THE JOURNAL OF BIOLOGICAL. CHEMISTRY Vol. 253, No. 16, Issue of August 25, pp. 5642-5646. 1978 Prrnted m U.S. A.

Localization of the Binding Site for Cell Attachment in the al(I) Chain of Collagen*

(Received for publication, December 23, 1977, and in revised form, January 30, 1978)

Hynda K. Kleinman,+ Ermona B. McGoodwin,$ George R. Martin,+ Robert J. Klebe,@ Peter P. Fietzek,l and David E. Woolley(( From the &aboratory of Developmental Biology and Anomalies, National Institute of Dental Research, Bethesda, Maryland 20014, the SDepartment of Human Biological Chemistry and Genetics, the University of Texas Medical Branch, Galveston, Texas 77550, the IDepartment of Biochemistry, Rutgers Medical School, Piscataway, New Jersey 08854, and the 11 University Hospital South Manchester, Manchester, England

Certain cells such as CHO (Chinese hamster ovary) and fibroblasts attach to a substrate of type I collagen via proteins that link the cell surface to collagen. Serum contains a glycoprotein, c-CAP (collagen cell attach- ment protein), that can mediate this adhesion. Previ- ously, we established that al(I)-CB’7 (cyanogen bromide peptide 7), which lies within residues 552 to 822 of collagen, contains a major binding site for c-CAP (al(I)- CB7 has been renumbered to account for an additional triplet recently identified at residue 613 (P. P. Fietzek and R. W. Glanville, manuscript in preparation)). Now we have examined the ability of various peptides de- rived by proteolytic digestion of al(I)-CB7 to bind to c- CAP based upon their ability to inhibit cell attachment to collagen. The binding site lies within residues 757 to 791. It is likely that this is the sole binding site in the al(I) chain since cleavage of the bond between residues 775 and 776 in the al(I) chain destroys cell attachment activity.

Collagen-coated surfaces are often used in vitro to culture cells, as collagen enhances cell attachment (Klebe, 1974), survival (Ehrmann and Gey, 1956), and differentiation (Hauschka and White, 1972). Recent studies (Klebe, 1974) have shown that trypsinixed fibroblasts do not attach directly to collagen but require a serum-derived glycoprotein, the cell attachment protein. Some studies (Pearlstein, 1976 and Engvall and Rouslahti, 1977) suggest that c-CAP’ is a normal component of the external cell surface of the large external transformation-sensitive protein type (Hynes, 1973). Such proteins are destroyed by the trypsin used during cell passage and serum serves as an exogenous source of the protein. It has been shown that c-CAP binds F?st to collagen; then the fibroblasts, in the presence of Ca” or Mg”‘, bind to the c- CAP. collagen complex.

Earlier we reported that the binding of CHO cells to colla- gen-coated dishes was inhibited by prior incubation of serum with certain collagenous proteins and peptides (Kleinman et al., 1976). Presumably, such peptides react with c-CAP and prevent its subsequent binding to the collagen substrate. The al(I) chain of type I collagen was found to be more potent than the n2 chain. Additional studies showed that ~1(1)-CB7, a cyanogen bromide-derived peptide from the al(I) chain,

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: c-CAP, collagen cell attachment protein; CHO, Chinese hamster ovary.

contained a major binding site for c-CAP. The homologous peptide in the al(II) chain of type II collagen was the only active peptide and showed similar levels of activity.

al(I)-CB7 is a large peptide (&& = 24,500) lacking carbo- hydrate. It has previously been shown to promote myogenesis (Hauschka and White, 1972) and contains, in addition, the peptide bond cleaved by animal collagenases (Gross et al., 1974). Here we describe studies which locate the region within al(I)-CB7 that reacts with c-CAP.

EXPERIMENTAL PROCEDURES

Preparation of Collagen Peptide al(l)-CB7-Type I collagen was extracted from lathyritic rats skins and also from fetal calf skins after incubation with pepsin (Miller, 1972) and purified according to the method of Bornstein and Piez (1966). The al(I) chains were separated from the 02 chains on CM-cellulose (Piez et al. 1963). The al(I) chains were digested with cyanogen bromide (Bornstein and Piez, 1966) and the resulting peptides were separated on a column of CM- cellulose using 20 mu sodium formate buffer, pH 3.8, containing 0.04 M NaCl and a linear salt gradient from 0 to 0.1 M NaCl over a total volume of 1600 ml at 45°C.

Proteolytic Cleavage-al(I)-CB7 was cleaved with chymotrypsin (a-chymotrypsin, CDI, 48 pg/ml, Worthington) as described by Rexrodt et al., 1973. A substrate to enzyme ratio of 50: 1 was employed and the incubation was carried out for 35 min at 37°C. The fragments produced were partially resolved on a Sephadex G-100 (fine) column (100 x 1.5 cm) equilibrated and eluted with 50 rnM ammonium bicarbonate buffer, adjusted to pH 8.5 with ammonium hydroxide, at a flow rate of 10 ml/h.

A proteolytic enzyme (Houmard and Drapeau, 1972) from Staph- ylococcus aureus, strain V8, that cleaves glutamyl bonds at both pH 4 and pH 7.8 and aspartyl-glycyl bonds at pH 4 was also used to produce peptides. al(I)-CB7 was cleaved with protease V8 (Miles) at pH 4 in 50 mu ammonium acetate buffer for 18 h or at pH 7.8 in 50 mM ammonium bicarbonate buffer for 3 to 18 h, both at 37°C. A substrate to enzyme ratio of 3O:l was employed. The peptides were fractionated on a column of Sephadex G-50 (superfine, 100 x 2.5 cm) using 50 mM ammonium bicarbonate at a flow rate of 15 ml/h.

al(I)-CB7, bearing arginyl residues modified with 1,2-cyclohex- anedione (as described below), was cleaved with tosylphenylalanyl chloromethyl ketone-treated trypsin (260 units/mg, Worthington). A substrate to enzyme ratio of 5O:l was employed and the incubation was carried out in 0.1 M sodium borate, pH 8, for 4 h at 37°C (Patthy and Smith, 1975). The resulting peptides were fractionated on the Sephadex G-50 column described above.

In these studies where arginine residues in the al(I)-CB7 were modified with 1,2-cyclohexanedione (Pierce) according to the method of Patthy and Smith (1975), 15 mg of the peptide was dissolved in 0.3 ml of 0.25 M borate buffer, pH 9.0, containing 10.4 mg of the reagent, and was incubated for 2 h at 37°C. The reaction was terminated by adding an equal volume of 30% acetic acid and the sample was desalted on a Bio-Gel P-2 (200 to 400 mesh) column (23 x 2.5 cm), equilibrated with 0.1% acetic acid, and lyophilized. In these studies, exposure to 1,2-cyclohexanedione was repeated three times to obtain maximum modification of the arginine residues. Since the modified

5642

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Binding Site of Collagen Cell Attachment Protein 5643

groups were resistant to hydrolysis, the number of residues modified was determined by difference before and after modification from the amino acid analyses.

Human collagenase was derived from rheumatoid synovium by tissue culture methods (Woolley et aZ., 1975 and 1976). The purified enzyme migrated as a single band in polyacrylamide gels both with and without SDS and was without nonspecific, neutral proteinase activity. In the experiment discussed here, 100 mg of type I collagen from rat skin was dissolved in 40 ml of 0.4 M NaCl in 50 mM Tris-HCl, pH 8, containing 10 mM CaCb and 0.1 M sucrose (to prevent fibril formation) and digested with 1 mg of pure enzyme dissolved in 2 ml of 50 mu Tris-HCl, 0.17 M NaCl, and 10 mM CaClz at 25°C for 16 h. The reaction mixture was then dialyzed exhaustively against distilled water and lyophihzed. Examination of the reaction products in 5% polyacrylamide gels containing SDS (Furthmayr and Timpl, 1961) confiied complete degradation of the collagen molecule into the characteristic % and ‘/4 fragments. An aliquot of the sample was applied to a 6% agarose column (100 x 2.3 cm) equilibrated with 0.5 M CaClz, 0.05 M Tris, pH 7.2, and was eluted at 10 ml/h to separate the % and % fragments of the a chains.

The amino acid composition of each peptide assayed was deter- mined and their molecular weights were estimated from their elution from a molecular sieve and, where possible, by 7.5% SDS-acrylamide gel electrophoresis. For amino acid analyses, samples were hydrolyzed in 6 N HCl in ampoules sealed under Nz for 24 h at 105°C (Miller, 1972) and analyzed on a Durrum D500 automatic amino acid analyzer. In some cases, partial amino acid sequences of peptides were obtained. Sequencing was carried out by the technique of Edman and Begg (1967) in a liquid phase sequenator, Beckman model 89OB, updated, using the dimethylallylamine program. The H system of Edman (1975) was used with thin layer chromatography to identify the phenylthiohydantoins.

Binding Assay of Collagen Peptides-The binding of the serum- derived cell attachment protein to collagen was assayed by measuring cell attachment (Kleinman et al., 1976). In this assay, prior binding of collagen peptides with c-CAP inhibits cell attachment. Various con- centrations of the test collagen peptides were incubated at 37°C in 2 ml of Eagle’s minimal essential medium containing 0.2% bovine serum albumin and 0.9% bovine serum for 1 h. During this incubation, peptides containing the c-CAP binding site would react with c-CAP. Subsequently, each mixture was poured onto a bacteriological Petri dish (60 x 15 mm) coated with 25 pg of type I collagen and incubated for 1 h at 37°C. During this hour, the c-CAP that had not reacted with the peptide would hind to the collagen coating. Then, freshly dissociated CHO cells were added to the plates and allowed to attach for 1.5 h at 37°C. The media containing the unattached cells were removed and the plates washed three times with phosphate-buffered saline. The attached cells were removed with phosphate-buffered saline containing 0.1% trypsin, 0.1% EDTA and were counted with a Coulter Counter (Coulter, ZBI).

RESULTS2

Chymotryptic Peptides-Chymotrypsin was the fit en- zyme we employed to cleave al(I)-CB7 because there are only three susceptible bonds in this peptide (Rexrodt et al., 1973). The major sites of chymotryptic cleavage of al(I)-CB7 are shown in Fig. 1. Chymotrypsin cleaves at residues 36,84, and 228 and produces one large (C3 = 147 amino acids) and three smaller peptides (Cl = 36, C2 = 48, and C4 = 43 ammo acids) as well as larger peptides arising from incomplete cleavage. These peptides were partially resolved by molecular sieve chromatography into an uncleaved peptide (C3,4) and C3, while Cl, C2, and C4 eluted together (Fig. 2). Each of these fractions was tested for c-CAP binding activity (Fig. 2, inset). Neither C3 nor the fraction containing Cl, C2, and C4 together was active, while al(I)-CB7, the starting material, produced complete inhibition at the concentrations tested. C3,4, the uncleaved peptide, containing residues 85 to 271 of al(I)-CB7,

‘The material describing the sequence of al(I)-CB7 and the amino acid analyses of &(I)-CB7 and its peptide fragments are presented as a miniprint supplement at the end of this paper. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Document MS7-2029, cite au- thors, and include a check or money order for $1.00 per set of photocopies.

was active and produced complete inhibition of cell attach- ment at the levels also tested. Identification of the active fragment as C3,4 was established by its chromatographic behavior, size on gel electrophoresis (-16,000), and its amino acid content. The purity of the peptide was confirmed by its migration as a single band on gel electrophoresis as shown in Fig. 2. Amino acid analyses revealed that it contained amino acids characteristic of the C3,4 fragment including 1 hydroxy- lysine (residue 133) and 1 homoserine (residue 271).

Protease V8 Peptides-Protease V8 was next employed to cleave al(I)-CB7 at pH 4 because under these conditions this enzyme cleaves rapidly at glutamyl bonds and only weakly at

PKmEAsE MPN4

p.1

molE4sE MPH7d 18 mlms

72 73 ltwFslN- 11 -

oEsAuRAl

FIG. 1. Schematic summary al(I)-CB7 peptides assayed for bind- ing c-CAP. The peptides designated by D were active in binding c- CAP while the peptides designated by mia were inactive. Dessau et al. (1978) indicates fragment active in binding fibronectin.

i! 2

G 0 f 9 ::

2 1

COLLAGEN Itno)

Cl&24

n FRACTION NUMBER

FIG. 2. Chromatography of the chymotryptic digest of al(I)-CB7 on Sephadex G-100 (line). Twenty-five milligrams of digested al(I)- CB7 was applied to the column and 0.8~ml fractions were collected. SDS-gel electrophoresis was carried out on a CNBr digest of the al(I) collagen chain, on purified al(I)-CB7, which was the substrate for chymotrypsin, and on the purified chymotryptic fragment C3,4. The inset shows the attachment assay carried out on the starting material al(I)-CB7 and on the fractions designated C3,4, C3 and Cl, C2,C4.

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5644 Binding Site of Collagen Cell Attachment Protein

0 Il.6 1 COLLAGEN Img,

P4’ P4Z P43 P44 m-H-

FRACTION NUMBER

‘by FIG. 3. Chromatography of the peptides produced from al(I)-CB7

digestion with protease V8 at pH 4 for 18 h on Sephadex G-50 (superfine). Thirty milligrams of digested al(I)-CB7 was applied to the column and 1.5~ml fractions were collected. SDS-gel electropho- resis was carried out on a CNBr digest of the nl(1) chain and on the fraction designated PJ which had been repurified on Sephadex G-50. The inset shows the attachment assay carried out on the starting material al(I)-CB7 and on the protease V8 fragments present in the fractions designated PJ, P42, P43, P44, and Ps5.

aspartyl-glycyl and at aspartyl-alanyl bonds. It was expected that a large fragment containing the chymotrypsin-sensitive bond at residues 228 and 229 would be obtained. The peptides produced were partially resolved by molecular sieve chroma- tography (Fig. 3). A peptide designated PJ that eluted early from the sieve was active in binding c-CAP (Fig. 3, inset). The fraction marked Prl was lyophilized and rechromatographed on the Sephadex G-50 column and its identity were estab- lished by amino acid analysis. The purity and size of the peptide (-14,000) were determined by gel electrophoresis (Fig. 3). This fragment was judged to include residues 103 to 241. However, amino acid analysis in combination with the molec- ular weight and known specificity of the enzyme were not adequate to establish whether the peptide began at residues 92, 103, or 109. Since amino acid analysis showed that the peptide contained 1 residue each of leucine and phenylalanine, the COOH terminus of the peptide must extend through residue 234 (leucine) but stop before residue 243 (phenylala- nine). The most likely terminal residue is the glutamyl at position 240. In addition, peptides in the region designated P42 (Fig. 3) were also active in binding c-CAP but their identity was not established. The fragments P43, P44 and Pa5 were not active in binding c-CAP (Fig. 3).

al(I)-CB7 was also incubated with protease V8 at pH 7.8 for 18 h producing a number of inactive fragments (profile not shown). The major peptide in the digest was identified by amino acid analyses as comprising residues 55 to 175. To reduce the number of cleavages, a 3-h incubation time was next employed and the fragments produced were separated by molecular sieve chromatography (Fig. 4). Fraction P73 actively inhibited cell attachment and comprised residues 205 to 240 as determined by amino acid composition and sequence analyses. Two of the inactive fragments were identified by

their molecular weights and ammo acid compositions. Frac- tion P71 consisted of residues 55 to 175, while P72 consisted of residues 26 to 91. Fraction P74 which was also inactive, prob- ably contained a mixture of small fragments.

Tryptic Peptides-Chemical modification of arginyl resi- dues in al(I)-CB7 was carried out to prevent cleavage by trypsin at these residues. With the arginyl residues modified, it was expected that trypsin would yield a fragment similar to P,3. As seen in Fig. 5, several cleavages were obtained with trypsin. One large fragment designated Tl and one smaller fragment designated T3 were active in binding c-CAP. Frag- ment Tl consisted of residues 107 to 255 and T3 consisted of residues 206 to 255. In addition, the inactive fragment T2 was found to include residues 1 to 97.

Collagenase CZeavage-Type I collagen from rat was cleaved as described under “Experimental Procedures”. Ex- amination of the reaction mixture by gel electrophoresis in- dicated that incubation with collagenase produced nearly quantitative cleavage to 3/ and ‘/4 fragments (Fig. 6). Neither the total collagenase digest nor the isolated % and ‘/4 fragments of the collagen chains were active (Fig. 6) in binding the cell attachment protein. In comparison, a cyanogen bromide digest of denatured collagen effectively inhibited cell attachment.

The peptide fragments of al(I)-CB7 that have been isolated, identified, and assayed are summarized in the scheme of Fig. 1. Fig. 7 contains the sequence of amino acids present within the active binding region. The two peptide bonds required for activity determined by chymotrypsin and collagenase are indicated.

FIG. 4. Chromatography of the peptides produced from al(I)-CB7 by digestion with protease V8 at pH 7.8 for 3 h on Sephadex G-50 (superfine). Fifty milligrams of digested al(I)-CB7 was applied to the column and 2.5~ml fractions were collected.

I 2.L 2- An

:

I l-

FIG. 5. Chromatography of the trypsin digest of al(I)-CB7 with modified arginyl residues on Sephadex G-50 (superfine). Fifty milli- grams of digested al(I)-CB7 modified with 1,2-cyclohexanedione was applied to the column and 2.5~ml fractions were collected.

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Binding Site of Collagen Cell Attachment Protein 5645

chain, a region homologous to the binding site in the al(I) chain. In addition, P73 contains the collagenase-sensitive site that, when cleaved in the entire native collagen molecule, results in loss of activity. By comparison, the seven cleavages produced by cyanogen bromide (see Fig. 6) diminished but did not abolish activity. Clearly, the residues around the collagenase site and localized within the residues in P73 rep- resent the only binding site in the al(I) chain.4 The collagen- ase site is within 4 residues of the apparently essential se- quence cleaved by chymotrypsin, that is uncleaved in C3,4. This segment of the al(I) chain (Fig. 7) lacks carbohydrate and thus cell attachment must involve the recognition and binding to a sequence-specific determinant. While glycine occurs at every 3rd residue, proline and hydroxyproline are lacking from residues 772 to 785. Proline and hydroxyproline are known to stabilize the collagen helix (Ramachandran and Ramakrishan, 1976). Since such enzymes as pepsin and chy- motrypsin do not cleave bonds in the region of 772 to 785 in the native protein, it is likely that this segment is helical. However, this portion of the molecule may be sufficiently unstable to allow a sequential determinant to be recognized and bound.

I I .Ol .10 1.0

TYPE I COLLAGEN lmgl

FIG. 6. Assay of type I collagen with and without digestion by CNBr and collagenase. Native rat skin collagen (M), after CNBr digestion (A- - -A) and after collagenase cleavage (o---O) were assayed for cell attachment activity. In addition, the isolated colla- genase fragments TC* (A-----A) and TCB (Cl- - El) were tested. Gel electrophoresis of the collagen before (2) and after ( 2 ) enzyme treatment is shown in the inset.

Gln-Gly-Ile-Ala-Gly-Gln-Arg-Gly-Val-~al-Val-Gly-Leu-Hyp-Gly-Gln-Ar~-Gly~l~ + t co11agenase chymorrypain

FIG. 7. Sequence within al(I)-CB7 containing binding site for c- CAP (from Fietzek et aZ., 1973).

DISCUSSION

In our initial study (Kleinman et al., 1976), we established that al(I)-CB7 was the most potent of the CNBr-derived peptides in inhibiting the serum-dependent binding of cells to collagen. Further, if bacteriological plates were coated with al(I)-CB7, they also served as a substrate for cell attachment with sermn3 These results suggest that the attachment of cells to collagen occurs at a specific site on the collagen molecule. Since al(I)-CB7 contains 271 residues and repre- sents one of the largest cyanogen bromide-derived fragments, we assayed various peptides produced from al(I)-CB7 by proteolytic digestion.

It should be noted that the smaller peptides P73 and 1‘3 are less active (with 50% inhibition values of 4.0 mg and 1.5 mg, respectively) than CB7 or C3,4 (with 50% inhibition values of 0.5 mg and 0.3 mg, respectively). Dessau et al. (1978), carrying out related studies on the interaction of CNBr-derived pep- tides with “antigelatin factor” (c-CAP) from serum, used immunological methods to assay binding and employed a longer incubation period than we did. Under their conditions, the smaller active peptides retained activity on a molar basis. Their studies also differ, in that binding of “antigelatin factor” to collagen is measured in the absence of cells. It is possible that proteases associated with the cells used in our studies are degrading the active site of such peptides as Pr3 faster than the larger peptides. Also, the interaction between c-CAP and small peptides may be less stable than that with large pep- tides.

Pearlstein (1976) recently reported that urea extracts of cultured cells promote cell attachment to collagen in the absence of serum. Since these extracts contain such proteins as cell surface protein (Yamada and Weston, 1974), he sug- gested that the cell surface protein and the cell attachment protein (Klebe, 1974) are identical.

None of the unique peptides produced by chymotryptic cleavage of al(I)-CB7 (i.e. Cl, C2, C3, or C4) was active. However, we isolated an active peptide, C3,4, resulting from incomplete cleavage of the peptide bond between residues 228 and 229 of al(I)-CB7. These results indicate that the binding site is located between residues 85 and 271 in al(I)-CB7 and suggest an important role for the residues in the region of the bond cleaved by chymotrypsin.

Using other enzymes we produced and assayed a variety of other peptides. Two relatively small peptides, P73 and T3, inhibited the serum-dependent binding of cells to collagen. P73, a peptide produced from al(I)-CB7 by digestion with protease V8 at pH 7.8, contains 36 amino acids (residues 205 to 240 of al(I)-CB7) that correspond to residues 756 to 791 of the al(I)-CB7 chain. 1’3, a tryptic peptide, is somewhat longer at the COOH-terminal end of the peptide and includes resi- dues 205 to 255 of al(I)-CB7 (residues 757 to 816 of al(I)- CB7). Those peptide so far found to be active have contained the sequence present in P73. While we did not investigate the binding site on the cr2 chain, Dessau et al. (1978) reported that fibronectin binds between residues 693 and 1101 of the a2

Several laboratories have studied similar cell surface pro- teins which can change cell shape and attachment and are altered by trypsin treatment and transformation. These pro- teins include cell surface protein, (Yamada and Weston, 1974), large external transformation-sensitive protein (Hynes, 1973), and fibronectin (Vaheri et al., 1976). Similarities have been observed in the size of these proteins suggesting that they may be identical or related materials. Fibronectin has also been reported (Rouslahti and Vaheri, 1975) to be identical to cold-insoluble globulin, a protein found in the cryoprecipitate of serum. Dessau et al. (1978) have recently found that “an- tigelatin factor” and cold-insoluble globulin are identical. Des- sau et al. reported that cold-insoluble globulin binds to colla- gen and that the binding sites include residues 646 to 822 of the crl(1) chain. This region contains the binding site for the cell attachment protein. We have found that both cell surface protein (from Dr. Yamada, National Institutes of Health) and

3 Unpublished observations.

4 Previously, we had reported (Kleinman et al., 1976) that al(I)- CB8 contained one-fourth of the activity of ul(I)-CB7. This activity was probably due to contamination of al(I)-CB8 with some al(I)- CB7 which is similar in molecular weight. In addition, al(IWB7 elutes heterogeneously on ion exchange chromatography (Butler et az., 1967).

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5646 Binding Site of Collagen Cell Attachment Protein

cold-insoluble globulin (from Dr. Harmann, MPI, Munich) are active in promoting cell attachment. Cell attachment to collagen may be a major function of these proteins.

Acknowledgments-We thank Jack Folk and Rupert Timpl for their helpful suggestions and Guy Hawkins for the amino acid anal- yses.

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Bornstein, P., and Piez, K. A. (1966) Biochemistry 5, 3460-3473 Butler, W. T., Piez, K. A., and Bornstein, P. (1967) Biochemistry 6,

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Biochem. J. X9,55-59 Edman, D. (1975) in Protein Sequence Determination (Needelman,

S. B., ed) pp. 232-475, Springer Verlag, New York Edman, D., and Begg, G. (1967) Eur. J. Biochem. 1,80-91 Ehrmann, R. L., and Gey, G. 0. (1956) J. Natl. Cancer Inst. 16,

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WoolleyH K Kleinman, E B McGoodwin, G R Martin, R J Klebe, P P Fietzek and D E

collagen.Localization of the binding site for cell attachment in the alpha1(I) chain of

1978, 253:5642-5646.J. Biol. Chem. 

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