Abstract. Two mAbs raised against the 64-kD core protein of a membrane heparan sulfate proteoglycan from human lung fibroblasts also recognize a nonhydrophobic proteoglycan which accumulates in the culture medium of the cells. Pulse-chase studies suggest that the hydrophobic cell-associated forms act as precursors for the nonhydrophobic medium-released species. The core proteins of the medium-released proteoglycans are slightly smaller than those of the hydrophobic cell-associated species, but the NH2-terminal amino acid sequences of both forms are identical. The characterization of human lung fibroblast cDNAs that encode the message for these core proteins and the effect of bacterial phosphatidylinositol-specific phospholipase C suggest that the hydrophobic proteoglycan is membrane-anchored through a phospholipid tail. These data identify a novel membrane proteoglycan in human lung fibroblasts and imply that the shedding of this proteoglycan may be related to the presence of the phospholipid anchor.

Human lung fibroblasts (HLF) were labeled with 3 5 S 0 2 -for 48 h and extracted with a guanidinium chloride buffer. A fraction of the extracted heparan sulfate proteoglycan (HSPG) appeared micelle-associated. In the absence of detergent these HSPG eluted in the void volume of Sepharose CL2B columns. In the presence of detergent these HSPG were included in Sepharose CL2B (Kav = 0.55) and 4B (Kav = 0.3) columns. This type of HSPG was specifically associated with isolated HLF cell membranes, suggesting that it may represent a fraction of integral membrane proteoglycans.Most of the HSPG in the HLF monolayers, however, eluted in the included volume of Sepharose CL2B (Kav = 0.4) and CL4B (Kav = 0.1) columns in the absence of detergent. This type of HSPG was not affected by detergent and was specifically retained in 'extracellular matrix' preparations. The medium of HLF monolayers contained HSPG of similar M , as the membrane-associated HSPG.Of these three distinct HSPG fractions only the membrane-associated form could be incorporated in liposomes, confirming that the HSPG in this fraction may be integral membrane components.Structurally distinct heparan sulfate proteoglycans (HSPG) have been isolated from many different tissues and cells in culture. Moreover, even within a single tissue distinct HSPG forms appear to coexist. Some cell-surface-associated forms occur as integral membrane molecules [l, 21. Others appear to be peripherally membrane-bound through receptors for the glycosaminoglycan moiety [2], or through interaction with other cell-surface-associated HSPG [3]. Still other forms may not be directly cell-associated, but appear to be constituent parts of the extracellular matrix proper, e.g. as components of basement membranes [4,5]. At these different sites, and under these different modes of association with the cell surface, HSPG appear to be involved in a variety of functions, such as cell attachment and spreading [6,7], maintenance of cell shape [8], growth control [Y, lo], anticoagulation [ll], ultrafiltration [12] and matrix assembly [13]. The relevance, however, of the different HSPG forms for these diverse functions has still to be established.In search of systems allowing one to define such structurefunction relationships, several reports have dealt with the production, turnover and localization of HSPG in cultured fibroblasts, including human skin [3,[14][15][16] and lung [17-191 fibroblasts, and have investigated the mode of association of HSPG with these cells. Both detergent-susceptible and -insensitive forms have been described, suggesting that some HSPG may be integral membrane molecules [lY].In the present report we extend these studies on the mode of association of HSPG with these cells and describe the HSPG copurifying with membranes of human lung

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Human lung fibroblasts produce heparan sulphate proteoglycans (HSPG) that are associated with the plasma membrane. A monoclonal-antibody (Mab)-secreting hybridoma, S1, was produced by fusion of SP 2/0-AG 14 mouse myeloma cells with spleen cells from mice immunized with partially purified cellular HSPG fractions. The HSPG character of the material carrying the epitope recognized by Mab S1 was demonstrated by: (i) the co-purification of the S1 epitope with the membrane HSPG of human lung fibroblasts; (ii) the decrease in size of the material carrying the S1 epitope upon treatment with heparinase or heparitinase, and the resistance of this material to heparinase treatment after N-desulphation. The S1 epitope appears to be part of the core protein, since it was destroyed by proteinase treatment and by disulphide-bond reduction, but not by treatments that depolymerize the glycosaminoglycan chains and N-linked oligosaccharide chains. Polyacrylamide-gel electrophoresis of non-reduced heparitinase-digested membrane HSPG followed by Western blotting and immunostaining with Mab S1 revealed a single band with apparent molecular mass of 64 kDa. Membrane proteoglycans isolated from detergent extracts or from 4 M-guanidinium chloride extracts of the cells yielded similar results. Additional digestion with N-glycanase lowered the apparent molecular mass of the immunoreactive material to 56 kDa, suggesting that the core protein also carries N-linked oligosaccharides. Fractionation of 125I-labelled membrane HSPG by immuno-affinity chromatography on immobilized Mab S1, followed by heparitinase digestion and polyacrylamide-gel electrophoresis of the bound material, yielded a single labelled band with apparent molecular mass 64 kDa. Treatment with dithiothreitol caused a slight increase in apparent molecular mass, suggesting that the core protein of this membrane proteoglycan of a single subunit containing (an) intrachain disulphide bond(s).