Isolation of a novel complement regulatory factor (GCRF) from glomerular epithelial cells.
Cultured rat glomerular epithelial cells (GEC) are able to prevent both antibody-directed and spontaneous (alternative pathway) complement activation. In this study, a novel complement regulatory factor (GCRF) was isolated from GEC. The ability to accelerate the decay of alternative pathway C3/C5 convertases formed on sheep erythrocytes (EC3bBbP) was used to guide purification. GEC were solubilized in Triton X-114 and GCRF was recovered in the aqueous phase. Complement inhibitory material also was present in the culture supernatant, which likely represented GCRF. By Mono Q anion exchange chromatography, GCRF eluted at greater than or equal to 0.6 M NaCl and by Superose 6 size-exclusion chromatography, it had a Kav less than or equal to 0.3. GCRF reduced the t1/2 of EC3bBbP from 128 minutes in buffer alone to 41 minutes in 3 micrograms/ml GCRF protein, and also prevented formation of EC3bBbP in a dose-dependent fashion. Digestion with chondroitinase ABC, neuraminidase, or trypsin, but not with heparitinase or chondroitinase AC significantly reduced the activity and size of GCRF, demonstrating that it is a sialic acid-containing dermatan sulfate proteoglycan. Thus, cultured rat GEC synthesize and secrete into the medium, GCRF, a dermatan sulfate proteoglycan with complement inhibitory activity.
Quigg
R J
RJ
Department of Internal Medicine, Medical College of Virginia, Richmond.
D000818:Animals; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000302:isolation & purification* / Q000494:pharmacology; D002850:Chromatography, Gel; D003169:Complement Inactivator Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D003871:Dermatan Sulfate / Q000302:isolation & purification* / Q000494:pharmacology; D004588:Electrophoresis, Cellulose Acetate; D004798:Enzymes; D004848:Epithelium / Q000737:chemistry / Q000276:immunology; D007678:Kidney Glomerulus / Q000737:chemistry / Q000276:immunology*
D011508:Chondroitin Sulfate Proteoglycans; D003169:Complement Inactivator Proteins; D004798:Enzymes; C032649:dermatan sulfate proteoglycan; D003871:Dermatan Sulfate