Skipping of exon 30 in C5 gene results in complete human C5 deficiency and demonstrates the importance of C5d and CUB domains for stability.

The deficiency of complement C5 is rare and frequently associated with severe and recurrent infections, especially caused by Neisseria spp. We observed the absence of component C5 in the serum of 3 siblings from a Brazilian family with history of consanguinity. The patients had suffered from recurrent episodes of meningitis and other less severe infections. Sera from these patients were unable to mediate hemolytic activity either by the classical or alternative pathways and presented extremely low levels of C5 protein (1.3, 0.9 and 1.0 microg/ml-normal range: 45-190 microg/ml). Hemolytic activity could be restored by the addition of purified C5 to deficient serum. Sequencing of sibling C5 cDNA revealed a homozygous 153 bp deletion that corresponds precisely to exon 30. The parents carried the same deletion but only in one allele. Sequencing of the corresponding region in the genomic DNA revealed a C to G substitution within intron 30 and, most significantly, the substitution of GAG(4028) for GAA(4028) at the 3' end of exon 30 which is most likely responsible for skipping of exon 30. The resulting in-frame deletion in the C5 mRNA codes for a mutant C5 protein lacking residues 1289-1339. These residues map to the CUB and C5d domains of the C5 alpha chain. This deletion is expected to produce a non-functional and unstable C5 protein which is more susceptible to degradation.

Deficiency of the human complement regulatory protein factor H associated with low levels of component C9.

We identified a 4-year-old Brazilian boy from a family of Japanese descent and history of consanguinity, who suffered from severe recurrent pneumonia. He carries factor H (FH) deficiency associated with reduced levels of component C9 and low serum levels of C3 and factor B. His mother also presented low levels of these proteins and factor I, while his father and sister had only lower levels of FH. Western blot assays confirmed the complete absence of FH and FHL-1 polypeptides in this patient. Sequencing of the proband's FH cDNA revealed a homozygous G453A substitution, encoding an Arg(127)His change. His mother, father and sister are heterozygous for this substitution. Despite the absence of FH in the plasma, this protein was detected in the patient's fibroblasts, suggesting that Arg(127) may be important for FH secretion. Low concentrations of C9 were detected in the proband serum but no mutations in the patient's C9 gene or promoter have been identified, suggesting that this is a consequence of uncontrolled complement activation and high C9 consumption.

Inherited complete factor I deficiency associated with systemic lupus erythematosus, higher susceptibility to infection and low levels of factor H.

Here we describe two new cases of complete deficiency of factor I (fI) in two sisters from a consanguineous Brazilian family. The eldest sibling (20-year-old) developed systemic lupus erythematosus (SLE) early during childhood while the youngest had been committed on several occasions owing to repeated infections although she was asymptomatic for auto-immune diseases. We also detected lower concentrations of C3 and factor B in both sisters. Biological functions dependent on complement activation such as the production of opsonins and killing of phagocytozed micro-organisms, chemotactic factors and haemolytic activity were all significantly reduced in both probands. Consistent with the absence of fI and low levels of fH, a deregulated production of C3b was observed by bidimensional electrophoresis in sera of both the probands.

Hereditary human complement C3 deficiency owing to reduced levels of C3 mRNA.

An 8-year-old son (L.A.S.) of consanguineous parents, presented recurrent bacterial infections, vasculitis and extremely low levels of serum C3 (0.15 microg/ml). The classical and alternative pathway haemolytic activities and the generation of opsonins and chemotactic factors derived from the activation of the complement system were markedly affected in the proband's serum. An in vitro addition of purified C3 restored the classical pathway-dependent haemolytic activity of his serum. Autoradiographs of the proband's lipopolysaccharide (LPS)-stimulated and 35S-labelled fibroblast supernatants after that the SDS-PAGE revealed no C3 alpha or beta chains. The amount of C3 mRNA synthesized by the proband's fibroblasts, as evaluated by reverse transcription-polymerase chain reaction (RT-PCR) assays, was greatly reduced.

Deficiency of human complement factor I associated with lowered factor H.

Deficiencies of factor I and/or factor H result in an increased consumption of C3 and higher susceptibility to recurrent infections. Here we describe a case of human factor I deficiency and lowered factor H levels. C3 concentration was 50% lower than normal, the classical pathway-dependent hemolytic activity was reduced to almost 30% of normal, and alternative pathway-dependent activity was completely absent. The killing by peripheral leukocytes of Candida albicans treated with deficient serum and the production of complement-dependent chemotactic factors were reduced in the proband's serum when compared with normal serum. Finally, we observed that C3 antigen present in the proband's serum has a different electrophoretic mobility than native C3 (most likely C3b), confirming the deregulation of complement activation due to the lack of regulatory proteins factors I and H. The impaired complement system described in this case, the first of its kind described in a Chile, explains the higher susceptibility to infections found in the proband.

Murine alpha-2-macroglobulin increase during inflammatory responses and tumor growth.

OBJECTIVE AND DESIGN: To determine the alpha-2-macroglobulin (alpha2M) levels in mice during acute and chronic inflammatory responses. MATERIALS AND METHODS: Inflammation was induced by one of the following stimuli: carrageenin, zymosan, lipopolysacharide, thioglycollate, bacilli Calmette Guerin, PPD (in pre-immunized and non-immunized animals) and tumor cells. The concentration of alpha2M was determined in plasma or peritoneal liquid by electroimmunoassay. RESULTS: In all the treatments employed, the plasma levels of alpha2M were higher than in untreated animals. This increase varied from 9%, 24 h after injection up a maximum of 66% 72 h post-injection. When compared to animals injected only with saline, the increases were significant 48 h after treatment with either zymosan or LPS, and 72 h after treatment with either thioglycollate or carrageenin. Treatment with BCG triggers an increase in alpha2M levels after 24 h (18.60%) and 48 h (27.90%). Immunized mice presented higher levels of this protein than non-immunized animals after challenge with PPD. The growth of Ehrlich tumor cells in the peritoneal cavity was directly correlated with the local levels of alpha2M which increased 3.5 fold, 10 days after injection. CONCLUSIONS: These results strongly indicate that in mice, the concentration of alpha2M can increase during acute and chronic inflammatory reactions with kinetics dependent on the particular kind of inflammatory agent.

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.