Gelatin-binding domain-specific anti-human plasma fibronectin Fab' inhibits fibronectin-mediated gelatin binding but not cell spreading.

Antisera against a Mr = 60,000 peptide containing the gelatin-binding domain of human plasma fibronectin (McDonald, J. A., and Kelley, D. G. (1980) J. Biol. Chem. 255, 8848-8858) bound the Mr = 60,000 peptide and intact fibronectin but not three other fragments released by leukocyte elastase proteinolysis (the Mr = 25,000 amino-terminal sequence, Mr = 140,000 sequence containing cell adhesive activity, and a Mr = 31,000 fragment). Affinity-purified Fab' blocked Mr = 60,000 peptide binding to gelatin and inhibited plasma and cellular fibronectin gelatin binding without affecting fibronectin-mediated cell spreading. In contrast, antifibronectin Fab' absorbed with the gelatin-binding fragment completely blocked fibronectin-mediated cell spreading. These data indicate that the gelatin-binding domain of fibronectin is immunogenic, and antisera against this domain recognize cellular fibronectin gelatin-binding sites. Inhibition of gelatin binding but not cell spreading by anti-gelatin binding domain Fab' confirms the hypothesis that fibronectin has separate sites mediating these activities. Selective inhibition of fibronectin-collagen binding by domain-specific antisera may help elucidate the role of fibronectin in organization of the extracellular matrix.

(1) is implicated in extracellular matrix organization. In vitro, fibronectin is a major component of fibroblast extracellular matrix co-distributed with procollagen types I and I11 (2, 3), and fibronectin binds to two major components of the extracellular matrix, collagen and hyaluronic acid (4-6). Addition of fibronectin to differentiated chondrocytes results in altered patterns of collagen synthesis ( 7 ) .
Although specific antiserum inhibits fibronectin-mediated cell adherence (8,9), selective inhibition of binding to individual matrix components by polyvalent antisera is unlikely since fibronectin has separate binding domains for collagen, glycosaminoglycans, and cells (4, 5, [10][11][12][13][14][15]. We have utilized proteolytic cleavage to isolate the gelatin-binding domain of fibronectin, and in this report we describe the effects of antiserum specific for the gelatin-binding domain of human plasma fibronectin (10) upon fibronectin-mediated cell spreading and gelatin binding.
* This work was supported in part by Grant HL26009 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article with 18 U.S.C. Section 1734 solely to indicate this fact. must therefore be hereby marked "advertisement" in accordance + To whom reprint requests should be addressed.

EXPERIMENTAL PROCEDURES
Gelatin-binding Fragment Antibody-Purified 60 k' fragment (10) (1.0 mg) in 1.0 ml of complete Freund's adjuvant was injected into two New Zealand rabbits at multiple intradermal sites in the back. Rabbits were boosted intradermally with 1 mg of 60 k fragment in incomplete adjuvant at 6-week intervals. Antiserum was titered by radioimmunoassay. Lactoperoxidase-glucose oxidase '2511-labeled 60 k fragment (10) (20,000 cpm, approximately 10 ng) was incubated with antiserum in polystyrene tubes (11 X 75 mm) in a total volume of 0.2 ml of buffer A (7.5 mM Tris-HC1-150 mM NaCl-0.5% Tween 20-0.1 mM Na2EDTA-0.1 mM phenylmethylsulfonyl fluoride, pH 7.4, at 25 "C). After 16 h at 4 "C, 50 pl of a 10% suspension of IgSORB (The Enzyme Center, Boston, MA) was added and incubated at 37 "C for 30 min. Bound '251-probe was separated from free by adding 3 ml of buffer A, vortexing, sedimenting (2,500 X g, 10 min) the IgSORB, and aspirating the supernatant. Bound ' *' I was determined by y counting. Specificity of anti-60 k antiserum was assessed by incubating 12'1labeled 60 k with an antiserum dilution giving 50% of maximal binding plus varying amounts of fibronectin or the 60 k, 25 kZ (previously designated 29 k fragment in Ref. lo), 140 k, and 31 k fragments (10).
Isolation of Preimmune and Anti-60 k ZgG and Fab'-Pure 60 k fragment was coupled to CNBr-activated Sepharose CL-4B (16) (Pharmacia Fine Chemicals, Piscataway, NJ) using 2 mg of fragment/ ml of gel. Before use, columns were washed with 8 M urea in 50 mM Tris-HC1, pH 7.4, 0.2 M glycine-HC1, pH 2.3, and PBS. Antiserum (50 m l / l O ml of gel) containing 0.02% NaN:, was circulated over the column overnight at 25 "C. The column was washed with PBS until the AZRO nm was base-line, then with 0.5 M NaCl in 50 mM Tris-HC1, pH 7.4, and bound proteins were eluted with 0.2 M glycine-HC1, pH 2.3. Identity of the eluted fraction as IgG was verified by SDSpolyacrylamide gel electrophoresis (10, 17) with and without reduction. Fab' fragments were obtained by pepsin digestion, reduction, and alkylation with 2-mercaptoethanol and iodoacetic acid (18). F a b was isolated by chromatography on Sephadex G-100, its identity was confirmed by gel electrophoresis, and its binding activity by competition for antiserum binding of "'1-labeled 60 k fragment was as described above. Preimmune Fab' was obtained from rabbit IgG (18) as described above and analyzed by SDS-gel electrophoresis.
Isolation ofAnti-fibronectin Fab' Absorbed with 60 k Fragment-Rabbit anti-FN IgG, affinity purified on FN-Sepharose CL-4B as described above, was cycled over 60 k fragment-Sepharose CL-4B. Fab' obtained from the IgG fraction not binding to the 60 k fragment column (designated anti-FN-minus 60 k) was used to test the effect of anti-FN-Fab without 60 k binding activity in biological assays.
Gelatin-binding Assay-Binding of '"I-labeled fibronectin and 60 k fragment to heat-denatured rabbit skin type 1 collagen was determined as previously described (10).

Effects of Domain-specific Anti-FN
Fab' coated with 25 pg/ml of gelatin, rinsed with PBS, and incubated with 25 pg/ml of fibronectin alone or after incubation with F a b (1 h, 37 "C). Trypsinized CHO cells washed with MEM plus soybean trypsin inhibitor were then plated in the presence of Fab' preparations or medium alone. After incubation for 1 h at 37 "C, the plates were rinsed, and cell spreading was determined by morphologic scoring (9,10). Cell spreading assays were blinded to avoid observer bias. Cell spreading on fibronectin-coated plastic substrate was carried out similarly, as described previously (10).
Effect of Anti-60 k Fub' upon Fibroblast Fibronectin Gelatin Binding-Human lung fibroblasts (IMR-90), passage 12, were seeded at a 1:3 subcultivation ratio in DMEM + 5% fetal bovine serum depleted of fibronectin-gelatin binding activity by gelatin-Sepharose chromatography (IO) and cultured 5 days. On day 5, culture medium was replaced with DMEM + 5 pCi of [''C]proline/ml (specific activity, 260 pCi/pmol) and 100 pg/ml of F a b , and the cells were incubated for an additional 40 h. After labeling, the medium was removed, proteinase inhibitors (10 mM Na2EDTA, I mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride) were added, and the medium was dialyzed for 4 h against 3 changes of buffer B (10 m~ 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-I50 m~ NaC1-10 mM NaZEDTA-1 mM phenylmethylsulfonyl fluoride-1 mM N-ethylmaleimide, pH 7.5) a t 4 "C. The cell layers were extracted with 1.0 ml of 2 M deionized urea in buffer B for 1 h at 37 "C, the extract was centrifuged (15,000 X g, 15 min, 0 "C), and the supernatant was dialyzed as described above. Total nongelatin binding and gelatin binding labeled polypeptides were displayed as previously described (10,17,19). Quantitative densitometry of autofluorograms was performed using a Zieneh soft laser scanning densitometer. Fibronectingelatin binding was quantified by summing the density of the fibronectin band in nonbound and bound fractions and calculating the percentage bound.

RESULTS
Specificity of Antisera and Fab"Titers of 1/1,200 to 1/ 8,000 (antiserum dilution giving 50% specific binding of probe) of anti-60 k antiserum developed by 2-3 months after initial injection. About 0.5 mg of IgG/ml of antiserum was obtained by affinity chromatography. SDS-polyacrylamide gel electrophoresis of affinity-purified and preimmune IgG gave one band of Mr = 150,000 (unreduced) and bands at M , = 50,000 and 25,000 (reduced), while Fab' preparations had one band at M, = 50,000 (unreduced) and at 25,000 (reduced).
As shown in Fig. 1, anti-60 k antiserum bound both 60 k fragment and intact fibronectin identically using a monomeric M, = 220,000 for intact fibronectin. No other elastase-released FN fragment competed with 60 k antiserum for 60 k binding even at 50-fold excess molar concentration. Identical results were obtained with affinity-purified anti-60 k IgG. Human plasma depleted of fibronectin by sequential gelatin-and antifibronectin-IgG affinity chromatography did not compete for fibronectin or 60 k probe binding by anti-60 k-IgG, demonstrating lack of cross-reaction of anti-60 k-IgG with other plasma proteins. Anti-60 k Fab' inhibited binding of '251-labeled 60 k probe by anti-60 k antiserum, while anti-FN-minus 60 k Fab' did not compete for binding of 60 k fragment by anti-60 k serum (Fig. 2), demonstrating the removal of 60 k binding activity by affinity chromatography.
Effect ofAnti-60 k Fab' Upon Fibronectin and 60 k Gelatin Binding-Anti-60 k Fab' competitively blocked both 60 k and fibronectin binding to gelatin, while preimmune Fab' had no effect (Fig. 3). Anti-60 k Fab' appeared to block 60 k fragment binding more effectively than fibronectin binding. Affinitypurified anti-FN-minus 60 k Fab' had no effect upon 60 k fragment binding to gelatin but did block fibronectin-gelatin binding (Fig. 3).
Effect of Preimmune, Anti-60 k, and Anti-FN-minus 60 k Fab' Upon Fibronectin-mediated Cell Spreading-Fab' preparations were incubated with gelatin-coated substrate, with fibronectin before addition to substrate, and with CHO cells during attachment and spreading on fibronectin-gelatin. Cell adhesion and spreading was only inhibited when anti-60 k Fab' was incubated with fibronectin, and the mixture was added to the gelatin-coated substrate prior to the addition of cells, demonstrating selective inhibition of fibronectin-gelatin binding, without affecting cell spreading (Table I, Fig. 4). Identical results were obtained with IMR-90 fibroblasts. To verify these results, similar experiments were carried out on substrates coated with fibronectin alone. Preimmune and anti-60 k Fab' had no effect upon CHO attachment or spreading    Table I. tin " Gelatin-coated dishes (25 pg/ml) were rinsed and incubated (30 min, 37 "C) with the indicated additions, rinsed again, and CHO cells plated. Thus, anti-60 k Fab' inhibited CHO attachment and spreading when incubated with fibronectin prior to addition to gelatin-coated plates ( C ) , but not when incubated with gelatin-coated plates prior to addition of fibronectin ( E ) , nor when incubated with CHO cells added on fibronectin, while anti-FN-minus 60 k Fab' completely blocked cell spreading when incubated with cells and substrate (Table I). Thus, anti-60 k Fab' did not bind to the gelatin substrate nor did it prevent cells from spreading on fibronectin bound to gelatin or to fibronectin plastic.

Inhibition of Newly Synthesized Fibronectin-Gelatin Bind-
ing by Anti-60 k Fab' in Vitro-Anti-60 k Fab' did not grossly alter fibroblast protein synthesis and secretion as the labeled polypeptides secreted into medium by preimmune and anti-60 k FaW-treated fibroblasts were similar. However, anti-60 k Fab' inhibited both medium and cell fibronectin-gelatin binding (Fig. 5). We cannot comment upon the relative amounts of fibronectin and procollagens synthesized in the presence of anti-60 k Fab' in the absence of more deflnitive biochemical analysis, although clearly both were synthesized and secreted. Quantitative densitometry (Table 11) revealed that maximal inhibition of medium fibronectin-gelatin binding was achieved to fibronectin-gelatin substrate ( F ) . " Spreading was scored by published criteria (10) by observation of three microscopic fields, and the scale 1, 2, 3, 4 equals 0-25, 25-50, 50-75, and 75-1008 of cells spread, respectively.
' CHO cells were plated on fibronectin-coated dishes in medium containing the indicated amount of Fab'. by 25 pg/ml of anti-60 k Fab', and urea-extracted fibronectingelatin binding was also decreased a t this level. IMR-90 maintained normal morphology and parallel alignment when labeled in DMEM in the presence of preimmune or anti-60 k Fab'.

DISCUSSION
Unlike previous investigators (ll), we obtained useful antisera in rabbits immunized with a fibronectin-gelatin binding fragment. Balb/c mice also developed antibody titers to 1/ 12,800, so human plasma fibronectin 60 k fragment is immunogenic in two mammalian species. Anti-60 k antiserum gave no precipitin lines on immunodiffusion; thus monitoring of antibody production by radioimmunoassay was essential. Only intact fibronectin and the 60 k fragment were recognized by anti-60 k antiserum, supporting the hypothesis that elastase fragments represent discrete domains of fibronectin (10). Anti-

TOTAL
NONBOUND BOUNC   Lower, densitometric scans of total, nonbound, and gelatinbound fractions of labeled medium polypeptides from preimmune and anti-60 k fragment F a b cultures. The position of fibronectin is indicated by the arrow, and the following two prominent bands represent procollagens. Note that all medium fibronectin from preimmune Fab' cultures bound to gelatin-Sepharose, while anti-60 k Fab' inhibited medium fibronectin binding (100 versus 27% binding). These scans were taken from the autofluorogram shown in the upper panel.
The significance of inhibiting fibronectin-gelatin binding in vitro by anti-60 k Fab' may be questioned. However, fibronectin has less avidity for native than denatured collagens in vitro (3). Thus, our assay for inhibition of high affinity gelatin binding may well underestimate the potential for inhibition of fibronectin binding to newly synthesized native collagens. Indeed, in preliminary studies, fibroblasts cultured with anti-60 k F a b maintained normal growth but exhibited marked alterations in organization of fibrillar extracellular fibronectin and collagens (21).
The demonstration that domain-specific anti-plasma FN recognizes and inhibits specific functions of cellular FN is not surprising in view of similar recognition of plasma and cellular FN by monoclonal antibodies (22). However, polyvalent domain-specific antisera to easily obtained fragments of plasma FN should prove useful for selective inhibition of cellular FN activities. In addition, such domain-specific antisera allow the quantification of FN in the presence of other FN fragments capable of competing with FN binding.