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Mapping of the complement regulatory domains in the human factor H-like protein 1 and in factor H1
1Department of Molecular Biology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Insights
Human factor H-like protein 1 (FHL-1) and factor H share critical N-terminal domains. All four N-terminal short consensus repeats (SCRs) are essential for FHL-1’s cofactor activity in complement regulation.
Area of Science:
- Complement system biology
- Protein structure-function relationships
- Immunology
Background:
- Human factor H-like protein 1 (FHL-1) comprises seven short consensus repeats (SCRs), mirroring the N-terminal SCRs of complement regulatory protein factor H.
- FHL-1 is a 42-kDa human plasma protein, distinct from the 43-kDa factor H-related 1 beta protein, though they co-migrate.
Purpose of the Study:
- To identify the specific SCRs of FHL-1 responsible for its cofactor activity.
- To elucidate the structural requirements for FHL-1’s function in factor I-mediated cleavage of C3b.
Main Methods:
- Recombinant expression of FHL-1 and its truncated forms using the baculovirus system.
- Immunoblotting to identify and characterize native FHL-1 in human plasma.
- Functional assays to assess cofactor activity in C3b cleavage.
Main Results:
- Recombinant FHL-1 exhibits cofactor activity, similar to factor H, in the factor I-mediated cleavage of C3b.
- Truncated FHL-1 forms containing SCRs 1 to 4 demonstrated cofactor activity.
- Deletion mutants lacking any of the N-terminal SCRs 1-4, or altered spacing between SCRs, were functionally inactive.
Conclusions:
- The four N-terminal SCRs of FHL-1 are essential for its cofactor activity in C3b cleavage.
- The precise spacing between these SCR domains is critical for maintaining FHL-1’s functional integrity.
Abstract:
The human factor H-like protein 1 (FHL-1) is composed of seven repetitive domains (short consensus repeats; SCRs) that are identical in sequence to the seven NH2-terminal SCRs of the complement regulatory protein factor H. We have identified the native FHL-1 protein as a 42-kDa human plasma protein by immunoblotting and by comparing the mobility to that of a recombinant FHL-1 protein. Here, we demonstrate the existence of two distinct co-migrating human plasma proteins that represent the 42-kDa FHL-1 protein and the previously identified 43-kDa factor H-related 1 beta protein. Similar to factor H, the recombinant FHL-1 protein displays cofactor activity in factor I-mediated cleavage of C3b. To identify relevant SCRs of factor H and FHL-1, we recombinantly expressed the domains shared between the two proteins in the baculovirus expression system. Recombinant FHL-1 and all truncated forms that include SCRs 1 to 4 displayed cofactor activity. All four NH2-terminal SCRs are essential, as deletion mutants composed of SCR 1 and 4 only; of SCRs 1, 2, and 4 only, or of SCRs 1, 3, and 4 only were functionally inactive. Similarly, the distance between these individually folding domains is critical for function, as a recombinant protein that had two and four amino acids inserted between SCRs 1 and 2 or between SCRs 3 and 4, respectively, had no activity. These results demonstrate that all four NH2-terminal SCRs of FHL-1 (and of factor H) are required for cofactor activity in factor I-mediated cleavage of C3b, and that the distance between these SCRs is essential.