Genetic, molecular and functional analyses of factor I - an inhibitor of the complement system
Sammanfattning: Factor I (FI) is a serine protease that inhibits the complement system by cleaving activated C3 and C4 complement proteins, in the presence of cofactors. Mutations in the gene coding for FI have been identified in complete FI deficient- and atypical hemolytic uremic syndrome (aHUS) patients.
The mutations were introduced in recombinantly expressed proteins, purified and analyzed in functional assays. The mutations are homozygous or compound heterozygous in patients with complete FI deficiency and the mutations mostly affect the folding of the FI protein, thereby hindering secretion. In contrast, the mutations in aHUS patients are heterozygous. One of the mutations, G243D, identified in three unrelated aHUS patients did not have any effect on the expression, secretion or function of FI. It was shown later that these three patients have additional mutations, deletions or autoantibodies against other complement proteins. In the other two studies we detected five mutants that showed impaired function for degrading C3b present on cell surfaces. Only two of these five mutants also had reduced activity in the cleavage of C3b and C4b in the fluid-phase. The rest of the mutations, with pre-mature stop codons and some missense mutations, resulted in no secretion of the protein.
So far, no crystal structure of FI is available and therefore we have predicted the structures of the individual domains of the heavy and light chains of FI using homology-based modeling. Based on these models, putative binding patches have been analyzed experimentally regarding their involvement in binding and cleavage of C3b. We found, in addition to the C-terminal serine protease domain, that all domains of the heavy chain are involved, especially the FIMAC and LDLr2 domains, but also the CD5 and LDLr1 domains. These results demonstrate the complexity of the FI protein; all domains contribute to its function.
Overall our data indicate that the mutations found in patients suffering from a complete FI deficiency and aHUS are located in all the domains of FI. The main difference between these two patient groups is that the mutations in complete FI deficient patients lead to no secretion of FI, which results in C3 depletion. In contrast, aHUS patients that still have FI, even at reduced concentrations, or have FI with impaired function, but no C3 depletion. This difference could result in the different clinical presentation observed in these patients. Complete FI deficient patients mainly suffer from recurrent bacterial infections due to lack of C3b opsonization, whereas aHUS patients have renal failure because of impaired regulation of complement in their kidneys.
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