Native conformations of human complement components C3 and C4 show different dependencies on thioester formation

Area of Science:

• Biochemistry
• Immunology
• Molecular Biology

Background:

• The thioester bond in complement proteins C3 and C4, and alpha2-macroglobulin, is traditionally believed to be crucial for covalent attachment and maintaining native conformations.
• Previous studies showed human C3 mutants (E1012Q, C1010A) lacking a thioester bond retained a native-like conformation, cleavable by C3 convertase, unlike the C3b-like C3(H2O) species.
• A transient species, C3(NH3)*, formed via aminolysis, was proposed to explain these observations, exhibiting distinct HPLC mobility and convertase cleavability.

Purpose of the Study:

• To investigate whether C3 thioester-region mutants resemble the C3(NH3)* species or native C3.
• To assess the general applicability of these findings by engineering equivalent mutations into human C4.
• To determine if the native conformational state can be adopted and maintained in C3 and C4 mutants lacking a thioester bond.

Main Methods:

• Utilized cation-exchange HPLC as a conformational probe for C3 mutants (C1010A, E1012Q).
• Engineered equivalent mutations (C1010A, E1012Q) into human C4.
• Assessed C4 mutant conformation by their cleavability with C1s.

Main Results:

• C3 C1010A and E1012Q mutants exhibited chromatographic behavior indistinguishable from native C3, not resembling C3(NH3)*.
• Human C4 mutants C1010A and C4 E1012Q did not form thioester bonds.
• Unlike C3 mutants, C4 mutants showed no evidence of stable native-like conformation, as assessed by C1s cleavage.

Conclusions:

• The native conformational state can be adopted during biosynthesis of C3 and C4, even without thioester bond formation.
• This native-like conformation is stable in mature C3 but unstable in mature C4.
• The instability in C4 may be related to its additional post-translational cleavage before secretion.