NATIVE CONFORMATIONS OF HUMAN-COMPLEMENT COMPONENTS C3 AND C4 SHOW DIFFERENT DEPENDENCIES ON THIOESTER FORMATION

The thioester bond in complement components C3 and C4 and the protease inhibitor alpha(2)-macroglobulin have traditionally been thought of a s fulfilling the dual roles of mediating covalent attachment and maint aining the native conformational states of these molecules. We previou sly reported that several human C3 thioester-region mutants, including variants E1012Q and C1010A, in the latter of which thioester-bond for mation is precluded, display an unexpected phenotype. Despite the lack of a thioester bond in these mutants, they appear to adopt a nativeli ke conformation as suggested by the finding that they are cleavable by the classical pathway C3 convertase, C4b2a, whereas the C3b-like C3(H 2O) species is not. Subsequently, a species referred to as C3(NH3) wa s described which potentially could account for the observations with the above mutants. C3(NH3) is a transient species formed on aminolysi s of native C3 that can spontaneously re-form the thioester bond. Impo rtantly, it has a mobility on cation-exchange HPLC that is distinct fr om both native C3 and C3(H2O), but like the native molecule, it is cle avable by an alternative-pathway C3 convertase. In this study we showe d by using cation-exchange HPLC as an additional conformational probe that C3 C1010A and E1012Q mutant proteins did not resemble C3(NH3). I nstead they displayed a chromatographic behaviour that was indistingui shable from that of native C3. To assess the general applicability of these observations, we engineered the equivalent mutations into human C4, specifically C4 C1010A and C4 E1012Q. As expected, thioester-bond formation did not occur in either of these C4 mutants, but in contrast with the results with C3 we found no evidence for the formation of a stable native-like conformation in either C4 mutant, as assessed using cleavability by Cls as the conformational probe. A possible interpret ation of our data is that the adoption of the native conformational st ate during biosynthesis of C3 and C4 is an energetically permissible p rocess, even if it is not locked in via thioester-bond formation. Wher eas this conformational state is stable in mature C3, it is unstable i n mature C4, perhaps reflecting the additional post-translational clea vage of C4 before its secretion.