Skipping of exon 27 in C3 gene compromises TED domain and results in complete human C3 deficiency.

Primary deficiency of complement C3 is rare and usually associated with increased susceptibility to bacterial infections. In this work, we investigated the molecular basis of complete C3 deficiency in a Brazilian 9-year old female patient with a family history of consanguinity. Hemolytic assays revealed complete lack of complement-mediated hemolytic activity in the patient's serum. While levels of the complement regulatory proteins Factor I, Factor H and Factor B were normal in the patient's and family members' sera, complement C3 levels were undetectable in the patient's serum and were reduced by at least 50% in the sera of the patient's parents and brother. Additionally, no C3 could be observed in the patient's plasma and cell culture supernatants by Western blot. We also observed that patient's skin fibroblasts stimulated with Escherichia coli LPS were unable to secrete C3, which might be accumulated within the cells before being intracellularly degraded. Sequencing analysis of the patient's C3 cDNA revealed a genetic mutation responsible for the complete skipping of exon 27, resulting in the loss of 99 nucleotides (3450-3549) located in the TED domain. Sequencing of the intronic region between the exons 26 and 27 of the C3 gene (nucleotides 6690313-6690961) showed a nucleotide exchange (T→C) at position 6690626 located in a splicing donor site, resulting in the complete skipping of exon 27 in the C3 mRNA.

Nonsense-codon-mediated decay in human hereditary complement C3 deficiency.

C3 occupies a central position in the complement pathway, mediating such diverse functions as convertase activity, opsonization and anaphylotoxin production. The deficiency of this protein is a rare autosomal recessive inherited disease, characterized by severe recurrent infections and immune complex disorders. We looked for molecular alterations that could explain the C3 deficiency present in a Brazilian boy of consanguineous parents who suffered from recurrent bacterial infections. Using reverse-transcriptase polymerase chain reaction to amplify C3 mRNA from LPS-stimulated fibroblasts from the patient, we demonstrated that his C3 gene has no large structural aberrations. However, after sequencing the amplified and cloned products we found: (1). a L314P amino acid substitution; (2). silent mutations at codons P577, S798 and A1437; and finally, (3). an R848STer substitution that results in the production of a truncated protein. Densitometry studies revealed a lower C3 mRNA concentration in the patient's fibroblasts, suggesting an inherent instability of his C3 mRNA. Our results indicate the presence of a premature termination codon in the C3 gene that results in a lack of the protein in patient's serum, which correlates with the acceleration of C3 mRNA decay in the patient's fibroblasts. This mRNA instability is consistent with a nonsense-codon-mediated decay process that ensures the elimination of possible deleterious truncated proteins, which, in the case of constitutively expressed abundant proteins such as C3, may otherwise accumulate to significant levels, leading to toxicity.

Native conformations of human complement components C3 and C4 show different dependencies on thioester formation.

The thioester bond in complement components C3 and C4 and the protease inhibitor alpha2-macroglobulin have traditionally been thought of as fulfilling the dual roles of mediating covalent attachment and maintaining the native conformational states of these molecules. We previously reported that several human C3 thioester-region mutants, including variants E1012Q and C1010A, in the latter of which thioester-bond formation is precluded, display an unexpected phenotype. Despite the lack of a thioester bond in these mutants, they appear to adopt a native-like conformation as suggested by the finding that they are cleavable by the classical pathway C3 convertase, C4b2a, whereas the C3b-like C3(H2O) species is not. Subsequently, a species referred to as C3(NH3)* was described which potentially could account for the observations with the above mutants. C3(NH3)* is a transient species formed on aminolysis of native C3 that can spontaneously re-form the thioester bond. Importantly, it has a mobility on cation-exchange HPLC that is distinct from both native C3 and C3(H2O), but like the native molecule, it is cleavable by an alternative-pathway C3 convertase. In this study we showed by using cation-exchange HPLC as an additional conformational probe that C3 C1010A and E1012Q mutant proteins did not resemble C3(NH3)*. Instead they displayed a chromatographic behaviour that was indistinguishable from that of native C3. To assess the general applicability of these observations, we engineered the equivalent mutations into human C4, specifically C4 C1010A and C4 E1012Q. As expected, thioester-bond formation did not occur in either of these C4 mutants, but in contrast with the results with C3 we found no evidence for the formation of a stable native-like conformation in either C4 mutant, as assessed using cleavability by C1s as the conformational probe. A possible interpretation of our data is that the adoption of the native conformational state during biosynthesis of C3 and C4 is an energetically permissible process, even if it is not locked in via thioester-bond formation. Whereas this conformational state is stable in mature C3, it is unstable in mature C4, perhaps reflecting the additional post-translational cleavage of C4 before its secretion.

C3: a versatile and multifunctional complement protein

The major complement component, C3, is the substrate for C3 convertases which emerge by activation of the classical and alternative complement pathways; fragments C3b and C3a are the resulting split products. The C3b becomes a constituent of the amplification C3-convertase in the alternative pathway, and of the C5 convertases responsible for the organization of the potentially cytolytic complex C5b-C9, being also able to interact with numerous serum proteins, cell surface molecules and foreign protein. The C3a functions as mediator of the early events of teh inflamatory process. Recent observations on the molecular features involved in the multiple interaction of C3 characterize this proteins as a most versatile and multifunctional molecule which is also an important participant of both the immune and monimmune surveillance mechanism