Multiple distinct membrane heparan sulfate proteoglycans in human lung fibroblasts.
Heparitinase digestion of the hydrophobic membrane-associated heparan sulfate proteoglycans (HSPG) of fetal human lung fibroblasts yields core proteins of various sizes: i.e. monomeric core proteins of 125, 90, 64, 48, and 35 kDa and a disulfide-linked dimeric core protein composed of approximately 35-kDa subunits. By immunizing BALB/c mice with liposome-incorporated HSPG, we have obtained a total of five anti-HSPG monoclonal antibodies (Mabs, i.e. Mabs S1, 1C7, 2E9, 6G12, and 10H4) with different specificities. Polyacrylamide gel electrophoresis of 125I-labeled membrane HSPG immunoprecipitated with these Mabs revealed that Mabs 1C7 and 2E9 bind only membrane HSPG which yield a 125-kDa core protein after heparitinase digestion, whereas Mab S1-bound HSPG yield a 64-kDa core protein, and Mabs 6G12 and 10H4 retain membrane HSPG with a 48-kDa core protein. Western blotting of the heparitinase-digested proteoglycans and immunostaining with the Mabs confirmed this pattern of reactivity. However, in this assay, Mabs 6G12 and 10H4 also detected a minor approximately 90-kDa core protein in addition to the 48-kDa core protein. Except perhaps for the 10H4 epitope, the epitopes recognized by these Mabs appear to be part of the peptide moieties as they resisted complete deglycosylation of the HSPG with trifluoromethanesulfonic acid. Since these data were inconsistent with a direct relationship between the major core proteins, the 48-, 64-, and 125-kDa core proteins were immunopurified and further compared by peptide mapping with Staphylococcus aureus protease V8, trypsin, and CNBr cleavage. Clearly distinct peptide patterns were obtained for the three different core proteins. These results imply that the 48-, 64-, and the 125-kDa membrane HSPG core proteins of human lung fibroblasts are derived from distinct proteoglycans.
Lories
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V
Center for Human Genetics, University of Leuven, Belgium.
Cassiman
J J
JJ
Van den Berghe
H
H
David
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G
D053674:Aggrecans; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000032:analysis*; D016326:Extracellular Matrix Proteins; D005347:Fibroblasts / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis* / Q000276:immunology; D006025:Glycosaminoglycans / Q000032:analysis*; D006031:Glycosylation; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000032:analysis*; D006801:Humans; D037181:Lectins, C-Type; D008081:Liposomes; D008168:Lung / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis; D008970:Molecular Weight; D010449:Peptide Mapping; D011133:Polysaccharide-Lyases / Q000494:pharmacology; D011509:Proteoglycans / Q000032:analysis*
D053674:Aggrecans; D000911:Antibodies, Monoclonal; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D037181:Lectins, C-Type; D008081:Liposomes; D008565:Membrane Proteins; D011133:Polysaccharide-Lyases; D011509:Proteoglycans; C021349:heparitinsulfate lyase