Heparan sulfate proteoglycans made by different basement-membrane-producing tumors have immunological and structural similarities

Differentiation

Differentiation (1985) 3 0 : 6 1 4 7

i c

Springer-Verlag 1985

Heparan sulfate proteoglycans made by different basement-membrane-producing tumors have immunological and structural similarities *, Reidar Albrechtsen', and John R. Hassell Laboratory of Pathology, National Cancer Institute, NIH, go00 Rockville Pike, Bethesda, M D 20892, USA The University Institute of Pathological Anatomy, Frederik V's vej 11, DK-2100 Copenhagen, Denmark Laboratory of Developmental Biology and Anomalies, National Institute of Dental Rcsearch, NIH, Bethesda, MD 20892, USA

Ulla M. Wewer

Abstract. Using immunological assays, we determined the relationship between the heparan sulfate proteoglycans produced by two different murine basement-membrane-producing tumors, i.e., the mouse Engelbreth-Holm-Swarm (EHS) tumor and the L2 rat yolk-sac tumor. Antibodies prepared against the heparan sulfate proteoglycans obtained from these two sources immunoprecipitated the same precursor protein with a molecular mass of 400,000daltons from "S-methionine pulse-labeled cells of both tumors. Immunohistochemistry showed the heparan sulfate proteoglycan to -be distributed in the extracellular matrix and also in the native basement membrane of surrounding normal murine tissues. Blocking and ELISA assays demonstrated that the antibodies recognized both antigens. Using techniques involving the chemical and enzymatic degradation of 35S-sulfate-labeledglycosaminoglycans, the mouse EHS tumor cells were found to produce mainly heparan sulfate (75%) along with smaller amounts of chondroitin sulfate (19%), whereas the L2 rat yolk-sac tumor produced mainly chondroitin sulfate (76%) with smaller amounts of heparan sulfate (21%). We conclude that these two murine basement-membrane-producing tumors elaborate an immunologically and structurally similar type of high-molecularweight heparan sulfate proteoglycan which subsequently becomes incorporated into basement-membrane-like material.

Introduction Proteoglycans are a class of sulfated macromolecules that have a core protein' with at least one covalently bound glycosaminoglycan chain [l 11. Among the different groups of glycosaminoglycansare hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparan sulfate, and heparin. Proteoglycans are constituents of various extracellular matrices, and they have important biological functions, such as resisting compression in the cartilage [20], providing optical transparency in the cornea [12, 23, 331,

*

and producing an ionic filtration barrier in the glornerular basement membrane [17,44]. Heparan sulfate proteoglycans are found in association with cells either on the cell surface [15, 22, 34, 39, 40,431 or as an integral component of the basement membrane [13, 16, 19, 381. Cell-surface-associated heparan sulfate proteoglycans have been isolated from different cell types, e.g., rat liver hepatocytes [22, 341. This proteoglycan is relatively small, having an estimated molecular mass of 80,000 daltons, and contains four 14,000-dalton heparan sulfate chains. Evidence has been presented which suggests that the hydrophobic part of the core protein interacts with the cell membrane, and thus may be intercalated in the lipid layer of the plasma membrane itself [21, 341. Antibodies raised against this heparan sulfate proteoglycan stained the sinusoidal plasmalemmal domain of rat hepdtocytes but not basement membranes [49]. A number of basement-membrane-producing tumors and cell lines have been useful in the study of the structure of the heparan sulfate proteoglycan found in the basement membrane [6, 9, 10, 13, 14, 30, 36, 42, 501. Recently, a 400,000-dalton protein has been identified as the precursor protein (see Footnote 1) of the heparan sulfate proteoglycan produced by the Engelbreth-Holm-Swarm (EHS) tumor [28]. Another basement-membrane-producing tumor, the L2 rat yolk-sac carcinoma [53, 541, produces chondroitin sulfate proteoglycans [2, 351 as well as a high-molecularweight heparan sulfate proteoglycan [9]. Antibodies raised against the heparan sulfate proteoglycans isolated from these two basement-membrane-producing tumors have been shown to react with various authentic basement membranes of normal tissues [6, 9, 26, 321. In the present study, we compared the relative abundance of the heparan sulfate proteoglycans and the nature of the precursor protein produced by the EHS and the L2 tumors. The precursor proteins of the heparan sulfate proteoglycans were found to be identical in size (400,000daltons) and to be immunologically closely related. Methods

To whom offprint requests should be sent Precursor protein is the initial protein translation product of the proteoglycan as synthesized in the rough endoplasmic reticulum. There are no glycosaminoglycan side chains. Core protein is the protein part of the complete proteoglycan after the addition of glycosaminoglymn side chains in the Golgi apparatus, along with other post-translational modifications

The EHS tumor This tumor apparently arose spontaneously in an ST/Eh mouse at the University Institute of Pathological Anatomy, Copenhagen, and was originally described as being a chondrosarcoma by Swarm [51]. However, it was later charac-

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terized as a basement-membrane-producing tumor by Orkin et a]. [37] and may be considered to be a murine parietal yolk-sac tumor. In the present study, short-term cultures of EHS tumor cells were prepared as described by Ledbetter et al. [28] by 2.4% dispase (protease, neutral grade 11; Boehringer Mannheim Biochemicals, Indianapolis, Ind) treatment of freshly harvested tumors. The culture medium was NCTC 109 containing 10% fetal bovine serum (Gibco Laboratories, Grand Island, NY).

methionine (1,470 mCi/mmol; Amersham Corporation, Arlington Heights, Ill). After a brief rinse, the cell layers were solubilized in 1 ml 0.02 M Tris-HC1, pH 7.4, containing 0.15 A4 NaCI, 1 YONonidet P-40, 1 % desoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), and 1 % aprotinin. The cell lysates were briefly sonicated, and the debris and insoluble material were removed by centrifugation at 4" C in an Eppendorf microfuge. The cell lysates were stored at -70" C until use.

The L2 rat yolk-sac carcinoma model system was experimentally induced by puncturing the uterine wall of a pregnant rat during midgestation [54]. It was established as a transplantable tumor line in Lewis rats and was characterized as a continuous cell line in culture [53]. The cells used in the present study had been passaged 55-70 times, and they were maintained as previously described [53, 541. The cells were fed every day and passaged three times per week. The culture medium was Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Gibco).

Overnight labeling with "S-sulfate. The cells were radiolabeled overnight in 1 ml medium containing 100 pCi/ml carrier-free 35SO;2 as the sodium salt (35 mCi/mmol; Amersham). The cell layers were extracted as previously described [52] using 4 M guanidine-HC1 (Ultrapure; Bethesda Research Laboratories, Gaithersburg, Md) and 5% Zwittergent 31 2 (Calbiochem-Behring, San Diego, Calif.). Solid guanidine-HC1was added to the media at a final concentration of 4 M. Unincorporated radioactivity in both the cell layers and media was removed by chromatography on Sephadex G-25 (Pharmacia, Piscataway, NJ) in 4 M guanidine-HCI.

Antisera

Enzymatic and chemical degradation

Rabbit antisera against the low-density heparan sulfate proteoglycan isolated from the EHS tumor [13, 141 and against the L2 heparan sulfate proteoglycan 191have been described elsewhere. An enzyme-linked immunosorbent assay (ELISA) was performed to ascertain that antisera against both laminin and type-IV collagen did not react with the purified heparan sulfate proteoglycan used for immunization, and that the rabbit antisera obtained did not react with known matrix proteins of the tumors.

35S-Sulfate-labeledmedia and cell layers were combined and digested with 0.5 mg/ml papain (P 3125; Sigma, St. Louis, Calif.) in 1 M sodium acetate (pH 6.5). 5 mM ethylenediaminetetraacetic acid (EDTA), and 5 mM cysteine for 24 h at 55" C. Nitrous acid digestion was performed using the low-pH method of Shively and Conrad [46]. Nondegraded material was separated from the degradation products on a column (1 x 50 cm) of Sephadex G-50 equilibrated with 0.02 A4 Tris-HC1, pH 7.0, containing 4 M guanidineHCI and 0.1 % CHAPS (Calbiochem-Behring). Resistant material present in the exclusion volume was treated for a further 1 h with chondroitinase ABC (Miles Laboratories, Naperville, Ill) at 37" C according to the method of Saito et al. [45]. Again, the nondegraded material was isolated and quantitated.

The L2 rat yolk-suc carcinoma

ELISA The antisera were titrated using an ELISA [7]. The wells in flat-bottomed microtiter plates (Immulon 2; Dynatech Laboratories, Alexandria, Va) were coated with 0.2 ml puritied proteoglycans (100 ng in 0.1 M NaHC03/Na2C03, pH 9.5) overnight at room temperature in a moist chamber. Following several washes, the wells were incubated for 2 h with serial dilutions of the antisera in phosphate-buffered saline (PBS) containing 0.05% Tween 20. Bound antibodies were detected using peroxidase-conjugated swine anti-rabbit IgG (P217; Dakopatt, Copenhagen, Denmark) that had been diluted (1:500) and incubated for 1 h. The reaction product was visualized using o-phenylendiamine dihydrochloride (16.5 mg per 30 ml) in citrate-phosphate buffer (pH 5.6) containing 100 p1 3% HzOz. The reaction was stopped by adding 4 N H2S04 after 5-1 0 min. All reactions were carried out at room temperature. The washing buffer consisted of 9 g/l NaCl containing 0.05% Tween 20. Radiolabeling and extraction procedures Confluent cultures in 35-mm dishes were metabolically radiolabcled at 37" C. The EHS tumor cell cultures were labeled after 18 h of culture. Pulse labeling with j5S-methionine. The cells were radiolabeled for 20 min in 1 ml methionine-free medium containing 2% dialyzed fetal bovine serum and 500 pCi/ml 35S-

Immunoprecipitation of the precursor protein of heparan sulfate proteoglycan The procedure employed for the cell layer was adapted from the method of Bumol and Reisfeld [3] as described in detail by Ledbetter et al. [28]. In brief, the cell lysate was first preabsorbed on protein-A sepharose (Pharmacia) which had previously been preabsorbed with nonimmunized rabbit serum. Specific antisera or nonimmunized rabbit serum (50 pl) were incubated with protein-A sepharose for 1 h at 4" C, and this IgG protein-A complex was then reacted with the preabsorbed lysate for 2 h at 4" C; this was followed by five washes with PBS containing 0.5% Tween 20, 0.05% SDS, 0.1 % bovine serum albumin, and 0.02% sodium azide. The precipitates were recovered from the proteinA sepharose by boiling in 0.01 M Tns-HC1 (pH 6.8), 1% SDS, and 10 mM dithiothreitol. The culture media were preabsorbed and immunoprecipitated as described previously [55]. The washing procedure consisted of alternate washes in (1) 0.05 M Tris-HC1 (pH 7.2), 0.15 M NaC1, 0.05% Tween 20, 0.05 M EDTA, and 10 pg/ml soybean-trypsin inhibitor, and (2) the same buffer with the addition of 0.5 M NaCI. The radioactivity

63

was measured in a Beckman liquid-scintillation counter using Hydrofluor scintillation solvent (National Diagnostics, Sonneville, NJ). SDS-polyacrylamide gel electrophoresis (PAGE) and fluorography The immunoprecipitates were separated by gel electrophoresis according to the procedure of Laemmli [25l using chemicals obtained from Bio-Rad (Bio-Rad Laboratories, Richmond, Calif.). In most cases, a 4% stacking gel and a 5% separating gel were used. Following staining with Coomassie Brillant Blue G250 and destaining, the gels were processed for fluorography using Autofluor (National Diagnostics) according to the manufacturer's instructions. The gels were dried and exposed to Kodak X-Omat AR film (Eastman Kodak Company, Rochester, NY) at - 70" C. Immunohistochemistry Small pieces of freshly harvested EHS and L2 tumors were fixed overnight at 4" C in 96% ethanol/glacial acetic acid (99: 1 v/v) as previously described [l]. The specimens were embedded in paraffin at 58" C using routine methods. Fourmicron-thick sections were cut and then stained with hematoxylin and eosin or processed (with or without a brief pretreatment of the tissue sections with protease [l]) for immunoperoxidase staining using the unlabeled-antibody peroxidase-antiperoxidase (PAP) technique [47. The antisera against the heparan sulfate proteoglycans were diluted (1 :100) in 0.05 M Tris-HC1, pH 7.3, and the sections were incubated for 18 h at 4" C in a moist chamber. Swine antirabbit IgG and PAP (Dakopatt) at dilutions of 1 :50 were used as the second and third layers, respectively, for 30 min at room temperature. The peroxidase reaction product was finally visualized using 0.5 mg/ml 3,3'diaminobenzidine in 50 m M Tris-HC1 (pH 7.2) and 0.03% HzOz. For washing between the steps, 50 mM Tris-HC1 containing 0.25 M NaCl was used. As controls, the specific antisera were replaced by preimmune sera. In some experiments, the purified heparan sulfate proteoglycans were added to the incubating antisera medium at a concentration of 50 pg/ml to test for competition. The staining reaction was scored as being negative (-), slightly positive (+), or intensely positive (+ +). Unless otherwise specified, the reagents used were of the best commercially available grade. Results

Characterization of the glycosaminoglycans of the EHS and of the L2 proteoglycans We first determined the amount and type of glycosaminoglycans present in the EHS and L2 tumors. Treatment with nitrous acid followed by selective enzyme degradation with chondroitinase ABC was used to characterize the 35S-labeled glycosaminoglycans.Undegraded material was separated from the degradation products by gel chromatography on Sephadex G-50. The material degraded by treatment with nitrous acid was considered to be heparan sulfate. Material that was resistant to degradation by nitrous acid but was degraded to disaccharides by chondroitinase ABC

Table 1. Comparison of the percentage of 35S-sulfate-labeledglycosaminoglycans synthesized by EHS and L2 tumor cells in culture

EHS tumor L2 tumor

Heparan sulfate

Chondroitin sulfate

Resistant

75 21

19 76

6 3

Data are expressed as a percentage of the total amount of glycosaminoglycans. Synthesized proteoglycans were labeled by incubating EHS and L2 cells in medium containing "S-sulfate. The radiolabeled glycosaminoglycans were isolated and identified as heparan sulfate or chondroitin sulfate on the basis of their susceptibility to nitrous acid deamination or chondroitinase ABC, respectively (see Methods). Radiolabeled material that was not degraded by either treatment was designated as being 'resistant'

was considered to be chondroitin (dermatan sulfate) sulfate. The results showed that the principal 35S-labeledglycosaminoglycan produced by the L2 tumor cells was chondroitin sulfate (76%), but significant amounts of heparan sulfate (21%) were also present (Table 1). In contrast, most of the 35S-labeledglycosaminoglycan obtained from the EHS tumor cells was heparan sulfate (75%), with 19% being chondroitin sulfate (Table 1). Immunological similarities between the EHS and the L2 proteoglycans Immunological cross-reactivity between the heparan sulfate proteoglycans of the EHS and L2 tumors was first indicated by the results of immunoperoxidase staining of paraffin sections of tumor tissues. Antisera raised against the EHS proteoglycan applied to the L2 tumor tissue, or antisera against the L2 proteoglycan applied to the EHS tumor tissue produced moderately to intensely positive immunostaining that was primarily confined to the extracellular matrix of the tumors (Fig. 1). The basement membrane of blood vessels also exhibited positive immunostaining. When the tissue sections had been pretreated with protease, the cytoplasm of the tumor cells was devoid of any positive staining (Fig. 1). However, when protease treatment was not employed, a few tumor cells exhibited a positive cytoplasmic staining reaction (not shown). No differences in the staining pattern of the two antisera were observed. The positive staining could be abolished or diminished by absorbing the antisera with the antigens. When the EHS proteoglycan antibody was absorbed with excess amounts of the L2 proteoglycan antigen, no positive staining reaction was seen on the L2 tumor tissue, and the staining was significantly reduced on the EHS tumor, as compared to that Seen using nonabsorbed antibody. Likewise, when the L2 proteoglycan antibody was absorbed with excess amounts of the EHS proteoglycan antigen, no positive reaction was visible on the EHS tumor, and the reaction on the L2 tumor sections was also diminished (Fig. 1). The interspecies cross-reactivity of the antisera raised against the two different heparan sulfate proteoglycans from mouse and rat yolk-sac tumors was further confirmed by ELISA. Antibodies to EHS and L2 heparan sulfate proteoglycans reacted with both the EHS proteoglycan (Fig. 2A) and the L2 proteoglycan (Fig. 2B).

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Fig. 1A-D. Localization of heparan sulfate proteoglycan in the extracellular matrix of the EHS and L2 tumors using thc immunoperoxidase staining technique. A EHS tumor; D L2 tumor with anti-EHS proteoglycan antibody. Staining is also visible in the basement-membrane area of the vessels in the surrounding connective tissue (arrowheah).When anti-EHS proteoglycan was absorbed with its corresponding antigen, the staining reaction was blocked B, and when the same antibody was absorbed with the L2 proteoglycan antigen, the reaction was significantly reduced C. Sections were counterstained with hematoxylin. A x 280; B x 300; C x 300; D x 250

65 A REACTION WITH EHS HEPARAN SULFATE PROTEOGLYCAN

1

3

2

4

-200K

SERUM CONCENTRATION

8: REACTION WITH I2 HEPARAN SULFATE PROTEOGLYCAN 1.51

SERUM CONCENTRATION

Fig. 2A, B. Enzyme immunosorbant assays (ELISA) demonstrating immunological similarities between the EHS and L2 proteoglycans. Antisera to the EHS proteoglycan (o), the L2 proteoglycan (o), and nonimmune rabbit serum (A) were titrated in microtiter wells coated with the two different proteoglycans. A The EHS proteoglycan; B the L2 proteoglycan

Identification of the precursor proteins of the L2 and the EHS proteoglycans

As recently described, antiserum to the EHS proteoglycan will precipitate a 400,000-dalton precursor protein from EHS cells pulse labeled with 35S-methionine[28]. The same high-molecular-weight band was obtained from EHS cells using the anti-12 proteoglycan antibody (Fig. 3, lane 2). Antisera to the EHS proteoglycan immunoprecipitated protein of similar size from radiolabeled L2 cells (Fig. 3, lane 3). The substitution of the specific antisera with preimmune rabbit serum did not reveal any precipitated proteins (Fig. 3, lane 4). Immunoprecipitation with anti-laminin antibodies yielded a band with M , of 370,000, and a doublet with M , of 210,000 and 200,000 under reducing conditions, as would be expected for laminin (Fig. 3, lane 1).

Discussion Murine parietal-endoderm-like cells, such as the EHS and L2 tumors, have previously been shown to elaborate significant amounts of laminin, type-IV collagen, entactin, and heparan sulfate proteoglycan, and to organize these constit-

Fig. 3. Immunoprecipitation of pulse-labeled EHS and L2 cells. The cells in culture were labeled for 20 min with 3SS-methionine, and the cell layers were harvested, extracted, and incubated with specific antisera. Immunoprecipitates were separated on SDSPAGE (5% separating gel) and subjected to fluorography. Lane I , EHS cells with anti-rat laminin; lune2, EHS cells with anti-12 proteoglycan; lane 3, L2 cells with anti-EHS proteoglycan; lane 4, L2 cells with preimmune rabbit serum. The precursor protein to the heparan sulfate proteoglycan from both the EHS and the L2 tumor had an apparent molecular mass of 400,000 daltons. The arrows indicate the top and the bottom of the separating gel. The molecular weight of Coomassic-stained, purified murine laminin is marked

uents and possibly other macromolecules into a basementmembrane-like material. In the present study, we demonstrated that the heparan sulfate proteoglycans from two different basement-membrane-producing tumors, i.e., the EHS and L2 tumors, are immunologically closely related and have a similarly sized precursor protein with a molecular mass of -400,OOO daltons. This conclusion was reached from result obtained using several immunochemical techniques. Using antibodies produced against these two heparan sulfate proteoglycans, strong immunoreactivity was seen in the extracellular matrix of both tumors. Immunoabsorption studies with the antisera and antigens showed that the two proteoglycans have many antigenic sites in common. Each proteoglycan, however, has additional antigenic sites not present in the other proteoglycan. The L2 heparansulfate-proteoglycan antigen could not totally abolish the staining reactivity of the anti-EHS heparan-sulfate-proteoglycan on sections of EHS tumor tissue. Both antisera showed cross-reactivity in ELISA assays in which the purified proteoglycans were oated on microtiter wells, and the two individual antisera ere found to react with both antigens. Finally, in immunoprecipitation studies of pulse-labeled EHS and L2 cells, we demonstrated that the antisera recognized a similarly sized, high-molecular-weight (400,000daltons) proteoglycan precursor protein. These observations are consistent with the resufts of our previous ultrastructural studies of purified EHS and L2 proteogly-

3

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cans, in which we found the length of the core protein to be 180-200nm ([27]; U.M. Wewer, I Margulies, and L.A. Liotta, unpublished results), as well as being consistent with our previous biochemical results which indicated the intact heparan sulfate proteoglycan to have a molecular mass of approximately 750,000daltons [9, 13, 14, 281. There is evidence to suggest that basement membranes also contain chondroitin sulfate proteoglycan [5, 18, 24, 29, 481. In the present study, we demonstrated that EHS and L2 tumor cells synthesize both heparan and chondroitin sulfate. However, it was conspicious that the mouse EHS tumor and the L2 rat tumor differ considerably with regard to the ratio of heparan and chondroitin sulfate proteoglycans produced. The major proteoglycan synthesized by the L2 rat yolk-sac carcinoma is a chondroitin sulfate proteoglycan (core protein with a molecular mass of 10,OOO daltons [2, 351. In addition, rat yolk-sac tumors experimentally induced at day 8 of gestation [31] produce almost exclusively a chondroitin sulfate proteoglycan (C.C. Clark, R.V. Iozzo, and A. Martinez-Hernandez, personal communication). Results reported by Clark and Iozzo [4] show that normal parietal endoderm cells from rat embryos (day 14.5 of gestation) likewise produce much more chondroitin sulfate proteoglycan than heparan sulfate proteoglycan. In contrast, the mouse EHS tumor, as confirmed in the present study, and a number of mouse parietal endoderm cell lines produce mainly heparan sulfate proteoglycan and smaller amounts of chondroitin sulfate proteoglycan [8, 30, 36, 521. There are at least two explanations for this diversity in the stoichiometry of proteoglycan biosynthesis; first, the tumors/cell lines express the pattern of marker protein synthesis, perhaps indicating the time of embryogenesis at which they developed; second, there might be a true species-specific difference between the mouse and the rat with regard to the biosynthesis of proteoglycans by parietal endoderm cells. A range of different-sized heparan sulfate proteoglycans from basement-membrane sources have been documented [6, 9, 10,13, 14,16,19,28,30,36,38,42, 501. Most metdbolic-labeling and biochemical studies of heparan sulfate proteoglycans obtained from native basement membrane have used glomerular basement membrane as the proteoglycan source. These proteoglycans have been estimated to have molecular masses in the range of 130,0W2OO,OOO daltons, with the molecular masses of the heparan sulfate chains ranging from 12,000 to 26,000 daltons, i.e., considerably smaller than the 750,000 daltons estimated for the EHS or L2 proteoglycans [9, 13, 14, 281. However, antibodies to heparan sulfate proteoglycans isolated from both tumor cells and glomerular basement membrane recognize determinants that are associated with a variety of native basement membranes [6,9,26,32, 501. Whereas the immunological cross-reactivity of native heparan sulfate proteoglycan with tumor proteoglycan has not previously been established, similarities between cartilage proteoglycan obtained from a chondrosarcoma and proteoglycans obtained from native sources have been clearly documented [41]. It is likely that there are common epitopes between tumor and native basement-membrane heparan sulfate proteoglycans as well. It is possible these large and small basement-membrane proteoglycans are related. Ledbetter et a]. [28] have presented evidence indicating that the 400,000-dalton precursor protein is used for the synthesis of the large, low-density proteoglycan that has a protein core with a molecular mass

of 400,000 daltons, and that some of these macromolecules are converted into a small, high-density proteoglycan with a range of core proteins of 95,000-130,OOO daltons. Therefore, they have suggested that the small proteoglycan might be derived from the large proteoglycan as a result of posttranslational modifications and/or proteolytic degradation. Thus, it is conceivable that the basement-membrane heparan sulfate proteoglycans constitute a group of antigenically similar macromolecules and that they represent one gene product with different-sized ‘fragments’. Alternatively, the different-sized heparan sulfate proteoglycans may be encoded for by different but related gene products. Acknowledgements. Part of the study was supported by the Danish Medical Research Council and the Danish Cancer Society (to R. Albrechtsen).

References 1. Albrechtsen R, Nielsen M, Wewer U, Engvall E, Ruoslahti E (1981) Basement membrane changes in breast cancer detected by immunochemical staining for laminin. Cancer Res 41:507&5081 2. Bourdon MA, Oldberg A, Pierschbacher M, Ruoslahti E (1985) Molecular cloning and sequence analysis of a chondroitin sulfate proteoglycan cDNA. Proc Natl Acad Sci USA 82: 1321-1325 3. Bumol TF, Reisfeld RA (1982) Unique glycoprotein-proteoglycan complex defined by monoclonal antibody on human melanoma cells. Proc Natl Acad Sci USA 79: 12451249 4. Clark CC, Iozzo RV (1984) Biosynthesis of proteoglycans by rat embryo yolk sacs in organ culture. J Cell Biol99:175a 5 . Cohn RH, Banerjee SD, Bernfield MR (1977) Basal lamina of embryonic salivary epithelia. Nature of glycosaminoglycan and organization of extracellular materials. J Cell Biol 70:464-478 6. Dziadek M, Fujiwara S,Paulsson M, Timpl R (1985) Immunological characterization of basement membrane types of heparan sulfate proteoglycan. EMBO J 4:905-912 7. Engvall E (1980) Enzyme immunoassay ELISA and EMIT. Methods Enzymol70 :419439 8. Engvall E, Oshima RG, Brennan MJ, Ruoslahti E (1984) Clonal tumorigenic endodermal cell lines producing basement membrane components. Exp Cell Res 150:258-267 9. Fengcr M, Wewer U, Albrechtsen R (1984) Basement membrane heparan sulfate proteoglycan from the L2 rat yolk sac carcinoma. FEBS Lett 173:75-79 10. Fujiwara S, Wiedemann H, Timpl R, Lustig A, Engel J (1984) Structure and interactions of heparan sulfate proteoglycans from a mouse tumor basement membrane. Eur J Biochem 143:145-157 11. Hascall VC, Kimura JH (1982) Proteoglycans: isolation and characterization. Methods Enzymol 82 :770-800 12. Hassell JR, Newsome DA, Krachmer JH, Rodrigues MM (1980) Macular corneal dystrophy: Failure to synthesize a mature keratan sulfate proteoglycan. Proc Natl Acad Sci USA 77 :3705-3709 13. Hassell JR, Robey PG, Barrach HJ, Wilczek J, Rennard S1, Martin G R (1980) Isolation of a heparan sulfate-containing proteoglycan from basement membrane. Proc Natl Acad Sci USA 77 :4494-4498 14. Hassell JR, Leyshon WC, Ledbetter SR, Tyree B, Suzuki S, Kato M, Kimata K, Kleinman HK (1985) Isolation of two forms of basement membrane proteoglycan. J Biol Chem 260:8098-8105 15. Iozzo RV (1984) Biosynthesis of heparan sulfate proteoglycan by human colon carcinoma cells and its localization at the cell surface. J Cell Biol 99:403-417

67 16. Kanwar YS, Farquhar MG (1979) Isolation of glycosaminoglycans (heparan sulfate) from glomerular basement membranes. Proc Natl Acad Sci USA 76 :4 4 9 3 4 9 7 17. Kanwar YS, Linker A, Farquhar MG (1980) Increased permeability of the glomerular basement membrane to ferritin after removal of glycosaminoglycans (heparan sulfate) by enzyme digestion. J Cell Biol 86:688-693 18. Kanwar YS, Hascall VC, Farquhar MG (1981) Partial characterization of newly synthesized proteoglycans isolated from the glomerular basement membrane. J Cell Biol90: 527-532 19. Kanwar YS, Veis A, Kimura JH, Jakubowski ML (1984) Characterization of heparan sulfate proteoglycan of glomerular basement membranes. Proc Natl Acad Sci USA 81 :762-766 20. Kempson G E (1980) The mechanical properties of articular cartilage. In: Sokoloff L (ed) The joints and synovial fluid. Academic Press, New York, pp 177-238 21. Kjelltn L, Oldberg A, Hook M (1980) Cell surface heparan sulfate : Mechanisms of proteoglycan-cell association. J Biol Chem 255:1O407-1O413 22. Kjellkn L, Petterson I, H G k M (1981) Cell-surface heparan sulfate : An intercalated membrane proteoglycan. Proc Natl Acad Sci USA 78: 5371-5375 23. Klintworth GK, Smith C F (1983) Abnormalities of proteoglycans and glycoproteins synthesized by corneal organ cultures derivcd from patients with macular corneal dystrophy. Lab Invest 48:603612 24. Kobayashi S, Oguri K, Kobayashi K, Okayarna M (1983) Isolation and characterization of proteoheparan sulfate synthesized in vitro by rat glomeruli. J Biol Chem 258: 12051-12057 25. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 68&685 26. Laurie GW, Leblond CP, Martin G R (1982) Localization of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin to the basal lamina of basement membranes. J Cell Biol95 :34&344 27. Laurie GW, Hassell JR, Kleinman HK, Martin G R (1984) Ultrastructure and interactions of heparan sulfate proteoglycan from basement membrane. J Cell Biol99 :78 a 28. Ledbetter SR, Tyree B, Hassell JR, Horigan EA (1985) Identification of the precursor protein to basement membrane heparan sulfate proteoglycan. J Biol Chem 260: 8106-81 13 29. Lemkin MC, Farquhar MG (1981) Sulfated and nonsulfated glycosaminoglycans and glycopeptides are synthesized by kidney in vivo and incorporated into glornerular basement membranes. Proc Natl Acad Sci USA 78: 1726-1730 30. Lowe-Krentz LJ, Keller JM (1983) Multiple heparan sulfate proteoglycans synthesized by a basement membrane producing murine embryonal carcinoma cell line. Biochemistry 22:4412419 31. Martinez-Hernandez A, Miller EJ, Damjanov I, Gay S (1982) Laminin-secreting yolk sac carcinoma of the rat. Biochemical and electron immunohistochemical studies. Lab Invest 47: 247-257 32. Mynderse LA, Hassell JR, Kleinman HK, Martin GR, Martinez-Hernandez A (1983) Loss of heparan sulfate proteoglycan from glomerular basement membrane of nephrotic rats. Lab Invest 48 :292-302 33. Nakazawa K, Hassell JR, Hascall VC, Lohmander LS, Newsome DA, Krachmer J (1984) Defective processing of keratan sulfate in macular corneal dystrophy. J Biol Chem 259: 13751-13757 34. Oldberg A, Kjelltn L, Hook M (1979) Cell-surface heparan sulfate. Isolation and characterization of a proteoglycan from rat liver membranes. J Biol Chem 254:850%8510 35. Oldberg A, Hayman EG, Ruoslahti E (1981) Isolation of a chondroitin sulfate proteoglycan from a rat yolk sac tumor and immunochemical demonstration of its cell surface localization. J Biol Chem 256: 10847-10852 36. Oohira A, Wight TN, McPherson J, Bornstein P (1982) Bio-

chemical and ultrastructural studies of proteoheparan sulfates synthesized by PYS-2, a basement membrane-producing cell line. J Cell Biol92: 357-367 37. Orkin RW, Gehron P, McGoodwin EB, Martin.GR, Valentine T, Swarm R (1977) A murine tumor producing a matrix of basement membrane: J Exp Med 145:204-220 38. Parthasarathy N, Spiro RG (1 984) Isolation and characterization of the heparan sulfate proteoglycan of the bovine glomerular basement membrane. J Biol Chem 259: 1274F12755 39. Rapraeger AC, Bernfield M (1983) Heparan sulfate proteoglycans from mouse mammary epithelial cells. A putative membrane proteoglycan associates quantitatively with lipid vesicles. J Biol Chem 258 :3632-3636 40.Rapraeger A, Bernfield M (1985) Cell surface proteoglycan of mammary epithelial cells. Protease releases a heparan sulfaterich ectodomain from a putative membrane-anchored domain. J Biol Chem 260:4103-4109 41. Rennard SI, Kimata K, Dusemund B, Barrach HJ, Wilczek J, Kimura JH, Hascall VC (1981) An enzyme-linked immunoassay for cartilage proteoglycan. Arch Biochem Biophys 207 : 399-406 42. Robinson J, Gospodarowin D (1984) Effect ofpnitrophenylPD-xyloside on proteoglycan synthesis and extracellular matrix formation by bovine corneal endothelial cell cultures. J Biol Chem 259: 3818-3824 43. Robinson J, Viti M, Hook M (1984) Structure and properties of an under-sulfated heparan sulfate proteoglycan synthesized by a rat hepatoma cell line. J Cell Biol98: 9 6 9 5 3 44. Rohrbach DH, Wagner CW, Star VL, Martin GR, Brown KS, Yoon J-W (1983) Reduced synthesis of basement membrane heparan sulfate proteoglycan in streptozotocin-induced diabetic mice. J Biol Chem 258 :1 1 672-1 1677 45. Saito H, Yamagata T, Suzuki S (1968) Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J Biol Chem 243: 15361542 46. Shively JE, Conrad HE (1976) Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry 15 3932-3942 47. Sternberger LA (1979) Immunocytochemistry, 2nd edn. John Wiley and Sons, New York 48. Stow JL, Glasgow EF, Handley CJ, Hascall VC (1983) Biosynthesis of proteoglycans by isolated rabbit glomeruli. Arch Biochem Biophys 225 :950-957 49. Stow JL, Kjellen L, Unger E, Hook M, Farquhar MG (1985) Heparan sulfate proteoglycans are concentrated on the sinusoidal plasmalemmal domain and in intracellular organelles of hepatocytes. J Cell Biol100:975-980 50. Stow JL, Sawada, J, Farquhar MG (1985) Basement membrane heparan sulfate proteoglycans are concentrated in the laminae rarae and in podocytes of the rat renal glomerulus. Proc Natl Acad Sci USA 82: 32963300 51. Swarm R L (1963) Transplantation of a murinc chondrosarcoma in mice of different inbred strains. J Natl Cancer lnst 31 :953-974 52. Tyree B, Horigan EA, Klippenstein DL, Hassell JR (1984) Heterogeneity of heparan sulfate proteoglycans synthesized by PYS-2 cells. Arch Biochem Biophys 231 :328-335 53. Wewer U (1982) Characterization of a rat yolk sac carcinoma cell line. Dev Biol93 :41 6 4 2 1 54. Wewer U, Albrechtsen R, Ruoslahti E (1981) Laminin, a noncollagenous component of epithelial basement membranes synthesized by a rat yolk sac tumor. Cancer Res 41 : 1518-1524 55. Wewer U, Albrechtsen R, Manthorpe M, Varon S, Engvall E, Ruoslahti E (1983) Human laminin isolated in a nearly intact, bioilogically active form from placenta by limited proteolysis. J Biol Chem 258: 12654-12660 Received August / Accepted in revised form Septpber 1985