EXPRESSION AND CHARACTERIZATION OF RECOMBINANT SUBUNITS OF HUMAN-COMPLEMENT COMPONENT C8 - FURTHER ANALYSIS OF THE FUNCTION OF C8-ALPHA ANDC8-GAMMA

Human C8 is composed of three nonidentical subunits (C8 alpha, C8 beta , and C8 gamma) that are encoded in separate genes. In C8 isolated fro m serum, these are arranged as a disulfide-linked C8 alpha-gamma dimer that is noncovalently associated with CSP, In this study, a recombina nt form of C8 alpha-gamma was expressed independently of C8 beta in in sect cells and COS-7 cells and was shown to be equivalent to serum-der ived C8 alpha-gamma with respect to its ability to combine with C8 bet a and form functional C8, Also expressed separately were mutant (mut) forms of C8 alpha and C8 gamma in which the single interchain disulfid e bond was eliminated, MutC8 alpha exhibited the ability to combine wi th C8 beta and express hemolytic activity, although at a lower level t han human C8. Addition of purified mutC8 gamma increased this activity , presumably by binding to mutC8 alpha, A possible role for C8 gamma a s a retinol binding protein was also investigated. Absorbance spectros copy and fluorescence emission and quenching revealed no specific bind ing of retinol to mutC8 gamma, Together, these results indicate that 1 ) the biosynthesis and secretion of C8 alpha-gamma is not dependent on C8 beta, which is consistent with in vivo observations in C8 beta-def icient humans; 2) C8 alpha can be synthesized independently of C8 gamm a; therefore, protection of C8 alpha from premature membrane interacti ons during biosynthetic processing is not a likely function of C8 gamm a; 3) C8 gamma enhances but is not required for expression of C8 activ ity; and 4) C8 gamma does not bind retinol; therefore, it cannot funct ion as a retinol transport protein.