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 a 0.6 M NaCl and by Superose 6 size-exclusion chromatography, it had a K_av ≤ 0.3. GCRF reduced the t_1/2 of EC3bBbP from 128 minutes in buffer alone to 41 minutes in 3 μg/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.