Expression and Characterization of Recombinant Subunits of Human Complement Component C8: Further Analysis of the Function of C8α and C8γ

Abstract

Human C8 is composed of three nonidentical subunits (C8α, C8β, and C8γ) that are encoded in separate genes. In C8 isolated from serum, these are arranged as a disulfide-linked C8α-γ dimer that is noncovalently associated with C8β. In this study, a recombinant form of C8α-γ was expressed independently of C8β in insect cells and COS-7 cells and was shown to be equivalent to serum-derived C8α-γ with respect to its ability to combine with C8β and form functional C8. Also expressed separately were mutant (mut) forms of C8α and C8γ in which the single interchain disulfide bond was eliminated. MutC8α exhibited the ability to combine with C8β and express hemolytic activity, although at a lower level than human C8. Addition of purified mutC8γ increased this activity, presumably by binding to mutC8α. A possible role for C8γ as a retinol binding protein was also investigated. Absorbance spectroscopy and fluorescence emission and quenching revealed no specific binding of retinol to mutC8γ. Together, these results indicate that 1) the biosynthesis and secretion of C8α-γ is not dependent on C8β, which is consistent with in vivo observations in C8β-deficient humans; 2) C8α can be synthesized independently of C8γ; therefore, protection of C8α from premature membrane interactions during biosynthetic processing is not a likely function of C8γ; 3) C8γ enhances but is not required for expression of C8 activity; and 4) C8γ does not bind retinol; therefore, it cannot function as a retinol transport protein.