglycans [10] presented in ECM and on eukaryotic cell surface. Bacteria possess specific binding molecules which interact with distinct sites of the proteins in the ECM of the host [11]. It has been shown earlier that this adherence is of vital importance for the infectious process. Thus these binding functions are suitable targets for antimicrobial therapy and prophylactic treatment. In earlier studies it has been demonstrated that cells of Staphylococcus aureus strain V8 bind ljtJI-labelled Vn in a receptor-ligand type of interaction [12, 9]. A protein with a molecular mass of 60 kDa has now been identified as a high- affinity staphylococcal Vn-binding protein . This protein was purified on a quinquedecapeptide (AKKQRFRHRNRKGYR) column. The peptide is derived from the heparin binding domain of vitronectin (Ala347 - Arg361) . Another procedure for its purification is based on an FPLC Mono-Q column to separate the staphylococcal cell surface proteins, and to obtain the 60 kDa bacterial protein retaining Vn-binding ability. A 60 kDa protein that bind to vitronectin is already known [9]. However the protein has neither been purified nor characterized.
During the course of infectious disease, bacteria colonise body sites by sequentially engaging their surface-bound adhesins with specific substances availabe on epithelial cells, endothelial cells, leukocytes, or the ECM. It is generally accepted that this recognition process is required to establish bacteria at a given site. Aside from the ability to recognise a host component, these binding proteins play a substantial role in determining the outcome of the host-parasite interacion. They may initiate invasion by the pathogen, either themselves or by engaging a cascade of secondary molecules. Undoubtedly many bacterial cell surface binding proteins have not yet been discovered, identified, isolated or characterized. It is important that these studies are carried out at a molecular level, because not only is it likely that a high level of sophistication of the microbial interactions with host components will be seen, but detailed characterization of these mechanisms may provide important information on cell adhesion strategies in general and in microbial colonization processes in particular.
Thus, there is a general need for understanding the mechanism of interaction between pathogenic bacteria and extracellular matrix components, such as vitronectin. The present invention provides both a new protein that binds to vitronectin and a new quinquedecapeptide that is derived from the heparin binding domain of vitronectin. The quinquedecapeptide constitutes the vitronectin region that is responsible for binding the new protein. Thus the present invention partially elucidates the above mentioned interaction mechanism.
Summary of the invention
A first aspect of the invention consists of a vitronectin binding protein having an apparent molecular weight of 60 kDa and having affinity to the quinquedecapeptide AKKQRFRHR RKGYR and having the N-terminal sequence MNKTDLINAVAEVADLVGKV, or variants, subfragments, multiples or mixtures thereof. A part of the vitronectin binding region of the protein has been found to be encoded by the nucleotide sequence 5'- CCCTCAACTGTTTCAAACAATATAATTGATGAACTTAAACAAGTTGG TGAATACAATCAAATTTTCACAACTGAAGTTGACGGTACAGTCATAAACAATTTATGT AAATNCAAAATGCCAAGAAGACATTGGATTACTTCCATTGCTTCTTGGCATTTTTTCA GGG -3'. With the reading frame begining at the first nucleotide this corresponds to the amino acid sequence
PSTVSNNIIDELKQVGEYNQIFTTEVDGTVINNLCKXKMPRRHWITSIASWHFFR. The letter N in the nucleotide sequence is an unidentified nucleotide, corresponding to X in the amino acid sequence, which is an unidentified amino acid. The four possible nucleotides give the four possible amino acids: phenylalanine, serine, tyrosine or cysteine. A clone was obtained and the nucleotide sequence was determined according to the general method described in K. Jacobsson and L. Frykberg, 1995, BioTechniques, Cloning of Ligand-Binding Domains of Bacterial Receptors by Phage Display, Vol. 18. No. 5, pages 878-885.
By subfragment is in the description and claims meant a part- fragment of the given domains or fragments which include parts from the various domains having mutually the same binding properties. By variant is in the desciption and claims meant proteins or peptides in which the original amino acid sequence has been modified or changed by insertion, addition, substitution, inversion or exclusion of one or more amino acids, although while retaining or improving the binding properties, also including functional derivatives. The invention also relates to those proteins which contain several arrays (multiples) of the binding domains or mixtures of the binding domains with retained binding properties. The invention also relates to mixtures of the various domains of amino acid sequences having mutually the same binding properties.
A second aspect of the invention consists of a method for preparing a vitronectin binding protein according to the first aspect. This can be done by a method comprising culturing a staphylococcal bacterial strain, preferably Staphylococcus aureus, strain V8, preparing a cell surface protein extract of the bacteria and either applying said extract to an affinity purification step using the peptide AKKQRFRHRNRKGYR or applying said extract to an ion-exchange purification step. From a culture of 100 ml 0.2 mg protein extract of cell wall proteins was obtained and therefrom 2-3 μq of the protein according to the invention was obtained, as in the Examples described below.
Other bacteria than S. aureus can be used for the production of the protein, such as other bacteria from the genus Staphylococcus. Various media can be used, such as Brain-Heart Infusion, Luria Broth or Todd-Hewitt Broth (THB) . A preferred medium is especially THB. The medium used should preferably be a rich and complex medium. The bacteria can be grown at temperatures between 33 and 42°C, preferably between 35 and 39°C and most preferably at 37°C. The pH is suitably ranging from 6.7 to 7.8, preferably from 7.0 to 7.5 and most preferably the culture is performed at the physiological pH, which is 7.3. As an alternative to LiCl-extraction, enzymatic digestion can be used. The protein according to the invention may also be produced by cultivation of a genetically engineered host, e. g. E. coli. that has been provided with the gene for the protein of the invention. In this case the protein may be provided as an extracellular or intracellular protein. An alternative to removing the protein from the cell surface is to disintegrate the cells and then clarifying the disintegrate by for example centrifugation, filtration or extraction. The disintegration can be performed by chemical methods using for example lysozymes, lysostaphin, alkalis, solvents or surfactants, physical methods such as osmotic shock, drying or freeze-thawing, mechanical methods such as high pressure, bead milling or ultrasonication. In a preferred method for disintegration lysostaphin is used. The method described in Example 2 below can be altered by substituting one or more steps or adding one or more steps by one or more of the following steps: precipitation, extraction of different kinds, adsorption, isoelectric focusing and evaporation. As further alternatives to the chromatography mehods described in Example 3 and 4 below can be mentioned gel filtration, reverse phase HPLC, hydrofobic chromatography etc.
A third aspect of the invention consists of the quinquedecapeptide AKKQRFRHRNRKGYR or a variant thereof, a pharmaceutical composition comprising an effective amount of said quinquedecapeptide or a variant thereof and pharmaceutically acceptable excipients, diluents and carriers. The excipients, diluents and carriers may include all known agents suitable for use as such in lotions, pastes, creams, ointments, rinsing fluids, e.g. for use in the eyes or in the nose, and other compositions for topical use. The composition may also be administered rectally or on a plaster. Those examples of additives and administration forms are not to be understood as limiting, but merely illustrative. A purpose with the third aspect is to inhibit bacterial adhesion to stationary vitronectin in connection with infections. The aspect defines a peptide that can be used for such a purpose. A fourth aspect of the invention consists of an antibody, such as a monoclonal or polyclonal antibody, against a vitronectin binding protein according to the first aspect.
The antibodies could be produced by immunizing an animal, e. g. as in Example 8 a rat, by administration of the protein of interest. It is well known how to immunize an animal with an antigen, in this case the protein according to the invention, collect the blood, isolate the serum and use the antibodies that react with the protein.
Also, monoclonal antibodies could be produced by a) immunizing mice with the protein according to the invention, and further by a method known per se b) isolating a lymphocyte mixture from the mice, c) fusing B-lymphocytes with B-myeloma mouse cells to produce a hybridoma, d) transferring the mixture of produced hybridomas and non- hybridized B-myeloma cells to a culture medium for the produced hybridomas, and cultivating and selecting the produced hybridomas over the non-hybridized B-myeloma cells, e) subcloning the hybridoma that produces the antibody that is directed specifically against the protein according to the invention, and at will f) isolating the antibody.
For example Kohler et al., Nature (London), 256 (1975) 495-497, Kohler et al., Eur. J. Immunol., 6 (1976) 511-519, Hochkeppel et al., Eur. J. Immunol., 118 (1981) 437-442, Secher et al.. Nature (London), 285 (1980) 446-450, Vernon et al. in: Mishell & Shiigi, Selected Methods in Cellular Immunology: 373-397, Freeman Se Co., San Fransisco 1980, Havell et al., J. Antimicrob. Ag. Chemotherap,. 2 (1972) 476-484, Milstein, Scientific American, 243 (October 1980) 56-64 and Groth & Scheidegger, J. Immunological Methods, 35 (1980) 1-21 describe the production of monoclonal antibodies. The antibody according to the invention can be used in a diagnostic or analytical kit as described below. They can also be used as a prophylactic or therapeutic agent for e.g. intravenous, intramuscular or subcutaneous administration. Administered in this way, the antibodies according to the invention can provide passive immunization to the organism being treated.
The third and fourth aspects concern providing means to inhibit the in vivo invasion of the human or animal body, of particularly S . aureus , although other microbial infections where the microbe binds to vitronectin are included in the invention. Many micoorganisms do bind to vitronectin when initiating invasion of an organism. Those microbes may interact with the quinquedecapeptide according to the third aspect and the interaction of such microbes with the substances according to the invention is therefore included in the invention. All such microorganisms could also cross-react with the antibodies according to the fourth aspect of the invention. Thus these aspects concern all pathogens that interact with vitronectin.
A fifth aspect of the invention consists of a pharmaceutical composition comprising an effecive amount of a vitronectin binding protein according to the first aspect and pharmaceutically acceptable excipients, diluents or carriers, and a vaccine composition comprising an effecitive amount of a vitronectin binding protein according to the first aspect, and possibly pharmaceutically acceptable adjuvants and excipients. The excipients, diluents and carriers for the pharmaceutical composition may include all known agents suitable for use as such in lotions, pastes, creams, ointments, rinsing fluids, e.g. for use in the eyes or in the nose, and other compositions for topical use. The composition may also be administered rectally or on a plaster. Those examples of additives and administration forms are not to be understood as limiting, but merely illustrative. Suitable adjuvants are those conventionally used in the field. Examples of suitable excipients are mannitol, lactose, starch, cellulose, glucose etc., only to mention a few. The examples given are not to be regarded as limiting the invention.
A purpose with the fifth aspect is to competitively inhibit the adhesion of bacteria. The pharmaceutical composition can be applied locally or adminstered intravenously, intramuscularly, subcutaneously, etc. to inhibit bacterial invasion.
The vaccine can be used to immunize a mammal including the human body, or other vertebrates.
A sixth aspect of the invention consists of a diagnostic or analytical kit comprising a vitronectin binding protein according to the first aspect or the quinquedecapeptide according to the third aspect, or a diagnostic or analytical kit comprising an antibody. The kit may for instance be used for assaying antibodies against the VnBP according to the first aspect. In this way it could be indicated that the organism has been infected with the bacteria of interest, or that the organism has sufficient immunological protection against future infections. For example it is possible to determine if a pregnant woman has antibodies enough to protect her baby from such an infection. Various samples could also be assayed for the presence of the VnBP according to the first aspect. Antibodies or the quinquedecapeptide can be used for this purpose. In the kit serum or solutions containing the protein according to the first aspect may be present for use as a standard.
A seventh aspect of the invention consists of a method for purifying vitronectin by using immobilized vitronectin binding protein in an affinity column. Vitronectin is unstable and difficult to purify, e. g. because of its high molecular weight and the fact that it tends to aggregate. With this aspect of the invention vitronectin could be produced more easily and cheaper. Brief Description of the Drawings
Fig. 1 represents the isolation of a bacterial protein recognising the quinquedecapeptide. A 60 kD staphylococcal protein was isolated using a peptide affinity column. Left lane shows molecular weight markers.
Fig. 2 illustrates the separation of staphylococcal cell surface proteins using FPLC on a Mono-Q column. The putative vitronectin- binding protein was indicated as VnBP.
Fig. 3 represents the results of SDS-PAGE and western blot analysis of the putative VnBP. Lane a, molecular weight markers; Lane b, silver staining of the fraction having the ability to bind immobilised Vn, revealing a single band having a molecular mass of 60 kDa. The protein transblotted onto a nitrocellular membrane was able to bind soluble Vn, which was subsequently detected using rabbit anti-Vn polyclonal antibodies (lane c) , or monoclonal antibody against Vn (lane d) . Lanes e and f were respective controls when Vn was ommitted.
Fig. 4 represents the detection of the putative VnBP from the staphylococcal cell surface protein extract. Lane a, the LiCl- extract profile was stained with Coomassie Blue on a nitrocellular membrane after SDS-PAGE separation; Lane b, the separated proteins were probed with Vn and was subsequently detected using rabbit anti-Vn polyclonal antibodies; Lane c, control experiment of the rabbit antibodies when Vn was ommitted; Lane d, high concentration of normal rabbit serum (1:200) was used to detect staphylococcal protein A (SpA) from the strain V8.
Fig. 5 illustrates the effects of synthetic peptides on binding of -Η-labelled S . aureus cells to immobilised Vn, fibronectin or fibrninogen on microtiter plates. The results of the peptide competition are expressed as relative inhibition compared with a PBS control. 15-mer: quinquedecapeptide; 13-mer: tridecapeptide. Fig. 6 illustrates the detection of the putative VnBP using a microtiterplate assay. Captured bacterial protein by immobilised Vn on the microtiterplates was detected using rat antibodies against the LiCl-protein extract, which was then probed with secondary antibodies against rat Igs.
Examples
The following Examples are merely illustrative in nature. Other methods in accordance with the present invention may occur to those skilled in the art.
Example 1. Bacterial strains and culture conditions
Staphylococcus aureus prototype strain V8, a well-known protease producer, was obtained from Dr. Staffan Arvidsson, Karolinska Institute, Stockholm, Sweden. The bacteria were grown in Todd- Hewitt Broth (THB) (Difco, Detroit, MI, USA) with 100 ml inoculum in 2 liter flasks at 37°C for 20 h on a gyratory shaker (New
Brunswick, USA) with vigorous agitation [12]. Bacterial cells were harvested at the late stationary phase by centrifugation (5,000 X g for 30 min at 4°C) and washed twice with 0.1 M phosphate buffer saline (PBS, pH 7.0). All common chemicals were of analytical grade, purchased from KEBO (Spanga, Sweden) .
Constituents of THB had no side-effects on bacterial adherence interactions [15].
Example 2. Preparation of bacterial cell surface protein extract
To achieve minimal cell lysis, LiCl-extract of staphylococcal cell surface protein was prepared as described elsewhere [10]. Briefly, every 1 g of bacteria was resuspended in 5 ml 1 M LiCl and the mixture was incubated at 37°C for 2 h on a gyratory shaker (New Brunswick, USA) with vigorous agitation. After centrifugation (5000 x g for 15 min at 4°C) sedimenting the bacteria, the supernatant was dialysed (membrane tubing MWCO: 12- 14 000, Spectrum Medical Industries Inc., Los Angeles, CA, USA) against 0,02 M Tris-HCl buffer pH 9.0 over night to remove LiCl. After dialysis the sample was centrifuged (5000 x g for 15 min at 4 °C) , and the protein concentration in the clear supernatant was determined using Bio-Rad protein assay. The extract was then stored at -20°C as 2 ml aliquots.
Example 3. Purification of the vitronectin binding protein
The VnBP was purified on a HiTrap NHS column (Pharmacia) coupled to the quinquedecapeptide (AKKQRFRHRNRKGYR) comprising the heparin binding consensus sequence of human vitronectin. The VnBP purified this way is shown in Figure 1. Bacterial protein recognising the synthetic peptide (Ala347-Arg-*"1) was isolated by the following procedure.
One millilitre HiTrap™ N-hydroxysuccinimide (NHS)-activated affinity columns were purchased from Pharmacia, Uppsala, Sweden. Five milligrams of the synthetic peptide was used to couple to a 1 ml column followed by washing and deactivation according to the manufacturer's instruction. Coupling efficiency was generally higher than 85%. About 20 ml LiCl-extract containing approximately 2 mg protein (Bio-Rad protein assay) was applied to the peptide-column through a P-3 peristaltic pump (Pharmacia) , followed by equilibration with PBS at 4°C. The column was washed by 1.0 M NaCl to remove any loosely bound material and was then eluted with 0.1 M glycine-HCl pH 3.0. Eluent was collected and neutralised immediately by a few crystals of Tris (Sigma) , and then subjected to SDS-PAGE with 12.5% separating gel on a PhastSystem™ (Pharmacia) . The SDS-PAGE gel was subsequently stained using Silver Plus Kit (Bio-Rad) and dried in a 65°C incubator. SDS-PAGE and silver staining procedures were as described in the manufacturers ' instructions. 20-30 μq purified protein was obtained from the column. Example 4. Alternative method for the purification of the vitronectin binding protein
Staphylococcal cell surface proteins were separated using FPLC on
® a Mono-Q column. Ion-exchange chromatography with FPLC System
® (Pharmacia, Uppsala, Sweden) was performed on a Mono Q High
Resolution 5/5 column (Pharmacia) . Preliminary runs optimized flow rate at 0.5 ml/min, paper-recording speed at 0.2 cm/min, time-based collection at 1 min/fraction, and OD2gQ nm at 0.2 as an optimal sensitivity range using UV-M monitor (Pharmacia) .
Fractionation of staphylococcal surface protein LiCl-extract was performed as follows. The FPLC ® System was first equilibrated with optimal 20 mM Tris-HCl pH 9.0 (buffer A), then 0.5 M NaCl in buffer A (buffer B) and buffer A again. In pilot experiments extract sample (2 ml) containing approximately 0.2 mg protein was loaded using a 2 ml loading loop (Pharmacia) , followed by washing using the buffer A till a stable base line. The chromatography procedure was as follwed: 5 min buffer A equilibration, 4 min sample loading onto the Mono-Q column, again 5 min buffer A returning to base-line, 30 min gradient 0-70 % buffer B, 10 min gradient 70-77.5 % buffer B, 13 min gradient 77.5-100 % buffer B, 5 min 100 % buffer B, and 2 min 100 % buffer A. In scale-up runs, a 10-ml super-loading loop (Pharmacia) was used with 20 min sample loading time, and the protein separation pattern was proved to be reproducible.
A separation profile of an LiCl-extract of S . aureus is shown in Fig. 2. The chromatography achieved optimal resolution of a number of bacterial proteins from staphylococcal cell surface. Occasionally some peaks drifted earlier or later with a margin of 1-2 min (0,5-2 ml). In both our pilot experiments and later scale-up runs the chromatogram was proved to be easily reproducible, and was therefore complementary to the SDS-PAGE profile of the staphylococcal cell surface proteins published previously [10]. As such there has been developed a convenient, inexpensive and reliable method to release and analyze bacterial cell surface proteins, many of which are yet to be identified and characterized.
Example 5. SDS-PAGE and western blotting analysis
Human Vn was purified as described by Yatohgo et al [16]. The purified preparation was homogeneous as judged by SDS-PAGE (two bands corresponding to 65 and 75 kDa) and western blot analysis with mouse anti-human Vn monoclonal antibody (Boehringer Mannheim GmbH, Germany) and did not cross-react with antibodies against fibronectin.
The fractions from Example 4 having the significant ability to bind to immobilised Vn, indicated in the Fig. 2 as the VnBP, were pooled and subjected to SDS-PAGE (PhastSystem ® Homogenous 12.5% gel, Pharmacia) using the PhastSystem ® (Pharmacia) , and the gels were stained with Silver Stain Plus (Bio-Rad) . The LiCl-protein extract was separated using the Mini-PROTEAN II cell (Bio-Rad) with 4% stacking gel and 7.5% separating gel. In western blot experiments, the separated protein was tranferred to nitrocellulose membranes (Schleicher Sc Schuell) at 65°C for 30 min following SDS-PAGE. After transblotting the membranes were blocked with 3% BSA in PBS for aproximatly 3h at room temperature, and then incubated with 5μg/ml Vn in PBST for 1 h at room temperature. The membranes were then incubated with 5 μg/ml rabbit anti-Vn polyclonal antibodies (Calbiochem) or monoclonal antibody aganist Vn (Boehringer Mannheim Biochemica) in PBS for 1 h at room temperature with gentle agitation. Secondary antibodies against rabbit or mouse Igs conjugated with alkaline phosphatase in 1:1000 dilution were added to the membranes. Finally the membranes were developed with bromochloroindolyl phosphate/nitro blue tetrazolium (Bio-Rad) according to the manufacturers' recomendation. Between each step the membranes were washed twice with 5 ml PBST for 5 min. As shown in Fig. 3, a single protein band having a molecular mass of 60 kDa was revealed after silver staining (lane b) , and in the western blot experiments. This protein, transblotted onto a nitrocellular membrane, had the ability to bind soluble Vn at a concentration of 5 μg/ml in PBS, which was subsequently detected using either rabbit anti-Vn polyclonal antibodies or monoclonal antibody directed against Vn (lane c and e) . Control experiments with both anti-Vn mentioned above, omitting Vn were performed (lane d and f) . In parallel experiments, an LiCl-extract transblotted onto a nitrocellular membrane was stained with Coomassie blue (Fig. 4, lane a). The separated proteins were probed with Vn (5 μg/ml in PBS) and rabbit anti-Vn revealing a single band having a molecular mass of 60 kDa (Fig. 4, lane b) , which confirmed our earlier published observation using H χ- labelled Vn as a probe. Results of control experiments using rabbit anti-Vn polyclonal antibodies is presented in lane c, which did not even detect the most abundant staphylococcal surface protein A. Sequence analysis reported by Finck-Barbancon et al [13] indicated that protein A from S . aureus strain V8, the strain employed in the present study, has a novel structure lacking an IgG-binding domain (58 amino acid) and two octapeptide repetitions located in the membrane attaching region. Using high concentration of normal rabbit serum (1:200), a faint band indicated staphylococcal protein A from the strain V8 having a molecular mass slightly above 45 kDa (lane d) , which coincided with published data [13]. Apparently no other components in the bacterial cell surface protein extract had the ability to bind to normal rabbit immunoglobulins.
Example 6. Amino acid analysis
Amino acid analysis was performed as follows. About 50 μg of the purified 60 kDa Vn-binding protein was hydrolyzed with 6 M HCl containing 2 mg/ml phenol for 24 h at llθ°C. The amino acid composition was determined by the Protein and Peptide Unit at the Deptartment of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm. Approximately 1.5 nmol purified protein was used for N-terminal sequence determination after methanolic HCl de-blocking treatment [18]. Briefly, the protein sample was dissolved in 1 N HCl in anhydrous methanol and the mixture was incubated at 37°C for 5 h followed by dialysis against deionized water overnight at 4°C and lyophilization. The freeze-dried sample was dissolved in 30 % Acetonitrile and sequencing was performed by the Biomolecular Resource Facility at Lund University, Lund, Sweden.
The amino acid analysis (Table I) shows that the 60 kDa Vn- binding protein has a high content of aspartic acid/asparagine (12.0%) and glutamic acid/glutamine (14.9%) corresponding to 26.9% of the total residues. By comparing amino acid compositions we here show that the 60 kDa VnBP described in this study is distinct from a 60 kDa/72 kDa promiscuous staphylococcal lectin- like protein [14]. Table II shows the N-terminal amino acid sequence of VnBP.
TABLE I
Amino acid composition of the purified 60 kDa vitronect in-binding protein of S. aureus
Amino acids Composition (mol %) Residues/molecules
Cysteine 2,1 12
Aspartic acid/iasparagine 12,0 66 T Thhrreeoonniinnee 6 6,,00 33
Serine 5,0 27
Glutamic acid/glutaraine 14,9 81
Proline 3,0 16
Glycine 8,6 47 A Allaanniinnee 9 9,,99 54
Valine 4,9 27
Methionine 1,2 7
Isoleucine 5,7 31
Leucine 6,4 35 T Tyyrroossiinnee 1 1,,88 10
Phenylalanin 3,1 17
Tryptophan 0,0 0
Lysine 10,3 56
Histidine 0,8 4 A Arrggiinniinnee 4 4,,22 23
Total residues 100 546
Example 7. Amino terminal sequencing
S . aureus has the ability to bind to heparin and heparan sulphate [10]. A heparin binding protein was purified from an S . aureus extract by passing it through a heparin column. The purified protein was subjected to N-terminal amino acid analysis according to Example 6, and as shown in Table II the heparan sulphate binding protein and the VnBP seem to be the same protein.
TABLE II
N-terminal amino acid sequence analysis
S . aureus V8 cell surface N-terminal amino acid protein sequence
60 kDa vitronectin-binding MNKTDLINAVAEVADLVGKV
60 kDa heparan sulphate- ML*T*LI*A binding
♦Difficult to interpret.
Example 8. Preparation of antibodies against the LiCl-extract
Antibodies aganist the LiCl-extract were produced in Wistar rats according to standard methods. Briefly, each of four rats was immunized with 100 μg staphylococcal protein extract subcutaneously at three occasions with a three-week span in between. The antigens were given with Freund's complete adjuvant (Sigma) at the first and with incomplete adjuvant (Sigma) at the following two injections. The antibody titer aganist the LiCl- extract was monitered using ELISA on icrotiterplates coated with the bacterial protein extract and detected using anti-rat Igs conjugated with horseradish peroxidase (The Binding Sites, Birmingham, UK) as secondary antibodies. The rat sera were pooled and stored at -20°C as 0.5 ml aliquots.
Example 9. Synthesis of the guinguedecapeptide
A 15 amino acid long peptide (Ala 47-Arg 61) was synthesised using t-butoxycarbonyl-protected amino acids with an automated peptide synthesiser (model 430A, Applied Biosystems, Inc.), and was subsequently purified on FPLC. The amino acid sequence of the synthetic peptide was determined according to the manufacturer's instructions from Applied Biosystems. A 13 amino acid long peptide (Asn371-Leu3°3) was kindly provided by Dr. W. Stϋber (Behringwerke. Marburg, Germany) .
Example 10. Interaction between S . aureus and the guinguedecapeptide
The specificity of the quinquedecapeptide (AKKQRFRHRNRKGYR) in its interaction with S . aureus binding to vitronectin was tested in an experiment where radiolabelled S . aureus cells were added to either immobilised vitronectin, fibronectin or fibrinogen. Figure 5 shows that binding to vitronectin was blocked by the quinquedecapeptide (AKKQRFRHRNRKGYR) but not by a control peptide (NQNSRRPSRATWL) . Binding to fibronectin or fibrinogen was unaffected.
Half a millilitre of an overnight culture of bacteria was diluted
-a in 5 ml THB containing 50 μCi of (methy1—Η)thymidine and grown for a further 5 h on a gyratory shaker with vigorous agitation.
-a
The JH-labelled bacteria were washed extensively with PBS and resuspended to a final density of 10° cells per ml (specific activity: approximately 200 CFU/cpm) and used promptly for microtiter plate adhesion assays. Microtiter plates (96-well) (Costar, Cambridge, MA, USA) were coated with 0.2 μg/well of Vn 1 preparation, fibronectin (Sigma Chemical Co) of fibrinogen (Kabi, Stockholm, Sweden) in lOOμl PBS overnight at 4°C and additional binding sites were blocked using 3% BSA in PBS containing 0.05% Tween-20 for 2 h at 37°C, similar to a published procedure [17]. Fifty microliter of 3H-labelled bacteria (approximately 5 x 10" CFU, or 2.5 x IO4 cpm) were added to the plates together with 50 μl of competing peptides in a series of concentrations with PBS as control. The mixtures were incubated for 1 h at 37"C and the final amount of bacteria associated with the microtiter plate was released by incubating with 150 μl 3% SDS for 1 h at room temperature and washing with another 150 μl 3% SDS. Radioactivity was determined using 5 ml of Ready-Safe™ Liquid Scintillation Cocktail (Beckman) in Liquid Scintillation System LS 1801 (Beckman) . Three washes of the microtiter plates with PBS-Tween were essential between each of the above steps.
Example 11. Detection of the vitronectin binding protein
For the purpose of detecting the vitronectin binding protein (VnBP) microtiter plates (96-well, Costar, Cambridge, MA, USA) were coated with Vn (0.20 μg/well) in lOOμl PBS at 4 °C over night, and non-specific binding sites were blocked with 3% BSA in PBS containing 0.1% Tween-20 (PBST) for 2 h at 37 °C. In 2-ml sample (containing 200 μg protein) pilot experiments, one and half ml fractions (3 min/ml) from FPLC obtained as in Example 4 were collected, dialyzed against a large volume of deionized water overnight at 4°C, freeze-dried and finally resuspended in 200 μl PBS, and were promptly subjected to the microtiter wells coated with Vn to test for Vn-binding activity. The mixtures were incubated for 1 h at 37 °C. Bound bacterial cell surface protein was detected using rat antibodies (1:1000) raised against LiCl extract and secondary antibodies against rat Igs (1:1000) conjugated with peroxidase (The Binding Sites, Birmingham, UK) . Three washes of the microtiter plate with PBST were essential between each of above steps. The plates were developed with 1.2- phenylendiamine (DAKO, Glostrup, Denmark) in 0.1 M citric acid- phosphate buffer pH 5.0 and approximately 0.01% hydrogen peroxide (KEBO) as recomended by the manufacturer. The plates were read at 490 nm with a Bio-Rad ELISA reader. Several repetitions of duplicate determinations were made and averaged in all cases.
A typical result of such an assay is shown in Fig.6. At about 30 % of the elution buffer B, corresponding to 0.15 M NaCl in 20 mM Tris-HCl pH 9.0 or 9 ml elution, the Vn-binding activity peaked. At 18 ml elution, or just before 70 % of the buffer B, a rather high background was detected, which was probably due to a considerably dominating protein content of the fraction.
Evidently, high background to a lesser extent occurred at the peaks at 13.5 ml and at the two peaks between 22.5- 27 ml elution (Fig. 6) . From a 2 ml run (containing 200 μg bacterial protein) approximately 2-3 μg VnBP could be recovered, suggesting approximately 1-1.5% of the staphylococcal cell surface protein was the 60 kDa VnBP. Such estimation was also true in the 10 ml runs.
References
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(1) GENERAL INFORMATION:
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(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: SE 9402490-8 (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: SE 9402490-8
(B) FILING DATE: 15-JUL-1994
(2) INFORMATION FOR SEQ ID NO: 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Staphylococcus aureus
(B) STRAIN: V8 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :
Met Asn Lys Thr Asp Leu lie Asn Ala Val Ala Glu Val Ala Asp Leu 1 5 10 15
Val Gly Lys Val 20
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ala Lys Lys Gin Arg Phe Arg His Arg Asn Arg Lys Gly Tyr Arg 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 166 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Staphylococcus aureus
(B) STRAIN: V8
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
CCCTCAACTG TTTCAAACAA TATAATTGAT GAACTTAAAC AAGTTGGTGA 50
ATACAATCAA ATTTTCACAA CTGAAGTTGA CGGTACAGTC ATAAACAATT 100
TATGTAAATN CAAAATGCCA AGAAGACATT GGATTACTTC CATTGCTTCT 150 TGGCA I I I I I TCAGGG 166
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Staphylococcus aureus
(B) STRAIN: V8
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Pro Ser Thr Val Ser Asn Asn lie Ile Asp Glu Leu Lys Gin Val Gly 1 5 10 15
Glu Tyr Asn Gin lie Phe Thr Thr Glu Val Asp Gly Thr Val lie Asn 20 25 30
Asn Leu Cys Lys Xaa Lys Met Pro Arg Arg His Trp Ile Thr Ser Ile 35 40 45
Ala Ser Trp His Phe Phe Arg 50 55

Claims

Claims :
1. A vitronectin binding protein that can be derived from staphylococci having an apparent molecular weight of 60 kDa and having affinity to vitranectin and the quinquedecapeptide
AKKQRFRHRNRKGYR (SEQ.ID.2), as well as variants, subfragments, multiples or mixtures of said protein having essentially the same binding characteristics.
2. A vitronectin binding protein according to claim l having the N-terminal sequence MNKTDLINAVAEVADLVGKV (SEQ.ID.l).
3. A method for preparing a vitronectin binding protein according to claim 1 comprising culturing a staphylococcal bacterial strain, preferably Staphylococcus aureus. strain V8, preparing a cell surface protein extract of the bacteria and purifying the protein preferably by applying said extract to an affinity purification step using the peptide AKKQRFRHRNRKGYR (SEQ.ID.2).
4. The quinquedecapeptide AKKQRFRHRNRKGYR (SEQ.ID.2) and variants thereof having essentially the same binding characteristics as the quinquedecapeptide.
5. A pharmaceutical composition comprising a pharmaceutically effective amount of the quinquedecapeptide AKKQRFRHRNRKGYR
(SEQ.ID.2) according to claim 4 and pharmaceutically acceptable excipients, diluents and carriers.
6. An antibody against a vitronectin binding protein according to claim 1, such as a polyclonal or monoclonal antibody.
7. A pharmaceutical composition comprising a pharmaceutically effective amount of an antibody according to Claim 6 or a vitronectin binding protein according to claim 1, including variants, subfragments, multiples or mixtures of said protein having essentially the same binding characteristics, and pharmaceutically acceptable excipients, diluents and/or carriers.
8. A vaccine composition comprising a pharmaceutically effective amount of a vitronectin binding protein according to claim 1, and pharmaceutically acceptable adjuvants and/or excipients.
9. Use of an antibody according to Claim 6 or a vitronectin binding protein according to claim 1 for the preparation of a pharmaceutical composition against staphylococcal infections.
10. A diagnostic or analytical kit comprising an effective amount of a vitronectin binding protein according to claim 1 and/or the quinquedecapeptide according to claim 4 and/or an antibody according to claim 6.
11. A method for purifying vitronectin by using the vitronectin binding protein according to claim 1 in an affinity column.
PCT/SE1995/000861 1994-07-15 1995-07-14 Vitronectin binding protein WO1996002566A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP95926088A EP0776336A1 (en) 1994-07-15 1995-07-14 Vitronectin binding protein
JP8504958A JPH10505581A (en) 1994-07-15 1995-07-14 Vitronectin binding protein

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9402490-8 1994-07-15
SE9402490A SE9402490D0 (en) 1994-07-15 1994-07-15 Vitronection binding protein

Publications (2)

Publication Number Publication Date
WO1996002566A1 WO1996002566A1 (en) 1996-02-01
WO1996002566A9 true WO1996002566A9 (en) 1997-02-13

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE1995/000861 WO1996002566A1 (en) 1994-07-15 1995-07-14 Vitronectin binding protein

Country Status (4)

Country Link
EP (1) EP0776336A1 (en)
JP (1) JPH10505581A (en)
SE (1) SE9402490D0 (en)
WO (1) WO1996002566A1 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6680195B1 (en) * 1997-11-26 2004-01-20 Inhibitex, Inc. Extracellular matrix-binding proteins from staphylococcus aureus
EP1125507A1 (en) * 2000-02-15 2001-08-22 Tiense Suikerraffinaderij N.V. (Raffinerie Tirlemontoise S.A.) Inulin products with improved nutritional properties
SE0000514D0 (en) * 2000-02-17 2000-02-17 Biostapro Ab A 52 kDa protein from coagulase negative staphylococci and fragments

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4837193A (en) * 1992-09-21 1994-04-12 Alfa-Laval Agri International Aktiebolag Fibrinogen binding protein
NZ261149A (en) * 1993-02-05 1997-07-27 Smithkline Beecham Plc Staphylococcus aureus polypeptides and monoclonal antibodies therefrom, their production and use

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