Mouse complement component C4 is devoid of classical pathway C5 convertase subunit activity.

Abstract

It has long been known that mouse C4 has unusually low hemolytic activity relative to the C4 of other mammalian species (e.g. human and guinea pig), the measurements being done in most cases using a C4-deficient guinea pig serum reagent in a one-step assay with EA. This low activity for mouse C4 previously had been attributed to "technical" difficulties such as lability of the protein during blood collection and partial species incompatibilities with guinea pig components. Recently, we presented evidence for the involvement of human C4 beta-chain residues 455-469, a putatively exposed hydrophilic segment, in contributing to a C5 binding site in the C4b subunit of the classical pathway C5 convertase, C4b3b2a. Given that there were five sequence differences between the human and mouse protein within this segment, we hypothesized that these substitutions may have compromised the C5 convertase subunit activity of mouse C4, thereby resulting in its low hemolytic activity. Using a multi-step hemolytic assay which was totally dependent upon C5 cleavage by the classical pathway, we found that mouse C4 was completely devoid of classical pathway C5 convertase subunit activity. We have been able to rule out the most obvious potential species incompatibilities (e.g. between C4mo and C5gp) as being responsible for this lack of activity. Moreover, we found that the low level of hemolytic activity of mouse C4 measured in the one-step assay can be ascribed totally to C5 cleavage, and subsequent terminal component assembly, by the alternative pathway C5 convertase, (C3b)2Bb. However, the assembly of the latter enzyme complex is dependent upon the presence of C3b molecules deposited initially via the classical pathway C3 convertase in which mouse C4b is a subunit. Finally, whereas conversion of human residues 458RP to the mouse-like sequence PL was sufficient to abrogate classical pathway C5 convertase subunit activity in human C4, the five substitutions which "humanized" the 452-466 segment of mouse C4 (corresponding to human residues 455-469) were on their own insufficient to impart this activity to mouse C4. This implies that, in addition to the 455-469 beta-chain segment of human C4, there are other regions of the molecule contributing to C5 binding which are also non-conserved between human and mouse C4.