[Expression of the starfish complement component C3 gene homologue under the influence of bacterial lipopolysaccharide].

The fragment of a homologue of complement component C3 gene has been cloned and sequenced from the starfish, Asterias rubens. Phylogenetic analysis of ArC3-like gene demonstrates that ArC3-like gene has close similarity to C3 gene homologues of Deuterostomia invertebrate animals. High level of ArC3-like gene expression was identified in circulating cells (coelomocytes), in a gut's derivate (hepatopancreas) and in male gonada but not in stomach, female gonad and rectal gland of A. rubens starfish. ArC3-like gene expression was shown in all types of starfish coelomocytes: in lymphocyte-like cells, granular and nongranular amebocytes. Injection of bacterial lipopolysaccharide (LPS) solution into the coelomic cavity of starfish leads to the increase of ArC3-like gene expression in coelomocytes and hepatopancreas over the control level of sterile sea water injection. The level of ArC3-like gene expression increased in response to LPS reaching the maximum 6 h after the stimulation, and decreased to basal level 24 h after the stimulation. Injection of LPS solution stimulated the increase of ArC3-like gene expression level in hepatopancreas reaching the maximum 6-12 h after the stimulation, and the level of mRNA of ArC3-like gene had still been increased 48 h after LPS injection. The data demonstrates sustained positive regulation of ArC3-like gene expression under the influence of LPS.

Sequence information, ontogeny and tissue-specific expression of complement component C3 in Indian major carp, Labeo rohita (Hamilton).

The complement system is one of the first line of immune defence mechanisms as well as a modifier of acquired immunity. C3 is the central complement component primarily synthesized in liver. The local synthesis of C3 in tissues other than liver may play an important role in local inflammatory processes. The present study aims at looking into ontogeny of C3 in Labeo rohita and its tissue-specific expression that is yet to be explored for Indian carps. Unfertilised eggs, and eggs after 0, 1, 3, 6, 12 h post-fertilization and hatchlings at 24 h, and 3 and 7 days post-fertilization were collected from three brood fish of L. rohita (rohu). Total RNA was extracted from approximately 50 mg of tissue and subjected to RT-PCR using heterologous carp primers to amplify C3 fragment. A product of 155 bp size of rohu C3 was amplified, the deduced amino acid sequence of which had 91.1% similarity to that of Cyprinus carpio C3. C3 mRNA was not detected in unfertilized and 6 h post-fertilised eggs. C3 transcripts were detected 12 h post-fertilisation. Similarly, tissues from liver, spleen, kidney, muscle, brain, gonads, intestine, blood, heart and gills collected from juveniles of rohu were subjected to detection of C3 transcripts by RT-PCR and C3 mRNA was detected in all the tissues. Thus, it is concluded that there is extra-hepatic synthesis of complement (C3) in L. rohita and the synthesis of this component occurs only 6 h post-fertilisation.

Identification and characterization of CPAMD8, a novel member of the complement 3/alpha2-macroglobulin family with a C-terminal Kazal domain.

We have identified and characterized a novel member of the complement 3/alpha(2)-macroglobulin (C3/alpha(2)M) family named CPAMD8 (complement 3 and pregnancy zone protein-like, alpha2-macroglobulin domain-containing 8). The gene maps to chromosome 19p13.2-p13.3 and spans approximately 130 kb. The gene partially overlaps with the protease-activated receptor-4 (PAR4) gene in the reverse orientation. The cDNA consists of 40 exons ( approximately 6 kb) and encodes a protein of 1885 amino acids. Similar to other proteins in this family, CPAMD8 contains a signal sequence, an RXXR processing site, and a thioester motif. In addition, CPAMD8 has a Kazal-type serine proteinase inhibitor/follistatin-like domain at the C-terminus. The intact CPAMD8 protein generated by in vitro transcription and translation resolved as a single band of about 200 kDa on SDS-PAGE. RT-PCR and immunoblot assays showed that CPAMD8 is expressed in a number of human tissues, most abundantly in the kidney, brain, and testis and at lower levels in heart, liver, and small intestine. CPAMD8 is also expressed in several types of cells in culture, in which it is proteolytically processed into two chains of about 70 and 130 kDa. The Kazal domain of CPAMD8 binds to heparin, and subcellular fractionation shows that CPAMD8 is membrane associated via ionic interaction. In response to immune stimulants, CPAMD8 expression is markedly up-regulated in cells in culture. Thus, CPAMD8 may, like other members of the C3/alpha(2)M family, function in innate immunity but in a localized manner.

Detection of two variants of complement component C3 in C3-deficient guinea pigs distinguished by the absence and presence of a thiolester.

The complement system is an essential part of the innate defense, and C3 is an integral part of this powerful system. In previously identified complement C3 deficient guinea pigs only approx. 5% of the normal serum C3 level is detectable. No differences were found between in vitro C3 protein synthesis and C3 mRNA levels of cells from C3-deficient and wild-type animals and the amino acid sequences of both C3 proteins are identical as deduced from cDNA sequencing. Previously, the principal inability to form a C3 thiolester was discussed as a possible reason for this C3-deficiency. Here we report the isolation of two functionally different C3 species from the C3-deficient animals. Only one of these C3 proteins exhibits normal hemolytic activity and contains a thiolester group. The second C3 species is exclusively present in C3-deficient animals and lacks a thiolester, explaining its failure to express hemolytic activity. The presence of a second C3 species lacking a thiolester structure only in C3-deficient animals indicates that the stability of the thiolester may play a role in C3 deficiency. However further analysis of the in vitro stability of the thiolesters of C3 from normal and C3-deficient guinea pigs revealed no differences. A decreased in vivo thiolester stability might lead to the presence of C3 with and without a thiolester or alternatively the expression of two isoforms of C3 in these animals. Considering the central role of C3 in host defense, the mechanisms of C3 thiolester formation require further analysis.

Reference typing report for complement component C3.

A comparison of five C3 variant samples has been performed by conventional high-voltage gel electrophoresis in three laboratories (Palermo, Berlin and Mainz). Local designation was shown within SD = +/-0.75 mm migration distance in the Mainz laboratory. Methodological modifications by laboratories were not accounted for (cooling temperature, relative mobilities between runs). In parallel, all reference samples were also sequenced after exon-specific amplification. As a result, two variants with identical final designations and two variants with different mobilities were shown to conform at the molecular basis exhibiting an amino acid exchange that causes the corresponding change in relative mobility as compared to normal C3F and C3S.

Frequency of complement third component (C3) and properdin factor (BF) phenotypes in patients with various clinical manifestations of HBV infection.

Four groups of children were tested for the distribution of C3 and BF phenotypes: HBV-infected patients with Gianotti-Crosti syndrome, with CAH and with glomerulonephritis, and healthy children. The frequency of the phenotype C3F was significantly higher in children with Gianotti-Crosti syndrome in comparison with healthy children. The frequency of the phenotype BFS was significantly higher in patients with glomerulonephritis than in individuals with CAH. The difference between the frequency of the BFS phenotype in glomerulonephritis patients and that in healthy subjects neared significance. We suggest that the carriers of these phenotypes are characterized by susceptibility to some immune complex diseases associated with HBV infection.

C3 reference typing report and nomenclature revision.

As the result of reference typing, two 'new' variants could be provisionally accepted (C3F045 and C3F015). The list of variants of the C3 polymorphism includes now 2 common and 29 rare variants.

Complement C3 proteins in psoriasis.

A new polymorphism of the complement factor C3 in human plasma was demonstrated by isotachophoresis in agarose gels followed by immunodetection with rabbit anti-human C3c and C3d immunoglobulins. Four bands were detected in the immunoprint of freshly drawn EDTA-plasma, which were C3s1, C3s2, C3f1 and C3f2. At least four additional C3 components in Mg2+ -zymosan activated plasma were present, which were C3b1 to C3b4. The different forms of C3 in frozen and thawed heparin-plasma from 20 patients with psoriasis and 20 healthy individuals were studied from the immunoprint. The total content of C3 components was 29% greater in the patients with psoriasis than controls. The major difference was in the C3b components which were increased by 46%. In psoriatic patients, the two slow C3 components C3s1 and C3s2 were increased by 24 and 56% respectively, when compared with controls. The two fast C3 components C3f1 and C3f2 were decreased to 29 and 37%. The results suggest a direct involvement of the complement factor C3 in psoriasis.