of heparan sulfate proteoglycan - Europe PMC

The EMBO Journal vol.4 no.4 pp.905-912, 1985

Immunological characterization of basement membrane of heparan sulfate proteoglycan

Marie Dziadek, Sakuhei Fujiwaral, Mats Paulsson and Rupert Timpl Max-Planck-Institut fir Biochemie, D-8033 Martinsried, FRG 'Present address: 2nd Internal Medicine, Kobe University Hospital, Kobe, Japan Communicated by R.Timpl

Antibodies were raised against a small high-density and a large low-density form of heparan sulfate proteoglycan from a basement membrane-producing mouse tumor and were characterized by radioimmunoassays, immunoprecipitation and immunohistological methods. Antigenicity was due to the protein cores and included epitopes unique to the low density form as well as some shared by both proteoglycans. The antibodies did not cross-react with other basement membrane proteins or with chondroitin sulfate proteoglycans from interstitial connective tissues. The heparan sulfate proteoglycans occurred ubiquitously in embryonic and adult basement membranes and could be initially detected at the 2-4 cell stage of mouse embryonic development. Low levels were also found in serum. Biosynthetic studies demonstrated identical or similar proteoglycans in cultures of normal and carcinoembryonic cells and in organ cultures of fetal tissues. They could be distinguished from liver cell membrane heparan sulfate proteoglycan, indicating that the basement membrane types of proteoglycans represent a unique class of extraceliular matrix proteins. Key words: heparan sulfate proteoglycan/radioimmunoassays/biosynthesis/tissue localization/embryonic basement membranes Introduction Heparan sulfate proteoglycans are a special class of sulfated glycosaminoglycan-protein complexes within the large family of proteoglycans (Heinegard and Paulsson, 1984). They are typical constituents of basement membranes (Kanwar and Farquhar, 1979; Hassel et al., 1980) where they appear to play a central role in controlling filtration and other biological properties (Farquhar, 1981). Structural studies of heparan sulfate proteoglycans from the basement-membrane producing mouse EngelbrethHolm-Swarm (EHS) tumor have demonstrated a high- and lowdensity form of the proteoglycan (Fujiwara et al., 1984). The high-density form (mol. wt. 130 000) consists of a small protein 10 000) connected to four, 30 nm long heparan core (mol. wt. sulfate chains (mol. wt. 29 000). The low-density proteoglycan (mol. wt. 2400 000) contains >50% protein and thus a substantially larger protein core. Similar data were obtained in other studies (Martin et al., 1984) and for a large proteoglycan from rat yolk sac carcinoma (Fenger et al., 1984). A considerable structural similarity to the EHS tumor high-density form is found for a proteoglycan from glomerular basement membranes (Kanwar et al., 1981, 1984) which may, in addition, contain a proteinrich heparan sulfate proteoglycan (Parthasarathy and Spiro, 1984). Numerous other studies based on metabolically labelled IRL Press Limited, Oxford, England.

types

proteoglycans obtained from basement membrane-producing cells and organs indicate a considerable structural diversity among heparan sulfate proteoglycans (Oohira et al., 1982, 1983; Oldberg et al., 1982; Buonassisi and Colburn, 1983; Lowe-Krentz and Keller, 1983; Hampson et al., 1984; Heathcote and Orkin, 1984; lozzo, 1984; Tyree et al., 1984). The precise relationship between these proteoglycans and the high- and low-density forms found in tumors remains to be elucidated. Other types of heparan sulfate proteoglycans may also exist, such as a cell membranebound form identified initially in rat liver (Oldberg et al., 1979; Kjellen et al., 1981) and larger variants from fibroblasts (Carlstedt et al., 1983) which are possibly related to the transferrin receptor (Fransson et al., 1984). The diversity of heparan sulfate proteoglycans in tissues has become increasingly studied using immunological methods (Hassell et al., 1980; Oohira et al., 1983; Buonassisi and Colburn, 1983; Woods et al., 1984; Fujiwara et al., 1984; Fenger et al., 1984). Here we describe quantitative immunochemical assays for proteoglycans from the EHS tumor and the properties of affinity-purified antibodies. Using these approaches we could detect similar proteoglycans in a variety of biological samples and distinguish them from other types of proteoglycans. Results Production and specificity of antibodies against heparan sulfate proteoglycan The low-density form of the proteoglycan purified from a mouse tumor basement membrane produced high titer antisera in rabbits (50% antigen binding in radioimmunoassay at dilutions 1:15 x 104). Approximately 100-fold lower titers were found in these sera for the high-density proteoglycan and laminin, and negligible reactions with other basement membrane proteins such as collagen IV, nidogen and fibronectin. The high-density proteoglycan was a much weaker immunogen and only moderate titers of 1:50 - 100 were obtained after multiple antigen injections. However, much stronger reactions were found in this antiserum for low-density proteoglycan and laminin (titers 1: 103- 104) presumably due to low contaminations of these antigens in the high-density proteoglycan used for immunization. Antibodies were purified from the sera by immunoadsorption on columns of the low- or high-density proteoglycan after a preadsorption step on a laminin column. Both sets of purified antibodies retained a stronger binding (30- to 100-fold) to the lowdensity proteoglycan than the high-density form, but failed to react with other basement membrane proteins (Figure 1). It was interesting, however, that antibodies raised against the low-density proteoglycan showed a higher antigen binding capacity when compared with those against high-density proteoglycan indicating a 30-fold difference in affinity constants (Engel and Schalch, 1980). Purified antibodies against both high- and low-density forms were used for immunoprecipitation and immunofluorescence analyses. Several antisera against laminin, collagen IV and nidogen showed negligible binding for the proteoglycans in radioimmuno-

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assays (titers 80% of the heparan sulfate from the protein core by heparitinase digestion did not change antigenic activity. In addition, lack of activity was found for purified heparan sulfate chains, indicating that the major epitopes are located on the protein core. The high-density proteoglycan showed a 1000-fold lower inhibitory activity. A similar low activity was found for laminin, collagen IV and nidogen (Figure 2a), presumably due to a low contamination ( 0.2%) by the low-density form rather than immunological

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