Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. Isolation, purification, and core protein characterization.
Confluent cultured human lung fibroblasts were labeled with 35SO4(2-). After 48 h of labeling, the pericellular matrix was prepared by Triton X-100 and deoxycholate extraction of the monolayers. Heparan sulfate proteoglycan (HSPG) accounted for nearly 80% of the total matrix [35S]proteoglycans. After solubilization in 6 M guanidinium HCl and cesium chloride density gradient centrifugation, the majority (78%) of these [35S] HSPG equilibrated at an average buoyant density of 1.35 g/ml. This major HSPG fraction was purified by ion-exchange chromatography on Mono Q and by gel filtration on Sepharose CL-4B, and further characterized by gel electrophoresis and immunoblotting. Intact [35S]HSPG eluted with Kav 0.1 from Sepharose CL-4B, whereas the protein-free [35S]heparan sulfate chains, obtained by alkaline borohydride treatment of the proteoglycan fractions, eluted with Kav 0.45 (Mr approximately 72,000). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, core (protein) preparations, obtained by heparitinase digestion of 125I-labeled HSPG fractions, yielded one major labeled band with apparent molecular mass of approximately 300 kDa. Reduction with beta-mercaptoethanol slightly increased the apparent Mr of the labeled band, suggesting a single polypeptide structure and the presence of intrachain disulfide bonds. Immunoadsorption experiments and immunostaining of electrophoretically separated heparitinase-digested core proteins with monoclonal antibodies raised against matrix and cell surface-associated HSPG suggested that the major matrix-associated HSPG of cultured human lung fibroblasts is distinct from the HSPG that are anchored in the membranes of these cells. Binding studies suggested that this matrix HSPG interacts with several matrix components, both through its glycosaminoglycan chains and through its heparitinase-resistant core. Core (protein) interactions seem to be responsible for the association of the proteoglycan with the extracellular matrix.
D000911:Antibodies, Monoclonal; D002458:Cell Fractionation; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification*; D000939:Epitopes / Q000032:analysis; D005109:Extracellular Matrix / Q000032:analysis* / Q000648:ultrastructure; D005347:Fibroblasts / Q000032:analysis; D006025:Glycosaminoglycans / Q000302:isolation & purification*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D008168:Lung / Q000032:analysis*; D008970:Molecular Weight; D011509:Proteoglycans / Q000302:isolation & purification*; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes
D000911:Antibodies, Monoclonal; D011508:Chondroitin Sulfate Proteoglycans; D000939:Epitopes; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans; D013431:Sulfates; D013462:Sulfur Radioisotopes