topological arrangement of the major structural features of

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed m U.S.A. Vol. 255. No. 9, Issue of May 10, pp. 4304-4312. 1980

Topological Arrangement of the Major Structural Features of Fibronectin*

(Received for publication, November 7, 1979, and in revised form, January 7, 1980)

Denisa D. Wagner and Richard 0. HynesS From the Department of Biology, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Using tryptic cleavage coupled with biosynthetic labeling, we have established the order along the polypeptide chain of the major tryptic fragments derived from dimeric fibronectin. The dimer is held together by interchain disulfide(s) all of which are very close to the COOH-terminal. This COOH-terminal region is very readily removed by proteolysis. The NH2-terminal region of the molecule is extremely cystine-rich and is released in a 25,000-dalton fragment. The rest of the molecule remains as a 200,000-dalton internal fragment.

By the use of cyanide cleavage, we have shown that the large fragment contains a disulfide-rich region at its NHz-terminal, adjacent to the 25-kd fragment, and contains a free sulfhydryl group which is 170,000 daltons from the NHz-terminal of the intact 230,000-dalton chain. There may be another free sulilydryl group 30,000 to 40,000 daltons further toward the COOH-terminal. The free sulfhydryl groups are necessary for binding of fibronectin to the cell surface and apparently participate in the formation of high molecular weight aggregates which contain fibronectin and are held together by disulfide bonds.

These results, together with others in the literature, have allowed us to locate within the molecule the sites at which fibronectin binds to collagen and fibrin and the region at which these proteins are cross-linked to fibronectin by factor XIII transglutaminase. The cross- linking region is in the NHz-terminal 25-kd fragment and the binding site is immediately adjacent in the cystine-rich region of the 200-kd fragment.

Fibronectin is a large glycoprotein present on the surfaces of many cell types (1-3). A related molecule is present in plasma (4). The major role of fibronectin is probably adhesion of cells to the substratum (5-7), and cell-surface fibronectin appears to be connected to actin microfilament bundles inside cells (8,9). These properties could explain some of the changes in behavior of malignant cells which have lost fibronectin on transformation (1-3). Fibronectin also appears to be involved in cell migration in vitro (10) and conceivably also in vivo (11, 12). Fibronectin binds strongly to gelatin and less strongly to

Public Health Service, National Cancer Institute (ROI CA 17007 to * This research was supported by grants from the United States

R. 0. H. and PO1 CA 14051 to S. E. Luna, Massachusetts Institute of Technology), and the American Cancer Society (CD-5A). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. * Recipient of National Institutes of Health Research Career De- velopment Award.

collagen (13-15) and fibrin (4, 14, 15) and can be cross-linked to each of these by Factor XI11 transglutaminase (16, 17). Fibronectin also interacts with heparin (18) and can be cross- linked by chemical cross-linkers to proteoglycans at the cell surface (19). This glycoprotein therefore has a number of apparent biological functions and several specific binding sites for other macromolecules. In order to elucidate the function of fibronectin, it is obviously necessary to learn more about its structure.

Biophysical analysis shows that fibronectin is a molecule composed of globular domains connected by flexible linking regions of polypeptide (20, 21). Different fragments can be obtained by cleavage of fibronectin with a variety of proteases (22-32). The fragments may correspond with the globular domains and presumably arise by cleavage of the flexible regions of the polypeptide chain. Mild tryptic cleavage or plasmin cleavage produces two fragments of 25,000 and 200,000 daltons. The smaller one is very cystine-rich and has no carbohydrate. The large one has fewer cystines and all or most of the carbohydrate and also contains 1 (or 2) cysteines whose presence is necessary for binding of fibronectin to the cell surface (24). The gelatin-binding site is located in the 200- kd’ fragment (24,28).

Other reports have described fragments of a variety of different sizes (25-32) but these have not been positioned on the intact polypeptide chain. The relative order of the 25-kd and 200-kd fragments is controversial since one report places the NHz-terminal of fibronectin in the 200-kd fragment (33), whereas another places it in the 25-kd fragment (28). The exact positions of the interchain disulfide bonds and the free sulfhydryl group(s) also remain uncertain. In this paper, we describe experiments which, together with other results (24, 25, 28, 31) allow positioning of the fragments, and of several of the other features mentioned, on the polypeptide chains of the fibronectin d k e r . These results were reported at the recent meeting of the American Society for Cell Biology (34).

EXPERIMENTAL PROCEDURES’ cells; cells “Ped were the hameter 6-11 l ine N1L.8, a normal fibroblaetic

line mey -re cultured in mlbecco‘a m3difiad mgle’s E d i w vzth high g l y ~ o s e and auppl-ntad w i t h 5\ fet.1 c d f SP-.

elin Qf cell*: mr preplration Of u n i f o d y labpled f ib-ectin, cells were 1abeled9meullholica11y w i t h [35s] lethionine 125vCi/ml, 1022 c i /mol , Nlv

m o s e labsllng with c-lZ-3H1, mannose ~ l o o ~ C i / ~ , 14.1 ci/-l, New -gland mgland mclear) in medium oontaininq 10% of the usual level of lathlonine. mr

b t h cases. ~ u c l e a ~ l g l w o s e -8 reduced to io% of w u a i level. Tnbpling was for 24 hours in

’ The abbreviations used are: kd, kilodaltons; DTNB, 5,5”di- thiobis(2-nitrobenzoic acid); MalNEt; N-ethylmaleimide; SDS; sodium dodecyl sulfate.

* The “Experimental Procedures” are presented in miniprint. Min- iprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chem- istry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Document No. 79M2248, cite author(s), and include a check or money order for $1.00 per set of photocopies.

4304

Structure of Fibronectin 4305

The Relative Position of the 200,000- and 25,000-dalton Fragments-In order to determine the order of fragments in fibronectin, we employed a modification of the procedure of Dintzis (35). Since protein synthesis proceeds from NH2- to COOH-terminal, then at times short relative to the time for completion of the polypeptide, the COOH-terminal regions of completed molecules would be expected to label preferentially whereas, with increasing labeling times, the labeling of any two different regions would tend towards a constant ratio dependent on their content of the labeled amino acid precur- sor.

NIL.8 cells were labeled for variable lengths of time at room temperature with [35S]methionine. After removal of the radioactive medium, cells were lysed rapidly on ice with 1% deox- ycholate containing emetine, a protein synthesis inhibitor. The newly synthesized fibronectin was purified by passing each cell lysate through a small gelatin-Sepharose column and

eluting the bound fibronectin with urea. Urea was then di- alyzed away and fibronectin was subjected to trypsin cleavage giving two major fragments of 200,000 and 25,000 daltons, as described previously (24). For each time point, the same number of dishes was used, so that the conditions for process- ing and cleavage were closely sindar. The tryptic fragments were analyzed on SDS gels (Fig. 1).

By scanning autoradiographs such as that in Fig. 1, we have determined for each labeling time the ratio of label in 25,000/ 200,000 fragments and have normalized to the ratio of 25,000/ 200,000 found in uniformly labeled molecules. By plotting the normalized ratio against the time of labeling, we have obtained curves such as that shown in Fig. 2. The fact that the ratio 25,000/200,000 increases with labeling time until the molecule of fibronectin becomes uniformly labeled shows that the 25,000-dalton fragment is to the NH2-terminal side of the 200,000-dalton fragment (see "Experimental Procedures").

Similar comparisons were also made for a minor 40,000- dalton fragment which we have reported earlier (24) as arising from further cleavage of the 200,000-dalton fragment and containing the free sulfhydryl group of the 200-kd fragment. Comparison of 25 kd/40 kd ratios confiis that the 40-kd fragment is COOH-terminal to the 25-kd fragment and analysis of 40 kd/200 kd ratios is consistent with the 40-kd fragment's being somewhere within the 200-kd fragment.

Hence, one can deduce the order: NH2-25 kd-200 kd (including 40 kd and "SH group). It has been shown previously that the interchain disulfides holding together the two chains of fibronectin are not in either the 25-kd or the 200-kd fragment but are very close to one end of the intact chains (24). The present experiment does not distinguish which end this is.

Cleavage by Cyanylation-We have previously reported that each fibronectin chain contains one (or at most two) free sulfhydryl groups per chain as well as 38 to 46 half-cystines

2 5 10 20 90 90C

200 -

40- 2 6

""

FIG. 1. Tryptic digestion of short term labeled fibronectin. Fluorograph of 10% gel. Labeling was for minutes indicated. Incu- bations of 2 to 20 min were at 22OC; 90-min incubation was at 37°C. All samples were treated with trypsin except 9OC, which shows fibronectin purified from cell lysates after 90 min of labeling. Note the appearance of label in the 200-kd fragment but very little in the 25-kd fragment a t 2 min and subsequent increase in label in the 25-kd fragment relative to the 200-kd fragment. Also note switchover in relative labeling of the 25-kd and 40-kd fragments as a function of time.

4306 Structure of Fibronectin

involved in disulfide bonds. Specific cleavage of polypeptide chains at sulfhydryl groups modified by reaction with Ell- mann’s reagent (DTNB) or at disulfides can be accomplished by reaction with cyanide which is followed by cyclization of the resulting thiocyanate and concomitant cleavage of the peptide backbone (36-38). We have used this cleavage to provide further evidence on the topology of the fibronectin chains using the sulfhydryl group(s) as fixed points of refer- ence. Certain complications arise in the use of this procedure on a molecule as large as fibronectin which contains a small number of cysteines and a much larger number of cystines. The analysis relies on the difference in rate of cleavage of different disulfides (39). If free SH groups in fibronectin are reacted with DTNB, the mixed disulfides formed in this way will be more susceptible to nucleophilic displacement by cyanide than the cysteines. The rate of cleavage of a disulfide

i i ib m I io m s

FIG. 2. Quantitation of radioactivity in the 25-kd and 200-kd fragments as a function of labeling time. Fluorographs of gels such as that shown in Fig. 1 were scanned and the relative incorporation into the 25-kd and 200-kd fragments was determined for each time. The ratios were normalized to the ratio observed after 90 min of labeling. Points show mean and standard deviation of the normalized ratios at each time (n = 4 to 6). The increase in ratio as a function of time of labeling indicates that radioactivity appears first in the 200- kd fragment which is therefore the more COOH-terminal of the two fragments. The data are from a single experiment but a similar curve was obtained in a second experiment and less complete results from several other experiments were consistent.

0 10 30 120ON 0 10 30 120 ON

23(

17(

ll(

190

150 130

FIG. 3. Cyanide cleavage of [“Slmethionine-labeled fibronectin. Fluomgraph of 5% gel. Cleavage was with 20 mM cyanide for minutes indicated; ON, overnight incubation (16 h). Left hand side of gel, samples were run reduced; right hand side of gel, samples were run without reduction. Note early appearance of the 170-kd fragment in both halves of the gel although this fragment migrates a t lower apparent molecular weight (-150,OOO) when nonreduced. Also note the later appearance of a group of fragments a t 190 kd derived by gradual cleavage of the 230-kd chain, and of smaller mass bands (150, 130 and 110 kd) derived by gradual cleavage of the 170-kd fragment.

230- 170-

0 10 30210 0 - 2 4 9

230: -190 17th 4130

*33

- A. B.

FIG. 4. Cyanide cleavage of ra*Slmethionine-labeled

-190 4 3 0

-33

fibronectin (A) - a d of [SH]ma&ose~lab;Jled fibronectin (B). Fluo- rographs of 10% gels. A, cleavage was with 10 m cyanide for hours indicated. All samples were reduced. Note appearance of the same large fragments as in Fig. 3 and one at 33 kd at early times. Also note that cleavages occur more slowly with 10 m than with 20 m cyanide (c6 Figs. 3 and 4B). B, cleavage was with 20 m cyanide for minutes indicated. Samples were run reduced. Note that the 170-kd fragment as well as all the late-appearing fragments (190, 150, 130 and 110 kd) are labeled but that no mannose label was detected in the 33-kd fragment (position marked by arrow).

TABLE I Generation of fragments early in cyanide cleavage

Results obtained by scanning autoradiographs. Pooled data from five experiments (three with “C and two with =S). Predicted ratio is 0.20 for parallel appearance of 33-kd and 170-kd fragments, assuming uniform labeline. ~~ ~ ~~

Label Min

10 15 30 ratio 33/170

“C-labeled amino acid mix 0.11 0.23 0.20 rS5S1Methionine 0.24 0.18

bond (and consequently the cleavage of the polypeptide chain) is controlled by the anionic stability of the leaving group (40, 41), and the thionitrobenzoate leaving group derived from DTNB is highly stabilized and is therefore a very good leaving group. So, the first cleavages observed after submitting DTNB-reacted fibronectin to cyanide should be next to cysteine residues and only later should cleavages due to cystines occur, some being more rapid than others, depending on their immediate environment (36,39).

Cleavage of Radioactive Fibronectin by Unlabeled Cya- nide-Metabolically labeled fibronectin was reacted with DTNB during purification on gelatin-Sepharose. Then SDS was added to 0.1% and samples were reacted with sodium cyanide for varying periods of time. After quenching of the reaction in acid and dialysis, samples were run on gels either reduced or nonreduced. The first fragments which appeared were 170,000 daltons (Figs. 3 and 4) and 33,000 daltons (Fig. 4A). Longer incubations with cyanide produced several further cleavages. The rate of these secondary cleavages was dependent on cyanide concentration. Both the 230,000-dalton chain and the 170,000-dalton fragment decreased progres- sively in size, generating smaller fragments. These smaller fragments were in two groups. The 230-kd chain generated a group of bands of around 190,OOO daltons, while the 170-kd fragment generated fragments of 150,OOO, 130,000, and 110,000 daltons (Figs. 3 and 4). The 170-kd fragment was labeled with mannose (Fig. 4B) and therefore contains carbohydrate residues, as do the late-appearing fragments (190, 150, 130, and 110 kd). No carbohydrate label could be detected in the 33-kd fragment. We have reported previously (24) that the 25-kd


fragment is carbohydrate-free and that all or most of the carbohydrate residues are in the 200-kd fragment consistent with the results reported here.

Analysis on nonreduced gels (Fig. 3, right) showed that the 170-kd fragment ran somewhat faster when not reduced, suggesting the presence of intrachain but the absence of interchain disulfides. The nonreduced gels also showed that the slowly generated final cleavage fragments (190, 150, 130, and 110 kd) were all free of interchain disulfide bonds, suggesting that the latter had been cleaved by cyanide.

Returning to the rapidly appearing 170-kd and 33-kd fragments, quantitative analysis showed that they appeared together and in parallel at early times (Table I). Their early appearance suggested that they arise by cleavage at free sulfhydryl groups modified by reaction with DTNB and that the slower subsequent cleavages arise from cleavage at cystine disulfides. Consistent with this supposition, cyanide cleavage of fibronectin, which was treated with N-ethylmaleimide prior to DTNB treatment, did not generate 170-kd and 33-kd fragments but did lead to slow reduction in sue of the 230-kd chain to a final broad band with a molecuar weight of about 190,OOO (Fig. 5A). As expected, the 150-, 130-, and 110-kd fragments which arise by secondary cleavage of the 170-kd fragment also did not appear when the fibronectin was blocked with N-ethylmaleimide (Fig. 5A), nor did the 33-kd fragment (not shown). Thus, if modification of free sulfhydryl groups by DTNB is prevented, the early cleavages generating 170-kd

0 5 15 30 l h 2h 4h 9h

19 0

A.

0 5 15 30 l h 2h 4h 9h

and 33-kd fragments are blocked, whereas slower cleavages leading to reduction in size of the 230-kd chain to about 190 kd proceed as usual via cleavage at cystines.

In a related control experiment, cyanide cleavage was carried out without prior DTNB treatment. One would predict results similar to the N-ethylmaleimide blocking experiment. In fact, we observed that cleavage of the 230-kd chain pro- ceeded as expected (Fig. 5 0 but generation of the 170-kd fragment (and the 33-kd fragment), while slower than in the control experiment (with DTNB, Fig. 5B), was not completely blocked (Fig. 50 . An explanation for this unexpected result is provided by a previously puzzling observation. Fibronectin in the culture medium of NIL.8 hamster cells exists as a dimer and runs as a single band on nonreduced gels (42). After purification, it frequently runs as a closely spaced double band when run nonreduced (23.24). If N-ethylmaleimide is added during purification of fibronectin, then the purified dimer remains as a single band. The slower member of the doublet found in control preparations is absent (Fig. 6). Thus, formation of this slower migrating form of dimeric fibronectin requires free sulfhydryl groups during the purification procedure (which involves treatments with urea and high pH). So, when fibronectin is purified without blocking its sulfhydryl groups, some of the cysteines can apparently react to give cystine residues which are then susceptible to cyanide cleavage exactly as observed in Fig. 5C. Hence, this result is consistent with earlier ones and also provides an explanation

0 5 15 30 l h 2h4h 9h

4 7 0

” ” B.

FIG. 5. Cyanide cleavage of [a6S]methionine-labeled fibronectin under various conditions. Fluorographs of 8% gels. AU

a 190 samples were incubated with 20 mM cyanide for minutes or hours (h) as indicated, and analyzed after reduction. A, fibronectin was treated

170 with N-ethylmaleimide before and during purification. Subsequently it was incubated with DTNB as usual. B, fibronectin was reacted with DTNB as in Figs. 3 and 4. The same fragments were produced as previously. Panel C, fibronectin was reacted without prior treatment with DTNB. Note low level of the 170-kd fragment compared with B and relatively larger yield of 190-kd fragments. The track at lefr in each panel contains [35S]methionine-labeled culture medium as molecular weight marker. The three marked bands have molecular weights of 230,000, 180,000, and 130,000 (see Ref. 4 under “Experi- mental Procedures”).

C.


for the two forms of fibronectin dimer present in purified fibronectin. The slower DTNB-independent generation of 170-kd can be explained by disulfide bond formation (intra- or interchain) by the free cysteine(s) of fibronectin. It remains clear from the N-ethylmaleimide blocking experiment (Fig. 5A) that the 170-kd and 33-kd fragments arising in the standard cyanide experiments (Figs. 3 and 4) arise by cleavage at DTNB-modified cysteine.

Cleavage of Unlabeled Fibronectin by Radioactive Cya- nide-If the cyanide used for cleavage is radioactive, then the incorporated CN group remains as part of the 2-iminothiazo- lidine-Ccarboxylic acid residue formed on the COOH-terminal side of the cleaved peptide bond. Hence, all fragments produced by cyanide cleavage will be radioactive with the excep- tion of the one containing the true NH2-terminal of the molecule. Radioactivity will be most rapidly incorporated into fragments which are COOH-terminal to a cysteine and more slowly into other fragments via other reactions. Cleavage with radioactive cyanide should, therefore, give information on whether the 170-kd or 33-kd fragments generated by cleavage at cysteine residues are NH2- or COOH-terminal to those residues.

Unlabeled fibronectin was cleaved by [I4C]NaCN using the same protocol as with normal cyanide, except that dialysis was done against 50% acetic acid to avoid solubility problems due to higher protein concentrations. After freeze-drying, samples were run reduced on gels. These were stained for proteins and scanned and, after drying and exposure of x-ray film, autoradiographs were scanned and specific activities of the 170-kd and 33-kd fragments were calculated. At early times, very little radioactivity was incorporated into the uncleaved fibronectin molecule or the 170,000-dalton fragment, while the

C N

FIG. 6. Dimeric forms of fibronectin. Fluorograph of 5% gel. Samples were run without reduction. C, fibronectin purified on gelatin-Sepharose and eluted with 4 M urea in pH 11 buffer (24). N, fibronectin purified with N-ethylmaleimide added during purification. Note double band in Sample C and absence of slower band in Sample N.

a8

nullvt 170

SPICIIIC IC111111

-

M

w

¶O

6 i6 ab mmns

FIG. 7. Cleavage of fibronectin by [“C]cyanide. Quantitation of specific activity was by scanning of Coomassie Blue-stained gels and of fluorographs (see “Experimental Procedures”). Graph shows ratio of specific activities of 33-kd and 170-kd fragments as a function of time of cleavage. Horizontal line indicates predicted ratio for one cyanide incorporated per fragment. The high ratios indicate prefer- ential incorporation of cyanide into the 33-kd fragment, especially at early times.

33,000-dalton fragment labeled strongly (Fig. 7). As the incubation continued, label began to appear both in the 170-kd fragment and in the intact 230-kd chain. If both the 170-kd and 33-kd fragments (which appear in parallel; Table I) had arisen by a cyanide cleavage at their NH2-terminal ends, then at early times a specific activity ratio (33:170) of about 5 would have been expected (see “Experimental Procedures” and Fig. 7). The observed ratio is more than 6-fold higher, suggesting that while the 33-kd fragment arises by a cyanide cleavage at its NH2-terminal, the 170-kd fragment does not and is therefore from the NH2-terminal of the fibronectin chain. Subse- quent incorporation of labeled cyanide into the 170-kd fragment leads to a fall in the 33:170 specific activity ratio. It is important to note that similar delayed labeling of the 230-kd chain also occurs (data not shown).

The slower incorporation of cyanide into 170-kd and 230-kd chains could arise either from cyanide insertion into disulfide bonds or from other side reactions of cyanide. Insertion of cyanide at disulfides is consistent with the slower cleavages of the 230-kd to 190-kd fragments and of the 170-kd to 150-, 130-, and 110-kd fragments (cf Figs. 3 to 5) and, as expected, these fragments are also labeled with [I4C]cyanide (data not shown).

Thus, the radioactive cyanide cleavage experiments lead to the following major conclusions: 1) The 170-kd fragment is from the NH2-terminal of fibronectin and is generated by cleavage at a sulfhydryl group which is 170,000 daltons from the NH&erminal. 2) The 33-kd fragment is COOH-terminal to a cysteine residue, perhaps the same one which generates the 170-kd fragment (see “Discussion”).

Combining the results of the cyanide cleavages with the earlier data on tryptic cleavages (24) and ordering of the tryptic fragments (Figs. 1 and 2), one obtains the model of fibronectin structure shown in Fig. 8 (see “Discussion”). Since the 170-kd cyanide-generated fragment overlaps with the 200- kd tryptic fragment and the latter is known to contain a free


sulfhydryl group (24), cyanide cleavage of the 200-kd tryptic fragment should generate a smaller fragment. Fig. 9 shows that this is indeed the case; cyanide cleavage after tryptic digestion produces a major fragment of 120 to 125 kd as well as cleavages of the 200-kd fragment to fragments of somewhat lower molecular weight. These results are consistent with the relative positioning of the different fragments as shown in Fig. 8.

mnlu C1111D1 I t m t n

I I I 110 I I I - 1ao

150 -I I I 170 'X-" I I I qla

NZfH'l' ' f ' f' I I or I f I I ' C

I IC

N n H HI !'I H M $8 f-$

I 125 I

40 - ao 40 "

FIG. 8. Diagram of cleavages of fibronectin dimer by trypsin and cyanide. The deduced structure of the dimer is shown in the center. Tryptic fragments are shown below this and cyanylation fragments above, with fragments arising from early cleavages at cysteines placed below fragments derived by later cleavages at cystines. The 190-kd fragments are not shown. The 125-kd fragment is derived by sequential trypsin digestion and cyanylation (see Fig. 9). Also shown at the bottom are the fragments described by Balian et al. (25) and positioned by comparison with our data and those of Mosher et at. (31). The ordering of the fragments is discussed in the text. Uncertainty about the position of the 33-kd fragment and about the nature of the second cleavage site giving rise to it are indicated by alternatives in the figure.

0 0 M Q )

0 0 o c + + a

230 * 200 *

* 230

* 170

- 125

FIG. 9. Cyanide cleavage of the 200-kd tryptic fragment. Fluorograph of 5% gel. [3SS]Methionine-labeled fibronectin (0) was subjected to trypsin giving the 200-kd fragment (2') which was then cleaved by cyanide (TC30 and TC90). Incubation was with 20 mM cyanide for 30 or 90 min. Note appearance of a 125-kd band and that the 200-kd band decreased in molecular weight. The 125-kd fragment is smaller than the 170-kd fragment obtained from intact fibronectin by reacting it with cyanide (0.

nkfnn n l x h

nbnn H ' n l n ? P

0

0 0

P 0 P r 0

t l 40 -e - V

A V -HBDlDlM Ctllll'

n CELL

I lCIDB CDlLY18 1111 fIDt11

SIlilCf

FIG. 10. Model of fibronectin showing major features and binding sites. Positioning of Factor XI11 and gelatin-binding sites is by comparison with Refs. 24,25, and 31 (see text). Carbohydrates (see Fig. 4B and Ref. 24) are shown as circles on stalks. Not all the disulfide bonds (-20 per chain) are shown.

DISCUSSION

For purposes of discussion, let us fmt state the major conclusions which we wish to draw from the results presented here and elsewhere.

1. Cleavage with trypsin or plasmin generates a small 25-kd fragment and a large 200-kd fragment from each chain. The order of these fragments is NH2-25 kd-200 kd-COzH.

2. The interchain disulfide bond(s) of the fibronectin dimer are within 10,OOO daltons of the COOH-terminal end of fibronectin. This region is readily removed by proteolysis.

3. Cleavage at the limited number (1 or 2) of cysteines per chain generates a 170-kd fragment from the NHg-terminal of the molecule and a 33-kd fragment which is to the COOH- terminal side of a cysteine residue. Therefore, the most NHz- terminal cysteine is 170,000 daltons from the NH&erminal. There may be another one further towards the COOH-ter- mind (see below). Free cysteine residues are necessary for binding of fibronectin to the cell surface (24) and therefore constitute a "cell binding site" which is located towards the COOH-terminal of the molecule.

4. The NH2-terminal region which is contained in the 25-kd fragment and also in the 170-kd fragment is extremely cystine- rich. Cleavage of cystine residues by cyanide leads to degradation of intact (230 kd) chains and 170-kd fragments each to a group of smaller fragments.

5. The gelatin-binding site which is located in the 200-kd fragment (24) and also in another 73-kd fragment generated by cathepsin D (25) contains functionally important disulfide bonds (24,25) and is located towards the NH2-terminal end of the 200-kd fragment adjacent to the 25-kd fragment.

6. The NHg-tenninal25-kd fragment is the site for reaction of fibronectin with Factor XI11 transglutaminase (31,32).

These conclusions are incorporated in the models shown in Figs. 8 and 10 and the evidence for each point is reviewed below.

Order of Major Domains (Tryptic Fragments)-The analysis of short term labeled fibronectin (Figs. 1 and 2) shows that the relative order of the 25-kd and 200-kd fragments is NH2-25 kd-200 kd-COzH. A minor fragment of 40 kd arising from the the 200-fragment by further cleavage contains a free cysteine, as does the 200-kd fragment (24), and can also be positioned to the carboxyl side of the 25-kd fragment and within the 200-kd fragment by these short term labeling results.

This order of fragments is consistent with the data of Fwie and Rifirin (28), who found that the smaller fragment obtained by thrombin cleavage of human plasma fibronectin (similar to our 25-kd trypsin fragment) retained the NHg-terminal pyroglutamate of intact fibronectin and has the same NH2- terminal amino acid sequence as does the intact molecule. On the other hand, the NH2-terminal analysis reported by Iwan- aga et al. (33) placed the NH&mninal on a 200,000-dalton cleavage product derived by plasmin cleavage of bovine


plasma fibronectin. This discrepancy could perhaps be explained by the use of different enzymes for cleavage by the two groups. However, our results obtained by a different approach place the 25-kd domain clearly in the NHz-terminal part of fibronectin.

The position of the interchain disulfides is deduced in the following way. Tryptic cleavage generates early fragments of 220 and 215 kd which are not interchain disulfide-bonded (24). ThBreforq, the interchain dislllfides are very close to one end. Boih we (24) and Furie and Rifkin (28) found that the smaller NHrterminal fragment behaves as a monomeric fragment and therefore does not contain the interchain disulfides. Therefore, the interchain disulfides must be at the COOH- terminal. This COOH-terminal linking region is removed very early during proteolytic digestion (24, 28). The cyanide cleavage results confirm the COOH-terminal location of the interchain disulfides since the 170-kd fragment (Figs. 3,4,5, and 7) is from the NHz-terminal of fibronectin (see below) and is not interchain disulfide-bonded (Fig. 3).

Position of Sulfhydryl Groups(s) and Disulfides-The gen- eratiop of the 170-kd fragment by cleavage with cyanide at a cysteine residue is shown by the fact that this cleavage does not occur if cysteine residues are blocked with N-ethylmaleimide (Fig. 5A) but does if the cysteines are modified by DTNB (Figs. 3,4, and 5B). The conclusion that the 170-kd fragment is NHz-terminal comes from the fact that it appears early during cyanide cleavage (Figs. 3,4, and 5B; Table I) but does not label rapidly with ['4C]cyanide (Fig. 7). Thus, it does not arise by a cyanide cleavage at its NHz-terminal (36, 37) and must therefore be an NHz-tenninal fragment.

The 33-kd fragment, which also arises early in cyanide cleavage (Figs. 4 and 7) appears in parallel with the 170-kd fragment (Table I) and does not appear if free cysteines are blocked with N-ethylmaleimide. It therefore also arises by cleavage at cysteine. In contrast with the 170-kd fragment, the 33-kd fragment does label rapidly with ['4C]cyanide, indicating that it arises by a cleavage at a cysteine residue at its NH2- terminal (Fig. 7; Refs. 36 and 37). These results position the 33-kd fragment to the carboxyl side of the 170-kd fragment but do not completely explain its origin. The two fragments do not account for the complete 230-kd chain. We have been unable to identify conclusively another fragment arising early in cyanide cleavage, despite numerous attempts involving a variety of metabolic labels including 14C-labeled amino acid mix (data not shown). The 33-kd fragment is not observed in gels which are run nonreduced. It must therefore be involved in some sort of disulfide-bonded complex, but attempts to characterize this complex further have so far been unsuccess- ful. Since it is known that interchain disulfides exist in the COOH-terminal region of the molecule, the 33-kd fragment could be involved in these. Alternatively, the 33-kd fragment could be attached to another unidentified fragment by intrachain disulfide bonds (of the intact 230-kd chain). The nature of the putative second site of cleavage required to generate a 33-kd fragment is also unknown; i.e. it could be a second cysteine residue or a particularly labile cystine disulfide bond. The uncertainties of interpretation of the cyanide cleavages in the COOH-terminal region of the molecule are indicated in Fig. 8. Further clarification is complicated by cleavages occur- ring at disulfide bonds known to be in this region and more work will be necessary to elucidate completely the structure of this region of the molecule. However, these uncertainties do not affect any of the conclusions drawn here.

Turning now to the NHz-terminal region of fibronectin, several lines of evidence indicate that it is extremely rich in disulfides. First, the 25-kd fragment is located at the NHz- terminal and was previously shown to be very rich in cystine

(24). Second, both the 230-kd chain and the 170-kd fragment are degraded by extended cyanide treatment (Figs. 3, 4, and 5). Degradation of the 230-kd chain is not affected by blocking of cysteines with N-ethylmaleimide (Fig. 5 ) , and is therefore presumably due to cyanide cleavage at cystine residues. The 125-kd fragment generated by cyanide cleavage of the 200-kd fragment shows the same gradual cleavage by cyanide (not shown). These results are best explained by a disulfide-rich region including the 25-kd fragment but extending into the 200-kd fragment for some distance. This region would be susceptible to cyanide cleavage of its cystines (36, 37, 39-41). Completely consistent with this interpretation of our results, Balian et al. (25) have reported a 73-kd fragment generated by cathepsin D which is cystine-rich. Further cleavage of the 73-kd fragment with plasmin generates a 30-kd fragment, which has properties very similar to the 25-kd fragment reported here, and a 40-kd fragment which is also cystine-rich. All of this information, which is summarized in Fig. 8, is entirely consistent.

While our cyanide cleavage experiments were in progress, Fukuda and Hakomori (43), using a cyanide cleavage protocol somewhat similar to ours, came to rather different conclusions. They reported fragments of 180, 125, and 90 kd and inter- preted their results in terms of cyanide cleavages entirely at modified cysteine residues. Their protocol differed from ours in several ways. They used higher concentrations of cyanide (50 mM) which were allowed to react for 30 min at pH 8 at room temperature. The reaction was quenched by acidifica- tion and the cyanide was removed by dialysis. Cleavage of the cyanylated protein was then carried out at pH 9 for 20 h at 37°C. Our protocol involved simultaneous cyanylation and cleavage at pH 9 but with 10 or 20 mM cyanide. We observed secondary cleavages as early as 2 h of incubation with 20 m~ cyanide (Figs. 3 to 5) which can be shown to be independent of cysteines (Fig. 5) and are clearly due to cleavage at cystine residues. Our interpretation of the results of Fukuda and Hakomori (43) is that they observed cleavages at cystine residues. Their cleavage fragments (180, 125, and 90 kd) appeared very similar to the secondary cleavage products we observed at late times (190, 150 + 130 and 110 kd). Consistent with this conclusion, when Fukuda and Hakomori (43) treated fibronectin with iodoacetic acid prior to DTNB (analogous to our N-ethylmaleimide blocking experiment, Fig. 5 ) , they obtained only the 180-kd fragment, whereas we obtained the 190-kd fragments. As we have shown, the 150-, 130- and 110- kd fragments arise by secondary cleavage of a primary cleavage fragment, of 170 kd, which arises from cleavage at a modified cysteine residue. Fukuda and Hakomori (43) presumably did not observe this fragment (or the 33-kd fragment) because they analyzed only a single time point. As discussed earlier, it is important in analyzing cyanide cleavage results to consider cleavages at a number of different times since cyanide can cleave modified cysteines or cystines, albeit at different rates (36-41). This is particularly pertinent in the case of a molecule such as fibronectin which has (per chain) about 20 disulfide bonds and only one or two sulfhydryl groups. Thus, in conclusion, our results do not conflict with those of Fukuda and Hakomori (43), but by looking at earlier times we were able to distinguish cleavage at cysteines from cleavage at cystines and we come to significantly different conclusions about the arrangement of these constituents.

Position of Binding Sites for Gelatin, Collagen and Fi- brin-The results of BaLian et al. (251, together with ours, also allow positioning of the gelatin-binding site, which Balian et al. showed to be in the 73-kd fragment and in the 40-kd subfragment and which we have reported to be in the 200-kd fragment (24). We both find that intact disulfide bonds are

Structure of Fibronectin 431 1

Id I

FIG. 11. Formation of aggregates from exogenously added dimeric fibronectin. Purified %3-labeled fibronectin was added to NIL.8 cells for times indicated. At each time point, cells were lysed and analyzed on nonreducing gels. Radioactivity in positions of fibronectin dimer and of high molecular weight aggregates was quantitated and expressed as percentage of total radioactivity bound. Fibronectin from both these fractions ran as 230-kd monomer on reduction, showing that the aggregates require disulfide bonds for their integrity and are not held together by other covalent bonds.

necessary for efficient binding to gelatin (24,25). These results would place the gelatin-binding site near the NHZ-terminal of the 200-kd fragment as shown in Fig. 10 and are consistent with the conclusion that this region of the molecule is disulfide-rich.

This conclusion is strongly supported by recent results of Mosher et al. (31). They have studied the reaction of fibronectin with Factor XI11 transglutaminase which can cross-link fibronectin to fibrin or collagen (16,17). They h d that Factor XI11 covalently couples amines to proteolytically derived fragments apparently identical with our 25-kd fragment, with the 73-kd fragment of Balian et al., and with the 29-kd fragment of Furie and Riflrin (28), and not into large fragments generated in the same digestions. These results, are consistent with earlier preliminary data (44)3 and confirm the arrangement of fragments shown in Figs. 8 and 10. Other data indicate that the gelatin-binding site is coincident with the collagen-binding site and probably also with the fibrin-binding site (lS), all of which are, therefore, in a region of the molecule adjacent to the NH2-terminal 25-kd fragment which reacts with Factor XI11 (see Fig. 10).

Disulfide Bonding through the Free Sulfhydryl Group(s)- We have reported earlier that blockage of free sulfhydryl groups on fibronectin prevents its binding to the cells (24). The results presented here (Figs. 5 and 6) indicate that the free sulfhydryl groups can form disulfide bonds during purification, some of which can be intramolecular. Since earlier evidence had indicated that fibronectin exists in disulfide- bonded aggregates (42, 45) and that disulfide bonds are necessary for binding to the cells (24,46), we have been interested in the possible importance of intermolecular disulfide bonds formed through the free sulfhydryl groups of fibronectin. Fig. 11 shows that when dimeric fibronectin with free sulfhydryl groups is added to cells, the bound fibronectin steadily becomes converted into disulfide-bonded aggregates. As reported earlier, this does not happen with dimeric fibronectin whose free sulfhydryl groups are blocked (24). A similar conversion of dimeric fibronectin into high molecular weight

D. D. Wagner and R. 0. Hynes, unpublished results.

aggregates held together by disulfide bonds has been observed for newly synthesized fibronectin which is exported to the surface as a dimer (47). These results further implicate the free sulfhydryl groups of fibronectin in intermolecular bonding at the cell surface, although they do not distinguish between homopolymers and heteropolymers, for instance with proteoglycans (19). The results reported here on the position of the sulfhydryl groups which appear to be involved in this process place one on each chain of the dimer, 170,000 daltons from the NH2-terminal or 100,000 daltons away from the collagen/ fibrin-binding site. At present, the results leave open the possibility that there may be a second sulfhydryl group on each chain 30,000 to 40,000 daltons further towards the COOH-terminal. These sulfhydryl groups (one or two per chain) appear to be involved in binding of fibronectin to the cell.

Thus, the detailed analysis of the structural organization of fibronectin has allowed us to position within the molecule several important binding activities; those for disulfide-bonding and for binding and cross-linking to collagen or fibrin. It will be of great interest also to locate the binding sites for heparin and other proteoglycan constituents (18, 19) and for glycolipids (48). Location of the various binding sites and structural features of fibronectin will be important in further understanding of its biological functions.

Acknowledgments-We would like to thank Mike Bohn, Harvey Lodish, and Frank Solomon for helpful suggestions during the course of this work, Naomi Foster for photography, and Janet Romaine for typing. We are also grateful to Deane Moeer and Martha Furie for communicating results prior to publication.

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