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WO1990012033A1 - Construction et utilisation de structures synthetiques codant pour le syndecane - Google Patents

Construction et utilisation de structures synthetiques codant pour le syndecane Download PDF

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Publication number
WO1990012033A1
WO1990012033A1 PCT/US1990/001496 US9001496W WO9012033A1 WO 1990012033 A1 WO1990012033 A1 WO 1990012033A1 US 9001496 W US9001496 W US 9001496W WO 9012033 A1 WO9012033 A1 WO 9012033A1
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Prior art keywords
sequence
peptide
dna
syndecan
oligonucleotide
Prior art date
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PCT/US1990/001496
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English (en)
Inventor
Merton R. Bernfield
Scott Saunders
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The Board Of Trustees Of The Leland Stanford Junior University
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Publication of WO1990012033A1 publication Critical patent/WO1990012033A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • This invention relates generally to the field of genetic engineering and more particularly to genes for proteoglycans, their insertion into recombinant DNA vectors, and the production of the resulting core proteins in recipient strains of microorganisms and the proteoglycan in recipient eukaryotic cells.
  • the cellular behavior responsible for the development, repair and maintenance of tissues is regulated, in large part, by interactions between cells and their extracellular matrix. These interactions are mediated by cell surface molecules acting as receptors that bind the large insoluble matrix molecules and induce responses that result in changes of cellular phenotype.
  • Several proteins associated with the cell surface can bind matrix components. These proteins differ in their specificity and affinity and in their mode of association with the cell surface. Some bind cells to single matrix ligands while others, such as some members of the integrin super family, appear to have multiple matrix ligands. Of the various matrix- binding proteins at the cell surface, only the integrins are known to be integral membrane proteins. The integrin fibronectin receptor codistributes both with extracellular fibronectin and with intracellular cytoskeletal components, apparently via an association of the receptor's cytoplasmic domain with the cytoskeletal protein talin.
  • the present inventors have studied a lipophilic proteoglycan containing both heparan sulfate and chondroitin sulfate that is found at the surface of mouse mammary epithelial cells and that behaves as a high affinity receptor specific for multiple components of the interstitial matrix.
  • This proteoglycan has been given the name syndecan in the mouse.
  • the proteoglycan binds the epithelial cells via its heparan sulfate chains to collagen types I, III, and V (Koda, J.E., Rapraeger, A., and Bernfield, M., J. Biol. Chem. (1985) 260; 8157-8162), fibronection (Saunders, S. and Bernfield, M.
  • Cultured epithelial cells shed the ectodomain from their apical surfaces as a non- lipophilic proteoglycan that contains all of the glycosar ⁇ inoglycan of the intact molecule and polarize the proteoglycan exclusively to their basolateral surfaces, a location consistent with its matrix receptor function. Upon suspension of these cells, the ectodomain is cleaved from the cell surface; the proteoglycan is not replaced while the cells are suspended (Jalkanen, M. , Rapraeger, A., Saunders, S., and Bernfield, M. , J. Cell Biol. (1987) 105: 3087- 3096).
  • the proteoglycan is mainly on epithelia in mature tissues (Hayashi, K., Hayashi, M. , Jalkanen, M. , Firestone, J.H., Trelstad, R.L., and Bernfield, M. , J. Histochem. Cytochem. (1987) 35_: 1079-1088), and some of the present inventors have previously proposed that it is a matrix anchor that stablizes the morphology of epithelial sheets by linking the cytoskeleton to the extracellular matrix (Bernfield, M. , Rapraeger, Al, Jalkanen, M. , and Banerjee, S.D., Basement Membranes (1985) 343-352).
  • Syndecan undergoes substantial regulation; its size, glycosaminoglycan composition and location at the cell surface vary between epithelial types, and its expression changes during development.
  • the proteoglycan is located exclusively at the basolateral cell surface of simple epithelia but surrounds stratified epithelial cells. At basolateral cell surfaces, it appears to contain two heparan sulfate and two chrondroitin sulfate chains, but where it surrounds cells, it contains only a single heparan sulfate chain and a single small chrondroitin sulfate chain (Sanderson, R.D., and Bernfield, M. , Proc. Natl. Acad. Sci. USA (1987) 23J3: 491-497).
  • the proteoglycan is lost when the cells terminally differentiate (Hayashi, K., Hayashi, M. , Boutin, E., Cunha, G.R., Bernfield, M. , and Trelstad, R. ., J. Lab. Invest. (1988) J58_: 68-76).
  • the proteoglycan is transiently lost when epithelia change their shape and is transiently expressed by mesenchymal cells undergoing morphogenetic tissue interaction.
  • Heparan sulfate proteoglycans are ubiquitous on the surfaces of adherent cells and bind various ligands including extracellular matrix, growth factors, proteinase inhibitors, and lipoprotein lipase; see Fransson, L., Trends Biochem. Sci. (1987) _12: 406-
  • an isolated peptide having a molecular weight of from about 31 kD to about 35 kD and comprising a hydrophilic amino terminus extracellular region, a hydrophilic carboxy terminus cytoplasmic region, and a hydrophobic transmembrane region between said cytoplasmic and extracellular regions, a dibasic sequence extracellularly adjacent the transmembrane region of the peptide, and at least one glycosylation site in the extracellular region including an Xac-Xaa- Ser-Gly-Xac sequence, wherein Xac is an acidic amino acid and Xaa is any amino acid and wherein said peptide is capable of functioning as a core protein for attachment of a heparan sulfate chain at said Ser.
  • A is alanine
  • C cysteine
  • D is aspartate
  • E glutamate
  • F is phenylalanine
  • G is glycine
  • H histidine
  • I is isoleucine
  • K lysine
  • L leucine
  • M methionine
  • N is asparagine
  • P proline
  • Q glutamine
  • R arginine
  • S serine
  • T threonine
  • V valine
  • W tryptophan
  • Y is tyrosine.
  • DNA and RNA molecules, recombinant DNA vectors, and modified microorganisms or eukaryotic cells comprising a nucleotide sequence that encodes any of the peptides indicated above are also part of the present invention.
  • sequences comprising all or part of the following DNA sequence, a complementary DNA or RNA sequence, or a corresponding RNA sequence are especially preferred:
  • DNA and RNA molecules containing segments of the larger sequence are also provided for use in carrying out preferred aspects of the invention relating to the production of such peptides by the techniques of genetic engineering and the production of oligonucleotide probes.
  • Figure 1 is a formula showing the cDNA sequence for syndecan and the corresponding amino acid sequence.
  • Figure 2 is a restriction map showing sequencing strategy of syndecan cDNA clones.
  • Figure 3 is a table showing potnetial glycosylation sites of the syndecan core protein and homology of these regions to the glycosylation site of other proteins.
  • Figure 4 is a schematic diagram showing different regions of the syndecan core protein.
  • Figure 5 is a table showing DNA sequence similarities between murine syndecan and human insulin receptor.
  • the 311 amino acid core protein has a unique sequence that contains several structural features consistent with its role as a matrix anchor and as an acceptor of two distinct types of glycosaminoglycan chains.
  • the expression of its mRNA is tissue-type specific, and both the 5' and 3' untranslated regions of its cDNA show substantial sequence homology to those of the human insulin receptor cDNA.
  • This core protein cDNA defines a new class of matrix receptor, an integral membrane proteoglycan, for which we propose the name syndecan (from the Greek, syndein, to bind together).
  • Nucleotide sequence of one strand of syndecan cDNA The numbers refer to the amino acid sequence and corresponding DNA codon sequence beginning at the amino terminus of the protein. The stop codon is marked "end.”
  • the trinucleotides of Table 1, termed codons, are presented as DNA trinucleotides, as they exist in the genetic material of a living organism.
  • Complementary trinucleotide DNA sequences having opposite strand polarity are functionally equivalent to the codons of Table 1, as is understood in the art.
  • An important and well known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed. Therefore, a number of different nucleotide sequences may code for a given amino acid sequence.
  • nucleotide sequences are considered functionally equivalent since they can result in the production of the same amino acid sequence in all organisms, although certain strains may translate some sequences more efficiently than they do others. Occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship in any way.
  • the equivalent codons are shown in Table 2 below. TABLE 2
  • Each 3-letter triplet represents a trinucleotide of DNA having a 5' end on the left and a 3' end on the right.
  • the letters stand for the purine or pyrimidine bases forming the nucleotide sequence.
  • T thymine Since the DNA sequence of the gene has been fully identified, it is possible to produce a DNA gene entirely by synthetic chemistry, after which the gene can be inserted into any of the many available DNA vectors using known techniques of recombinant DNA technology. Thus the present invention can be carried out using reagents, plasmids, and microorganism which are freely available and in the public domain at the time of filing of this patent application. For example, nucleotide sequences greater than
  • oligonucleotides can readily be spliced using, among others, the techniques described later in this application to produce any nucleotide sequence described herein. For example, relatively short complementary oligonucleotide sequences with 3' or 5' segments that extend beyond the complementary sequences can be synthesized.
  • proteins that lack the amino terminus first 17 amino acids are preferred since the first 17 amino acids appear to represent a signal sequence.
  • additional amino acids can be absent from either or both terminals of the sequence given without losing ability to act as a core protein for synthesis of proteoglycans.
  • up to 10 additional amino acids can be present at either or both terminals.
  • preferred compounds are those which more closely approach the specific formulas given (or the corresponding sequence that lacks a signal sequence) with 10 or fewer, more preferably 5 or fewer, absent amino acids being preferred for either terminal and 7 or fewer, more preferably 4 or fewer, additional amino acids being preferred for either terminal.
  • Whether a change results in a functioning peptide can readily be determined by assessing the ability of the corresponding DNA coding for this peptide to produce this peptide in glycosylated form when introduced into eukaryotic cells. Examples of this process are described later in detail. If attachment of glycosaminoglycan chains occurs, the replacement is immaterial, and the molecule being tested is equivalent to those specifically described above. Peptides in which more than one replacement has taken place can readily be tested in the same manner. The number of replacements is not strictly limited, but 10 or fewer are preferred.
  • DNA molecules that code for such peptides can readily be determined from the list of codons in Table 2 and are likewise contemplated as being equivalent to the DNA sequence of Table 1.
  • any discussion in this application of a replacement or other change in a peptide is equally applicable to the corresponding DNA sequence or to the DNA molecule, recombinant vector, transformed microorganism, or transfected eukaryotic cells in which the sequence is located (and vice versa). Codons can be chosen for use in a particular host organism in accordance with the frequency with which a particular codon is utilized by that host, if desired, to increase the rate at which expression of the peptide occurs.
  • DNA (or corresponding RNA) molecules of the invention can have additional nucleotides preceeding or following those that are specifically listed.
  • poly A can be added to the 3'-terminal
  • short (e.g., fewer than 20 nucleotides) sequence can be added to either terminal to provide a terminal sequence corresponding to a restriction endonuclease site, stop codons can follow the peptide sequence to terminate transcription, and the like.
  • DNA molecules containing a promoter region or other control region upstream from the gene can be produced.
  • RNA molecules are said to correspond to DNA molecules if they encode the same amino acids and/or control sequences.
  • Peptides of the invention can be prepared for the first time as purified preparations, either by direct synthesis or by using a cloned gene as described herein.
  • purified is meant, when referring to a peptide or DNA or RNA sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type.
  • purified as used herein preferably means at least 95% by weight, more preferably at least 99% by weight, and most preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000, can be present).
  • the term “pure” as used herein preferably has the same numerical limits as “purified” immediately above.
  • isolated refers to a peptide, DNA, or RNA molecule separated not only from other peptides, DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule but also from other macromolecules and preferrably refers to a macromolecule found in the presence of (if anything) only a solvent, buffer, ion or other low molecular weight component normally present in a solution of the same. "Isolated” and
  • purified do not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acylamide gel) but not obtained either as pure substances or as solutions.
  • Two protein sequences are homologous (as this term is preferably used in this specification) if they have an alignment score of >5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 (or greater). See Dayhoff, M.O., in Atlas of Protein Sequence and Structure, 1972, volume 5, National Biomedical Research Foundation, pp. 101-110, and Supplement 2 to this volume, pp. 1-10.
  • the two sequences (or parts thereof— robably at least 30 amino acids in length) are more preferably homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program mentioned above.
  • Two DNA sequences are homologous if they hybridize to one another using nitrocellulose filter hybridization (one sequence bound to the filter, the other as a 3 2 p _ labeled probe) using hybridization conditions of 40-50% formamide, 37°-42° C, 4x SSC and wash conditions (after several room temperature washes with 2x SSC, 0.05% SDS) of stringency equivalent to 37° C with lx SSC, 0.05% SDS.
  • hybridization conditions 40-50% formamide, 37°-42° C, 4x SSC and wash conditions (after several room temperature washes with 2x SSC, 0.05% SDS) of stringency equivalent to 37° C with lx SSC, 0.05% SDS.
  • replacement by or replacement does not necessarily refer to any action that must take place but to the peptide that exists when an indicated “replacement” amino acid is present in the same position as the amino acid indicated to be present in a different formula (e.g., when leucine is present at position 5 instead of isoleucine). Salts of any of the macromolecules described herein will naturally occur when such molecules are present in (or isolated from) aqueous solutions of various pHs. All salts of peptides and other macromolecules having the indicated biological activity are considered to be within the scope of the present invention.
  • Examples include alkali, alkaline earth, and other metal salts of carboxylic acid residues, acid addition salts (e.g., HC1) of amino residues, and zwitter ions formed by reactions between carboxylic acid and amino residues within the same molecule.
  • acid addition salts e.g., HC1
  • zwitter ions formed by reactions between carboxylic acid and amino residues within the same molecule.
  • Hydrophobic and hydrophilic regions can be determined by standard procedures from amino acid sequences, for example by plotting hydrophobicity according to the procedure of Kyte and Doolittle, J_-_ Mol. Biol. (1982) 157: 105-132. Plotted values averaged over groups of seven contiguous residues that are positive indicate hydrophobic regions, while negative values indicate hydrophilic regions.
  • the invention has specifically contemplated each and every possible variation of peptide or nucleotide that could be made by selecting combinations based on the possible amino acid and codon choices listed in Table 1 and Table 2, and all such variations are to be considered as being specifically disclosed.
  • genetic information encoded as mRNA is obtained from cultured epithelial cells, preferably from mammalian sources, and used in the construction of a DNA gene, which is in turn used to produce a peptide of the invention.
  • An initial crude cell suspension is sonicated or otherwise treated to disrupt cell membranes so that a crude cell extract is obtained.
  • Known techniques of biochemistry e.g., preferential precipitation of proteins
  • the crude cell extract, or a partially purified RNA portion therefrom, is then treated to further separate the RNA.
  • crude cell extract can be layered on top of a 5 ml cushion of 5.7 M CsCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA in a 1 in. _ 3
  • SW27 rotor Beckman Instruments Corp., Fullerton, Calif.
  • RNA is precipitated from the aqueous phase with ethanol in the presence of 0.2 M Na-acetate pH 5.5 and collected by centrifugation. Any other method of isolating RNA from a cellular source may be used instead of this method.
  • RNA may be employed such as polyadenylated, crude or partially purified messenger RNA, which may be heterogeneous in sequence and in molecular size.
  • the selectivity of the RNA isolation procedure is enhanced by any method which results in an enrichment of the desired mRNA in the heterodisperse population of mRNA isolated. Any such prepurification method may be employed in preparing a gene of the present invention, provided that the method does not introduce endonucleolytic cleavage of the mRNA.
  • Prepurification to enrich for desired mRNA sequences may also be carried out using conventional methods for fractionating RNA, after its isolation from the cell. Any technique which does not result in degradation of the RNA may be employed. The techniques of preparative sedimentation in a sucrose gradient and gel electrophoresis are especially suitable.
  • the mRNA must be isolated from the source cells under conditions which preclude degradation of the mRNA.
  • the action of RNase enzymes is particularly to be avoided because these enzymes are capable of hydrolytic cleavage of the RNA nucleotide sequence.
  • a suitable method for inhibiting RNase during extraction from cells involves the use of 4 M guanidium thiocyanate and 1 M mercaptoethanol during the cell disruption step.
  • a low temperature and a pH near 5.0 are helpful in further reducing RNase degradation of the isolated RNA.
  • mRNA is prepared essentially free of contaminating protein, DNA, polysaccharides and lipids. Standard methods are well known in the art for accomplishing such purification. RNA thus isolated contains non-messenger as well as messenger RNA.
  • a convenient method for separating the mRNA of eukaryotes is chromatography on columns of oligo-dT cellulose, or other oligonucleotide-substituted column material such as poly-U or poly-T Sepharose, taking advantage of the hydrogen bonding specificity conferred by the presence of polyadenylic acid on the 3' end of eukaryotic mRNA. Hybridization with oligonucleotide probes prepared from DNA sequences set forth in this specification can then be used to isolate the particularly desired mRNA.
  • the next step in most methods is the formation of DNA commplementary to the isolated heterogeneous sequences of mRNA.
  • the enzyme of choice for this reaction is reverse transcriptase, although in principle any enzyme capable of forming a faithful complementary DNA copy of the mRNA template could be used.
  • the reaction may be carried out under conditions described in the prior art, using mRNA as a template and a mixture of the four deoxynucleoside triphosphates, dATP, dGTP, dCTP, and dTTP, as precursors for the DNA strand.
  • one of the deoxynucleoside triphosphates be labeled with a radioisotope, for example 32 P in the alpha position, in order to monitor the course of the reaction, to provide a tag for recovering the product after separation procedures such as chromatography and. electrophoresis, and for the purpose of making quantitative estimates of recovery.
  • a radioisotope for example 32 P in the alpha position
  • the cDNA transcripts produced by the reverse transcriptase reaction are somewhat heterogeneous with respect to sequences at the 5' end and the 3' end due to variations in the initiation and termination points of individual transcripts, relative to the mRNA template.
  • the variability at the 5' end is thought to be due to the fact that the oligo-dT primer used to initiate synthesis is capable of binding at a variety of loci along the polyadenylated region of the mRNA.
  • Synthesis of the cDNA transcript begins at an indeterminate point in the poly-A region, and variable length of poly-A region is transcribed depending on the inital binding site of the oligo-dT primer. It is possible to avoid this indeterminacy by the use of a primer containing, in addition to an oligo-dT tract, one or two nucleotides of the RNA sequence itself, thereby producing a primer which will have a preferred and defined binding site for initiating the transcription reaction.
  • the indeterminacy at the 3'-end of the cDNA transcript is due to a variety of factors affecting the reverse transcriptase reaction, and to the possiblity of partial degradation of the RNA template.
  • the isolation of specific cDNA transcripts of maximal length is greatly facilitated if conditions for the reverse transcriptase reaction are chosen which not only favor full length synthesis but also repress the synthesis of small DNA chains.
  • Preferred reaction conditions for avian myeloblastosis virus reverse transcriptase are given in the examples section of U.S. Patent 4,363,877 and are herein incorporated by reference.
  • the specific parameters which may be varied to provide maximal production of long-chain DNA transcripts of high fidelity are reaction temperature, salt concentration, amount of enzyme, concentration of primer relative to template, and reaction time.
  • the conditions of temperature and salt concentration are chosen so as to optimize specific base-pairing between the oligo-dT primer and the polyadenylated portion of the RNA template. Under properly chosen conditions, the primer will be able to bind at the polyadenylated region of the RNA template, but non-specific initiation due to primer binding at other locations on the template, such as short, A-rich sequences, will be substantially prevented.
  • the effects of temperature and salt are interdependent. Higher temperatures and low salt concentrations decrease the stability of specific base-pairing interactions.
  • reaction time is kept as short as possible, in order to prevent non-specific initiations and to minimize the opportunity for degradation. Reaction times are interrelated with temperature, lower temperatures requiring longer reaction times. At 42°C, reactions ranging from 1 min. to 10 minutes are suitable.
  • the primer should be present in 50 to 500- fold molar excess over the RNA template and the enzyme should be present in similar molar excess over the RNA template. The use of excess enzyme and primer enhances initiation and cDNA chain growth so that long-chain cDNA transcripts are produced efficiently within the confines of the short incubation times.
  • the cDNA prepared as described above may be used as a template for the synthesis of double- stranded DNA, using a DNA poly erase such as reverse transcriptase and a nuclease capable of hydrolyzing single-stranded DNA.
  • a DNA poly erase such as reverse transcriptase and a nuclease capable of hydrolyzing single-stranded DNA.
  • the cDNA can be purified further by the process of U.S. Patent 4,363,877, although this is not essential.
  • heterogeneous cDNA prepared by transcription of heterogeneous mRNA sequences, is treated with one or two restriction endonucleases.
  • the choice of endonuclease to be used depends in the first instance upon a prior determination that recognition sites for the enzyme exist in the sequence of the cDNA to be isolated. The method depends upon the existence of two such sites. If the sites are identical, a single enzyme will be sufficient.
  • the desired sequence will be cleaved at both sites, eliminating size heterogeneity as far as the desired cDNA sequence is concerned, and creating a population of molecules, termed fragments, containing the desired sequence and homogeneous in length. If the restriction sites are different, two enzymes will be required in order to produce the desired homogeneous length fragments.
  • restriction enzyme(s) capable of producing an optimal length nucleotide sequence fragment coding for all or part of the desired protein must be made empirically. If the amino acid sequence of the desired protein is known, it is possible to compare the nucleotide sequence of uniform length nucleotide fragments produced by restriction endonuclease cleavage with the amino acid sequence for which it codes, using the known relationship of the genetic code common to all forms of life. A complete amino acid sequence for the desired protein is not necessary, however, since a reasonably accurate identification may be made on the basis of a partial sequence.
  • the uniform length polynucleo- tides produced by restriction endonuclease cleavage may be used as probes capable of identifying the synthesis of the desired protein in an appropriate in vitro protein synthesizing system.
  • the mRNA may be purified by affinity chromatography. Other techniques which may be suggested to those skilled in the art will be appropriate for this purpose.
  • restriction enzymes suitable for use depends upon whether single-stranded or double- stranded cDNA is used.
  • the preferred enzymes are those capable of acting on single-stranded DNA, which is the immediate reaction product of mRNA reverse transcription.
  • the number of restriction enzymes now known to be capable of acting on single-stranded DNA is limited.
  • the enzymes Haelll, Hhal and Hin(f)I are presently known to be suitable.
  • the enzyme MboII may act on single-stranded DNA.
  • additional suitable enzymes include those specified for double-stranded cDNA.
  • double-stranded cDNA presents the additional technical disadvantages that subsequent sequence analysis is more complex and laborious. For these reasons, single-stranded cDNA is prefered, but the use of double-stranded DNA is feasible. In fact, the present invention was initially reduced to practice using double-stranded cDNA.
  • the cDNA prepared for restriction endonuclease treatment may be radioactively labeled so that it may be detected after subsequent separation steps.
  • a preferred technique is to incorporate a radioactive label such as ⁇ P in the alpha position of one of the four deoxynucleoside triphosphate precursors. Highest activity is obtained when the concentration of radioactive precursor is high relative to the concentration of the non-radioactive form. However, the total concentration of any deoxynucleoside triphosphate should be greater than 30 yM, in order to maximize the length of cDNA obtained in the reverse transcriptase reaction.
  • Fragments which have been produced by the action of a restriction enzyme or combination of two restriction enzymes may be separated from each other and from heterodisperse sequences lacking recognition sites by any appropriate technique capable of separating polynucleotides on the basis of differences in length.
  • Such methods include a variety of electrophoretic techniques and sedimentation techniques using an ultracentrifuge.
  • Gel electrophoresis is preferred because it provides the best resolution on the basis of polynucleotide length.
  • the method readily permits quantitative recovery of separated materials. Convenient gel electrophoresis methods have been described by Dingman, C.W., and Peacock, A.C., Biochemistry (1968) 1_: 659 , and by Maniatis, T., Jeffrey, A. and van de Sande, H., Biochemistry (1975) 1 :3787.
  • cDNA transcripts obtained from most sources will be found to be heterodisperse in length.
  • polynucleotide chains containing the desired sequence will be cleaved at the respective restriction sites to yield polynucleotide fragments of uniform length.
  • polynucleotide fragments of uniform length Upon gel electrophoresis, these will be observed to form a distinct band.
  • other discrete bands may be formed as well, which will most likely be of different length than that of the desired sequence.
  • the gel electrophoresis pattern will reveal the appearance of one or more discrete bands, while the remainder of the cDNA will continue to be heterodisperse.
  • the electrophoresis pattern will reveal that most of the cDNA is present in the discrete band.
  • Sequence analysis of the electrophoresis band may be used to detect impurities representing 10% or more of the material in the band.
  • a method for detecting lower levels of impurities has been developed founded upon the same general principles applied in the initial isolation method. The method requires that the desired nucleotide sequence fragment contain a recognition site for a restriction endonuclease not employed in the initial isolation.
  • the amount of material present in any band of radioactively labeled polynucleotide can be determined by quantitative measurement of the amount of radioactivity present in each band, or by any other appropriate method.
  • a quantitative measure of the purity of the fragments of desired sequence can be obtained by comparing the relative amounts of material present in those bands representing sub-fragments of the desired sequence with the total amount of material.
  • DNA ligase which catalyzes the end-to-end joining of DNA fragments
  • the gel electrophoresis bands representing the sub-fragments of the desired sequence may be separately eluted and combined in the presence of DNA ligase, under the appropriate conditions. See Sgaramella, V., Van de Sande, J.H., and Khorana, H.G., Proc. Natl. Acad. Sci. USA (1970) £7:1468. Where the sequences to be joined are not blunt-ended, the ligase obtained from E. coli may be used; Modrich, P., and Lehman, I.R., J. Biol. Chem. (1970) 245:3626.
  • the efficiency of reconstituting the original sequence from sub-fragments produced by restriction endonuclease treatment will be greatly enhanced by the use of a method for preventing reconstitution in improper sequence.
  • This unwanted result is prevented by treatment of the homogeneous length cDNA fragment of desired sequence with an agent capable of removing the 5'-terminal phosphate groups on the cDNA prior to cleavage of the homogeneous cDNA with a restriction endonuclease.
  • the enzyme alkaline phosphatase is preferred.
  • the 5'-terminal phosphate groups are a structural prerequisite for the subsequent joining action of DNA ligase used for reconstituting the cleaved sub-fragments.
  • ends which lack a 5'-terminal phosphate cannot be covalently joined.
  • the DNA sub-fragments can only be joined at the ends containing a 5'-phosphate generated by the restriction endonuclease cleavage performed on the isolated DNA fragment.
  • cDNA transcripts under the conditions described above, are derived from the mRNA region containing the 5'-end of the mRNA template by specifically priming on the same template with a fragment obtained by restriction endonuclease cleavage.
  • the above-described method may be used to obtain not only fragments of specific nucleotide sequence related to a desired protein, but also the entire nucleotide sequence coding for the protein of interest.
  • Double-stranded, chemically synthesized oligonucleotide linkers, containing the recognition sequence for a restriction endonuclease may be attached to the ends of the isolated cDNA, to facilitate subsequent enzymatic removal of the gene portion from the vector DNA.
  • the vector DNA is converted from a continuous loop to a linear form by treatment with an appropriate restriction endonuclease.
  • the ends thereby formed are treated with alkaline phosphatase to remove 5'-phosphate end groups so that the vector DNA may not reform a continuous loop in a DNA ligase reaction without first incorporating a segment of the syndecan DNA.
  • the cDNA, with attached linker oligonucleotides, and the treated vector DNA are mixed together with a DNA ligase enzyme, to join the cDNA to the vector DNA, forming a continuous loop of recombinant vector DNA, having the cDNA incorporated therein.
  • the closed loop will be the only form able to transform a bacterium. Transformation, as is understood in the art and used herein, is the term used to denote the process whereby a microorganism incorporates extracellular DNA and reproduces it stably from generation to generation. Plasmid DNA in the form of a closed loop may be so incorporated under appropriate environmental conditions. The incorporated closed loop plasmid undergoes replication in the transformed cell, and the replicated copies are distributed to progeny cells when cell division occurs. As a result, a new cell line is established, containing the plasmid and carrying the genetic determinants thereof.
  • Transformation by a plasmid in this manner occurs at high frequency when the transforming plasmid DNA is in closed loop form, and does not or rarely occurs if linear plasmid DNA is used.
  • cDNA clones encoding the syndecan polypeptide from a normal mouse mammary gland epithelial cell line as well as mouse liver tissue.
  • the cDNA derived protein sequence of syndecan is unique; comparisons with the National Biomedical Research Foundation and the translated NIH-Genebank databases detected no statistically significant similarities.
  • the nascent polypeptide sequence is 311 amino acids and has -a molecular mass of 32,868 daltons.
  • Treatment of syndecan with heparatinase and chondroitinase ABC generates a protein with relative mobility of ca. 69k daltons versus globular molecular weight markers on a gradient SDS-PAGE system.
  • This anomoly appears to be a charge effect and has been seen in other proteins rich in proline, alanine, and highly charged amino acides.
  • Syndecan is not a disulfide cross-linked dimer. Its migration on SDS-PAGE is unchanged following DTT treatment; its CNBr-cleavage product produces a single signal during amino acid sequencing; and its single cysteine in the predicted mature protein is located in the putative transmembrane domain. It also does not appear to be cross-linked by lysyl oxidase- or transglutaminase- mediated reactions because ⁇ -aminoproprionitrile and monodansylcadaverine treatments of NMuMG cells do not change its mobility on SDS-PAGE.
  • Proteins with regions rich in proline, alanine and highly charged amino acids have highly extended conformations and anomalously slow mobilities in SDS-PAGE, Guest, J.R., Lewis, H.M. , Graham, L.D., Packman, L.C., and Perham, R.N., J. Mol. Biol. (1985) 185: 743-754. These amino acids are abundant in syndecan, and a Chou and Fasman secondary structure prediction is consistent with large regions of extended conformation.
  • In vitro translation of synthetic mRNA corresponding to the coding region of syndecan (Sacl-Hindlll fragment of clone 4-19b) produces a nascent polypeptide of ca. 45k daltons.
  • the amino acid sequence derived from the syndecan cDNA shows three functional domains; an extracellular domain and, by inference, transmembrane and cytoplasmic domains.
  • the transmembrane domain was inferred from the physical properties of syndecan.
  • the derived C- terminal sequence of syndecan contains both a characterics transmembrane domain (amino acids 253 to 277 in Table 1) and a 34 amino acid putative cytoplasmic domain.
  • the cytoplasmic domain was inferred from properties already known for purified syndecan indicating that syndecan associates with the actin cytoskeleton.
  • An immune serum generated against a synthetic peptide from the C-terminus of the derived protein sequence reacts with native syndecan extracted from NMuMG cells but not with the ectodomain, providing direct evidence for the cytoplasmic domain.
  • the ectodomain of syndecan is released from
  • NMuMG cell surfaces during cell culture, rapidly in response to cell rounding, or by mild trypsin treatment.
  • the putative extracellular domain of syndecan contains a single dibasic site near the plasma membrane at which cleavage of syndecan from the cell surface undoubtedly occurs. Because the endogenously shed ectodomain of syndecan is indistinguishable from the trypsin-released form, a cell surface trypsin-like protease has been proposed. Shedding during cell culture is from the apical surface. However, when these cells are released from the substratum, destroying their polarity, the ectodomain is rapidly shed. These previously known results suggest that a cell surface protease is involved, but the structure of the site was not known. Identification of the putative cleavage site by the present invention will now allow more detailed investigation of this activity and will allow production of modified proteoglycans and other proteins that can be readily cleaved to release their extracellular regions for ready purification.
  • Syndecan isolated from several sources is a hybrid proteoglycan, containing both chondroitin sulfate and heparan sulfate. These chains are known to be linked via a xyloside to serine residues in proteins, Roden, L., The Biochemistry of Glycoproteins and Proteoglycans (1980) 267-371 and Dorfman, A., Cell Biology of Extracellular Matrix (1981) 115-138. Regulating the elaboration of both chondroitin sulfate and heparan sulfate chains on the same core protein is a significant problem because the intial four saccharides are identical.
  • Specific chain elongation subsequently involves the sequential action of an N-acetylgalactosaminyltransfer- ase and a glucuronosyltransferse for chondroitin sulfate, and an N-acetylglucosaminyltransferase and a glucuronosyltransferase for heparan sulfate.
  • This specific chain elongation must involve recognition of unique structural features of the core protein, indicating that distinct peptide sequences might exist at chondroitin sulfate versus heparan sulfate attachment sites.
  • chondroitin sulfate and heparan sulfate on syndecan provides the opportunity to assess the relationship between these attachment sites.
  • Syndecan contains three potential ser-gly glycosaminoglycan attachment sites that contain some features of this consensus acceptor sequence but also contain unique features (Figure 3B). Though each of these three sequences retains an acidic amino acid two residues N-terminal to the acceptor Ser-Gly, they lack the consensus glycine that is two residues C-terminal to the Ser-Gly. This omission does not preclude this sequence from serving as a xylosyltransferase acceptor because it is also omitted from the Gly-Ser site of type IX collagen, Huber, S., Winterhalter, K.H., and Vaughan, L., J. Biol. Chem. (1988) 26 ⁇ : 752-756.
  • An artificial peptide containing a heparan sulfate elongation site of the formula Xac-Xaa-Ser-Gly-Xac, where Xac is an acidic amino acid (aspartate or glutamate) and Xaa is any amino acid, can be prepared and used to produce heparan sulfate in eukaryotic cells as described herein.
  • the artificial peptide need not contain any of the remaining structure of the molecules described herein as long as it provides the indicated sequence at a location in the peptide that is available for glycosylation.
  • Such locations can be predicted, such as by using the algorithms developed by Chou and Fasman, or by empirically inserting a DNA sequence encoding this amino acid sequence into a gene and determing that the product functions as a recognition sequence for the elongation of heparan sulfate chains.
  • a simple artificial peptide might contain multiple copies of the recognition sequence either located directly adjacent to each other or being joined by from one to ten, preferably one to five, amino acids.
  • Another preferred embodiment involves producing a known polypeptide by genetic engineering that has been engineered to contain the attachment site of the invention at a location known to reside on an external surface of the polypeptide.
  • sequences from the natural syndecan amino acid sequences adjacent the Xac- Xaa-Ser-Gly-Xac sequences are not required, they may be retained if desired in order to produce a protein that more closely resembles syndecan. Accordingly, artifical peptides containing from 1 to 10, 20, 30, or even more naturally adjacent amino acids as shown in Table 1, located either C terminal or N terminal or both to the Xac-Xaa-Ser-Gly-Xac sequence, represent other viable embodiments of the invention. Proteins containing such longer sequences can be prepared in the same manner discussed above using corresponding longer DNA sequences encoding the desired region.
  • the number of chondroitin sulfate chains on syndecan apparently differs in cells of distinct cellular organization and changes in response to TGF- ⁇ , implying that each potential glycosaminoglycan • attachment site is not always utilized.
  • a possible novel regulatory mechanism for this variation is suggested by the location in syndecan of its single potential N-linked glycosylation site, Asn-Phe-Ser, at residues 43-45. This site is located within the putative chondroitin sulfate attachment sequence, and the attachment of an N-linked sugar at this site would likely prevent subsequent recognition by the xylosytransferase.
  • syndecan is expressed mainly in epithelia.
  • Northern blot analysis of mRNA revealed two mRNA species at 2.6 and 3.4kb (constant ratio 3:1 respectively) in NMuMG cells as well as skin, liver, and midpregnant mammmary gland, all containing immunoreactive syndecan.
  • these two mRNAs were undetectable in cardiac and skeletal muscle, tissues of mesenchymal origin that do not stain with 281-2.
  • primitive and embryonic mesenchymal cells also show the 2.6 and 3.4kb mRNA species.
  • the first hydrophobic stretch consists of 12 amino acids beginning shortly after the presumptive start methionine. Because syndecan is oriented with its N-terminus outside of the plasma membrane, this appears to be a signal sequence. The N-terminus of mature syndecan is blocked, and, therefore, it has not been possible to determine the N-terminus directly. A likely site for signal peptidase cleavage is following amino acid residue 17 ( Figure 1) in the predicted sequence. Cleavage at this site would generate an N- terminal glutamine which could readily cyclize forming a pyrrolidone carboxlyl residue and thus a blocked N- terminus, as exists in a number of eukaryotic proteins.
  • the second hydrophobic stretch is a sequence near the C-terminus which has characteristics of a transmembrane domain (thick underline. Figure 1).
  • This sequence is a highly hydrophobic stretch of 25 residues followed immediately by a series of highly charged residues, consistent with the stop transfer signals found following most membrane spanning domains.
  • This domain also contains the only cysteine and one of the four tyrosines in the apparant mature protein sequence.
  • the putative transmembrane domain defines two hydrophilic domains of the syndecan core protein, a putative extracellular domain consisting of approximately 235 amino acids, and a smaller putative cytoplasmic domain consisting of 34 amino acids.
  • the putative cytoplasmic domain contains three tyrosine residues, but the sequences adjacent to these tyrosines are not similar to the presently identified consensus sequences for tyrosine phosphorylation, Hunter, T., and Cooper, J.A., Ann. Rev. Biochem. (1985) 54: 879-930.
  • This domain presumably has protein binding activity because the intact proteoglycan but not the ectodomain co-sediments with F-actin, Rapraeger, A., and Bernfield, M. , Extracelluar Matrix (1982) 265-269, and because syndecan associates with the actin-containing cytoskeleton when cross-linked at the cell surface, Rapraeger, A., Jalkanen, M. , and Bernfield, M. , J. Cell Biol. (1986) 10_3: 2683-2696.
  • the putative extracellular domain has several sequence characteristics that correspond with the known properties of this proteoglycan.
  • the ectodomain of syndecan is shed by cleavage from its membrane anchor, Jalkanen, M., Rapraeger, A., Saunders, S., and Bernfield, M. , J. Cell Biol. (1987) 10_5: 3087-3096, and an indistinguishable molecule is released from the cell surface by mild trypsin treatment, Jalkanen, M. , Rapraeger, A., Saunders, S., and Bernfield, M. , J. Cell Biol. (1987) 105: 3087-3096.
  • the only dibasic sequence (Arg-Lys) in this extracellular domain is located adjacent to the putative transmembrane domain at residues 250-251 (identified in Figure 1 by arrows). This location places the cleavage site adjacent to the plasma membrane.
  • the putative extracellular domain lacks cysteine thus eliminating disulfide bridges as a means of generating secondary structure in this moleucle.
  • the ectodomain contains both heparan sulfate and chondroitin sulfate chains, Rapraeger, A., Jalkanen, M. , Endo, E., Koda, J., and Bernfield, M., J. Cell Biol. (1985b) 260: 11046-11052.
  • the serine hydoxyl group of ser-gly sequences are the attachment sites for these glycosaminoglycan chains, Roden, L., The Biochemistry of Glycoproteins and Proteoglycans. 267-371 and Dorfman, A., Cell Biology of Extracellular Matrix 115-138.
  • Syndecan possess five such potential glycosaminoglycan attachment sites, all within the putative extracellular domain; three such serines are clustered ar the N-terminus at residues 37, 45, and 47, and the remaining two are clustered near the membrane at residues 207 and 217 (open circles.
  • the ectodomain from NMuMG cells is insensitive to digestion by N-glycosidase F, as assessed by PAGE, Weitzhandler, M., Streeter, H.B., Henzel, W.J., and Bernfield, M. , J. Biol. Chem. (1988) 2 : 6949-6952.
  • the putative extracellular domain contains a single canonical sequence for the attachment of N-linked oligosaccharide (solid circle. Figure 1).
  • the serine in this Asn-Xaa-Ser sequence is a putative glycosaminoglycan attachment site.
  • syndecan or a molecule related to syndecan will be expressed when the DNA sequence encoding it is functionally inserted into a vector that is expressed in a eukaryotic cell containing an enzyme system capable of producing glycosaminoglycan chains.
  • functionally inserted is meant in proper reading frame and orientation, as is well understood by those skilled in the art.
  • Expression of syndecan can be enhanced by including multiple copies of the syndecan gene in a transformed or transfected host, by selecting a vector known to reproduce in the host, thereby producing large quantities of protein from exogeneous inserted DNA, or by any other known means of enhancing peptide expression.
  • U.S. Patent 4,419,450 discloses a plasmid useful as a cloning vehicle in recombinant DNA work.
  • U.S. Patent 4,362,867 discloses recombinant cDNA construction methods and hybrid nucleotides produced thereby which are useful in cloning processes.
  • U.S. Patent 4,403,036 discloses genetic reagents for generating plasmids containing multiple copies of DNA segments.
  • U.S. Patent 4,363,877 discloses recombinant DNA transfer vectors.
  • Patent 4,356,270 discloses a recombinant DNA cloning vehicle and is a particularly useful disclosure for those with limited experience in the area of genetic engineering since it defines many of the terms used in genetic engineering and the basic processes used therein.
  • U.S. Patent 4,336,336 discloses a fused gene and a method of making the same.
  • U.S. Patent 4,349,629 discloses plasmid vectors and the production and use thereof.
  • U.S. Patent 4,332,901 discloses a cloning vector useful in recombinant DNA.
  • Manipulation of the expression vectors will in some case produce constructs which improve the expression of the polypeptide in eukaryotic cells or express syndecan in other hosts. Furthermore, by using the syndecan cDNA or a fragment thereof as a hybridization probe, structurally related genes found in other organisms can be easily cloned. These genes include those that code for related core proteins of proteoglycans from other species, especially mammals such as humans and other primates.
  • oligo- nucleotide probes based on the principal and variant nucleotide sequences disclosed herein.
  • Such probes can be considerably shorter than the entire sequence but should be at least 14, preferably at least 20, nucleotides in length. Longer oligonucleotides are also useful, up to 30, 40, 50, 75, or 100 nucleotides and further up to the full length of the gene. Both RNA and DNA probes can be used.
  • Such probes can also be used in diagnostic tests that detect the presence of genetic material of a predetermined sequence in samples, e.g., as in a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the probes are typically labelled in a detectable manner (e.g., with 32p, 3 H, biotin, or avidin) and are incubated with single-stranded DNA or RNA from the organism in which a gene is being sought.
  • Hybridization is detected by means of the label after single-stranded and double-stranded (hybridized) DNA (or DNA/RNA) have been separated (typically using nitrocellulose paper).
  • Hybridization techniques suitable for use with oligonucleotides are well known.
  • oligonucleo ⁇ tide refers to both labeled and unlabeled forms and not just to labeled probes.
  • oligonucleotides corresponding to the segments of the gene that code for glycosaminoglycan attachment sites.
  • an oligonucleotide with high probability of success in the identification of other gene products is the 64-fold degenerate oligonucleotide of the form GANGGNTCTGGNGA, where N represents presence of all four nucleotides in degenerate sequences.
  • the complementary oligonucleotide having the degenerate sequence TCNCCAGANCCNTC is also particularly useful and has the added advantage of ability to identify messenger RNA of these gene products in Northern analysis.
  • the invention allows the production in large amounts of highly pure heparan sulfate proteoglycans that contain heparan sulfate chains that are characteristic of specific cell types.
  • the surface of endothelial cells is non-thrombogenic because of the anti-coagulant properties of the heparan sulfate chains in a proteoglycan on their surfaces.
  • Preparations of this highly anti-coagulant heparan sulfate proteoglycan in soluble form is now possible by transfection of cultured endothelial cells with a DNA construct defined by this invention. Expression of the contruct would produce syndecan containing endothelial cell-derived heparan sulfate chains.
  • Sydecan contains a unique protease-susceptible site adjacent to the plasma membrane, allowing the harvesting of this modified syndecan as a soluble product in high yield and purity.
  • This approach would produce an anti ⁇ coagulant proteoglycan with very high potency, potentially several thousand times more potent than commercially available heparin.
  • the soluble proteins or peptides containing cell-type-speteific heparan sulfate chains made possible by this invention, can be used in the prevention and therapy of certain viral diseases. Dextran sulfate and heparin have been shown to reduce infection and replication of certain retroviruses, including human immunodeficiency virus (HIV). However, these molecules are highly heterogenous and are probably non-specific.
  • a more specific inhibitor would be a soluble heparan sulfate peptide or proteoglycan derived from a cell type that interacts with the virus.
  • Peptides derived from this invention can also be used as highly specific competitive inhibitors of heparan sulfate (or chrondroitin sulfate) chain initiation. Because mutant transformed cells with reduced cell-surface heparan sulfate are substantially less turmorigenic, this invention has the potential of producing anti-tumor drugs that are non-cytotoxic.
  • a DNA construct derived from this invention can be used in fibroblasts that contain surface proteoglycans that bind various growth factors, including acidic fibroblast growth factor (FGF) and basic FGF. This bonding potentiates the action and prevents the proteolytic degradation of these growth factors.
  • FGF acidic fibroblast growth factor
  • PDGF Platelet-derived growth factor
  • the peptide sequences involved in heparan sulfate chain attachment identified by the present invention will allow production of large amounts of cell-type-specific heparan sulfate proteoglycans and enable this attachment site to be placed into other biological macromolecules that do not normally contain it, thereby providing products that are not otherwise available. These products will represent a singular molecular species, whereas the heparins and all other heparan sulfate proteoglycans heretofor described represent many molecular species.
  • the greater uniformity afforded by the present invention leads to greater potency and potentially to greater specificity of the materials being purified, thereby enhancing their therapeutic applications.
  • heparin from pig intestine or beef lung or dextran sulfate a synthetic product, that are polydispersed, of low potency, and of little specificity
  • Cell lines containing the genetic material necessary for the practice of the present invention can be obtained from a number of public sources, some of which are specifically identified in the following examples.
  • normal mouse mammary epithelial cells can be prepared from normal mouse tissue using the procedure described in the examples below. The same procedure can be used to obtain genetic material from other species.
  • NMuMG mouse mammary epithelial cells (passages 13-22) were maintained in bicarbonate-buffered Dulbecco's modified Eagle medium (Gibco) as described previously, David, G., and Bernfield, M., Proc. Natl. Acad. Sci. USA (1979) 7_6: 786-790.
  • Dulbecco's modified Eagle medium Gibco
  • cells were plated on 245 x 245 mm tissue culture plates (Nunc) at approximately one-fifth confluent density and grown to 80-90 percent confluency (3-4 days).
  • RNA extraction buffer (4 M guanidine isothiocyanate in 5 mM sodium citrate pH 7.0, 0.1M ⁇ -mercaptoethanol and 0.5% N-lauryl sarcosine) and total RNA prepared by CsCl density centrifugation, Chirgwin, J.M., Pryzybyla, A.E., MacDonald, R.J., and Rutter, W.J., Biochemistry (1979) 18: 5194-5299.
  • RNA was purified by chromatography on oligo(dT)-cellulose (type 3; Collaborative Research) and utilized in the commercial synthesis (Strategene) of cDNA by the SI method, Huynh, T.V., Young, R.A., and Davis, R.W., DNA Cloning: A
  • a primer extension cDNA library was prepared using the RNase H method, Gubler, U., and Hoffman,
  • First strand cDNA was synthesized from 10 yg of an 18-bp oligonucleotide containing sequence derived from near the 5' end of PM- 4 (see Example 2).
  • the second strand was synthesized using RNase H(BRL) and DNA polymerase Klenow fragment (Boehringer-Mannheim) .
  • the cDNA was methylated with EcoRI methylase and then ligated with synthetic EcoRI linkers (New England Biolabs). Excess linkers were removed by EcoRI digestion and the cDNA was purified on agarose gel electrophoresis and recovered by electroelution. The resulting cDNA was inserted into ⁇ gt-10 (Promega and packaged using Giga pack Gold (Stratagene) .
  • EXAMPLE 2 EXAMPLE 2
  • the cells were resuspended in 50 ml TBST (Tris buffered saline triton: 10 mM Tris pH 7, NaCl 150mM, Triton X-100 0.3%), sonicated, and following addition of 100 yl immunoserum (1:500 dilution), incubated overnight at 4 C. This mixture was centrifuged for 10 min at 4000 rpm and used to screen expressed ⁇ gt-11 cDNA clones. Young, R.A., and Davis, R.W., Science (1983) 22 : 778-782, by detection with alkaline phosphate-conjugated goat-anti- rabbit IgG (Promega).
  • syndecan purified from NMuMG cells reacted with an immunserum prepared against a synthetic peptide containing the C-terminal 7 amino acids (Lys-Gln-Gln- Glu-Glu-Phe-Tyr-Ala) of the PM-4 derived protein sequence.
  • This immunserum failed to react with the ectodomain which lacks the putative cytoplasmic domain.
  • this serum does not cross react with any other cellular proteins as assessed by Western blotting of total cell extracts.
  • Purified lambda DNA was prepared from positively selected clones by Lambdasorb immunoprecipitation (Promega). Fragments released by restriction endonuclease digestions were isolated by electrophoresis followed by excision from SeaPlaque agarose (FMC BioProducts). These isolated fragments were subcloned directly, in the presence of agarose, Struhl, K., BioTechniques (1985) 3_: 452-453, to either pGEM 3 and 4 for in vitro transcription, or M13 mpl8 and mpl9. Messing, J., Methods Enzymol. (1983) 101: 20- 78, for sequence analysis.
  • DNA sequencing was performed by the dideoxy chain termination method, Sanger, F., Nicklen, S., and Coulson, A.R., Proc. Natl. Acad. Sci. USA (1977) 74: 5463-5467, using a modified T7 DNA polymerase (Sequenase TM, U.S. Biochemical).
  • the strategy is summarized in Figure 2. Sequence was generated from both ends of subcloned restriction fragments using universal M13 sequencing primers. The internal sequence of large fragments as well as the complementary strands of all fragments were determined using oligonucleotide primers synthesized in accordance with preceding sequences.
  • the cDNA ( Figure 1) has the following features: The first AUG is at postion 240. This putative intiation codon is preceded by two inframe termination codons (TAA and TGA at positions 39 and 72 respectively) and followed by a 930 base open reading frame that ends at position 1173 with a TGA termination codon. Following the putative coding region are 1,243 bases of 3'-untranslated sequence that ends with the poly(A) stretch.
  • RNA for Northern analysis was prepared from the following: NMuMG cells, adult liver, newborn skin, mid-pregnant mammary gland, adult cerebrum, skeletal and cardiac muscle. Excised tissues were ground to a fine powder in the presence of liquid nitrogen and transferred directly to RNA exraction buffer (see above); the NMuMG cells were extracted after washing with PBS as described above. The samples were vigorously vortexed, an equal volume of lOmM Tris pH 8.0, ImM EDTA, and 1% SDS added, and subsequently extracted exhaustively with 24:24:1 Tris-saturated phenol:chloroform:isoamyl alcohol followed by a single extraction with 24:1 chloroform:isoamyl alcohol.
  • RNA was precipitated by addition of 1/3 volume of 10 M LiCl.
  • Poly(A) RNA was prepared by oligo d(T) chromatography as described above.
  • Hybridization probes were prepared by in vitro transcription of the 5' EcoRI-SacI fragment of PM-4 subcloned into pGEM3, Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K., and Green, M.R., Nucl. Acids Res. (1984) 12: 7035-7056. Blots were prehybridized at 61°C in 50% formamide, 1% SDS, 5X SSPE, 0.1% ficoll, 0.1% polyvinylpyrrolidone and 100 yg/ml denatured salmon sperm DNA.
  • Hybridization was for 16 hrs at 61°C in the same buffer containing 5 x 106 cpm/ml of RNA probe. Filters were washed 2 x 15 min at room temperature in 5% SDS/IX SSPE and 6 x 30 min at 67°C in 1% SDS/0.1X SSPE. Molecular sizes were determined relative to ethidium bromide stained molecular weight markers (BRL) and 18S and 28S riboso al RNA.
  • BTL ethidium bromide stained molecular weight markers
  • Northern blot analysis of the poly(A) RNA preparations reveals two mRNA bands in NMuMG cells as well as in skin, liver and mammary gland tissues; one band is at 2.6 and the other at 3.4kb.
  • Longer exposures of the Northern blot discussed above, as well as others containing larger quantities of poly(A) RNA verify that the mammary gland expresses both the 2.6 and the 3.4 kb messages (data not shown).
  • a seven amino acid (14C-labeled) synthetic peptide, corresponding to the predicted C-terminus of syndecan ( Figure 1) was prepared by direct synthesis.
  • the N-terminal lysine of this peptide was cross-linked by glutaraldehyde to keyhole limpet hemocyanin (KLH, Calbiochem) for immunization and bovine serum albumin (BSA, Fraction V, Sigma) for screening as described by Doolittle, R.F., Of URFS and ORFS: A Primer on How to Analyze Derived Amino Acid Sequences (1986) 85.
  • carrier protein was dissolved in 0.5 ml of 0.4 M phosphate, pH 7.5, mixed with 7.5 ymoles of peptide in 1.5ml water and 1.0 ml of 20 mM glutaraldehyde was added dropwise with stirring over the course of 5 min. After continuous stirring at room temperature for 30 min., 0.25 ml of 1 M glycine was added to block unreacted glutaraldehyde and the stirring resumed for an additional 30 min. The product was dialyzed exhaustively against phosphate-buffered saline and incorporation determined by TCA precipitation and liquid scintillation counting. This procedure resulted in the attachment of 17 moles of synthetic peptide per mole of carrier protein.
  • the native lipophilic form of syndecan and the nonlipophilic medium ectodomain form Jalkanen, M. , Rapraeger, A., Saunders, S., and Bernfield, M., J. Cell Biol. (1987) 105: 3087-3096, were isolated and purified as described elsewhere and assessed for their reactivity to the immune sera.
  • a cationic nylon membrane, Gene-Trans (Plasco Inc., Woburn, MA) was placed into an immunodot apparatus (V&P Scientific, San Diego, CA) and, samples of intact syndecan and the ectodomain (0.5, 5, 50 and 500 ng) were loaded on the membrane using mild vacuum.
  • the membrane was washed for 60 min at room temperture with ten changes of TBST and then incubated for 30 min with 1:7500 dilution of alkaline phosphatase goat-anti- rabbit IgG (Promega, Madison WI). Following washing for 60 min with ten changes of TBST, the immobilized alkaline phosphatase was visualized with nitro blue tetrazolium (NBT) 330 yg/ml and 5-bromo-4-chloro-3- indolyl phosphate (BCIP) 165 yg/ml in lOOmM Tris pH 9.5, 100 mM NaCl, and 5 mM MgCl 2 .
  • NBT nitro blue tetrazolium
  • BCIP 5-bromo-4-chloro-3- indolyl phosphate
  • Syndecan can be expressed within mammalian cells by transfection of a DNA contruct containing the syndecan core protein cDNA linked to a eukaryotic promoter that has the properties of both high-level expression and activity in a wide range of cell types.
  • a DNA contruct containing the syndecan core protein cDNA linked to a eukaryotic promoter that has the properties of both high-level expression and activity in a wide range of cell types.
  • the expression vector pH ⁇ APr-1- neo has been described (Gunning et al., PNAS 84:4831- 4835) which utilizes the human ⁇ -actin promoter and fullfills both of the above requirements.
  • This vector also contains the neomycin-resistance gene which allows selection of transfected cells with the antibiotic G- 418.
  • nucleotides 214-1379 of the sequence shown in Figure 1 which encompasses all of the coding region was inserted directionally between the Sall-BamHI sites of the pH ⁇ APr-1-neo vector and thus named p ⁇ -SSyn-neo.
  • this fragment was passed sequentially through pGEM 3Z (Promega), pGEM 7Zf (Promega), and Bluescript (Stratagene) .
  • This DNA construct was transformed into the bacterial strain TG-1 and prepared in large scale using routine plasmid preparation techniques including CsCl 2 density centrifugation.
  • the purified circularized plasmid DNA was transfected into Chinese Hamster Ovary (CHO) cells by standard calcium phosphate precipitation technique, and transfected clones were selected with G418.
  • CHO Chinese Hamster Ovary
  • the parental CHO (hamster) cells express mRNA which is cross-reactive with the murine syndecan cDNA, neither whole cells nor proteoglycan purified from these cells is reactive with the monoclonal antibody 281-2, a rat monoclonal antibody generated against murine syndecan. Therefore it has been possible to assess the function of the transfected murine syndecan gene using this antibody.
  • Anti-sense RNA produced from vectors of this type if expressed in sufficiently high levels, is capable of binding to endogenous message intracellularly and blocking its subsequent translation.
  • this vector the same coding region Sacl-Hindll fragment of syndecan described above was inserted into the BamHI-Hindlll site of the pH ⁇ Apr-1-neo vector to produce the vector p ⁇ -ASyn-neo.
  • the cDNA was inserted into the vector in the opposite orientation so as to produce mRNA from the transfected gene that is complementary to endogenous syndecan mRNA.
  • this fragment was sequencially passed through pGEM 3Z (Promega) and Bluescript (Stratagene) .
  • the 64 fold degenerate oligonucleotide of the form GAN GGN TCT GGN GA should statistically have the highest probability of success in the identification of other gene products which contain this putative signal for glycosaminoglycan attachment.
  • the complementary oligonucleotide of the form TCN CCA GAN CCN TC should have similar utility, with the added advantage of its ability to identify the messenger RNA of these gene products in Northern analysis.

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Abstract

La présente invention se rapporte à un peptide de mammifère purifié et à des informations génétiques de codage de tels peptides. Le peptide, d'un poid moléculaire compris entre environ 31 kD et environ 35 kD, comprend une région extra cellulaire de terminaison amino, une région cytoplasmique de terminaison carboxy et une région transmembranaire située entre la région cytoplasmique et la région extracellulaire, une séquence basique située à proximité extracellulairement adjacente de la région transmembranaire du peptide ainsi qu'au moins un site de glycosylation situé dans la région extracellulaire et comportant une séquence Xac-Xaa-Ser-Gly-Xac, dans laquelle Xac représente un acide aminé acide et Xaa représente n'importe quel acide aminé. Des peptides additionnels ayant ce site de glycosylation et des informations génétiques utiles dans la préparation d'un certain nombre de variantes basées sur un tel peptide sont également décrits.
PCT/US1990/001496 1989-03-29 1990-03-22 Construction et utilisation de structures synthetiques codant pour le syndecane WO1990012033A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5422243A (en) * 1991-01-15 1995-06-06 Jalkanen; Markku T. Detection of syndecan content in biological materials such as tissues and body fluids for indications of malignant transformations of cells
US5486599A (en) * 1989-03-29 1996-01-23 The Board Of Trustees Of The Leland Stanford Junior University Construction and use of synthetic constructs encoding syndecan
US5610148A (en) * 1991-01-18 1997-03-11 University College London Macroscopically oriented cell adhesion protein for wound treatment
US5629287A (en) * 1991-01-18 1997-05-13 University College London Depot formulations
US5726058A (en) * 1992-12-01 1998-03-10 Jalkanen; Markku Syndecan stimulation of cellular differentiation
US5851993A (en) * 1994-06-13 1998-12-22 Biotie Therapies Ltd. Suppression of tumor cell growth by syndecan-1 ectodomain
US6017727A (en) * 1994-03-07 2000-01-25 Biotie Therapies Ltd. Syndecan enhancer element and syndecan stimulation of cellular differentiation
US6699968B1 (en) 1989-03-29 2004-03-02 Children's Medical Center Corporation Construction and use of synthetic constructs encoding syndecan

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF CELL BIOLOGY, "Cell Surface Proteoglycan of Mouse Mammary Epithelial Cells in Shed by cleavage of its Matrix-binding Ectodomain from its membrane-associated Domain", Vol. 105, pp. 3087-3096, December 1987, JALKANEN et al., See Abstract. *
JOURNAL OF CELL BIOLOGY, "Molecular cloning of Syndecan, an integral membrane proteoglycan", Vol. 108, pp. 1547-1556, April 1989, SAUNDERS et al., See Fig. 1. *
JOURNAL OF CELL BIOLOGY, "Mouse Mammary Epithelial Cells Produce Basement Membrane and Cell Surface Heparan Sulfate Proteoglycans Containing Distinct Core Proteins", Vol. 106, pp. 953-962, March 1988, JALKANEN et al., See abstract. *
PROMEGA 1987/1988, Catalogue and Reference Guide, issued 1987. "Cloning Systems and Vectors", see pages 5 and 6. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5486599A (en) * 1989-03-29 1996-01-23 The Board Of Trustees Of The Leland Stanford Junior University Construction and use of synthetic constructs encoding syndecan
US6531295B1 (en) 1989-03-29 2003-03-11 Children's Medical Center Corporation Synthetic constructs encoding syndecan
US6699968B1 (en) 1989-03-29 2004-03-02 Children's Medical Center Corporation Construction and use of synthetic constructs encoding syndecan
US7183393B2 (en) 1989-03-29 2007-02-27 Children's Medical Center Corporation Construction and use of synthetic constructs encoding syndecan
US5422243A (en) * 1991-01-15 1995-06-06 Jalkanen; Markku T. Detection of syndecan content in biological materials such as tissues and body fluids for indications of malignant transformations of cells
US5610148A (en) * 1991-01-18 1997-03-11 University College London Macroscopically oriented cell adhesion protein for wound treatment
US5629287A (en) * 1991-01-18 1997-05-13 University College London Depot formulations
US5726058A (en) * 1992-12-01 1998-03-10 Jalkanen; Markku Syndecan stimulation of cellular differentiation
US6017727A (en) * 1994-03-07 2000-01-25 Biotie Therapies Ltd. Syndecan enhancer element and syndecan stimulation of cellular differentiation
US5851993A (en) * 1994-06-13 1998-12-22 Biotie Therapies Ltd. Suppression of tumor cell growth by syndecan-1 ectodomain

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