Synthesis and regulation of the two human complement C4 genes in stable transfected mouse fibroblasts.

N Miura,H L Prentice,P M Schneider,D H Perlmutter

doi:10.1016/s0021-9258(18)48236-1

Abstract

The major histocompatibility complex-linked human complement C4 genes are highly homologous in primary structure but give rise to products which differ in complement-activating function. In order to examine the synthesis, function, and regulation of these two genes independently, cloned C4A and C4B genes were transfected into mouse fibroblast L-cells. In the stable transfected cell lines, C4A and C4B are synthesized, undergo a complex series of post-translational modifications, and each functions appropriately in activation of the classical complement pathway. A marked difference in the kinetics of complement component C1-mediated cleavage of the C4A- and C4B-alpha chains was demonstrated in the transfectants and may contribute to the differences in the intrinsic functional activity of the two C4 isotypes. In contrast to the expression of other complement genes which are affected during the hepatic "acute phase response" (factor B, C3), the expression of C4 was not regulated by interleukin-1 or tumor necrosis factor. Interferon-gamma, however, mediated a dose- and time-dependent increase in the expression of the C4 genes. Moreover, interferon had a significantly greater and longer-lasting effect on the synthesis of C4A than that of C4B. Differences in the expression and regulation of these two genes provide insight into the control of complement activation during inflammation.

Highlights

The C4 protein is synthesized in liver as anapproximately 185-kDa single-chain hemolytically inactive precursor, proC4, composedof three disulfide-linked peptide chains, a, /3 (-78 kDa), and y
Arginine-rich intersubunit linking peptides are excised by the action of plasmin, or plasmin-like enzymes, and an approximately 5kDa fragment from the carboxyl terminus of the a chain is cleaved in the extracellular fluid by the action of a metalloelastase
The C4 genes are characterized by a high degree of polymorphism; more than 30 alleles, including null alleles, have been determined by electrophoretic mobility [22]

Summary

RESULTS

Synthesis of C4 in TransfectedL-cells and HumanHepatoma Cells-In previous studies Southern blot analysis of cosmid DNAs isolated from a human genomic library (JY cell line; Priesshuman lymphoblastoid cell line) indicated that the C4A gene and at least 5 kb of 5' and 3' flanking sequence was spanned by cosmid 4.1 [55] and that the C4B gene and at least 10 kb of5' flanking sequence was spanned by cosmid V1B (Ref. 34 and Fig. la). Genomic DNA from LC1, LC2, parent untransfected L-cells (Ltk-), and HepG2 cells was subjected to Southern blot analysis using radiolabeled human C4 cDNA (pC4 AL1) [56] as probe. A 6.5-kb KpnI and 14-kb BglII fragment was present in LC1,LC2, and HepG2 butnot in theparent untransfected cell line (Fig. lb). These fragmentsco-migrated with those of cosmids 4.1 and V1B. A 5.5-kb C4 mRNA was present in LC1, LC2, and HepG2 but not in Ltk- cells when total cellular RNA was subjected to RNA blot analysis with radiolabeled human C4cDNA pC4AL1. A 5.5-kb mRNAwas present in LC2 and HepG2 cells but not in LC1 and Ltk- cells probed with the 7300 a

HH CD kb

IOOr b

DISCUSSION

Although most individuals have approximately equivalent