Biosynthesis of a Single Chain PRO-C5 by Normal Mouse Liver mRNA: Analysis of the Molecular Basis of C5 Deficiency in AKR/J Mice

Abstract

Abstract An in vivo labeling technique has been used to probe the molecular lesions precipitating in C5 deficiency in the AKR/J mouse. 14C-labeled amino acids were administered i.p. into normal and C5-deficient mice, and the plasma was harvested 4 hr later. By using monospecific anti-C5, newly synthesized 14C-C5 was immunoprecipitated from the plasma and postmitochondrial supernatants (PMS) of a liver homogenate. By SDS-PAGE analysis it was demonstrated that normal mouse plasma (apparent m.w. 205,000) was composed of two dissimilar subunits, an α-chain (115,000 daltons) and a β-chain (82,000). However, nonsecreted C5 immunoprecipitated from the PMS was resolved into two nonreducible polypeptide chains of m.w. 200,000 and 170,000, respectively. By comparison to plasma C5, the 170,000 dalton peak polypeptide chain probably represents incompletely synthesized, partially degraded, or unglycosylated pro-C5. 14C-C5 immunoprecipitates from the plasma, and the PMS of AKR/J C5-deficient mice contained insignificant radioactivity and on SDS gels did not resolve into any distinct peaks, suggesting that C5 is not synthesized in this strain. 14C-C3 immunoprecipitated from the plasma of normal and AKR/J mice in each case was composed of covalently-linked α- and β-chains (m.w. 130,000 and 85,000, respectively), 14C-C3 immunoprecipitated from the PMS of normal and C5-deficient liver homogenates in each case migrated on SDS gels as a single polypeptide chain, pro-C3 (m.w. 200,000). Confirmation of these findings have been achieved by cell-free translation studies. Poly(A)-mRNA isolated from normal mouse liver stimulated the incorporation of 3H-leucine into protein in a time-dependent fashion in a reticulocyte lysate system under optimal conditions. 3H-C5 immunoprecipitated from the translation reaction mixture behaved as a single nonreducible polypeptide chain (m.w. 170,000). Poly A-mRNA from the liver of the AKR/J mouse displayed similar kinetics and dose-response stimulation of protein synthesis upon translation in the cell-free system, but failed to direct the synthesis of C5 or C5 immunoreactive peptides, although C3 was synthesized normally as pro-C3. Since the intact machinery for carbohydrate synthesis is not present in the reticulocyte cell-free system, it is envisaged that the 170,000-dalton C5 polypeptide chain is possibly unglycosylated pro-C5. Thus, C5 is synthesized as a single-chain pro-C5 and post-translationally converted to a two-subunit C5 molecule by limited proteolysis. In the AKR/J C5-deficient mouse C5 is not synthesized at all, suggesting the lack of a functional mRNA for C5 in this strain.