Human Keratinocytes and A-431 Cells Synthesize and Secret Factor B, the Major Zymogen Protease of the Alternative Complement Pathway

Biosynthetic radiolabeling studies demonstrate that human keratinocytes and A-431 cells a human epidermoid earn cell line, synthesize and secrete factor B as a meric 105 kD protein. Epithelial cell derived facto B comigrates in SDS PAGE with that produced by HepG2 cells, a human hepatoma cell line traditionally employed in studies complement component biosynthesis. Comparative chase studies in A 431 arid HepG2 cell show that this native pathway complement component is produced as co migrating 100-kD intracellular proteins that are processed in both cell types to 105 kD extracellular factor B. Quantitatively, immunoprecipitable factor B account for 0.005 of radiolabeled proteins in A 431 cell culture media. Treatment of biosynthetically radiolabeled A 431 cell culture media with cobra venom factor and factor D for 60 mm at 37degree C produces the specific factor Bcleavage products Ba and Bb. These fragments are not identifiable in control culture media subjected to similar treatment in the absence of alternative pathway activators. Northern blot analysis of total cellular RNA from human keratinocytes, A-431 cells, and HepG2 cells reveals qualitative identity of a 2.8 kb factor B mRNA species in these three cell types. The relative level of factor B mRNA expression in these cells parallels their level of factor B protein synthesis (i.e., HepG2 cells > A-431 cells > human keratinocytes). Epithelial cell derived factor B may play an important role in local inflammatory reaction and also directly interact with epithelial cell derived C3—a key classical and alternative pathway complement component recently shown to be produced by human keratinocytes