THE JOURN.41. OF BIOI.OGIC.41. CHEMISTRY Vol 254, No. IS. Iswe of .July IO. pp. 60546059, 1979 Pr’rmted m US A.

Isolation of a Tryptic Fragment Containing the Collagen-binding Site of Plasma Fibronectin*

(Received for publication, September 11, 1978, and in revised form, January 11, 1979)

Erkki Ruoslahti, Edward G. Hayman, Pentti Kuusela,$ John E. Shively, and Eva Engvall

From the Division of Immunology, City cf Hope National Medical Center, Duarte, California 91010

Fibronectin, a high molecular weight cell surface and plasma protein, has previously been shown to bind to collagen and to mediate adhesion of cells to collagen- coated culture dishes.

A collagen-binding peptide was isolated after exten- sive tryptic digestion of fibronectin from human plasma by affinity chromatography on gelatin-Sepharose and gel filtration. The peptide gave a single band with a molecular weight of 30,000 in sodium dodecyl sulfate- polyacrylamide gel electrophoresis and had alanine as the only NH&.erminal amino acid. A shorter digestion followed by similar fractionation yielded a group of three peptides with molecular weights of approxi- mately 70,000.

The M, = 70,000 (70K) peptide fraction and the M, = 30,000 (30K) peptide were immunochemically cross-re- active with intact fibronectin. A relatively small pro- portion of antibodies in anti-fibronectin and in an an- tiserum prepared against the 70K peptide fraction were directed against determinants present in the 30K pep- tide, and the 30K peptide gave a weak antibody re- sponse even after prolonged immunization. These re- sults suggest that the collagen-binding part of the fi- bronectin molecule is poorly immunogenic, possibly because of evolutionary conservation. The antigenic determinants detected by antisera prepared against the 30K peptide were also present in the 70K peptides and in whole fibronectin, suggesting that the 30K and 70K peptides are derived from the same part of the fibro- nectin molecule. Amino acid analysis of the peptides revealed a high content of glycine. The glycopeptide nature of the 30K and 70K peptides was indicated by their binding to concanavalin A.

These results show that a major functional property of the M, = 450,000 fibronectin molecule, its ability to bind to collagen, is preserved in tryptic fragments, one comprising less than 10% of the whole molecule.

Fibronectin is a major cell surface glycoprotein found in normal fibroblasts and several other cell types. It is usually absent from the surface of malignant cells (for review and references, see Ref. 1). It is also present in plasma at a concentration of about 300 pg/ml(2). Fibronectin is composed of two polypeptide chains which have molecular weights close to 220,000 and are covalently linked by disulfide bonds. The polypeptide chains of the cellular form may be slightly larger

* This work was supported by Grants CA 22108 and CA 16434 from the National Cancer Institute, Department of Health, Education, and Welfare. 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 USC. Section 1734 solely to indicate this fact.

$ Present address, Dept. of Immunology and Bacteriology, Univer- sity of Helsinki, Haartmaninkatu 3, 00290 Helsinki 29, Finland.

than those of the plasma form (3-6). The two forms of fibronectin are immunologically indistinguishable, and their amino acid and carbohydrate compositions and peptide maps are similar (7, 8). Fibronectin from plasma and cell cultures binds to collagen and can be purified by affinity chromatog- raphy on collagen insolubilized on Sepharose particles (9). It also mediates attachment of cells to collagen-coated plates (10, 11). Fibronectin from plasma has been shown to enhance the phagocytosis of gelatinized gold particles by the Kupffer cells in the liver (12).

To investigate the molecular structure responsible for these activities, we set out to study the functional properties of proteolytic fragments of fibronectin. In this report, we describe the isolation from fibronectin of a collagen-binding tryptic fragment with a molecular weight of about 30,000.

MATERIALS AND METHODS

Isolation and Digestion of Fibronectin-Fibronectin was purified from plasma essentially as described earlier (9). In brief, titrated human plasma was passed through a column of twice its volume of Sepharose CL-4B (Pharmacia, Inc., Piscataway, N. J.) to remove material binding to Sepharose and then was fractionated on a column one-third its volume of gelatin-Sepharose containing about 2 mg of gelatin/ml of Sepharose. The gelatin column was washed with phos- phate-buffered saline (NaCl/Pi), pH 7.2, followed by 1 M urea in 0.05 M Tris-HCl buffer, pH 7.5. Fibronectin then was eluted with 4 M urea in the Tris buffer. Peptides from the digestion of fibronectin were fractionated similarly using 1 ml of gelatimSepharose/2 mg of protein, except that the column was washed with NaCl/P,’ only, and phenyl- methane sulfonyl fluoride (PhCH2S02F), was added to all solutions to a final concentration of 10m4 M. In some experiments, heat-denatured collagen from calf skin (type I) (Sigma) instead of gelatin was used to prepare the affinity columns.

The fibronectin in the fractions eluted from the gelatin-Sepharose column was monitored by absorption at 280 nm. The peak fractions were pooled, made 0.1 M in ammonium bicarbonate by adding the solid salt, and digested with trypsin (tosyl phenylalanyl chloromethyl ketone-treated trypsin, Worthington). The fibronectin concentration in the pool was 0.5 to 1 mg/ml. The digestion was performed either by using 0.1% trypsin by weight of the amount of fibronectin for various times, or by using two additions of 1% of trypsin, 3 h apart, with a total digestion time of 6 h (prolonged digestion). The digestion was stopped in both cases by adding phenylmethanesulfonyl fluoride to the final concentration of 10m4 M. The digest was dialyzed against distilled water and lyophilized. It then was dissolved in NaCl/P, and fractionated on gelatin-Sepharose or by gel filtration.

Gel Filtration-Fragments of fibronectin were fractionated on a column (1.5 x 110 cm) of Sephacryl S-200 (Pharmacia) or, in some experiments, on a column (2.5 x 110 cm) of Sephadex G-200 (Phar- macia) equilibrated with NaCl/P,. The columns were calibrated with human immunoglobin (Mr = 150,000), albumin (Mr = 66,000), and ovalbumin (M, = 44,000).

Assays-Antibodies to fibronectin, iodination of fibronectin, and radioimmunoassays have been described (13). Antisera to the M, =

’ The abbreviations used are: NaCl/P,, phosphate-buffered saline; 30K and 40K peptides, collagen-binding fragments of M, = 30,000 and 40,000, respectively; SDS, sodium dodecyl sulfate.

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Collagen- binding Site of Fibronectin 6055

70,000 (70K) and M, = 30,000 (30K) peptides were prepared in rabbits. Each injection consisted of 0.1 mg of peptide in 0.5 ml of NaCl/P, thoroughly mixed with 1 ml of Freund’s complete adjuvant (GIBCO, Grand Island, N. Y.). Three injections were given at 2-week intervals, followed by monthly injections. The rabbits were bled 1 week after the third and each subsequent injection. The determination of colla- gen-binding capacity of fibronectin and its fragments was done as described (9, 14) utilizing a fibronectin-alkaline phosphatase conju- gate. Briefly, wells of polystyrene microtiter plates were coated with heat-denatured collagen and incubated with the fibronectin-enzyme conjugate. Binding of the conjugate to the collagen on the plates was detected and quantitated by measuring the enzyme activity. Unla- beled fibronectin or its collagen-binding fragments inhibit the binding of the conjugate and can be quantitated in the assay.

Concanavalin A binding was tested using concanavalin A-Sepha- rose (Pharmacia). Fibronectin or tryptic peptides, labeled with “‘I, were dialyzed against a 0.1 M sodium acetate buffer, pH 6.5, containing 0.5 M sodium chloride and applied to a concanavalin A-Sepharose column, equilibrated with this buffer. Elution was performed with 1 M a-methylmannoside (Sigma) in the same buffer.

Immunodiffusion was performed using 1% agarose in NaCl/P,. Antisera were concentrated for immunodiffusion by lyophilization followed by dissolving in smaller volume.

Gel Electrophoreszs-Polyacrylamide electrophoresis in the pres- ence of sodium dodecyl sulfate (SDS) with or without reduction of the sample with 1% p-mercaptoethanol was performed using slab gels according to Laemmli (15) and under nondenaturing conditions ac- cording to Davis (16). Albumin, ovalbumin, and carbonic anhydrase (MV = 29,000) were used as markers.

Amino Acid Analyszs-Duplicate samples (20 /zg) were hydrolyzed under reduced pressure at 110°C for 24,48, and 72 h with 0.5 ml of 3 up-toluenesulfonic acid containing 0.2% 3-(2-aminoethyl)indole. Hy- drolysates were analyzed on a Beckman 121M amino acid analyzer.

Sequenator Analysu-A Beckman model 890C Sequencer was used. The Sequencer program and the identification of the phenyl- thiohydantoin derivatives by high pressure liquid chromatography were as described by Hunkapiller and Hood (17).

RESULTS

Isolation of Tvptic Peptides with Collagen- binding Activ- ity-progressively smaller fragments of fibronectin were de-

a b cd ef gh

,66~

n44

29

FIG. 1. Polyacrylamide gel electrophoresis in the presence of SDS and 2-mercaptoethanol. a, purified fibronectin from human plasma; b to e, fibronectin digested using 0.1% trypsin for 5 min, 30 min, 2 h, and 4 h, respectively; k fibronectin digested for 6 h using two additions of 1% trypsin; g, purified 30K peptide; h, 70K peptide fraction. The standards used for molecular weight calibration were bovine serum albumin (SS,OOO), ovalbumin (44,000), and carbonic anhydrase

FIG. 2. Affinity chromatography on gelatin-Sepharose of human fibronectin digested with two additions of 1% of trypsin for 6 h. The arrow indicates where urea was applied to elute bound material from the column.

I . I . . /! ,-.-k. .

t l

50 100 150 200 ml

VOLUME OF ELUATE

n1

Volume of Eluote FIG. 3. Gel filtration on Sephacryl S-200 of the material bound to

gelatin-Sepharose in the experiment shown in Fig. 2. The arrows indicate (from left to right) the elution positions of immunoglobulin G, albumin, and ovalbumin. The flow rate was 12.5 ml/h, and the fraction volume was 2.5 ml. Material eluting between 118 and 130 ml was pooled for further experiments.

tected by SDS-gel electrophoresis with increased time of digestion and trypsin concentration (Fig. 1, b to t). Fraction- ation on gelatin-Sepharose showed that, depending on the extent of digestion, 10 to 25% of the material in such digests had retained collagen-binding activity.

Initial experiments in which the concentration of trypsin and the length of digestion were varied indicated that the digestion proceeded rapidly through a series of high molecular weight components to give a relatively stable collagen-binding fragment with an approximate molecular weight of 30,000.

To isolate this fragment, fibronectin was subjected to pro- longed digestion with trypsin. After such digestion, about 10% of the protein was found to bind to gelatin-Sepharose (Fig. 2). Gel filtration of the bound material on Sephacryl S-200 gave a major protein peak of M, less than 40,000 (Fig. 3). The material in this peak gave a single homogeneous band at M, = 30,000 on SDS-gel electrophoresis (Fig. lg) and was almost completely bound to gelatin-Sepharose when a sample of the peptide from gel filtration was again fractionated on such a column (Fig. 4). The yield of the isolated peptide was approx- imately 2% of the starting amount of fibronectin on a weight

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6056 Collagen- binding Site of Fibronectin

To gain information on the cleavages that resulted in the formation of the collagen-binding fragment with M, = 30,000 (hereafter called 30K peptide), collagen-binding fragments were isolated from less extensively digested fibronectin. When fibronectin was subjected to digestion with 0.1% trypsin for 30 min to 4 h (Fig. 1, c to e) and the collagen-binding peptides were subjected to gel filtration, a major portion of the protein eluted in the M, = 90,000 region. Fig. 5 shows such a fraction- ation of a 2-h digest. Gel electrophoresis of the material eluting in the major peak typically revealed the presence of a peptide at M, = 70,000, and two minor peptides, one with a slightly higher (79,000) and the other with a slightly lower (66,000) molecular weight than the main peptide (Fig. lh). Omission of reducing agent did not appreciably change the electrophoretic mobility of these or the 30K peptide; they migrated slightly faster than under reducing conditions (not shown). The yield of the 70K peptide fraction was 15 to 20% of the initial amount of fibronectin. Several independent iso- lations of the 30K and 70K components were performed with similar results. In some of these isolations, collagen-Sepharose was used instead of gelatin-Sepharose. When the order of the isolation steps was reversed (i.e. gel filtration was performed prior to affinity chromatography on gelatin-Sepharose), there was no effect on the final results.

Immunochemical Characterization of the 30K and 7OK

r 1 E 0.8

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g 0.6

k 0.5

l’yA -04. ’

’ -Au #

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5 10 15 20 25 ml

VOLUME OF ELUATE FIG. 4. Refractionation on gelatin-Sepharose of a sample of the

material pooled from a gel filtration experiment similar to the one shown in Fig. 3. The mainpeak represents material bound and eluted with urea.

Peptides-The peptides were characterized using antisera against whole fibronectin, the 70K fraction, and the 30K peptide. In immunodiffusion, anti-fibronectin gave one precip- itin line against fibronectin but did not react visibly with the peptides (Fig. 6A). Some anti-fibronectin sera, after concen- tration, showed partial cross-reactivity with the 70K fraction, but not with the 30K peptide (not shown). Anti-70K gave one line against the 70K peptide fraction (Fig. 6B). This line was continuous with the line this antiserum gave with fibronectin, indicating that fibronectin has all of the relevant antigenic determinants present in the 70K peptides. The anti-70K se- rum did not precipitate 30K peptide, and anti-30K serum did not give any precipitation reactions. However, when tested in a radioimmunoassay, the anti-30K serum did show binding of labeled fibronectin, 70K fraction, and 30K peptide (Table I). Anti-fibronectin and anti-70K also bound all three labeled compounds. Anti-7OK had a relatively higher titer against the “51-labeled 70K and 30K peptides than anti-fibronectin. Anti- 30K at low dilution bound all three labels equally well, Two antisera against 30K peptide were prepared. Both showed similar low titers which did not become higher even after the immunization had been continued for 4 months.

To study the relationship between the 30K and 70K pep- tides, a radioimmunoassay based on the binding of ““I-labeled 30K to anti-30K was employed. The 30K and 70K peptides and whole fibronectin were approximately equally active in-

I I

Volume of Ekmte

FIG. 5. Gel filtration on Sephacryl S-200 of the material bound to gelatin-Sepharose from 80 mg of human plasma fibronectin digested using 0.1% trypsin for 2 h. Details of the fractionation are the same as in Fig. 3. Material eluting between 85 and 95 ml was pooled for further experiments.

FIG. 6. Immunodiffusion in agarose. Fibronectin (I), 70K fraction (2), and 30K peptide (3) were tested against anti- fibronectin (center well, A), and anti- 70K (center well, B). The anti-7OK se- rum used was concentrated 5-fold. After diffusion at room temperature for 24 h, the plates were washed first with NaCl/ P, then with water, dried, and stained with Coomassie blue.

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Collagen- binding Site of Fibronectin 6057

TABLE I TABLE II

Binding of ‘2”I-labeled fibronectin, 70K fraction, and 3OKpeptide to anti-fibronectin. anti- 7OK. and anti-30K

Amino acid composition (mol per cent) of human plasma fibronectin and its 70K and 30K fragments

Labeled protein Antiserum titer”

Anti-fibronectin Anti-70K Anti-30K Amino acid

Fibronectin”

Sample

70K fraction” 30K peptide’

““I-fibronectin 30,000 2,400 40 ‘*“I-labeled 70K 4,000 5,000 50 ““I-labeled 30K 30 60 70

’ Reciprocal of the dilution of antiserum giving 50% binding of the added radioactivity.

I I I I I

01 1 10

Concentration of fibronectin or fragment (nM)

FIG. 7. Inhibition of binding of ‘?-labeled 30K to anti-30K by fibronectin (O---O), 70K peptide fraction (H), and 30K pep- tide (A-A,). The molar concentrations of fibronectin were based on M, = 220,000.

FIG. 8. Gel electrophoresis in Tris buffer using 10% polyacrylamide gel. a, 30K peptide, 10 pg; b, 30K peptide, 5 pg; c, 70K peptide fraction; d, fibronectin. The position of human albumin is shown for compari- son (arrow).

hibitors in this assay on a molar basis (Fig. 7). A mixture of fibronectin peptides which did not bind to gelatin-Sepharose was about 10 times less active as inhibitor than fibronectin on a weight basis in this assay.

T yr 4.2 5.7 5.2

Phe 2.5 3.1 2.2

'b 2.6 1.0 0.2

LYS 5.1 3.3 2.9

His 2.0 3.5 3.4

Arg 4.5 4.3 3.5

Asp 9.5 10.8 11.5 Thr 8.4 10.2 10.3

Ser 8.3 7.9 9.3

Glu 11.6 14.1 12.9 Pro 8.5 6.5 5.4

GUY 8.9 10.4 13.7

Ala 4.9 3.6 5.2

%-cys 2.8 1.7 1.1 Val 6.2 5.3 4.5

Met 0.9 2.0 3.0 Ile 3.6 2.6 2.0

Leu 5.5 3.9 3.6

HYP 0 0

a From Ref. 8. ’ Duplicate samples were hydrolyzed for 48 h. ’ Duplicate samples were analyzed following hydrolysis for 24, 48,

and 72 h. The values for serine and threonine were extrapolated to zero hydrolysis time, and the 72-h values were used for isoleucine and valine.

TABLE III

Binding of ‘2”I-labeled fibronectin and its collagen-binding fragments to concanavalin A-Sepharose and gelatin-Sepharose

Concanavalin-A Sepha- rose Gelatin-Sepharose

% w % o/o I”‘I-fibronectin 11 77 5 84 ““I-labeled 70K 12 73 28 68

‘*‘I-labeled 30K 10 83 27 70

n Eluted with 1 M o-methylmannoside. b Eluted with 4 M urea.

Partial Chemical Characterization of the 30K and 70K Peptides-As was the case with SDS-gel electrophoresis (Fig. l), the 30K peptide gave a single major band in polyacrylamide gel electrophoresis under nondenaturing conditions, while the 70K peptide fraction was resolved in three bands (Fig. 8).

Amino acid compositions of the peptides revealed a mark- edly high content of glycine in the peptides (Table II). No hydroxyproline was found in the 30K and 70K peptides.

Two independent preparations of the 30K peptide gave alanine as the only NHs-terminal amino acid and a single NH2-terminal sequence when analyzed by an automatic amino acid Sequencer. Amino acid sequence data on fibronectin and its peptides will be reported elsewhere.

The radiolabeled 70K and 30K peptides as well as intact fibronectin bound to concanavalin A-Sepharose and could be eluted with sugar (Table III), which indicates that they are glycosylated. Quantitatively similar binding of the labeled peptides to gelatin-Sepharose was observed. Some loss of the collagen-binding activity of the peptides was repeatedly found to occur upon radioiodination. No binding occurred to albu- min-Sepharose used as a control.

Collagen-binding Properties of the 70K and 30K Pep- tides-The isolation of the 70K and 30K peptides on gelatin and collagen-Sepharose shows that they have affinity to col-

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Collagen- binding Site of Fibronectin

I I I I I 1 I

10-Z 0' 100 10' 102 103 104

Concentration of fibronectin CT fragment (nM)

FIG. 9. Inhibition of binding of enzyme-labeled fibronectin to col- lagen-coated microtiter wells by unlabeled fibronectin (o--O), 70K peptide fraction (M), and 30K peptide (X-X). The molar concentrations of fibronectin were calculated on the basis of M, = 220,ooo.

lagen. The avidity of the collagen-binding of the peptides was studied in an assay in which the binding of enzyme-labeled fibronectin to collagen on a solid phase was inhibited by the material studied. Fibronectin and the 30K and 70K peptides inhibited in this assay (Fig. 9). The 70K fraction was almost 2, and the 30K peptide was more than 3, orders of magnitude less active than intact fibronectin in this assay.

DISCUSSION

Our results show that after extensive cleavage of the fibro- nectin molecule with trypsin, the collagen-binding activity is retained in tryptic fragments. Fractionation of such cleavage products has led to the isolation of a peptide with a molecular weight of 30,000 which possesses affinity for collagen. This fragment, which comprises less than 10% of the entire fibro- nectin molecule, seems to contain the collagen-binding site of fibronectin (or one of them if there are several). Depending on whether one or both of the polypeptide chains of fibronec- tin give rise to the 30K peptide, the theoretical yield of a fragment of this size would be 6 to 13% of the weight of the fibronectin used as starting material. We recovered the 30K peptide with a yield which was about 15 to 30% of the theoretical. Less than theoretical yields would be expected if the collagen-binding material were not uniformly digested. Collagen-binding fragments larger than the 30K peptide were present, and cleavage of the 30K peptide to fragments smaller than is compatible with the preservation of its collagen-bind- ing may also have occurred.

Immunochemical analysis of the collagen-binding tryptic fragments revealed several interesting features. While the 30K fragment comprises at least 6% of the whole molecule, only 0.1% of the antibodies in anti-fibronectin sera reacted with this fragment. It could be that many of the antigenic deter- minants in fibronectin are destroyed upon fragmentation. However, since hyperimmunization with the 30K peptide resulted in low titers of antibody, it seems that the collagen- binding region of the fibronectin molecule is poorly immuno- genic. This would be in agreement with the apparent conser- vation of the collagen-binding property of fibronectin, evi- denced by its presence and similar specificity in widely differ- ent species such as human, chicken, and fish (14).

Fibronectin and the 70K peptides were, on a molar basis, equally efficient as inhibitors of the binding of ‘“‘I-labeled 30K

to anti-30K as the 30K peptide itself. This was true when M, = 220,000 was used for fibronectin, which assumes that the two chains of fibronectin both contain the relevant fragment. This suggests that both chains give rise to the 30K peptide and that each contains a collagen-binding site. Similarly, the fact that the 70K peptide fraction was as active as the 30K shows that the 70K peptides contain the antigenic determi- nants present in the 30K peptide. These results, and the fact that the yields of the 70K and 30K peptides were inversely related, make it reasonable to assume that these peptides arise from the same or largely overlapping regions of the fibronectin polypeptide chain(s).

The 70K and 30K peptides inhibited binding of labeled fibronectin to collagen. The inhibition curves were parallel to those obtained with intact unlabeled fibronectin but were shifted towards higher molar concentrations. This suggests that the peptides, especially the 30K, bind less avidly to collagen than intact fibronectin but compete for the same site(s) on collagen as fibronectin. The difference in avidity could depend on the presence of more than one binding site for collagen in fibronectin with resultant multiple, high affinity binding of the intact molecule, while the peptides with fewer binding sites would bind less tightly. It is also possible that the integrity of the fibronectin molecule is important for the correct conformation of the binding site(s) and that the loss of parts of the molecule may result in an altered structure of this site in the peptides.

Relatively little is known about the structure of fibronectin, and its large size makes structural work difficult. While the apparent lowered affinity of the 30K peptide for collagen suggests that the collagen-binding site of fibronectin may not be contained in this peptide in an entirely intact form, its smaller size should make the peptide useful in elucidation of the structural requirements for the collagen-fibronectin inter- action. The apparent homogeneity of the 30K peptide with respect to molecular weight and NHz-terminal analysis should be helpful in this regard. Further structural work on this peptide is now in progress.

Fibronectin is highly susceptible to proteolysis. Plasma fibronectin is rapidly cleaved by plasmin, yielding fragments with molecular weights slightly lower than that of the intact polypeptide chains (18). The dimeric structure is lost upon such digestion, indicating that the interchain disulfide bond(s) are located close to one end of the molecule. Further digestion releases a fragment with a molecular weight of 27,000. Our 30K collagen-binding peptide seems to be different from this fragment. Tryptic digestion also gave a rapid release of a peptide moving in the 30,000 molecular weight range in SDS- gel electrophoresis (Fig. lb), but this peptide did not appear in the collagen-binding fractions, and a much more intensive proteolysis was needed to release the 30K peptide. Also, our 30K peptide was slightly smaller than the rapidly released peptide. These results suggest that the collagen-binding site of fibronectin is not located in the part of the mclecule involved in the interchain bonding.

While the interaction of fibronectin with collagen in vitro is well documented by several laboratories (9, 14, 19,20), little evidence is available that such interaction occurs in ho. At present, the co-distribution of collagen and fibronectin ob- served by immunofluorescence on the surface of cultured fibroblasts (21, 22) and the detachment of cell surface fibro- nectin by treatment with collagenase under some conditions (23) are the only evidence available to suggest that fibronectin may bind to collagen under conditions that approximate the in uiuo situation. Cell surface fibronectin, as detected by antibodies to the intact fibronectin molecule and by surface labeling, is extremely susceptible to release from the cell

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Collagen-binding Site of Fibronectin 6059

surface by proteases (24-26). Whether the collagen-binding portions of the fibronectin molecule remain associated with the cell layer is not known. Our collagen-binding fragments and antibodies to them may prove useful in the analysis of this and other interactions of fibronectin in cell cultures and in vivo.

Achnowledgments-We thank Aulikki Pekkala and Ursino Del Valle for technical assistance.

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E Ruoslahti, E G Hayman, P Kuusela, J E Shively and E Engvallfibronectin.

Isolation of a tryptic fragment containing the collagen-binding site of plasma

1979, 254:6054-6059.J. Biol. Chem. 

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