Immunochemical characterization of a 150 000 dalton human fibroblast surface glycoprotein

Biochimica et Biophysica Acta, 667 (1981) 1--14

© Elsevier/North-Holland Biomedical Press

BBA 38584

IMMUNOCHEMICAL CHARACTERIZATION OF A 150 000 DALTON HUMAN FIBROBLAST SURFACE GLYCOPROTEIN

JOS VERLINDEN, FRED VAN LEUVEN, JEAN-JACQUES CASSIMAN and HERMAN VAN DEN BERGHE Division of Human Genetics, Department of Human Biology, University of Leuven, Minderbroedersstraat 12, B-3000 Leuven (Belgium)

(Received June 23rd, 1980) Key words: Glycoprotein; Crossed immunoelectrophoresis; Papain treatment; (Human fibroblast)

Summary The characterization of a human fibroblast surface glycoprotein, visualized by crossed immunoelectrophoresis using rabbit antibodies against whole fibroblasts, is described. The antigen is synthesized by fibroblasts in culture and was localized both intracellularly and at the cell surface. It was highly antigenic and was detected only in h u m a n cells o f mesenchymal origin. The glycoprotein occurred in two different forms with a2 and /3 electrophoretic mobility. The slow migrating amphiphilic fi form was localized at the cell surface and showed a single protein band with an apparent molecular weight of 1 5 0 0 0 0 in SDS-polyacrylamide gel electrophoresis. By external papain t r e a t m e n t of intact viable cells, a water-soluble molecule was released with a reduced molecular weight (140 000) and an increased electrophoretic mobility as compared to the native membrane component. This hydrophilic form was also present intracellularly in fibroblasts not treated with exogeneous proteases. The observation that the detergent-solubilized/3 form was irreversibly converted to a more anodic form by incubation of whole cell extract at acidic pH, suggested that the intracellular protein represented a lysosomal degradation product of native internalized fibroblast surface glycoprotein. Introduction Interactions of cells with each other and with their environment are likely to be mediated by specialized, externally oriented molecules. In contrast to periphAbbreviations: Tes, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; PPO, 2,5-diphenyloxazole.

eral blood cells, whose membrane architecture has been extensively described, little is known about the structure, function and genetic control of surface molecules of cultured cells. In previous reports, we have described the application of crossed immunoelectrophoresis in the characterization of cellular antigens of cultured fibroblasts [1,2]. A polyspecific antiserum, produced by immunization of rabbits with whole human fibroblasts, contained specificities against at least 15 different cellular antigens. Several of these cellular proteins were localized at the outer cell surface since they were accessible to lactoperoxidase-catalyzed iodination of fibroblast cell layers. A major antigen, visualized with the antiserum, appeared in crossed immunoelectrophoresis as an immunoprecipitate composed of two completely fusing peaks, with respectively a2 and ~ mobility. This polymorphic antigen was the first protein against which detectable amounts of antibody were produced by the rabbits. Investigation of different cell lines and tissues demonstrated that the expression of the antigen was variable, depending on the origin of the cells. In the present report, the biochemical and biological characterization of this cellular protein will be described with particular reference to its polymorphic appearance in crossed immunoelectrophoresis. Materials and Methods Cell lines. Normal human fibroblasts were obtained from skin biopsies of the right forearm of healthy adult donors. Lung fibroblasts were derived from lung biopsies of two spontaneously aborted fetuses, aged 19 and 22 weeks. Amniotic fluid cells were grown from amniotic fluid obtained by amniocentesis between the 15th and 20th week of pregnancy. Endothelial cells were obtained from umbilical cord veins as described [3]. MG63 cells, derived from a human osteosarcoma, were a gift from H. Heremans [4]. Four human tumor-derived cell lines, CCL30 (squamous cell carcinoma), CCL98 (choriocarcinoma), CCL121 (fibrosarcoma) and CCL136 (rhabdomyosarcoma) were obtained from the American Type Culture Collection (Rockville, MD). Peripheral lymphocytes, erythrocytes and platelets were obtained from the Red Cross Blood Centre, Leuven. Cell culture. Diploid human fibroblasts, obtained from skin biopsies of healthy caryotyped volunteers, were cultured as previously described [1]. The medium consisted of Dulbecco's Modified Eagle's medium (Flow Co) containing 10% (v/v) heat-inactivated newborn calf serum, 1 g/1 NaHCO3, 15 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (Tes) and 15 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), buffered to pH 7.5 with 1 N NaOH. Between the 5th and the 15th passage (1 : 2 split ratio), a pool of cells of six donors was made, with an equal n u m b e r o f cells of each donor. This pool was cultured for another passage in medium containing newborn calf serum. After reaching confluency (about 2 . 1 0 6 cells per 75 cm 2 flask), the cells were either scraped from the plastic substratum or harvested by trypsinization as follows: the cell layers were washed once with 5 ml of a solution containing 0.02% EDTA in 2 mM Tris-buffered saline; 3 ml crystalline trypsin (0.05%, Sigma, 2X crystalline, in EDTA solution) was added to the cell

layer and incubation was continued for 5 min at 37°C. The collected cells were washed three times in Dulbecco's phosphate-buffered saline and stored as dry pellets at --200C until used. Radioactive labeling of cells. Cellular antigens were labeled either metabolically by endogenous incorporation of [ 3sS] methionine and [ 14C]fucose or exogenously by enzymatic iodination. Human fibroblasts, plated out at an initial density of 25 000 cells/cm :, were cultured in medium containing 10% serum for 24 h. The culture medium was removed and replaced by methionine-free medium containing 10% serum and 8 ~Ci/ml of [3SS]methionine (The Radiochemical Centre; 1240 Ci/mmol) for 24 h. Metabolic carbohydrate labeling was carried out by incubation of confluent fibroblast cell layers with serum-free medium supplemented with 1.2 ~zCi/ml of [14C]fucose (The Radiochemical Centre; 250 mCi/mmol) for 6 h. Lactoperoxidase-calatalyzed iodination with Na12SI (The Radiochemical Centre; 100 mCi/ml) was performed on confluent monolayer cultures as described [ 5]. Immunization procedures. Polyspecific antibodies against normal human fibroblasts (antifibroblast) were obtained by intradermal immunization of rabbits with whole scraped human fibroblasts as described [2]. A second antiserum was produced as follows. Whole scraped human fibroblasts, cultured during the last passage in medium containing rabbit serum instead of calf serum, were used as antigen. No homogenization by sonication or other means was done. Female rabbits of an inbred laboratory strain (Albino New Zealand) were injected intravenously with 2 • 106 cells during 10 consecutive days. 5 days after the last injection, the animals were bled by heart puncture. An antiserum predominantly directed against the fibroblast surface glycoprotein (anti-fibroblast surface glycoprotein) was prepared by injection of immunoprecipitated antigen. Crossed immunoelectrophoresis of fibroblasts extracted by sonication in acetate buffer (pH 5.5) containing 2% Berol (as described below), was performed with high concentrations of antigen and intravenously prepared anti-fibroblast antiserum in order to obtain immunoprecipitation peaks that were visible by dark-field illumination without staining of the gel. The agarose gel was then soaked for 48 h in saline and the fibroblast surface glycoprotein immunoprecipitate was carefully excised from the wet gel at positions that were free of other immunoprecipitates. The excised agarose pieces were homogenized in 0.5 ml saline, mixed with 0.5 ml complete Freund's adjuvant and irijected intradermally in different sites above the scapula. The rabbits were inoculated at 2-week intervals with an amount of antigen corresponding to 2 • 106 fibroblasts. 2 weeks after the fourth injection, the animals were bled by heart puncture. Further bleedings were obtained at 6-week intervals, while the animals received a booster dose of antigen 2 weeks before each bleeding. Gammaglobulins were prepared from the sera as described [2]. Crossed immunoelectrophoresis and autoradiography. The general procedures were as described [2]. Cell extracts were obtained by sonication of the cell pellet in electrophoresis buffer (Veronal 0.075 M, pH 8.6) containing 2% of

the nonionic detergent Berol EMU-043 (Berol Kemi AB, Sweden). Unsoluble material was removed b y centrifugation in a Beckman Airfuge at 130 000 × g for 60 min. After application of the samples, the first dimension was run at 10 V/cm for 90 min at 5°C. The second dimension was run, perpendicular to the first, at 4 V/cm and 15°C for 16 h. The gels were dried and stained with Coomassie Brilliant Blue in acetic acid/methanol/water (1 : 40 : 5) and destained in the same mixture. Charge-shift crossed immunoelectrophoresis was carried out as described, except that Berol was used as the nonionic detergent [6]. The affinity of the antigen for lectins was examined by mixing cell extracts with different lectins followed by crossed immunoelectrophoresis as described [7]. With ~4C-labeled cells, labeled peaks were visualized by scintillation autoradiography (fluorography). The procedure, described for polyacrylamide gels [8] was modified to give optimal results with agarose gels. Immediately after electrophoresis, nonprecipitated proteins were removed by soaking in water overnight, without pressing the gel. The gel was dehydrated by soaking in methanol for 2 h, with methanol changed after 1 h. The gel was then impregnated with PPO (2,5diphenyloxazole) b y soaking for 3 h in 5% (w/v) PPO in methanol. The gel was pressed, air-dried and exposed to LKB Ultrofilm at --70°C. With ass- and ~sIlabeled cells, labeled peaks were visualized by direct exposure of the stained gels to LKB Ultrofilm at --70°C. Exposed films were developed in a Kodak X-ray developer. Treatment o f cells with proteases. For papain-treatment, confluent cell layers were first washed with 5 ml of a solution containing 0.02% EDTA in 2 mM Tris-buffered saline. Papain (Papaya Latex, Sigma) was dissolved in phosphate-buffered saline containing 0.02% EDTA and 5 mM cysteine and activated by incubation at 37°C for 30 min. The washed cell layers were incubated with 3 ml 0.01% activated papain for 5 min at 37°C. The detached cells were collected by centrifugation (800 × g) and washed three times in phosphate-buffered saline. The cell-free supernatant and the first wash were concentrated under vacuum (Collodion thimbles, Schleicher and Schtill) after addition of 5 mg/ml iodoacetamide to inhibit papain activity. Treatment of fibroblast monolayers with 0.05% trypsin was carried out as described above (see cell culture). The cell-free supernatant and the first wash were pooled and concentrated under vacuum. Soybean trypsin inhibitor (Calbiochem, 0.8 mg/ml) was added to inhibit residual trypsin activity. Treatment of cells with neuraminidase. Neuraminidase-treatment was performed on both intact suspended cells and on cell homogenates. Whole fibroblasts, obtained by trypsinization of cell layers, were suspended in an isotonic sodium acetate solution (0.030 M) buffered to pH 5.5 with acetic acid containing 0.125 M NaC1 and 0.010 M CaC12 (1 • 106 cells/ml). After addition of 50 U/ml neuraminidase (Vibrio cholerae, Calbiochem-Behring Corp.) the cell suspension was incubated at 37°C for 60 min with regular agitation, washed three times in phosphate-buffered saline and analyzed by crossed immunoelectrophoresis. Cell homogenates were obtained b y sonication of trypsinized cells in pH 5.5 isotonic acetate buffer containing 2% Berol. The cell extract was incubated with 100 U/ml neuraminidase for 60 rain at 37°C and analyzed by crossed im-

munoelectrophoresis without further manipulations.

Immunoprecipitation and polyacrylamide gel electrophoresis. Because of the unavailability of completely monospecific antisera, the double-immunoprecipitation technique in solution was not suitable for the unambiguous determination of the polypeptide content and molecular weight of the antigen. Therefore, an alternative procedure has been followed as already described above (see immunization procedures) with minor modifications. After radioactive labeling of fibroblasts with [3SS]methionine or with 12sI by lactoperoxidase, crossed immunoelectrophoresis was carried out with anti-fibroblast surface antigen antiserum included in the immunogel. At the end of the electrophoresis, the gel was soaked for 48 h in saline. The individual immunoprecipitates were cut out from the wet gel, dissolved in sample buffer containing sodium dodecyl sulfate and 2-mercaptoethanol and boiled in water for 2 min. Electrophoresis was performed according to Laemmli [9] on 7% polyacrylamide slab gels with marker proteins in the peripheral slots. Autoradiographs were obtained by exposure of the dried gel to LKB Ultrofilm at --70°C. Results A representative example of the pattern obtained by crossed immunoelectrophoresis of human skin fibroblasts extracted with the nonionic detergent, Berol, is shown in Fig. 1A. The general characterization of the antigens visualized with the polyspecific antifibroblast antiserum, including their cellular localization, has been described [ 1,2 ] and will not be further discussed here. The immunoprecipitate close to the baseline, composed of two completely cross-reacting peaks with a2 and fi mobility, represents the antigen under investigation. Based on the mobility in agarose electrophoresis, the slow-migrating major peak was named fi form in contrast with the faster-migrating a form. Addition of Berol to the extraction buffer was necessary to obtain solubilization of the ~ form. The amphiphilic nature of the slow form was confirmed by charge-shift crossed immunoelectrophoresis; anionic (sodium deoxycholate) and cationic (cetyltrimethylammonium bromide) detergents had a strong influence on the electrophoretic mobility of the slow-migrating component of the polymorphic antigen, as compared to the nonionic detergent Berol (results not shown). The migration velocity of the a form, which was soluble without use of Berol, was not affected by the ionic detergents.

Surface localization Only the slow-migrating form was exposed to the outer cell surface as was demonstrated by external 12Si.labeling with lactoperoxidase in combination with crossed radioimmunoelectrophoresis (Fig. 1B). The antigen was not removed from the cell surface by trypsin treatment, since no difference in the amount of 12SI-label associated with the immunoprecipitate was observed between cells harvested by scraping or trypsinization. Incorporation of [3sS]methionine (Fig. 1C) and [14C]fucose (Fig. 1D) during metabolic labeling, excluded the possibility that the antigen was a serum component adsorbed to the cell surface from the culture medium.

6 A

C

D

Fig. 1. C r o s s e d i m m u n o e l e c t x o p h o r e s i s o f t r y p s i n i z e d f i b r o b l a s t s , e x t r a c t e d w i t h V e r o n a i b u f f e r ( p H 8 , 6 ) c o n t a i n i n g 2% Berol. T h e e q u i v a l e n t o f 8 " 1 0 5 cells w a s a p p l i e d o n t h e gel. T h e i m m u n o g e l c o n t a i n e d polyspecific anti-fibroblast antibodies (100 gg IgG/cm2). First dimension, 90 rain at 10 V]cm (anode to the left); second dimension, 16 h at 4 V/cm (anode to the top). The immunopreeipitates corresponding to t h e c~ a n d fl f o r m o f t h e s u r f a c e g l y c o p r o t e i n a r e i n d i c a t e d w i t h a n a r r o w . ( A ) P a t t e r n o b t a i n e d b y C o o m a s s i e B r i l l i a n t Blue s t a i n i n g . (B) F i b r o b l a s t s , l a b e l e d e x t e r n a l l y w i t h 1 2 5 I b y l a c t o p e r o x i d a s e - c a t a i y z e d i o d i n a t i o n ( a u t o r a d i o g r a m ) . (C) F i b r o b l a s t s l a b e l e d m e t a b o l i c a l l y w i t h [ 3 S S ] m e t h i o n i n e ( a u t o r a d i o g r a m ) . (D) F i b r o b l a s t s l a b e l e d m e t a b o l i c a l l y w i t h [ 14 C ] f u c o s e ( f l u o r o g r a m ) .

Production o f an antiserum predominantly directed against the fibroblast surface glycoprotein To facilitate the further biochemical and biological characterization of the polymorphic antigen, a more specific antiserum was prepared following a twostep procedure. The antigen was strongly immunogenic in rabbits since high titers of antib o d y were produced after only a few intradermal injections as opposed to other cellular antigens forming visible immunoprecipitates in crossed immunoelectrophoresis only after continued immunization of the same animals. This

observation led us to an alternative immunization procedure: rabbits were injected intravenously during 10 consecutive days with whole scraped human fibroblasts. Following this approach, an antiserum with limited specificity was obtained containing mainly antibodies against the fibroblast surface glycoprotein as well as antibodies towards three other antigens with fl mobility (results not shown). In a second step, the specificity of the antiserum was further increased by immunization with immunoprecipitated antigen, as described under Materials and Methods. Immunization with these immunoprecipitates resulted in an anti-

A

m

C

D

t Fig. 2. C r o s s e d i m m u n o e l e c t r o p h o r e s i s of cells (A, B) a n d callulax m a t e r i a l r e l e a s e d in the s u p e r n a t a n t (C, D ) o b t a i n e d b y d i s s o c i a t i o n o f f i b r o b l a s t m o n o l a y e r s w i t h 0 . 0 5 % t r y p s i n (A, C) or 0 . 0 1 % p a p a i n (B, D). T h e cens w e r e e x t r a c t e d w i t h V e r o n a ] b u f f e r ( p H 8 . 6 ) c o n t a i n i n g 2% Berol. T h e cellular m a t e r i a l r e l e a s e d b y p r o t e a s e t r e a t m e n t w a s c o n c e n t r a t e d and a d j u s t e d t o t h e s a m e v o l u m e as the c o r r e s p o n d i n g cell e x t r a c t . T h e e q u i v a l e n t o f 8 • 105 cells w a s a p p l i e d o n t h e gel, the i m m u n o g e l c o n t a i n i n g anti-flbroblast s u r f a c e g l y c o p z o t e i n a n t i b o d i e s ( 1 5 jug I g G / c m 2). E l e c t z o p h o r e t i c c o n d i t i o n s as in Fig. 1. T h e plates w e r e s t a i n e d w i t h C o o m a s s i e Brilliant Blue. T h e a r z o w i n d i c a t e s the m i g r a t i o n d i s t a n c e o f ~ 2 - m a c r o g l o b u l i n .

serum that visualized only the fibroblast surface glycoprotein in Coomassie Brilliant Blue-stained crossed immunoelectrophoresis (Fig. 2A) but which did still detect small amounts of two other antigens in crossed radioimmunoelectrophoresis o f 3SS-labeled cells. Treatment o f cells with papain The localization of the fl form at the cell surface, demonstrated by lactoperoxidase-catalyzed iodination, was illustrated further by its sensitivity to external papain digestion. Incubation of fibroblast cell layers with 0.01% papain resulted in a complete detachment of all cells from the plastic substratum within 5 min. Viability, measured by Trypan Blue exclusion, was greater than 80%. After papain dissociation, the a m o u n t of fl form associated with the cells was significantly decreased, whereas the a m o u n t of a form was not affected (Fig. 2B). The antigen removed from the cell surface by papain treatm e n t was recovered quantitatively in the cell-free supernatant. The amounts released were about 50-times greater than that released by trypsin dissociation (Fig. 2C and D). With both dissociation procedures, the solubilized antigen showed an increased electrophoretic mobility in crossed immunoelectrophoresis as compared to the Berol-solubilized form. Glycoprotein nature The interaction of the fibroblast surface glycoprotein with a number of lectins was investigated by mixing cell extract with the lectins before analysis in crossed immunoelectrophoresis. Significant changes in the precipitation pattern were observed with concanavalin A, phytohemagglutinin, Ricinus lectin and Robinia lectin. Wheat germ agglutinin and Dolichos lectin had no influence (results not shown). The glycoprotein nature of the polymorphic antigen was further corroborated by its ability to incorporate [14C]fucose, as was shown by fluorography of the crossed immunoelectrophoresis pattern of metabolically labeled cells (Fig. 1D). Glycoproteins containing sialic acid can be i d e n t i f e d by neuraminidase treatm e n t of the antigen followed by crossed immunoelectrophoresis [7]. Due to the removal o f the negatively charged neuraminic acid, the desialated glycoprotein will exhibit a cathodic shift in migration velocity compared to the native glycoprotein. Incubation of trypsinized whole fibroblasts with neuraminidase in isotonic acetate buffer (pH 5.5) resulted in a reduced electrophoretic mobility of the fl form (Fig. 3A and B). The fast-migrating c o m p o n e n t which is inaccessible to external iodination and papain digestion, showed an unaltered electrophoretic mobility after desialation o f whole cells. However, when the incubation was performed on cell homogenates, both forms were sensitive to neuraminidase. Trypsinized fbroblasts were extracted by sonication in pH 5.5 sodium acetate buffer containing 2% Berol and incubated for 60 min at 37°C with neuraminidase. The thus treated cells showed an immunoprecipitate in crossed immunoelectrophoresis with a reduced electrophoretic mobility as compared to the control experiment, indicating that both ~ and fl forms contained sialic acid (Fig. 3C and D). The control experiment (no addition of enzyme) revealed an interesting phe-

A

A

C

Fig. 3, C r o s s e d i m m u n o e l e c t r o p h o r e s i s o f t r y p s i n l z e d f i b r o b l a s t s . T h e e q u i v a l e n t o f 8 " 1 0 5 cells w a s a p p l i e d o n t h e gel, t h e i m m u n o g e l c o n t a i n i n g a n t i - f i b r o b l a s t s u r f a c e g l y c o p r o t e i n a n t i b o d i e s ( 1 5 / ~ g I g G / c m 2). E l e c t r o p h o r e t i c c o n d i t i o n s as i n Fig. 1. T h e p l a t e s w e r e s t a i n e d w i t h C o o m a s s i e B r i l l i a n t Blue. T h e arrow indicates the migration distance of ~2-macroglobulin. (A) Fibroblasts, extracted with Veronal b u f f e r ( p H 8 . 6 ) c o n t a i n i n g 2% Berol. (B) F i b r o b l a s t s , t r e a t e d w i t h 5 0 U / m l n e u x a m i n i d a s e f o r 6 0 m i n a t 3 7 ° C a n d s u b s e q u e n t l y e x t r a c t e d as s u b - A . (C) F i b r o b l a s t s , e x t r a c t e d w i t h a c e t a t e b u f f e r ( p H 5 . 5 ) c o n t a i n i n g 2% B e r o l a n d i n c u b a t e d f o r 6 0 r a i n a t 3 7 ~ C . ( D ) F i b r o b l a s t s , t r e a t e d as s u b - C a n d s u b s e q u e n t t y incubated with 100 U/ml nettraminidase for 60 rain at 37°C.

n o m e n o n : incubation of the cell extract at 37°C induced a quantitative shift of the ~ form to the anode, resulting in one peak with an area equal to the sum of the areas of the original two peaks. This peak started in the ~ region, although its average electrophoretic mobility was slightly slower than the peak of the form. The acid-induced conversion did only occur with acetate buffer of pH less than 6 and was irreversible, since titration of the extract to pH 8.6 with NaOH did n o t restore the original pattern. Activation of cell~ar proteases by

10 lowering the pH seemed to be responsible for the observed phenomenon, since no shift in migration velocity was observed when the cell extract was incubated at 4°C instead of 37°C (see also below). Molecular weigh t determination The polypeptide content and molecular weight of the polymorphic antigen was determined by SDS-polyacrylamide gel electrophoresis. Immunoprecipi1

220

2

3

~4

5

6

~i

165 155

135 -

S8

Fig. 4. S D S - p o l y a c r y l a m i d e gel c l e c t r o p h o r e s i s o f i m m u n o p r e c i p i t a t e d f i b r o b l a s t s u r f a c e g l y c o p r o t e i n ( a u t o r a d i o g r a m ) . I m m t t n o p r e c i p i t a t e s we're o b t a i n e d b y crossed i m m u n o e l e e t x o p h o r e s i s of [ 3 S S ] m e t h i o n i n e - l a b e l e d f i b r o b l a s t s . T h e e q u i v a l e n t o f 1 . 2 - 1 0 6 cells was a p p l i e d , t h e i m m u n o g e l c o n t a i n i n g antif i b r o b l a s t sttrface g l y c o p r o t e i n a n t i b o d i e s . T h e i m m u n o p r e c i p i t a t e s w e r e e x c i s e d a n d dissolved in s a m p l e b u f f e r c o n t a i n i n g SDS (2%), 2 - m e r c a p t o e t h a n o l (2%), g l y c e r o l ( 1 0 % ) a n d b r o m o p h e n o l blue as t r a c k i n g d y e . T h e s a m p l e s w e r e b o i l e d f o r 2 r a i n a n d e l e c t r o p h o r e s e d o n a 7% slab gel f o r 16 h at 50 V. T h e differe n t f o r m s o f t h e a n t i g e n w e r e p r e p a x e d as follows: ~ f o r m (lane 1) a n d fl f o r m (lanes 2 a n d 4) b y e x t r a c t i o n of f i b r o b l a s t s w i t h 2% Berol in V e r o n a l b u f f e r (pH 8.6); c~ f o ~ n b y e x t r a c t i o n of f i h r o b l a s t s w i t h 2% Berol in a c e t a t e b u f f e r ( p H 5.5) a n d i n c u b a t i o n for 60 m i n a t 3 7 ° C (lane 6); f i b r o b l a s t s u r f a c e g l y c o p r o tein s o l u b i l i z e d b y p a p a i n t r e a t m e n t o f w h o l e cells (lanes 3 a n d 5). T h e m o l e c u l a r w e i g h t m a r k e r s u s e d w e r e f i b r o n c c t i n ( 2 2 0 0 0 0 ) , R N A - p o l y m e r a s e ( 1 6 5 0 0 0 a n d 1 5 5 0 0 0 ) , fl-galactosidase ( 1 3 5 0 0 0 ) a n d bovine serum albumin (68 000).

11

tates of the different forms, obtained by crossed immunoelectrophoresis of [3SS]methionine-labeled fibroblasts, were excised from the unstained gel and applied in parallel slots of a 7% slab gel {Fig. 4). The slow-migrating fi form (lanes 2 and 4), extracted from fibroblasts with 2% Berol in Veronal buffer (pH 8.6), consisted of a single polypeptide chain with an M~ of 150 000. The papain-solubilized protein (lanes 3 and 5) showed one band migrating slightly faster than the fi form with Mr 140 000. The form, obtained by extraction of cells either at pH 8.6 (lane 1) or at pH 5.5 (lane 6), had an M~ intermediate between that of the amphiphilic ~ form and the papain-solubilized protein. The same molecular weights were obtained when the samples were not reduced with 2-mercaptoethanol. With cells labelled with 12sI by lactoperoxidase, the pattern was also unchanged. From these results, it appears that the shift to a higher electrophoretic mobility, as measured by agarose electrophoresis, is accompanied by a reduction in the Mr, the latter being most pronounced for antigen released by papain treatment of whole cells. In a control experiment, the 3SS-labeled fibroblast extract was analyzed by crossed immunoelectrophoresis following the same procedure, except that antihuman albumin (Dako) instead of anti-fibroblast surface glycoprotein antiserum was applied in the second dimension gel. Control samples, excised from this agarose gel at the positions of the polymorphic antigen, contained no radioactivity excluding the possibility that the 150 000 material was present at these positions even in the absence of anti-fibroblast surface glycoprotein antibodies.

Screening of cell lines Human cells of different origin were investigated by crossed immunoelectrophoresis for the presence of fibroblast surface glycoprotein. The results are summarized in Table I. Fibroblasts were the only tissue-derived cell type in TABLE

I

SCREENING PROTEIN

OF HUMAN

CELLS

FOR

THE PRESENCE

O r i g i n and t y p e o f the cells

Tissue~derived cells N o r m a l skin fibroblasts N o r m a l l u n g fibroblasts E n d o t h e l i a l cells f r o m u m b i l i c a l c o r d veins Cells derived f r o m a m n i o t i c fluid N-ormal f e t a l flbroblasts N o r m a l fetal epithelial cells N o r m a l b l o o d cells Peripheral l y m p h o c y t e s Erythrocytes Platelets T u m o r - d e r i v e d cell lines O s t e o s a r c o r n a - d e r i v e d cells S q u a m o u s cell c a r i n o m a - d e r i v e d cells C h o r i o c a r c i n o m a - d e r i v e d e n d o c r i n e cells F i b r o s a r c o m a - d e r i v e d cells E m b r y o n a l r h a b d o m y o s a r c o m a - d e r i v e d cells

OF THE

Code

FIBROBLAST

Presence of the antigen

+ + m +

F

MG63 CCL30 CCL9S CCL121 CCL136

SURFACE

+

+

GLYCO-

12 which the antigen could be demonstrated. There was no difference between fibroblasts from adult and fetal donors. Epithelial cells, endothelial cells and none of the different blood cells examined, did bear the polymorphic antigen. A few tumor-derived cell lines were investigated. MG63 (osteosarcoma) and CCL121 (fibrosarcoma), both of mesenchymal origin, had detectable amounts of the fibroblast surface glycoprotein. The three other CCL-cell lines, not derived from connective tissue mesenchyme, did not express the antigen. Fibroblasts of animal origin (mouse, rat, hamster, dog) showed no reactivity with the anti-fibroblast surface glycoprotein antiserum. Discussion

The present investigation describes the characterization of a human fibroblast surface glycoprotein using an immunochemical approach without prior purification of the antigen. The antigen was detected by the technique of crossed immunoelectrophoresis using a polyspecific antiserum prepared by intradermal immunization of rabbits with whole human skin fibroblasts. The capacity of the fibroblast surface glycoprotein to induce high titers of antibody within a short period of time was exploited to produce a nearly monospecific antiserum following a two-step procedure. The high antigenicity of the protein correlated well with the lack of immunological reaction of these antibodies with non-human fibroblasts. The polymorphic antigen was actively synthesized by fibroblasts in culture and was localized at the cell surface. The glycoprotein nature of the component was demonstrated by its affinity for different lectins, its capacity to incorporate [ 14C]fucose and the presence of sialic acid. These are known properties of the large external transformation sensitive glycoprotein (fibronectin). The insensitivity to trypsin and the difference in molecular weight, however, indicated that the fibroblast surface glycoprotein was not related to fibronectin. Finally, anti-fibronectin antibodies did not react with the antigen in crossed immunoelectrophoresis, but precipitated a protein of different mobility [2]. The fibroblast surface glycoprotein was also not sensitive to collagenase, suggesting its non-identity with collagenous proteins (results not shown). The amphiphilic nature, demonstrated by the affinity for detergents, indicated that the antigen is a membrane component penetrating the lipid bilayer. When solubilized by nonionic detergent extraction in alkaline buffer, the antigen appeared in crossed immunoelectrophoresis as a double immunoprecipitate consisting of two cross-reacting peaks with different electrophoretic mobilities. Only the major slow-migrating/3 form was localized at the cell surface as shown by its accessibility to 12SI-labeling by lactoperoxidase. By papain digestion of whole cells, it was released from the cell surface in a water-soluble form with a reduced molecular weight (140 000) and an increased electrophoretic mobility as compared to the native Berol-solubilized membrane protein (150 000). These results suggest that the glycoprotein is built up of a short hydrophobic peptide burried in the lipid bilayer and a much larger hydrophilic domain protruding outside the cell. Papain would split a peptide bound at the hydrophobic-hydrophilic junction thus cleaving off the hydrophilic part and probably leaving the hydrophobic part in the lipid core. Similar findings

13 were described for several other membrane proteins including the major sialic acid-rich glycoprotein of erythrocyte membranes (glycophorin) [10], the intestinal brush border enzyme aminopeptidase [11] and the human major histocompatibility complex [12]. The observation that the hydrophilic part is not degraded further into low molecular weight peptides by papain, indicates that it occurs in a highly folded globular shape. The removal of the detergentbinding hydrophobic peptide can explain the increased electrophoretic mobility of the papain-solubilized form. The water-soluble form of the fibroblast surface glycoprotein was also present intracellularly in fibroblasts not treated with exogenous proteases. This fast component of the typical double immunoprecipitate, with a molecular weight of 145 000, might be a lysosomal degradation product of the native internalized membrane component. This hypothesis is strengthened by the observation that the a form is secreted in low amounts by fibroblasts in the culture medium. Moreover, the Berol-solubilized form was irreversibly converted to the hydrophilic form by incubation of whole cell extract at acidic pH. Since lysosomal proteases are known to have their optimal activity at low pH, liberation of these enzymes during homogenization is probably responsible for the observed interconversion at pH 5.5. Only connective tissue cells and tumor cells derived from mesenchymal cells {sarcomas) had detectable amounts of the antigen. Although our screening of different cell types is not complete, the antigen seems to be a selective marker of cells derived from the mechanocyte (precursor of fibroblasts, chondroblasts and osteoblasts). Further study of various human tissues will confirm this. Recently, a human cell surface glycoprotein coded for by chromosome 7 has been described, using the technique of murine-human somatic cell hybridization [13]. This antigen, extracted from human cells with nonionic detergent, had a molecular weight of 165 000 and was present in all fibroblastic cell lines examined, but not in human peripheral blood lymphocytes. All these characteristics are shared by the fibroblast surface glycoprotein. However, not enough information about the chromosome 7 coded protein is available to determine whether these two surface glycoproteins are identical. The polymorphic antigen is one of the few fibroblast surface proteins which have been characterized. Since it can be extracted from the cell membrane in a form that retains antigenic activity, it is an excellent candidate for purification and further study of its functional significance.

Acknowledgements The expert technical assistance of Ms. M. Caems, L. Stas and M. Willems is gratefully acknowledged. This work was supported by Grant 3.0043.79 (Fonds voor Geneeskundig Wetenschappelijk Onderzoek) and by Research Fund OT/ VII/30 (K.U. Leuven). F. Van Leuven is a Post Doctoral Research Fellow of the American Cystic Fibrosis Foundation.

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