Journal of Cell Science 101, 625-633 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

625

Recognition of collagen by fibroblasts through cell surface glycoproteins

reactive with Phaseolus vulgaris agglutinin

HIROAKI ASAGA* and KATSUTOSHI YOSHIZATOt

Molecular Cell Science Laboratory, Zoological Institute, Faculty of Science, Hiroshima University, Kagamiyama 1-3-1, Higashihiroshima-shi,Hiroshima 724, Japan

•Present address: Cell Chemistry, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173, JapantAuthor for correspondence

Summary

The role of glycochains of cell surface glycoproteins inthe cell to collagen interaction was examined by studyingthe effect of lectins on the fibroblast-mediated collagengel contraction. Lectins of Phaseolus vulgaris agglutinin(PHA), concanavalin A (ConA), lentil seed agglutinin(LCA), pea agglutinin (PSA), Ricinus communis ag-glutinin-60 (RCA), and wheat germ agglutinin (WGA)dose-dependently inhibited gel contraction, while lectinsof mushroom agglutinin (ABA), peanut agglutinin(PNA), pokeweed mitogen (PWM), and soybean ag-glutinin (SBA) did not. Of these lectins, PHA seemed tobe worthy of further analysis, because PHA, but notother lectins, inhibited spreading of fibroblasts oncollagen fibrils but not on plastic or gelatin, suggestingthat cell-surface glycoproteins responsive to the lectin

are involved in the specific binding of fibroblasts tonative collagen fibrils. The inhibitory effect of PHA-E4,an isolectin of PHA, was more intense than that of PHA-L4, another isolectin of PHA. The collagen gel contrac-tion was also inhibited by tunicamycin and monensin in aconcentration-dependent and reversible manner. Theseresults strongly suggest that PHA-E4-reactive glyco-proteins of the fibroblast surface play an important rolein cell to collagen binding during the gel contraction.Five membrane proteins including fix subunits of theintegrin family were obtained by affinity chromatogra-phy with PHA-E4.

Key words: collagen, fibroblasts, collagen receptor,Phaseolus vulgaris agglutinin, glycoprotein.

Introduction

Collagen is a major component of extracellular matrices(ECM) of multicellular animals, and its roles can beclassified into: (1) supporting multicellular body organ-ization, and (2) regulation of physiological functions ofcells. Interactions of cells with ECM involve bindingbetween cell surface receptors and ECM components asligands. Recently, cell surface receptors for collagenwere identified and characterized (Wayner and Carter,1987; Kramer and Marks, 1989; Gullberg et al. 1989,1990b).

Collagen gel culture of fibroblasts (Elsdale and Bard,1972) has been a useful experimental system for thestudy of the cell to collagen interaction: fibroblastsrecognize, bind and reorganize collagen fibrils duringculture, resulting in the contraction of collagen gel (Bellet al. 1979; Steinberg et al. 1980; Harris et al. 1981;Stopak and Harris, 1982; Allen and Schor, 1983; Buttleand Ehrlich, 1983; Grinnell and Lamke, 1984; Yoshi-zato et al. 1985b; Guidry and Grinnell, 1985, 1986;Gillery et al. 1986; Guidry and Grinnell, 1987; Adamsand Priestley, 1988; Montesano and Orci, 1988; Schafer

et al. 1989; Kono et al. 1990; Gullberg et al. 1990a;Asaga et al. 1991). Recently, we proposed that there isan indirect interaction between fibroblasts and collagenfibrils via cellular fibronectin (cFN) but not plasmafibronectin (pFN), using monoclonal antibody A3A5(mAb A3A5), which specifically inhibits the fibroblast-mediated gel contraction (Asaga et al. 1991).

Monoclonal antibody A3A5 is a potent inhibitor ofcollagen gel contraction. However, complete inhibitionwas not obtained even in the presence of relatively highamounts of the antibody, when the gel culture wasextended to more than 3 days (Asaga et al. 1991). Thissuggests the involvement of other type of mechanism ofbinding between fibroblasts and collagen in the processof gel contraction.

There have been several studies showing that cellsurface glycoproteins act as receptors for ECM. In thepresent study, we have tested the possibility ofinvolvement of this type of receptor in the fibroblast-mediated collagen gel culture using several kinds oflectins as a probe. We showed that fibroblast-surfaceglycoproteins recognized by Phaseolus vulgaris ag-glutinin (PHA, especially PHA-E4) act as a collagen

626 H. Asaga and K. Yoshizato

receptor in the process of collagen gel contraction. Thechemical nature of the PHA-E4-reactive glycoproteinson cell membranes was analyzed and its function isdiscussed here in relation to the integrin family.

Materials and methods

Chemicals, reagents and culture materialsThese were obtained as follows: Dulbecco's modified Eagle'smedium (DMEM) from Kyokuto Pharmaceutical IndustrialCo., Ltd. (Tokyo); fetal bovine serum (FBS) from JapanBiotest Laboratory (Tokyo); ethylenediaminetetraacetic acid(EDTA) and Af-(2-hydroxyethyl)-piperazine-W-2-ethanesul-fonic acid (Hepes) from Dojin Chemical Institute (Kuma-moto, Japan); streptomycin and penicillin from Meiji SeikaCo. (Tokyo); trypsin from Difco (Detroit); Falcon culturedishes from Becton Dickinson Labware (Oxnard, CA);Millipore niters and nitrocellulose papers from MilliporeJapan (Yonezawa, Japan); Centricon-10 from W.R. Graceand Co. (Danvers, MA); human plasma fibronectin (pFN)from Hoechst Japan (Tokyo); concanavalin A (ConA), lentilseed (Lens culinaris) agglutinin (LCA), mushroom (Agaricusbisporus) agglutinin (ABA), pea (Pisum sativum) agglutinin(PSA), peanut agglutinin (PNA), Phaseolus vulgaris ag-glutinin (PHA), PHA-E4, PHA-L4, pokeweed mitogen(PWM), Ricinus communis agglutinin-60 (RCA), soybeanagglutinin (SBA), wheat germ agglutinin (WGA), PHA-E4-agarose, and peroxidase-PHA-E4 from Seikagaku Kogyo Co.Ltd. (Tokyo); iV-acetyl-D-galactosamine (GalNAc) andNonidet P-40 (NP40) from Sigma Chemical Co. (St. Louis.,MO); Pepstatin A from Peptide Institute Inc. (Osaka);tunicamycin, monensin (sodium salt) and Silver Stain KitWako from Wako Pure Chemical Industries (Osaka). Col-lagen (Type I) from Koken Co., Ltd. (Tokyo), which wasprepared from calf dermis by digestion with pepsin in a diluteHC1 solution and characterized as described previously(Yoshizato et al. 1985a). All other chemicals were of reagentgrade and purchased from Nacalai Tesque, Inc. (Kyoto) orWako Pure Chemical Industries.

Cell cultureHuman skin fibroblasts were obtained from explants of thenormal dermis and cultured as described previously (Yoshi-zato et al. 1981; Asaga et al. 1991). Fibroblasts were grownand maintained in 75 cm2 plastic dishes in DMEM containing10% FBS, 10 mM NaHCO3, 20 mM Hepes, 100 i.u./mlpenicillin and 100 jug/ml streptomycin in a moist atmosphereof 5% CO2/95% air at 37°C. The population doubling level ofcells used in the present study was within 18. Cells weredetached from dishes with calcium- and magnesium-freeHanks' solution (CMF-Hanks') containing 0.1% trypsin and 1mM EDTA, collected by centrifugation at 500 g for 5 min andwere used for experiments after washing twice with Hanks'solution.

Collagen gel culture of fibroblastsFibroblasts were populated three-dimensionally in hydratedcollagen gels by the method of Elsdale and Bard (1972) withslight modifications (Asaga et al. 1991). The following stocksolutions were prepared and kept at 4°C: 0.5% (w/v) collagensolution; 4x concentrated DMEM, which contains 80 mMHepes, 40 mM NaHCO3, 0.4 mg/ml streptomycin and 400i.u./ml penicillin. Fibroblasts were harvested from monolayercultures by treating with 0.1% trypsin and 1 mM EDTA inCMF-Hanks', counted, adjusted to the desired cell number

and collected by centrifugation in a plastic tube. The tube wasplaced on ice, and the cell pellet was resuspended in DMEMcontaining 0.1% collagen, 20 mM Hepes, 10 mM NaHCO3,0.1 mg/ml streptomycin and 10% FBS, which had beenprepared by quickly mixing the stock solutions and redistilledwater. Then pH was adjusted to 7.4 by adding 1 M NaOH.One milliliter of medium containing 2 x 105 fibroblasts wasinoculated in 25 mm bacteriological dishes and cultured at37°C. Collagen had gelled within 10 min and cells wereembedded three-dimensionally in gels. When necessary,lectin, tunicamycin or monensin was introduced into gelculture. The extent of contraction was quantified duringculture by measuring the diameter of collagen gels.

Spreading assay of fibroblastsCollagen-fibril-coated (100 /jg/cm2) plastic dishes were pre-pared according to Yoshizato et al. (1985a, 1988). Culturemedium was added to collagen-coated dishes and the disheswere kept at 37°C to polymerize collagen molecules into fibrilsprior to use.

Gelatin-coated (100 ̂ g/cm2) plastic dishes were prepared asfollows. Collagen solution (0.5%, w/v) was treated for 5 minat 100°C, poured into dishes, and dried at 60°C.

Fibroblasts harvested from monolayer cultures were sus-pended in DMEM containing 10% FBS at a concentration of2 x 104 cells/ml, inoculated on dishes to give a density of 105

cells/cm2, and cultured. Cells were allowed to attach for about30 min, and then medium was replaced with DMEMcontaining 10% FBS and lectins. Morphology of fibroblastswas observed by phase-contrast microscopy (Nikon, TMD)and recorded by taking photomicrographs.

Dot blot analysisIn order to examine whether PHA-E4 binds collagen and FNor not, the dot blot analysis was performed as follows. Onemicroliter of a solution of collagen (1 mg/ml) or human pFN(1 mg/ml) was dotted to nitrocellulose papers and dried atroom temperature. The papers were soaked in 2% bovineserum albumin for 1 h to block any remaining protein bindingsites, incubated for 1 or 2 h with PBS containing 10 /̂ g/mlperoxidase-PHA-E4, washed three times with PBS, andincubated in PBS containing 0.05% H2O2 and 0.5 mg/ml 4-chloro-1-naphthol.

Isolation of glycoproteins recognized by PHA-E4Fibroblasts (5 x 107) were harvested from culture dishes withrubber policemen, washed three times with PBS, andhomogenized with 5 ml of PBS containing 1 mM phenyl-methylsulfonyl fluoride (PMSF), 2 mM JV-ethylmaleimide(NEM) and 1 ^g/ml pepstatin A at 4°C. The homogenate wascentrifuged at 1,000 g for 10 min, and the supernatant wascentrifuged successively at 12,000 g for 20 min, and at 105,000g for 1 h. The pellet was washed once with 5 ml of thehomogenization medium described above and was homogen-ized in 0.5 ml of PBS containing 2% NP40, 1 mM PMSF, 2mM NEM and 1 j/g/ml pepstatin A, pH 8.0. The homogenatewas diluted three-fold with the same buffer solution and wascentrifuged at 12,000 g for 20 min. Affinity separation ofPHA-E4-reactive glycoproteins was performed according toFleischmann et al. (1985). The supernatant (membranefraction) was loaded onto a column (5 mm in diameter) ofPHA-E4-agarose (2 ml), which had been equilibrated withPBS (pH 8.0) containing 0.5% NP40, and was allowed tostand at 4°C for 12 h. The unbound fraction was collected. Thecolumn was washed with 20 ml of PBS (pH 8.0) containing0.5% NP40 and was eluted with PBS (pH 8.0) containing0.5% NP40, 0.2 mM K2B4O7 and 200 mM GalNAc. Both

Cell to collagen interactions via glycoprotein 627

unbound and bound fractions were concentrated by ultrafil-tration using Centricon-10 and were subjected to electrophor-etic analysis.

ElectrophoresisPolyacrylamide gel electrophoresis in the presence of sodiumdodecyl sulfate (SDS-PAGE) was performed by the methodof Laemmli (1970) with modifications described previously(Asaga et al. 1991). Samples to be analyzed were given SDSand 2-mercaptoethanol at a final concentration of 6% (w/v)and 5% (v/v), respectively, treated for 5min at 100°C, andwere subjected to SDS-PAGE. SDS-PAGE was carried outwith a stacking gel of 3% acrylamide and a separation gel of7.5% acrylamide. Lower gel buffer (0.4% SDS in 1.5 M Tris-HC1, pH 8.8) was used instead of upper gel buffer (0.4% SDSin 0.5 M Tris-HCl, pH 6.8) for stacking gels. Afterelectrophoresis, polypeptides in gels were stained with highlysensitive silver staining (Oakley et al. 1980; Morissey, 1981)using Silver Stain Kit Wako.

Results

Inhibition of fibroblast-mediated collagen gelcontraction by lectinsFibroblasts were cultured in collagen gels in thepresence of ConA, WGA, RCA, PHA, PSA, LCA,SBA, ABA, PNA or PWM at various concentrations(10, 20 or 50 ^g/ml). Lectins of ConA, WGA, RCA,PHA, PSA and LCA inhibited fibroblast-mediatedcollagen gel contraction in a concentration-dependentmanner, while other lectins did not affect the gelcontraction (Fig. 1). Fibroblasts cultured in collagengels spread three-dimensionally prior to gel contrac-tion. However, when the gel contraction was inhibitedby lectins, most fibroblasts remained round in shape(data not shown). The effect of lectins of ConA, WGA,PHA, PSA and LCA does not seem to be due tocytotoxicity, because viability of fibroblasts cultured onplain plastic was not affected by these lectins at aconcentration of 100 ^g/ml (data not shown). RCAshowed a strong toxicity for fibroblasts.

Lectins did not need to be present throughout cultureto show their effects on gel contraction. Fibroblastswere harvested by treating them with trypsin andEDTA, preincubated in Hanks' solution containing 100jig/ml of a lectin (ConA, PHA, WGA, LCA or PSA) at4°C for 30 min, washed three times with Hanks' solutionto remove free lectin molecules and then'introducedinto collagen gels that did not contain lectins. As shownin Fig. 2, all of the lectins tested significantly delayed gelcontraction.

PHA affects spreading of fibroblasts on collagen-fibrils but not on plastic or gelatinWe performed a spreading assay of fibroblasts on plainor collagen-fibril-coated (100 //g/cm3) plastics. Lectinsof ConA, WGA, LCA and PSA inhibited cell spreadingon plastic (Fig. 3). These lectins also inhibited spread-ing on collagen fibrils and on gelatin-coated dishes (datanot shown). The effect of PHA was noteworthy,because this lectin inhibited spreading only on collagen-fibrils, not on plastic (Figs 3, 4). In the experiment

shown in Fig. 4, cells were cultured on plastic, collagen-fibrils or within a collagen gel in the presence of PHA ata higher concentration (100 /zg/ml). PHA did not showany effect on fibroblast spreading on plain plastic, butinhibited the spreading on collagen fibrils or within acollagen gel. PHA appears to recognize cell surfaceglycochains that are specifically involved in the interac-tion with collagen fibrils. We examined also the effect ofPHA on fibroblast spreading on gelatin-coated plastic.PHA showed no effect on the spreading on gelatin as inthe case of plastic (data not shown), indicating that themode of cell binding to gelatin is different from that tonative collagen.

Tunicamycin or monensin inhibits gel contractionWe introduced into collagen gel cultures of fibroblaststunicamycin, which inhibits glycochain synthesis ofglycoprotein (Alonso-Caplen and Compans, 1983), ormonensin, which inhibits glycoprotein secretion (Tar-takoff, 1983) (Fig. 5). These antibiotics dose-depen-dently inhibited gel contraction. Gels containing eachinhibitor were rinsed three times with inhibitor-freemedium to remove unbound inhibitors and werecultured again in an inhibitor-free condition. The gelsstarted to contract, indicating that the effect of theseantibiotics is reversible and, therefore, that the drugsare not cytotoxic. These antibiotics could produce theirinhibitory effects at any time during contraction (datanot shown). This contrasts with the effect of lectins;lectins added after gels had started to contract did notinhibit the contraction.

PHA-E4 is more effective than PHA-L4The PHA molecule is a tetramer composed of two typesof subunits, called E and L, both of which have thesame molecular weights but differ in ability of glyco-chain recognition (Leavitt et al. 1977). PHA is amixture of five forms of molecules, which are namedE4, E3L1, E2L2, E1L3 and L4, respectively. In order toknow which kind of glycochain plays a key role in cell tocollagen binding we introduced PHA-E4 and PHA-L4into the collagen gel culture of fibroblasts and com-pared their effects on gel contraction. Both types oflectins dose-dependently inhibited gel contraction, theeffect of PHA-E4 being more intense (Fig. 6).

Affinity analysis of PHA-E4 receptorsIt has been shown that cFN is involved in fibroblast-mediated collagen gel contraction (Asaga et al. 1991).PHA-E4 did not bind glycochains of either collagen orfibronectin, which was shown by dot blot analysis usingperoxidase-PHA-E4 (data not shown).

To understand the chemical nature of glycoproteinsrecognized by PHA-E4, the membrane fraction offibroblasts was obtained as an extract with NP40, andsubjected to affinity chromatography with a PHA-E4-agarose column (Fig. 7). Five bands were obtained inthe eluate (Fig. 7, lane 4). When PHA-E4 was mixedwith membrane fraction, precipitates were formed. Thesame banding pattern of proteins was obtained whenthe precipitates were subjected to SDS-PAGE (data not

628 H. Asaga and K. Yoshizato

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Fig. 1. Effect of lectins onfibroblast-mediated collagengel contraction. Fibroblasts (2x 105) were cultured in 1 ml of0.1% collagen gel containingDMEM, 10% FBS and lectinsat concentrations indicated.(•) Without lectins, (T) with10 ug/ml, (•) 20 /ig/ml, (A) 50^g/ml. Each value representsthe average of twodeterminations, whose rangewas less than 5% of theaverage.

shown). The Mr values of three of five bands were 130 x103, 150 x 103 and 180 x 103, respectively. Mr values ofthe other two were larger than 230 x 10 . The 130 kDaprotein could be /3rintegrin, because this protein wasreported to be involved in the fibroblast-mediatedcollagen gel contraction (Gullberg et al. 1990a).

Discussion

The present study clearly demonstrates that PHAspecifically inhibits cell to collagen interactions shownby inhibition of the spreading of fibroblasts on collagen

fibrils or in collagen gels and fibroblast-mediatedcollagen gel contraction. PHA-E4, an isolectin of PHA,shows a relatively strong inhibition of collagen gelcontraction.

The interaction of cells with ECM, or artificialsubstrata, such as plastic, has been extensively studied.Kundsen et al. (1981) reported that a 140 kDamembrane glycoprotein is involved in cell to substratumadhesion. Lehto and his associates showed that a 140kDa glycoprotein of plasma membranes is involved incell spreading (Lehto, 1983; Virtanen et al. 1982).Oppenheimer-Marks and Grinnell (1981, 1982, 1984)showed that WGA inhibits FN receptor (FNR) func-

Cell to collagen interactions via glycoprotein 629

5 10 15 20 25

Hours In Culture

Fig. 2. Delay of gel contraction by ConA, PHA, WGA,LCA and PSA. (A) Effect of ConA and PHA. (B) Effectof WGA, LCA and PSA. Fibroblasts (2 x 105) weresuspended in 1 ml of Hanks' solution containing 100 ^g/mlof a lectin, preincubated at 4°C for 30 min, washedextensively with Hanks' solution, and embedded in 1 ml of0.1% collagen gel containing DMEM and 10% FBS.(•) Without lectin-treatment, (V) with ConA, (O) PHA,(A) WGA, (•) LCA and (O) PSA. Each point representsthe average of two determinations, the range of which wasless than 6% of the average.

tion, and that the antibodies against the membraneglycoproteins recognized by WGA also inhibit thespreading of cells on pFN-coated substrata. WGA wasused to identify or purify the glycoprotein in all of thesestudies, but PHA has not been a target of study.

The cell surface receptors for various ECM com-ponents were identified and characterized using affinitygels conjugated with ECM or monoclonal antibodiesagainst cell surface proteins (Wayner and Carter, 1987;Pytela et al. 1985a, 1985b; Horwitz et al. 1985; Akiyamaet al. 1986; Wayner et al. 1988; Kramer and Marks,1989; Gullberg et al. 1989,1990b). The majority of thesereceptors have structural relativity with one another.Therefore, these receptors are generally called "in-tegrin(s)" (Buck et al. 1986; Hynes, 1987). All integrins

Fig. 3. Effect of ConA, WGA,LCA, PSA and PHA onspreading of fibroblasts onplain plastic. Fibroblastssuspended in DMEMcontaining 10% FBS wereseeded at 105 cells/cm2 onplastic culture dishes andcultured for 30 min. Mediawere replaced with DMEMcontaining 50 Mg/ml lectins andthe culture was continued foradditional 6 h. (A) ConA, (B)WGA, (C) LCA, (D) PSA,(E) PHA, (F) without lectins.Photomicrographs were takenat the end of culture. Bar, 100

630 H. Asaga and K. Yoshizato

Fig. 4. Effect of PHA on the morphology of fibroblasts cultured on three different substrata. Fibroblasts were cultured for30 min on plain plastic dishes (A and D), collagen-fibril-coated (100 ^g/cm2) dishes (B and E), or within collagen gels (Cand F). Fibroblast were seeded at 105 cells/cm2 in experiments of A, B, D and E. Fibroblasts (2 x 105) were cultured in 1ml of 0.1% collagen gels in the experiments of C and F. Media were replaced with the fresh ones containing 100 ^g/mlPHA and the culture was continued for additional 7 h. Photomicrographs were taken at the end of culture. (A through C)in the presence of PHA; (D through F) without PHA. Bar, 100 /xm.

are plasma membrane-bound complexes with a and /3subunits noncovalently associated in a heterodimericstructure. These subunits showed apparent Mr values ofabout 110-180 (xlO3) by SDS-PAGE.

The significance of glycosylation of FNR was recentlystudied by Akiyama et al. (1989), who showed that theprocessing of oligosaccharide to a mature form of FNRis not important for subunits assembly and insertioninto the plasma membrane, but is important for its FN-binding function. However, to our knowledge, thefunctional significance of the glycochain of receptors inbinding has not been reported for other ECM com-ponents including collagen.

The results of the present study suggest the possibilitythat PHA-E4 recognizes the glycochain of collagenreceptor, and that the glycochain plays important rolesfor binding collagen fibril. Other lectins, ConA, WGA,LCA and PSA, may also recognize specific glycochains

ou

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Fig. 5. Effect of tunicamycin and monensin on fibroblast-mediated collagen gel contraction. Fibroblasts (2 x 105)were cultured in 1 ml of 0.1% collagen gel containingDMEM and 10% FBS. Tunicamycin (T) or monensin (M)was introduced into the culture medium at 4 ^g/n l̂ (A), 2jig/ml ( • ) , or 1 jig/ml (T). Control experiment (•) wasperformed without inhibitors. The arrows indicate thetimes when inhibitors were removed. Each point representsthe average of two determinations, the range of which waswithin 5% of the average.

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Fig. 6. Inhibition of contraction of collagen gel by PHA-E4and PHA-L4. Fibroblasts (2 x 10s) were cultured in 1 mlof 0.1% collagen gel containing DMEM, 10% FBS andPHA-E4 (E) or PHA-L4 (L) at the concentrationsindicated. (•) Without lectins, ( • ) with 10 fig/m\, (•) 20/ig/ml, (A) 50 ng/m\. Each point represents the average oftwo determinations, the range of which was less than 8%of the average.

involved in binding to collagen. However, these lectinsalso recognize other glycochains that are not involved inbinding to collagen, because their inhibition of fibro-blast-spreading seems nonspecific. There is a possibilitythat WGA inhibited FNR function in the experimentsdescribed in the present paper, because it was demon-strated that WGA inhibits FNR function (Oppen-heimer-Marks and Grinnell, 1981) and fibroblast-mediated collagen gel contraction requires cFN (Asagaet al. 1991).

The inhibition of cell spreading or gel contraction byConA, LCA and PSA may be an indirect effect, since itwas suggested that ConA indirectly inhibits cell spread-ing by modulating the cytoskeleton or preventingreceptor redistribution (Oppenheimer-Marks and Grin-nell, 1981).

The glycochain structure recognized by PHA-E4 ischaracterized in detail. High-affinity binding to PHA-E4-agarose occurs only with biantennary glycopeptidescontaining two outer galactose residues and a residue ofN-acetylglucosamine-linked /S(l,4)-linked mannoseresidue in the core (Cummings and Kornfeld, 1982).Only Asn-linked oligosaccharides interact strongly withPHA-E4 (Kornfeld and Kornfeld, 1970). In the present

pFN

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Fig. 7. SDS-PAGE analysis of the glycoproteins recognizedby PHA-E4. Proteins of fibroblasts were subjected to SDS-PAGE and were visualized by silver staining. Lane 1,3 figof whole proteins; lane 2, 3 fig of proteins of themembrane fraction; lane 3, 3 jig of proteins passed throughan affinity column of PHA-E4-agarose; lane 4, 0.5 fig ofglycoproteins eluted from the affinity column. Filledarrowheads in lane 4 indicate the position of the bandsobserved. An arrow in the lane on the right of the gelindicates the band of human pFN.

study, it was also demonstrated that tunicamycin, aninhibitor of synthesis of Asn-linked glycochain (Alonso-Caplen and Compans, 1983), suppresses fibroblast-mediated collagen gel contraction. Therefore, it may bereasonably suggested that Asn-linked glycochains playa critical role in fibroblast-mediated collagen gelcontraction.

The SDS-PAGE pattern of membrane glycoproteinsrecognized by PHA-E4 revealed five bands. Three ofthem have apparent Mr values of 130, 150 and 180(xlO3), respectively. The chemical nature of theseproteins has not been described further. However, the130 kDa band appears to be ft-integrin, becauseGullberg et al. (1990a) suggested the involvement of130 kDa ft-integrin in fibroblast-mediated collagen gelcontraction. Therefore, there is a possibility that PHA-E4 recognizes and blocks glycochains of collagenreceptors of integrin family. MT values of the other twobands were larger than 230 x 103, suggesting that PHA-E4 might block glycochains of unknown collagenreceptors.

We proposed that collagen gel contraction byfibroblasts requires cFN but not pFN, in the previousstudy (Asaga et al. 1991). It, therefore, appears thatthere are at least two binding mechanism betweenfibroblasts and collagen in the process of collagen gelcontraction, indirect binding via cFN and direct bindingvia the glycoprotein recognized by PHA-E4. Detailedcharacterization of the glycoprotein described in thepresent paper will be the subject of further study.

632 H. Asaga and K. Yoshizato

This work was supported in part by a grant-in-aid for specialproject research from the Ministry of Education, Science andCulture of Japan (01870034) to K.Y. and H.A. is the recipientof postdoctoral fellowship from the Japan Society forPromotion of Science.

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(Received 3 October 1991 - Accepted 18 November 1991)