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Physiologic inactivation of fluid phase C3b: isolation and structural analysis of C3c, C3d,g (alpha 2D), and C3g
Insights
This study characterizes complement component 3b (C3b) inactivation fragments, C3c and C3d,g, isolated from human blood. Findings clarify the order of peptides within the C3b alpha-chain, advancing understanding of complement system regulation.
Area of Science:
- Immunology
- Complement System
- Proteolysis
Background:
- The inactivation of complement component 3b (C3b) generates various fragments, but their complete characterization remains incomplete.
- Initial C3b inactivation involves factor I and its cofactors, leading to the formation of iC3b, C3c, and C3d,g.
- Understanding these fragments is crucial for comprehending complement system regulation and its role in immune responses.
Purpose of the Study:
- To isolate and structurally characterize C3c and C3d,g fragments generated from C3b inactivation in human blood.
- To compare C3 fragments generated in vitro with those isolated from patients with in vivo complement activation.
- To definitively order the C3c and C3d,g peptides within the alpha-chain of C3b.
Main Methods:
- Complement activation in whole blood using cobra venom factor.
- Isolation of C3 fragments using solid-phase monoclonal antibodies targeting C3g and C3c.
- Structural characterization via SDS-PAGE and amino-terminal sequence analysis.
Main Results:
- C3c isolated from normal blood comprised polypeptides of 75,000, 43,000, and 27,000 Da; C3d,g was a 40,000 Da single chain.
- Amino-terminal sequencing confirmed the origin of C3c peptides from the C3b alpha'-chain and C3g from C3d,g.
- Analysis of patient samples revealed complex C3 fragment patterns, including potential iC3b or iC3b-like products.
Conclusions:
- The study definitively orders C3c and C3d,g peptides within the C3b alpha-chain.
- The findings contribute to a more complete understanding of C3b inactivation pathways.
- Characterization of C3 fragments aids in diagnosing and understanding complement-mediated diseases.
Abstract:
The fragments that result from the inactivation of C3b have not been completely characterized. Initial inactivation is catalyzed by the protease factor I, which, in the presence of its cofactor (factor H), cleaves two peptide bonds in the alpha'-chain of C3b. This results in the release of a small peptide (C3f, Mr 3000) from iC3b, which consists of the C3 beta chain covalently bonded to two alpha'-chain-derived peptides (Mr 68,000 and Mr 43,000). Surface-bound iC3b is cleaved at a third site by factor I to produce C3c and C3d,g (or alpha 2D). The factor I cofactor for this cleavage is the C3b receptor that is present on erythrocyte and leukocyte membranes. This report describes the isolation and initial structural characterization of C3c and C3d,g generated in whole blood after complement activation with cobra venom factor. These fragments were compared with the C3 fragments isolated from the serum and plasma of a patient with complement activation in vivo. The fragments were isolated with two solid phase monoclonal antibodies, one of which recognizes a determinant on C3g (clone 9) and one of which recognizes a determinant on C3c (clone 4). C3c isolated from normal blood showed three polypeptides that had apparent m.w. of 75,000, 43,000, and 27,000. The C3d,g consisted of a single polypeptide chain with a m.w. of 40,000. Amino terminal sequence analysis showed that the Mr 27,000 peptide from C3c is derived from the amino terminal portion of the alpha'-chain of C3b, whereas the Mr 43,000 peptide is derived from the carboxy terminus of the same chain. Amino terminal sequence analysis showed also that C3g is derived from the amino terminus of C3d,g. The C3 fragments isolated from a patient with partial lipodystrophy, nephritic factor activity, low serum C3 levels, and circulating C3 cleavage products showed a more complicated pattern on SDS-PAGE. The fragment isolated with clone 9 had an apparent m.w. of 40,000, identical to C3d,g generated in vitro, and it had the same amino terminal sequence as C3d,g generated in vitro. The eluate from insolubilized clone 4, however, showed prominent bands with Mr of 75,000, 56,000, 43,000, and 27,000, together with a triple-banded pattern at 68,000 and a minor band at 80,000. This eluate thus appears to contain C3c, and iC3b or an iC3b-like product. The origin of the Mr 56,000 and Mr 80,000 peptides have not yet been determined. These studies, with previous data, definitively order the C3c and C3d,g peptides in the alpha-chain of C3.(ABSTRACT TRUNCATED AT 400 WORDS)