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Molecular cloning of rat C4b binding protein alpha- and beta-chains: structural and functional relationships among
Insights
Researchers found that rats possess a C4b-binding protein (C4BP) complex with anticoagulant protein S, a conserved interaction not seen in all mammals. This discovery highlights C4BP
Area of Science:
- Immunology
- Biochemistry
- Complement System Regulation
Background:
- C4b-binding protein (C4BP) regulates the complement system.
- Human C4BP interacts with anticoagulant protein S and serum amyloid P component (SAP).
- Previous studies indicated a lack of C4BP-protein S complex in rabbit and bovine plasma, with mouse beta-chain gene pseudogenization.
Purpose of the Study:
- To investigate the presence of a C4BP-protein S complex in rat plasma.
- To characterize the interaction between rat C4BP and SAP.
- To determine the sequence identity of rat C4BP alpha- and beta-chains with other species.
Main Methods:
- Gel filtration chromatography and Western blotting to detect protein complexes.
- Isolation of rat C4BP alpha- and beta-chain cDNA clones from a rat liver cDNA library.
- Amino acid sequence analysis and comparison between rat and other species' C4BP chains.
Main Results:
- A C4BP-protein S complex was detected in rat plasma, similar to humans.
- Rat C4BP did not form a complex with rat SAP but did with human SAP.
- Rat C4BP alpha-chain showed 64% identity with mouse and 60% with human counterparts.
- Rat C4BP beta-chain showed 68% identity with human beta-chain.
Conclusions:
- The C4BP-protein S interaction is conserved in rats, representing the first non-primate species with this conserved interaction.
- Rat C4BP exhibits species-specific interactions with SAP.
- Sequence analysis provides insights into the evolutionary conservation of C4BP function.
Abstract:
The C4b binding protein (C4BP) functions as a regulator of the complement system by interacting with the activated form of the fourth complement component, C4b. Human C4BP also interacts with the anticoagulant protein S and the serum amyloid P component (SAP). It is composed of seven identical 70-kDa alpha-chains and one 45-kDa beta-chain. The alpha-chain contains a binding site for C4b, whereas the beta-chain contains the protein S binding site. Recent studies have shown rabbit and bovine plasma to lack a C4BP-protein S complex, and the mouse beta-chain gene to have evolved into a pseudogene. Using a gel filtration chromatography system in combination with Western blotting, we detected a complex between C4BP and protein S in rat plasma, similar to the complex known in human plasma. Using purified rat C4BP and SAP we were unable to detect any complex between the two proteins, but rat C4BP was able to form a complex with human SAP. Rat cDNA clones encoding the C4BP alpha- and beta-chains were isolated from a rat liver cDNA library. The rat alpha-chain cDNA predicted a mature polypeptide chain of 545 amino acid residues, whereas the beta-chain cDNA predicted a mature polypeptide of 243 amino acid residues. The overall amino acid sequence identities between the rat alpha-chain and the mouse, human, rabbit, and bovine alpha-chains were 64, 60, 59, and 52%, respectively. The identities between the rat beta-chain and the human and bovine beta-chains were 68 and 57%, respectively. The rat represents the first non-primate species in which the C4BP-protein S interaction has been found to be conserved.