Identification of a 64 kDa heparan sulphate proteoglycan core protein from human lung fibroblast plasma membranes with a monoclonal antibody

Abstract

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).

Reversible Regulation of Tissue Factor–Induced Coagulation by Glycosyl Phosphatidylinositol–Anchored Tissue Factor Pathway Inhibitor

“…15 In pooled low-density fractions from large quantities of cells (from ten 150-cm 2 flasks) that were treated with heparinase, the glypican 1-reactive antibody S1 detected a 68-kDa band (data not shown) that had a molecular mass corresponding to the published size of the glypican 1 core protein. 27 These data are consistent with localization of GPI-anchored proteoglycan receptors in the glycosphingolipid-rich microdomains. Attempts to study in detail the cellular partitioning of glypican 1 and 3 protein between detergent-soluble and -insoluble fractions failed because of low sensitivity and considerable background staining of available antibodies.…”

“…After resuspension, the pool was treated with 25 mU heparinase (Calbiochem) in 50 mmol/L Tris, 100 mol/L NaCl, 1 mmol/L PMSF, and 20 g/mL leupeptin, pH 7.0, for 2 hours at 37°C, 26 followed by SDS-PAGE for Western blotting with monoclonal antibody S1. 27 Expression of GPI-Anchored TFPI-1 in CHO-K1 Cells…”

Characterization of Syntenin, a Syndecan-binding PDZ Protein, as a Component of Cell Adhesion Sites and Microfilaments

“…Immunization schemes and the protocols for cell fusion and hybridoma selection were as described (De Boeck et al, 1987). Five clones were selected, based on the antibody binding profiles obtained in BIAcore: 1C8, 9C10, 4D12, 4F6, and 9E7.…”

In vitro model for hematopoietic progenitor cell homing reveals endothelial heparan sulfate proteoglycans as direct adhesive ligands

“…Monoclonal antibodies (mAb) and GAGs mAb against HSPGs used were antisyndecan-1 (clone B-B4, Serotec, Oxford, UK), antisyndecan-3 (clone 1C7) [28], antisyndecan-4 (clone 8G3), and antiglypican-1 (clone S1) [29], all kindly provided by Guido David (Center of Human Genetics, Leuven, Belgium), and CD44v3 (clone 3G5, R&D Systems, Abingdon, UK). Anti-HS antibodies used were JM403 [30], 10E4 [31], and 3G10 [31] (Seikagaku, Tokyo, Japan).…”