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Phylogeny of C4b-C3b cleaving activity: similar fragmentation patterns of human C4b and C3b produced by lower animals
Insights
Complement system proteins C4b and C3b are cleaved by regulatory proteins across diverse species, indicating ancient structural similarities. This study reveals conserved proteolytic activity in vertebrate plasma, predating humans by 300 million years.
Area of Science:
- Immunology and Evolutionary Biology
- Complement System Proteolysis
- Biochemical Analysis of Vertebrate Plasma
Background:
- The complement system, a crucial part of innate immunity, involves a cascade of protein activations.
- Regulatory proteins like factor I, H, and C4b-binding protein (C4bp) control complement activation by cleaving C4b and C3b.
- Understanding the evolutionary conservation of these cleavage pathways provides insights into immune system development.
Purpose of the Study:
- To investigate the capacity of plasma and serum from various phylogenetic species to cleave human C4b and C3b.
- To identify and compare the resulting cleavage fragments with those generated by human serum.
- To explore the evolutionary origins and conservation of complement regulatory protein activity.
Main Methods:
- Incubation of fluid-phase and cell-bound human C4b and C3b with plasma/serum from mammals, reptiles, amphibia, and fishes.
- Analysis of cleavage products using gel electrophoresis to identify specific fragments.
- Investigation of ion dependencies (Ca2+, Mg2+, Ba2+) for proteolytic activity in fish serum/plasma.
Main Results:
- Plasma/serum from mammals, reptiles, amphibia, and fishes effectively cleaved human C4b and C3b, producing similar fragments as human serum.
- Birds showed limited C4b degradation and no C3b cleavage activity; primitive vertebrates (chondrichthyes, agnatha) lacked this activity.
- Fish serum/plasma exhibited a unique Ca2+ ion requirement for C4b and C3b degradation, with Mg2+/Ba2+ yielding an intermediate product.
Conclusions:
- Proteolytic activity against complement activation products is present in species predating humans by 300 million years.
- Human C4b and C3b share structural characteristics with their evolutionary ancestors, as evidenced by conserved cleavage patterns.
- The findings highlight the ancient evolutionary origins of complement regulatory mechanisms and protein recognition.
Abstract:
Functional and structural studies of the activated proteins of the complement system C4b and C3b have led to the identification of cleavage products resulting from the effect of the regulatory proteins, factor I, H, and C4b binding protein (bp). In this paper we report the results of studies that investigated the capacity of plasma or serum from a wide range of phylogenetic species to yield similar cleavage products. Sera and plasma from mammals, reptiles, amphibia, and fishes are capable of cleaving fluid phase human C4b and C3b, generating apparently the same fragments as observed using normal human serum: alpha 2, alpha 3, alpha 4 from the alpha' chain of C4b: and alpha-68, alpha-46, alpha-43, and alpha-30 from the alpha' chain of C3b. When C3b bound to a cell membrane is used C3c and C3dg are generated. The generation of these fragments from C3bi is a dose-dependent reaction. There is no correlation between the evolution of the species and the quantitative capability to degrade the substrates. Birds possess only a limited capability to degrade the alpha' chain of C4b and have no cleaving activity for C3b, whereas sera from more primitive vertebrate species (chondrichthyes and agnatha) fail to participate in the reaction. Contrary to other species, the proteins in fish serum or plasma responsible for the degradation of C4b and C3b show a unique requirement for Ca2+ ions. Magnesium and barium are less effective, and in their presence a 65,000 dalton intermediate product is observed. These results demonstrate that protein(s) displaying proteolytic activity for products of complement activation, probably related to I, H, and C4bp, are present in plasma of species whose evolution have preceded humans by 300 million years. Moreover, the recognition of human substrates and the generation of fragments identical to those produced by human serum suggests that human C4b and C3b share structural characteristics with their evolutionary ancestors in the serum or plasma of the species studied.