IN-VITRO EXPRESSION OF THE BETA-SUBUNIT OF HUMAN-COMPLEMENT COMPONENTC8

Human C8 is one of five components of the cytolytic C5b-9 complex of c omplement. It is an oligomeric protein composed of three subunits (alp ha, beta, gamma) encoded in separate genes. These are arranged as a di sulfide-linked alpha-gamma dimer and a noncovalently associated beta c hain. Biosynthesis studies and analyses of humans with hereditary Cs d eficiencies suggest that C8 alpha-gamma synthesis and secretion can oc cur independently of C8 beta, but that serum levels of C8 beta are dep endent on C8 alpha-gamma. One aim of the present study was to determin e if functional human C8 beta could be synthesized in the absence of C 8 alpha-gamma. Human C8 beta expression constructs were prepared and u sed to produce recombinant C8 beta (rC8 beta) in insect and COS-7 cell s. Both cell types secreted rC8 beta that was similar in size to human C8 beta and exhibited similar ability to associate with human C8 alph a-gamma and form functional Cs. A mutant form of C8 beta in which N-gl ycosylation sites were eliminated was also expressed and found to be f unctionally similar to rC8 beta and human C8 beta. These results indic ate that C8 alpha-gamma is not required for intracellular processing a nd secretion of C8 beta. Furthermore, N-linked carbohydrate on C8 beta is not necessary for association with C8 alpha-gamma or for Cs activi ty. (C) 1997 Elsevier Science Ltd.