Basement membrane heparan sulfate proteoglycan is the main proteoglycan synthesized by glomerular epithelial cells in culture.
The production and distribution of basement membrane-type heparan sulfate proteoglycans (BM HSPG) were investigated in a mouse glomerular epithelial cell line. Confluent cell monolayers were radiolabeled with [35S]sulfate or [35S]cysteine. Proteoglycans were isolated from the medium and cell layers by ion exchange chromatography and their nature determined by enzyme digestion (chondroitinase ABC) or degradative treatment (nitrous acid). It was found that more than 80% of the proteoglycans in both the cell layer and medium were heparan sulfate proteoglycans (HSPG) based on their susceptibility to nitrous acid degradation. More than half of the HSPG in the cell layer could be precipitated with an antiserum that specifically recognizes BM HSPG; only 10% of those released into the medium were precipitated with this antiserum. When immunoprecipitates of [35S] sulfate-labeled proteoglycans were analyzed by SDS-PAGE, the mature proteoglycans ran as a broad band at the top of the gel. When immunoprecipitates of [35S]cysteine-labeled proteoglycans were similarly analyzed, a 250 kd precursor core protein band was seen in addition to the mature proteoglycan. When BM HSPG were localized by immunofluorescence and immunoelectron microscopy (immunoperoxidase), they were found intracellularly in biosynthetic compartments (ER and Golgi cisternae) and extracellularly in deposits of basement membrane-like matrix located beneath and between the cells. These results indicate that l) BM HSPG are the predominant type of proteoglycans made by glomerular epithelial cells in culture; 2) these HSPG are assembled into a loosely organized matrix that is deposited beneath and between the cells; and 3) this cell type produces a higher proportion of BM HSPG than other cultured epithelial cells studied previously.
Stow
J L
JL
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Soroka
C J
CJ
MacKay
K
K
Striker
L
L
Striker
G
G
Farquhar
M G
MG
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001485:Basement Membrane / Q000032:analysis / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism / Q000648:ultrastructure; D005109:Extracellular Matrix / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism*; D006497:Heparan Sulfate / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D007150:Immunohistochemistry; D007678:Kidney Glomerulus / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D011509:Proteoglycans / Q000032:analysis / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans