10 pg/ml heparin, 10 pg/ml chondroitin sulfate, and 50 mM sodium acetate, pH .... (heparitin-sulfate lyase, EC 184.108.40.206) (2 h, 37 "C) or heparinase (EC. 220.127.116.11) (2 h, ..... mAb S1; lanes a-c, electrophoresis under nonreducing conditions; lanes d-f ...
THEJOURNAL OF BIOLOGICAL CHEMISTRV 8 1987 by The American Society of Biological Chemists, Inc
Vol. 262, No. 2, Issue of January 15, pp. 854-859, 1987 Printed in U.S.A.
Heparan Sulfate Proteoglycans of Human Lung Fibroblasts STRUCTURALHETEROGENEITY CELL-ASSOCIATED FORMS*
OF THE CORE PROTEINS OF THE HYDROPHOBIC
(Received for publication, June 23, 1986)
Veerle Lories$, Hilde De Boeck, Guido David$, Jean-Jacques Cassiman, and Herman Van DenBerghe From the Center for Human Genetics, University of Leuven, Campus Gasthuisberg Onderwijs en Navorsing, Herestraut, B-3000 Leuven, Belgium
Heparan sulfate proteoglycans(HSPG) were solubi- tions for exercising these multiple functions, occurring both lized from human lung fibroblast monolayers with de- as cell-surface constituents (14, 15) and as integral compotergent. Presumptive membrane-associated formsdis- nents of the extracellular matrix (10, 11).The glycosaminoplaying hydrophobic properties were purified by gel glycan chains play a crucial role in the biological properties filtration on SepharoseCL-4B, by ion-exchange chro- of these proteoglycans, but the core proteins also have impormatography on Mono Q and by incorporation in lipid tant contributions. First, the structure of the core protein vesicles. The HSPG preparations were ’“1-iodinated may be an important determinant of the type of glycosamiand treated with heparitinase before sodium dodecyl noglycan chain that is incorporated in the proteoglycan (16). sulfate-polyacrylamide gel electrophoresis. Five radio-Furthermore, the core proteins of some of the HSPG which labeled proteins with apparent molecular weights of occur at the cell surface could supply a hydrophobic anchor 125,000, 90,000, 64,000, 48,000, and 35,000 were that links the proteoglycan to the membrane (14, 15, 17, 18). visualized by autoradiography. A sixth protein, iden- Other forms, present in the extracellular matrix of the cells, tified in nonreduced 1261-HSPGpreparations, appeared also appear to be retained in their positions by interactions as a non-HS chain-bearing M , 35,000 peptide which was disulfide-linked to an HS chain-bearing peptide of that involve the polypeptide rather than the polysaccharide similar size. This multiplicity of core proteins did not chains (19), which implies that these proteoglycans may be targeted to theirultimatedestinations through molecular seem to result from proteolysis during the heparitinase treatment itself, since some of the core proteins mi- addresses that arecontained within the core protein. Finally, the HSPG of the cell surface appear to be involved in transgratedindependentlyduringgelfiltrationbefore heparitinase digestion. Moreover, heparitinase diges- membrane interactions with the cytoskeleton (5, ZO),indicattion of ”‘1-HSPG purified by affinity chromatography ing that some functions of the core proteins may beregulatory. Some of the membrane-associated forms resemble or may on an immobilized monoclonal antibody yielded only the M , 64,000 protein. Alternative depolymerizations even be cell-surface receptors, and thus may be involved in of the HS chains by heparinase or HNOz also yielded the transduction of specific messages into the cell interior multiple protein bands. These results imply that het- (21). Substantial core protein structural heterogeneity seems erogeneity of the core protein moiety maybe a genuine to underlie this functional diversity. Yet, so far, none of the property of the hydrophobic HSPG of human lung fi- HSPG core proteins has been sufficiently characterized to broblasts. The occurrence of multiple integral mem- allow a precise evaluation of this heterogeneity or a determibrane HSPG forms may be relevant for the multiple nation of structure-function relationships in this moiety of functionsthathavebeenascribedtocell-surface the molecules. HSPG. In prior characterizations of the HSPG of human lung fibroblasts (18),we could discriminate three forms of HSPG on the basis of hydrodynamic size, hydrophobic character, and culture distribution: large HSPG which were recovered Heparan sulfate proteoglycans (HSPG)’ have been impli- from the extracellular matrix of the cells, smaller HSPG cated in a variety of cellular processes such as cell attachment which were secreted in the culture medium, and small HSPG and spreading (1-4), maintenance of cell shape (5, 6), growth which wereassociated with the cellular membranes. The latter control(7, 8), anticoagulation (9),ultrafiltration (lo), and had unique protease-sensitive hydrophobic properties and matrix assembly (11-13). The HSPG occupy strategic posi- could be incorporated in liposomes. These data confirmed and complemented results obtained previously byothers in human * This investigation was supported by Grant 3.0030.81 ofthe Fonds fibroblasts (21, 22) and other tissues (14, 15, 17). The coexvoor Geneeskundig Wetenschappelijk Onderzoek, Belgium and by United States Public Health Service Research Grant HL-31750 (to istence of hydrophobic and nonhydrophobic forms implied G. D.). The costs of publication of this article were defrayed in part that HSPG isolated from a single tissue were heterogeneous by the payment of page charges. This article must therefore be hereby in their peptide moiety. In the present report we describe the marked “advertisement” in accordance with 18 U.S.C. Section 1734 purification of the hydrophobic membrane HSPG from culsolely to indicate this fact. tured human lung fibroblasts and thepartial characterization $ Specialisatiebursaal of the Instituut tot aanmoediging van het of their core proteins. The results suggest that these memWetenschappelijk Onderzoek in Nijverheid en Landbouw, Belgium. brane proteoglycans themselves also occur in multiple forms.
5 Onderzoeksleider of the Nationaal Fonds voor Wetenschappelijk Onderzoek, Belgium. To whom correspondence should be addressed. The abbreviations used are: HSPG, heparan sulfate proteoglycan(s); HLF, human lung fibroblasts; GdnHC1, guanidine hydrochloride; SDS, sodium dodecyl sulfate; mAb S1, monoclonal antibody S1.
Cell Culture-Fetal human lung fibroblasts (HLF) were grown on plastic substrata in Dulbecco’s modified Eagle’s minimal essential
Proteoglycan Core Protein Heterogeneity
medium (Gibco) containing 10% (v/v) fetal calf serum (13). Confluent monolayers between passages 10 and 17 were incubated for 48 h with 5 pCiof H235S0, (carrier-free)/ml (New England Nuclear) in low sulfate culture medium (13). Buffers-Triton X-100 buffer contained 150 mM NaCl, 1%(v/v) Triton X-100, 10 mM Na,HPO,, 2 mM KHzP04, pH 7.4, 50 mM 6aminohexanoic acid, 10 mM EDTA, 5 mM N-ethylmaleimide, 5 mM benzamidine, 1 mM phenylmethanesulfonyl fluoride, and 1 pg/ml pepstatin A. Urea buffer consisted of 6 M urea, 0.5% (v/v) Triton X100, and 50 mM Tris-C1, pH 8.0. GdnHCl buffer was composed of 4 M GdnHCl, 100 mM 6-aminohexanoic acid, 10 mM EDTA, 10 mM Nethylmaleimide, 5 mM benzamidine, 50 pg/ml bovine serum albumin, 10 pg/ml heparin, 10 pg/ml chondroitin sulfate, and 50 mM sodium acetate, pH 5.8. Tris-Cl buffer contained 10 mM octyl glucoside and 100 mM Tris-CI, pH 7.5. Borate buffer was 50mM octyl glucoside and 100 mM borate, pH 8.5. Pyridine HCl buffer was 1 M pyridine HCl, pH 5.8, 6 M urea, 1 M GdnHC1, and 10% (v/v) Triton X-100. Heparinase buffer contained 50 mM Tris-C1, pH 7.0, 100 mM NaC1, 100 pg/ml bovine serum albumin, 50 mM 6-aminohexanoic acid, 2.5 pg/ml pepstatin A, 1mM phenylmethanesulfonyl fluoride, and 20 pg/ ml leupeptin. SDS buffer was composed of 1 mg/ml SDS, 350 mM NaC1, 1 mM EDTA, and 50 mM Tris-C1, pH 8.0. In all instances, the protease inhibitor phenylmethanesulfonyl fluoride was added to these buffers, from a concentrated fresh stock solution in isopropyl alcohol, a few seconds before use (23). Extraction of Cell-associated HSPG-After the incubation of HLF monolayers with "SOO:-, the medium was recovered from the culture flasks, the flasks were rinsed three times with 10 ml of cold phosphatebuffered saline, and the rinsed HLF monolayers were scraped from the substrate in Triton X-100 buffer (12 m1/150 cmz flask). The suspension was rapidly cooled to 4 "C and was cleared by centrifugation (10,000 X g; 60 min). The proteoglycans in this crude extract (usually -500 ml from 40 flasks) were concentrated by adsorption on a 5-ml DEAE-Sepharose Fast Flow (Pharmacia) column. Bound material was eluted with 5 volumes of urea buffer containing 750 mM NaCl. This eluate was filtered through a Minisart (Sartorius GmbH, Gottingen, West Germany) cellulose acetate membrane of0.20-pm pore size, diluted %fold in urea buffer and applied on a Mono Q column. After eluting the bulk of the bound proteins from the column with a 0-0.6 M linear NaCl gradient in urea buffer, the retained proteoglycans were eluted in a small volume (1.5-2 ml) of GdnHCl buffer containing 0.5% (v/v) Triton X-100. The "SO:- activity recovered at this stage was taken to represent 100% of the detergentextractable label present in the glycosaminoglycan. The hydrophobic cell-associated HSPG were further purified from this concentrated extractby gel filtration over Sepharose CL-4B, ionexchange chromatography on Mono Q, and incorporation of the HSPG into liposomes. Prior investigations have indicated that hydrophobic HSPG represent -15% of the total 35S-glycosaminoglycan and -30-40% of the total =S-heparan sulfate that can be extracted from the cells with detergent (18). Gel Filtration-Gel filtration over Sepharose CL-4B (Pharmacia) (1 X 100 cm) in GdnHCL buffer, in GdnHCl buffer containing 0.5% (v/v) TritonX-100, or in GdnHCl buffer without heparin, chondroitin sulfate, and bovine serum albumin was performed at 4 "C. The flow rate was 4 ml/h, and 1.6-ml fractions were collected. Gel filtration over Sepharose CL-4B (1 X 45 cm) in SDS buffer was performed a t room temperature. Before chromatography in SDS buffer, the samples were made 2% in SDS and were boiled for 5 min. The flow rate was 6 ml/h, and 0.8-ml fractions were collected. The V, and V, values of the columns were determined with blue dextran and 3H-glucosamine,respectively. Ion-exchange Chromatography-Samples in urea buffer were applied on a Mono Q column equilibrated in the same buffer. Bound materials were eluted by a linear NaCl gradient (from 0 to 1.2 M) in urea buffer, delivered by a fast protein liquid chromatography system (Pharmacia) ata flow rate of 30 ml/h, at room temperature. Incorporation of HSPG in Liposomes-After purification of the membrane HSPG by gelfiltration and ion-exchange chromatography, the samples were dialyzed against urea buffer and adsorbed on DEAETrisacryl M beads (LKB Instruments, Bromma, Sweden). After rinsing the beads with Tris-CI buffer, bound HSPG were eluted in GdnHCl buffer containing 75 mM octyl glucoside. Phosphatidylcholine was added to a concentrationof 5 mg/ml from a stock solution (25 mg/ml) in GdnHCl buffer containing 75 mM octyl glucoside. Liposomes were formed by dialyzing the HSPG-lipid mixture against GdnHCl buffer without detergent. lZ5I Iodination of the Core Proteins-Purified HSPG were dialyzed
against ureabuffer and adsorbed on a small volume ( 4 5 0 pl) DEAETrisacryl M beads. After careful rinsing with Tris-C1buffer to remove all Triton X-100, the pelleted beads were mixed with 10 pl (1 mCi) of Na1251 (Amersham Corp.) and 10 pl of chloramine T (1 mg/ml). After 5 min at room temperature the labeling was stopped by adding 100 plof K2SZ05(5 mM) and KI (10 mM). The DEAE suspension was extensively washed with Tris-C1 and with urea buffer to remove free label. The IZ5I-HSPGwere eluted from the DEAE beads in urea buffer containing 1 M NaCl. The presence of the DEAE matrix during the labeling procedure had no influence on the pattern of labeled bands obtained (not shown). Other HSPG samples were iodinated using the Bolton-Hunter reagent (Amersham).HSPG solubilized inborate buffer (40 pl) were added to dried labeling reagent, and the reaction mixture was agitated for 15 min a t 4 "C (24). Excess reagent was reacted with 0.2 M glycine in borate buffer (5 min at 4 "C) and was separated from the Iz5I-HSPGby adsorption of the latter on DEAE beads. To evaluate the labeling reactions, T - H S P G were again submitted to ion-exchange chromatography on Mono Q and gel filtration over Sepharose CL-4B, as described above. Finally, the fractions containing the Iz5I-HSPG were dialyzed against urea buffer and were adsorbed on a small volume of DEAE-Trisacryl M beads. Bound "'IHSPG were eluted in a small volume of 10-fold-concentrated heparinase buffer (without protease inhibitors) and were stored a t -20 "C until further use. Heparime andHeparitinase Digestion-After appropriate dilution and addition of protease inhibitors, the Iz5I-HSPGsamples in heparinase buffer were digested with 1 mIU (22 mIU/ml) heparitinase (heparitin-sulfate lyase, EC 18.104.22.168) (2 h, 37 "C) or heparinase (EC. 22.214.171.124) (2 h, 30 "C). Both enzymes were obtained from Miles Laboratories. Control samples were incubated in buffer in the absence of enzyme. Alternatively, and with similar results, the Iz5I-HSPGsamples were precipitated with 75% (v/v) ethanol in the presence of 50 pg/ml chondroitin sulfate, dissolved in heparinase buffer, and digested as described above. To test whether the heparitinase digestion was complete within 2 h (the time period for which it was normally performed), "'I-HSPG samples were treated with heparitinase for time periods ranging from 15 to 180 min. SDS-polyacrylamide gel electrophoresis and autoradiography of these treated samples showed that the reaction was complete already after 30 min. Moreover, the addition of fresh enzyme during the incubation did not affect the pattern of protein bands which was obtained (results not shown). Protease K Digestion-Heparitinase-digested '"I-HSPG were made 0.5% (w/v) in SDS and wereboiled for 5 min. Protease K digestion of the denatured samples was performed at 60 "C for 40 min at a concentration of 70 pg/ml protease K. A control sample was treated similarly, but in the absence of protease K. SDS-Polyacrylamide Gel Electrophoresis-SDS-polyacrylamide gel electrophoresis was performed on gradient (4-16% T, 10% C) polyacrylamide gels using the buffer system of Laemmli (25). [methylL4C]phosphorylase b (97 kDa), [methyl-14C]albumin (69 kDa), [methyl-14C]ovalbumin(46 kDa), and [methyl-"C]carbonic anhydrase (30 kDa) were used as molecular mass standards (New England Nuclear). Before electrophoresis, samples were made 2% (w/v) in SDS and boiled for 5 min. Samples run under reducing conditions were supplemented with 2% P-mercaptoethanol. After electrophoresis, the gels were stained with 0.2% Coomassie Brilliant Blue R-250 in 12.5% (w/v) trichloroacetic acid, 45% (v/v) ethanol, destained in ethanol/acetic acid/water (265:69), and dried under vacuum. Kodak XAR-5 film was used for autoradiography. N-Des~lfation-'*~I-HSPGwere applied to a small DEAE-Trisacry1 M column and were eluted in pyridine HCl buffer. After the addition of 19 volumes of dimethyl sulfoxide, the mixture was incubated at 37 "C for 4 h. Under these conditions, dimethyl sulfoxide selectively removes N-SOg- from the pyridinium salts of HS (26). The reaction was terminated by removing the dimethyl sulfoxide by dialysis. Reduction and Alkylation of Disulfide Bonds-HSPG samples were taken up in GdnHCl buffer without N-ethylmaleimide and buffered with 50 mM Tris-C1 at pH8.0. Reduction with dithiothreitol (10 mM, 4 h at room temperature) was followed byalkylation with iodoacetamide (40 mM, overnight at room temperature). Nitrous Acid Degrudation-1261-HSPG were treated with 0.4 M HNOZ,pH 1.5, for 10 min at room temperature. The reaction was stopped by the addition of excess ammonium sulfamate and neutral-
Heparan SulfateProteoglycan Core Protein Heterogeneity
3 1 Z 9,
FIG. 1. Purification of the membrane HSPG by gel filtration over Sepharose CL-4B. Metabolically 35SOi--labeled HLF monolayers were extracted with Triton X-100 in the presence of protease inhibitors. Proteoglycans in the extract were concentrated (cf. "Materials and Methods") and chromatographed on Sepharose CL4B in GdnHCl buffer containing0.5% (v/v) Triton X-100. Under these conditions, membrane HSPG elute with a Kavof -0.27 (18). FIG. 2. Purification of the membrane HSPG by ion-exchange chromatography on Mono Q. After gel filtration on Sepharose CL-4B,the fractions containing HSPGwere pooled as indicated by the bar in Fig. 1 and applied on Mono Q. Bound material was eluted with a linear NaCl gradient from 0 to 1.2 M NaCl in urea buffer. FIG. 3. Liposome incorporation of themembrane HSPG.After ion-exchange chromatography,the HSPGcontaining fractionswere pooled as indicatedby the bar in Fig. 2, transferred to GdnHCl buffer containing 75mM octyl glucoside, mixed with phosphatidylcholine (5 mg/ml), dialyzed against GdnHCl buffer without detergent, and chromatographed on Sepharose CL-4B in GdnHCl buffer to separate liposome-incorporated proteoglycans (indicated by bar) from nonincorporated forms. ization with NaOH. At pH 1.5 nitrous acid specifically cleaves HS chains at the site of N-SO$--hexosamine residues (27). Affinity Chromatography on Immobilized mAb SI-The monoclonal antibody (mAb)S1 is directed againstthe core protein moiety of some HSPG of HLF.' mAb S1 (15 mg), purified from ascites fluid, was immobilized on CNBr-activated Sepharose4B (1 g) in 100 mM NaHC03, pH 8.3, 500 mM NaCl. lZ5I-HSPGin Tris-C1 buffer were incubated withmAb S1-Sepharosebeads (overnight,4 "C) before and after digestionwithheparitinase.Unboundmaterials were eluted with 10 volumes of Tris-C1 buffer. S1-boundIz5I materials were eluted in GdnHCl buffer.
remove the lipid and the carrier material (i.e. heparin, chondroitin sulfate, andbovine serum albumin) from the purified hydrophobic HSPG, liposome-associated HSPG were solubilized with 0.5% (v/v) Triton X-100 and rechromatographed on Sepharose CL-4B in carrier-free GdnHCl buffer containing 0.5% Triton X-100 (not shown). This purified preparation contained 9.9 f 3.1% ( n = 7 ) of the total 35Sactivityin glycosaminoglycan originally extracted from the fibroblasts. Based on these results and onprevious investigations (18) it was calculated that, onaverage, -63% of the total hydrophobic HSPG were recovered. RESULTS These preparationsof HSPG were radiolabeled with N a ' T Isolation of the Hydrophobic Cell-associated HSPG Formsaccording tothechloramine-T procedure. The material Approximately 30% of the total 35S-HSPG present ina con- behaved likethe purified metabolically labeled"SOO," material fluent monolayer of cultured human lung fibroblasts appear when chromatographed on Sepharose CL-4B and on Mono to be membrane-anchored througha hydrophobic moiety (18). Q , and could be incorporated in liposomes (results not shown). T o isolate these components, rinsed HLF monolayers were To check whether all the '1 material was of HSPG nature, extractedwithTriton X-100in thepresence of protease a '"I sample was digested with heparinase in the presenceof inhibitors. This procedure preferentially solubilizes the HSPG protease inhibitors, either before or after N-desulfation, and which are associated with the plasma membrane and leaves subsequently chromatographed on Sepharose CL-4B in SDS the matrix proteoglycans behind (18). The concentrated exbuffer. WithoutN-desulfationthematerial was heparintract (cf. "Materials and Methods")was chromatographed on ase-sensitive. Indeed, compared toundigested samples (Kav= Sepharose CL-4B in the presence of detergent (Fig. 1).The 0.19-0.25), digested samples (Kav = 0.45-0.52) were more fractions containing HSPG (Kav 0.27) were pooled as inretarded (Fig. 4A).After N-desulfation with dimethylsulfoxdicted (bar in Fig. 1) and were submitted to ion-exchange chromatography on Mono Q (Fig. 2). HSPG eluted at-0.8 M ide, however, nearly all the lZ5I material resisted heparinase digestion (Fig. 4B), which confirmed that the effect of hepaNaCl and were separated from some contaminating chonwas indeed due to the degradation droitin sulfate proteoglycans eluting a t -0.9 M NaCl. The rinase on the native sample the label was associated with isolated HSPG (pooled as indicated in Fig. 2) were incorpo- of HS chains and, thus, that lz5I HSPG. (Heparinase cleaves HS chains at the site of glucosarated in liposomes and chromatographed on Sepharose CL4B in the absence of detergent (Fig. 3). Liposome-associated mine residues carrying both 0- and N-SOi- groups). Characterization of the Core Protein Moiety-To charactermaterial eluted in thevoid volume of the column. Finally, to ize the core protein moiety of the hydrophobic cell-associated H. De Boeck, V. Lories, G. David, and J.-J. Cassiman, manuscript HSPG, lz5I-HSPGwere treated with heparitinase. Undigested and treated samples were analyzed by SDS-polyacrylamide in preparation.
Heparan Sulfate Proteoglycan
Core Protein Heterogeneity
ditions. Heparitinase digestion yielded again five bands as described above for unreduced proteoglycans (Fig. 6, lane d). Heparinase (Fig. 6, lane b ) and HNO, (Fig. 6, lane a ) treatment gave rise to three diffuse and incompletely resolved bands with-on average-higher apparent molecular weights than after heparitinase treatment. Thus, alternative enzymatic and chemical degradations of the glycosaminoglycan moiety of the proteoglycans also yielded heterogeneous, yet different, core proteins preparations. The latter was not unexpected, since cleavage of the HS chains by heparitinase is not limited to N-sulfated glucosamine residues (as cleavage by HN02 and heparinase is) and also occurs at the site of N-acetylated residues. To test whether any chemical damage might be inflicted on the core protein moiety by the chloramine-Tlabeling, purified FIG.4. Heparinase digestion of '2611-membraneHSPG. "'1nonreduced membrane HSPG were "'I-iodinated with the labeled HSPG sampleswere submitted to heparinase digestion before Bolton-Hunter reagent and digested with heparitinase. The ( A ) andafter (H)selective N-desulfation. Digested (U and ) undigested (o"-o) samples were made 2% (w/v) in SDS, ))oiled, pattern of protein bands that appeared after autoradiography and chromatographed on SepharoseCL-4R in SDS buffer. from (Fig. 6, lane e ) was very similar to the one obtained preparations which were iodinated according to the chloraa b c d e mine-?' procedure (e.g.Fig. 5, lane c ) . Slight differences in the relative densities of the different bands could, however, be Mr x 10-3 observed. Such differences may be the consequence of differencesinthe relativelysine andtyrosinecontents of the different core protein forms. Origin of the Core Protein Heterogeneity-To investigate further the origin of the multiple protein bands which appearedafterheparitinase digestion of "'I-HSPG, the I2"I-97 HSPG were fractionated by affinity chromatography on immobilized anti-HSPG mAb S1 and by gel filtration on Seph-69 arose CL-4Bbefore heparitinase digestion. -46 mAb S1 recognizes an epitope situated on the core protein (or at least on the non-glycosaminoglycan) moietyof a HSPG -30 of HLF.' When "'I-HSPG were applied to immobilized mAb SI, andbound materials were heparitinase-digested after eluFIG. 5. Effect of disulfidebond reduction on '261-membrane tion, only a 64-kDa band showed on autoradiography (Fig. 7 , HSPG. "sI-membrane HSPC were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography before (lanes a and d ) and lanes b and e ) . When heparitinase-digested "'I-HSPG were after (lanes b and c ) heparitinase treatment. Lane e shows hepariti- applied, and thebound materials were eluted andanalyzed by nase-treated "'1-HSPG after digestion with protease K. Lunes a and SDS-polyacrylamide gel electrophoresis, again only the 64b, electrophoresis under non-reducing conditions; lanes c, d , and e, kDa protein band was observed (Fig. 7, lanes c and d ) . Reducelectrophoresis under reducing conditions. tion of disulfide bonds did not affect the migration of this 64kDa peptide (Fig. 7: compare lanes b and c to lanes d and e ) . gel electrophoresis and autoradiography. Under nonreducing Thus, of the five protein bands that result from the hepariticonditions, undigested '"I-HSPG migrated as a smear in the nase treatment of the HSPG, only the 64-kDa protein was highmolecularweight region (Fig. 5, lane a ) . Heparitinase treatment of the nonreduced ""I-HSPG yielded five bands, a b c d e four prominent bandswith an apparentM , of 35,000,48,000, v l r x 10-3 64,000 and 125,000 and a fifth weak band of 90,000 (Fig. 5, lane b ) . The five bands represented proteins, since no label was retained in the gel when the heparitinase-treated ""'1HSPG were further digested with protease K (Fig. 5, lane e ) . When undigested "'I-HSPG were reduced before electrophoresis, a peptide with an apparentM , of 35,000, migrating asa sharp band, was released from the HSPG complex (Fig. 5, 97 lane d ) . Reduced, heparitinase-treated "'I-HSPG yielded ap69 parently the samefive bands as the nonreduced, heparitinasetreated HSPG (Fig. 5; compare lane c to lane b). Relative 46 densities of thebands, however, were different: reduction 30 decreased the relative density of the bandat 64 kDa, whereas the relative density of the band at35 kDa was increased. To facilitate the interpretation of the results, the HSPG FIG.6. Comparison of heparinase, heparitinase, and nitrous were reduced and rechromatographed over Sepharose CL-4B acid treatment of ""I-membrane HSPG. Reduced HSPG were '"1 radiolaheled (chloramine-T method) and treated with nitrous acid to remove the disulfide-linked35-kDa peptide before "'I(lane a ) , heparinase (lane b ) , heparitinase (lane d), or left untreated iodination. These "'I-iodinated, reduced HSPG were treated (lane c).Nonreduced membraneHSPC were iodinated (Boltonwith heparitinase, heparinase, and HNO, and analyzed by Hunter reagant) anddigested with heparitinase (lane e ) . ElectrophoSDS-polyacrylamide gel electrophoresis under reducing con- resis was performed under reducing conditions.
Heparan Sulfate Proteoglycan a
Core Protein Heterogeneity
Mr x 10-3
f Mr x 10-3
59 46 30
30 FIG. 7. Fractionation of 1251-membraneHSPG by mAb SI. Nonreduced 1251-membraneHSPG were applied on CNBr-Sepharose immobilized mAb S1 before (lanes b and e ) or after (lanes c and d ) heparitinase digestion. The 12511-HSPG that were applied and bound as nondigested HSPG were treated with heparitinase before electrophoresis. Lanes a and f, starting sample, applied onmAb S1 without prior heparitinase digestion; lanes b-e, bound samples, eluted from mAb S1; lanes a-c, electrophoresis under nonreducing conditions; lanes d-f, electrophoresis under reducing conditions.
/Ir x 10-3
97 recognized by mAb SI. Since heparitinase digestion of the HSPG whichwas bound to mAb S1 as native HSPG also 69 yielded only a 64-kDa band, it was concluded that the64-kDa 46 core proteinformsare derived from a distinctsubset of membrane proteoglycans. This conclusion was supported by 30 results observed from the digestion of 9 - H S P G t h a t had t been fractionated bygel filtration on Sepharose CL-4B in FIG. 8. Fractionation of 1251-membrane HSPG by gel filtraGdnHCl buffer containing 0.5% Triton X-100. The broad 0.27; not shown) was subdi- tion over Sepharose CL-4B. Purified "'1-HSPG, chromatoHSPG peak (maximum a t K,, vided in six pools,ranging from materials elutinga t K., 0.1 graphed over Sepharose CL-4B in GdnHCl buffer containing 0.5% (v/v) Triton X-100 elute as a broad peak with maximum a t K., to materials eluting a t Kay 0.5. When equal amounts of 0.27 (not shown). Such an HSPG peak was subdivided in six pools: material of these different pools were digested with hepariti- Kav= 0.083-0.150 (lane a), K., = 0.150-0.217 (lane b), Kav = 0.217nase andanalyzed by SDS-polyacrylamide gel electrophoresis 0.283 (lane c), K., = 0.283-0.350 (lane d), K., = 0.350-0.417 (lane e), under nonreducing (Fig. 8A) and reducing (Fig. 8 B ) condi- and K. = 0.417-0.483 (lane f). Equal amounts of radiolabel were tions, autoradiography showed that the composition of the precipitated with ethanol (75% v/v) and digested with heparitinase HSPG fractions changed with increasing Kav.The 125-, 90-, before SDS-polyacrylamide gel electrophoresis and autoradiography. electrophoresis under nonreducing conditions; R, electrophoresis and 48-kDa protein bands decreased in relative density with A, under reducing conditions. increasing Kav,whereas the 64- and 35-kDa bandsincreased. Thus, at least some of the different core protein forms mierogeneity, rather thanincompleteness of the reaction, seems grated independently duringgel filtration before heparitinase to account for the 64-kDa band remaining after reduction. digestion. It was concluded,also in view of the results obtained Indeed, the migration of the 64-kDa core protein which is with mAb S1, that the multiplicityof the core protein bands recognized by the monoclonal antibody S1 is not affected by is not simply explained by proteolytic activity during the reduction of disulfide bonds (Fig. 7). In the reduced samples heparitinase treatment. the 35-kDa protein band became more prominent. This sug-
gested that in addition to the five core protein formsdescribed above, a dimeric complex consisting of two disulfide-linked Exploiting thetypical solubility characteristics, high charge peptides, each with an apparent M, of about 35,000, was density, hydrodynamic size, and hydrophobic properties of present in nonreduced samples. Some of these 35-kDa pepthe membrane HSPGof human lung fibroblasts, we were able tides did not carry H S chains, since they were released from the HSPG complex by reduction of disulfide bonds (Fig. 5, to obtain sufficientlypurified preparations to analyze the protein moiety of these molecules after '"1 radiolabeling. The lane d ) and since heparitinase treatment apparently did not results indicate extensive structural heterogeneity and imply reduce their size. The structure of the membrane HSPG of HLF appears that the membrane HSPG of human lung fibroblastsoccur in multiple forms. distinct from that reported for HSPG isolated from other Part of the membrane HSPG of human lung fibroblasts cultured cells or tissues. Disulfide-bondedmultimers of HSPG consists of single polypeptide core proteins of varying size have also been described for human skin fibroblasts (28) and carrying one or more HS chains. Others, in contrast, appear for mouse 3T3 cells (29), but the subunit structure and the to have a disulfide-bonded dimer structure. Indeed, hepariti- participation of a non-HS chain-bearing polypeptide in the nase treatment of the "'I-HSPG yielded five iodinated pro- formation of adisulfide-bonded HSPG dimer, seem to be teins with apparent M,of 125,000, 90,000,64,000, 48,000, and unique features of the HSPG of human lung fibroblasts. In 35,000 (Fig. 5 , lanes b and c). Comparing nonreduced to human skin fibroblasts thecore protein moiety of the HSPG reduced core protein patterns (Fig. 5, lane b to lane c; Fig. 7, is formed by a disulfide-bonded homodimer of M, 180,000 lane a to lane f; Fig. 8, A and H),it was obvious that the (21). Although one of the core peptides of the HSPG of HLF relative density of the 64-kDa bandwas higher in the nonre- had an apparent M, of 90,000 and the 64-kDa band may duced samples than in the reduced samples. Structural het- harbor disulfide-bondedhomodimers, no evidence for the DISCUSSION
Proteoglycan Core Protein Heterogeneity
occurrence of a form mimicking the organization described for skin fibroblasts was obtained here. In Swiss mouse 3T3 cells, in contrast, HSPG monomers of M , 20,000 form disulfide-linked aggregates of M , > 700,000 (29). HSPG isolated from other in vitro cultured tissuesalso seem to be structurally distinct in theircore protein moiety. Characterization studies have led to variable core protein M , estimates: 240,000 for human colon carcinoma cells (30), 80,000 for PC-12 cells (31), and 53,000 for mouse mammary epithelial cells (32). A core protein of 53-kDa has also been identified in HSPG directly isolated from bovine lungs (33). Finally, structural heterogeneity occurring within a single tissue has been reported for the core protein moiety of HSPG isolated from baby hamster kidney cells (34). Nitrousacid degradation of the cell-associated HSPG of these fibroblasts revealed three main polypeptides having apparent molecular weights of 65,000, 85,000, and 120,000. Interestingly, rather similar M, estimates were obtained here for nitrous acid-degraded IZ5I-HSPGof HLF (Fig. 6, lane a ) . The high variability of reported size estimates might be an indication of species and tissue specificities of the HSPG structure. On the other hand, methodological differences and disulfide bridge formation between HSPG monomers may account for some of these variations (29). Alkylation of the thiol groups during extraction, a precaution which was taken here, should minimize this possibility of artificial cross-linking. The origin and significance of the occurrence of multiple core protein forms among membrand proteoglycans isolated from a single tissue remains unclear. Although nitrous acid could cleave some core proteins, the resultobtained after chemical cleavage of the glycosaminoglycan chains (Fig. 6, lane a) is consistent with the multiplicity of core proteins obtained after heparinase (Fig. 6, lane b ) and heparitinase (Fig. 6,lune d ) treatments. Moreover, at least part of the core protein moiety appeared to be heterogenous before heparitinase digestion. Heparitinase treatment of mAb Sl-immunopurified HSPG yielded only a 64-kDa protein band (Fig. 7), and some of the different core protein forms migrated independently during gel filtration before heparitinase digestion (Fig. 8).Thus, proteolysis of a single proteoglycan during the heparitinase treatment (if any proteolysis occurs in the presence of several protease inhibitors) is not likely to be the origin of the multiple protein bands.The possibility that some of the multiple protein bands were generatedduring the isolation of the HSPG, however, could not definitely be excluded. If so, we would have isolated the hydrophobic terminal of the core protein (by the criterion of incorporationin liposomes) but degraded t o different degrees. This seems unlikely because the heparitinase digestions of five preparations of independently purified HSPG yielded all very similar results. When the core protein moiety was radiolabeled with the Bolton-Hunter reagent, again very similar results were obtained (Fig. 6, lane e ) , indicating that this part of the procedure can be excluded in induction of artifacts. Thus, the heterogeneity of the membrane HSPG might occur in situ. This heterogeneity could be relevant for the multiplicity of the functions that have been ascribed to these HSPG. For example, specialized core protein structures may target distinct proteoglycans into specific membrane domains or specific supramolecular complexes. Alternatively, the different forms may represent different subunits or different metabolic
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