Carp Complement Research Articles

Molecular cloning of Gibel carp (<i>Carassius auratus gibelio</i>) complement component C3 and its expression profile after Cyprinid herpesvirus 2 infection

The complement systems play an important role in innate and adaptive immunity. In this study, the complement C3 gene, designated CagC3, was cloned and sequenced from Gibel carp (Carassius auratus gibelio). The expression pattern of CagC3 in different tissues of healthy Gibel carp and after challenge with Cyprinid herpesvirus 2 (CyHV-2) were evaluated using quantitative real-time PCR. The full-length CagC3 cDNA was 5131 bp with an ORF of 4950 bp, encoding a predicted protein of 1649 amino acids. The deduced amino acid sequence showed that CagC3 has conserved domains and residues known to be critical for C3 function. Phylogenetic analysis demonstrated that CagC3 clustered with homologs from common carp and grass carp (Ctenopharyngodon idella). CagC3 is expressed in all examined tissues of healthy Gibel carp, with the highest expression in liver. In vivo, after CyHV-2 challenge, CagC3 transcription was significantly upregulated in liver, spleen and kidney with the peaks at 24 hr, 2 d, and 2 d, respectively. In vitro, CagC3 expression in the Gibel carp brain cell line showed the same pattern as that in vivo after stimulation with CyHV-2 or poly(I:C). However, CagC3 expression was downregulated at 24 hr after induction with lipopolysaccharide (LPS), and then reached the peak at 2 d. These results suggest that CagC3 is involved in the innate immune response of Gibel carp to viral infection.

A CD46-like molecule functional in teleost fish represents an ancestral form of membrane-bound regulators of complement activation.

In the complement system, the regulators of complement activation (RCA) play crucial roles in controlling excessive complement activation and in protecting host cell from misdirected attack of complement. Several members of RCA family have been cloned from cyclostome and bony fish species and classified into soluble and membrane-bound type as in mammalian RCA factors. Complement-regulatory functions have been described only for soluble RCA of lamprey and barred sand bass; however, little is known on the biological function of the membrane-bound RCA proteins in the lower vertebrates. In this study, a membrane-bound RCA protein, designated teleost complement-regulatory membrane protein (Tecrem), was cloned and characterized for its complement-regulatory roles. Carp Tecrem, an ortholog of a zebrafish type 2 RCA, ZCR1, consists of four short consensus repeat modules, a serine/threonine/proline-rich domain, a transmembrane region, and a cytoplasmic domain, from the N terminus, as does mammalian CD46. Tecrem showed a ubiquitous mRNA expression in carp tissues, agreeing well with the putative regulatory role in complement activation. A recombinant Chinese hamster ovary cell line bearing carp Tecrem showed a significantly higher tolerance against lytic activity of carp complement and less deposition of C3-S, the major C3 isotypes acting on the target cell, than control Chinese hamster ovary (mock transfectant). Anti-Tecrem mAb enhanced the depositions of carp C3 and two C4 isotypes on autologous erythrocytes. Thus, the present findings provide the evidence of complement regulation by a membrane-bound group 2 RCA in bony fish, implying the host-cell protection is an evolutionarily conserved mechanism in regulation of the complement system.

Molecular cloning, characterization and expression analysis of the complement component C6 gene in grass carp

The complement system, as a representative of innate immunity, plays a key role in the host defense against infections. C6 is the member of complement components creating the membrane attack complex (MAC). In this study, we cloned and characterized the grass carp complement component C6 (gcC6) gene. Our data showed that gcC6 gene contained a 2724 bp open reading frame (ORF), a 237 bp 5′-untranslated region (UTR) and a 219 bp 3′-UTR. The deduced amino acid sequence of gcC6 showed 77.6% and 58.9% identity to zebrafish C6 and rainbow trout C6, respectively. GcC6 gene was expressed in a wide range of grass carp tissues, and the highest expression level of gcC6 was detected in the spleen and liver. Upon challenge with Aeromonas hydrophila, its expression was significantly up-regulated in muscle, trunk kidney, liver, head kidney, spleen, heart and intestine, whereas it was down-regulated in the brain and skin. The expression level of gcC6 was high at the unfertilized egg stage. It was significantly increased at 1 day post-hatching, but it was decreased at 10 days post-hatching. This result suggested that the complement C6 transcripts in early embryos were of maternal origin.

Complement expression in common carp ( Cyprinus carpio L.) during infection with Ichthyophthirius multifiliis

A real-time PCR assay for determination of the complement response to infection with the ectoparasite Ichthyophthirius multifiliis in carp is presented. Specific primers were designed for selected genes representing the three pathways of the carp complement system. The investigated complement molecules were C1r/s, C3, C4, C5, factor I, factor B/C2-A (Bf/C2-A), mannose-binding lectin (MBL) and MBL-associated serine protease (MASP). The expression of the selected genes was analyzed on RNA extracts from skin, liver, and whole blood from carp at 3, 12, 24, 36, and 48 h post-infection (pi) with I. multifiliis. A pronounced up-regulation of Bf/C2-A, in skin, blood, and liver (250-, 60-, and 4-fold respectively), was observed at later sampling points pi (24–48 h). In addition, an intermediate (from 5 to 13-fold) down-regulation of MASP was observed in skin and liver samples at 36 and 48 h pi with respect to control fish. MBL was expressed only in liver and no variation in the transcription level of this lectin was observed. Complement factor C3 was significantly up-regulated in liver (4-fold up-regulation, 24 h pi). The presented results indicate that infection with the parasite I. multifiliis in carp to a large extent stimulates the expression of complement molecules. Moreover, the dramatic and early up-regulation of Bf/C2-A in skin indicates a role of this molecule as an acute-phase reactant. Furthermore, our study confirms the role of fish skin as an important extra-hepatic site of expression of complement molecules as well as an active regulator of complement expression. Expression of some of the components of the complement system in blood suggests that leukocytes in carp act as an important extra-hepatic source of complement molecules.

Purification and functional assessment of C3a, C4a and C5a of the common carp ( Cyprinus carpio) complement

Promotion of inflammatory response is an important role of the complement system, but this kind of function is poorly documented for the lower vertebrates. Here we report chemotactic activity of purified anaphylactic fragments derived from the complement components C3, C4 and C5 of the common carp. The purified anaphylatoxins are two C5a-desArg peptides derived from the C5-I isotype, an intact form and a desArg form of C4a from C4-2 isotype, and an intact form and a desArg form of C3a from C3-H1 isoform. These were identified by N-terminal sequencing, mass spectrometry, and peptide mass fingerprinting. In the chemotaxis assay using carp kidney neutrophils, the two C5a-desArg fragments, which are probably allotypic variants, showed a potent chemotactic activity at 0.5–1 nM, whereas C3a or C4a showed no significant activity. The results suggest that C3a, C4a and C5a of bony fish have functionally diverged to the state similar to their mammalian homologs.

Isolation of Carp Complement Factor B and Formation of the C3-Convertase

Isolation of Carp Complement Factor B and Formation of the C3-Convertase

Characterization of the Soluble Membrane Attack Complex (SMAC) of Carp (Cyprinus carpio) Complement

The membrane attack complex (MAC) of mammalian complement, which is composed of complement components, C5b, C6, C7, C8 and C9, shows cyiolyiic activity by disturbing the structure of cyioplasmic mambrane. The soluble form of the membrane attack complex, termed SMAC, of the complement system is innert unlike the membrane attack complex formed on target cell membranes. Thus formation of SMAC is inferred as a regulatory mechanism of the cytotoxic activity of the complement system. In the present study, a complex homologous to mammalian SMAC was purified from zymosan-activated carp serum by gel filtration through Sepharose Cl;6B and affinity chromatography using anti-carp C9-Toyopearl. The purified carp SMAC showed a partial antigenicity of C9 and behaved as a 1,020kDa a -globulin. Two-dimensional SDS-PAGE of carp SMAC revealed polypeptide spots of C5b, C6, C7, C8 and C9, in agreement with those previously identified in carp MAC. In addition, three spots specific to SMAC were detected on the two-demensional gel, which may represent proteins responsible for solubilization of MAC, such as SP40,40 and S-protein present in mamJnalian SMAC. These results suggest that structure and function of SMAC have been conserved through the evolution of vertebrates .

Expansion of genes encoding complement components in bony fish: biological implications of the complement diversity

The complement system is a major humoral component of vertebrate defenses for tagging and killing target microorganisms. Recent molecular analyses have uncovered a striking feature of bony fish complement, namely that several complement components are encoded by multiple genes. In this review, the structural diversity of C3, C4, C5, factor B, C2, C1r/s and MASP are discussed with special reference to their functional differentiation, mainly focusing on the common carp ( Cyprinus carpio), a tetraploidized teleost. In carp, all the members (C3, C4, C5 and a non-complement protein α2-macroglobulin) of the thioester-containing protein family are present in multiple isotypes, differing in the primary structures of various functional sites. Three factor B/C2-like isotypes identified in carp showed distinct expression pattern (sites and inducibility), with one behaving as an acute-phase reactant. Two C1r/C1s/MASP2-like isotypes also contain an amino acid substitution that likely affects their substrate specificity. Overall, the present data suggest that the expanded genes of the carp complement system produce more diversified functional components than are known for mammals. The biological significance of this diversity is discussed.

コイ補体の構造と機能に関する研究

コイ補体の構造と機能に関する研究

H3 10:45 Sequence diversity of cDNA encoding the third component (C3) of carp ( [formula omitted]) complement

H3 10:45 Sequence diversity of cDNA encoding the third component (C3) of carp ( [formula omitted]) complement

CDNA cloning of the third component (C3) of carp complement

cDNA cloning of the third component (C3) of carp complement

Terminal components of carp complement constituting a membrane attack complex

The membrane attack complex (MAC) of carp complement was extracted with deoxycholate from rabbit erythrocytes lysed by carp serum and purified by a two-step chromatographic procedure. On two-dimensional SDS-PAGE of carp MAC, eight bands were detected. The band of M r 91 000 was identified as carp C9 by western blotting using anti-carp C9, and two bands of M r 62 000 and one band of M r 22 000 were confirmed as those of carp C8α, C8β and C8γ, respectively, by their N-terminal amino acid sequences. The bands of M r 102 000 and 73 000, which generated from a 180 000 band under reducing conditions, were those corresponding to human C5bα and C5bβ, respectively. The remaining bands of M r 115 000 and 106 000 were identified as those corresponding to human C6 and C7, as determined by their molecular size, single-chain structures and similarities in N-terminal amino acid sequences to their mammalian counterparts. Densitometric scan of the gels showed the molar ratio of C5b, C6, C7, C8 and C9 in carp MAC to be 1:1:1:1:4. Based on these results, it appears that, as with mammals, the cytolytic pathwat of bony fish complement is composed of five terminal components from C5 to C9.

Purification and characterization of the eighth and ninth components of carp complement

Complement components corresponding to mammalian C8 and C9 were isolated from carp ( Cyprinus carpio) serum. Carp C8 ( M r 146 000) proved to be a γ-globulin composed of three polypeptide chains (α-chain, M r 62 000; β-chain, M r 62 000); γ-chain, M r 22 000). The α-chain was disulfide-linked to the γ-chain and the β-chain was non-covalently associated with the α-γ chain, in fair agreement with mammalian C8. However, the N-terminal amino acid sequences of the three subunits showed no homology with those of human C8. Carp C9 was an α-globulin composed of a single polypeptide ( M r 91 000) and the N-terminus was blocked. Carp serum depleted of C8 did not hemolyse either carp antibody-sensitized sheep erythrocytes or non-sensitized rabbit erythrocytes, while C9-depleted carp serum did not hemolyse the former, but did hemolyse the latter target cells, as in the case of C9-depleted human serum.

Monoclonal Antibodies against the Third Component (C 3) of Carp Complement

Monoclonal Antibodies against the Third Component (C 3) of Carp Complement

Isolation of a carp complement protein homologous to mammalian factor D

Factor D of carp ( Cyprinus carpio) complement was purified to apparent homogeneity by a 4-step Chromatographie procedure and examined for physicochemical and functional properties. Carp factor D proved to be an α-globulin with a molecular mass of 29 kDa and the serum concentration was estimated to be 6 μg/ml. The NH 2-terminal amino acid sequence (30 residues including five unidentified positions) of carp factor D showed high homologies (57–60%) to those of mammalian factor D. Neither functional compatibility nor common antigenicity was observed between carp and human factor D. This report is apparently the first description on the factor D molecule in a non-mammalian species.

An Improved Method for the Preparation of EAC14, an Intermediate in Immune Hemolysis by Carp Complement

TitleAn Improved Method for the Preparation of EAC14, an Intermediate inImmune Hemolysis by Carp ComplementAuthor(s) Uemura, Takeshi; Nakao, Miki; Matsuyama, Hiroko; Yano, TomokiCitation九州大学農学部紀要 ¦¦ 36(3/4) ¦¦ p273-278; Journal of the Faculty ofAgriculture, Kyushu University ¦¦ 36(3/4) ¦¦ p273-278Issue Date 1992-02URL http://hdl.handle.net/2324/23990Right

The Complement System of Carp Cyprinus carpio-VI. Isolation of the Second Component of Complement (C2) from Carp Serum.

The second component (C2) of carp complement was isolated from carp serum by a six-step purification procedure: 1) affinity chromatography on Blue-Cellulofine, 2) affinity chromatography on heparin-agarose, 3) gel filtration on Sephadex G-200, 4) TSKgel DEAE-5PW chromatography (HPLC), 5) TSKgel Heparin-5PW chromatography (HPLC), and 6) TSKgel G3000SWXL chro-matography (HPLC). Hemolytic activity of carp C2 in column eluates was measured using EAC14 (sheep erythrocytes sensitized with carp antibody, C1 and C4) and heat-inactivated carp serum (deficient in C1 and C2) as reagents for detecting C2. About 0.4mg of carp C2 was obtained from 100ml of serum. The final recovery was 9.4%, representing a 799-fold purification. Carp C2 was composed of a single polypeptide chain with molecular weight of 108, 000 and was susceptible to heat-inactivation at 45°C. These physicochemi-cal properties of carp C2 closely resemble those reported for mammalian C2.

Opsonic effect of the third complement component (C3) of carp ( Cyprinus carpio) on phagocytosis by neutrophils

The opsonic effect of carp complement on the phagocytosis by carp peritoneal neutrophils was investigated using antibody-sensitised sheep erythrocytes (EA) and baker's yeast (Y), which are the activators of the classical complement pathway (CCP) and the alternative complement pathway (ACP), respectively. Carp antibody (A) exerted little opsonic effect on the phagocytosis of sheep erythrocytes (E). The treatment of EA and Y with normal carp serum (C) greatly enhanced phagocytosis. Such opsonic effect is apparently due to the complement being activated via CCP and ACP, because the treatment of EA and Y with C3-depleted carp serum produced no effect. It was also found that the treatment of EAC and YC with the Fab fragment of anti-carp C3 (rabbit IgG) abolished the opsonising effect of carp complement deposited on the target cells. These results indicate that C3 is the major phagocytosis-promoting factor (opsonin). In addition, it was also demonstrated that pretreatment of the neutrophil surface with trypsin greatly decreased the phagocytosis of EAC, suggesting the presence of C3 receptors on the neutrophil surface.

The complement system of carp Cyprinus carpio-V. Isolation of the third component of complement (C3) from carp serum.

The third component (C3) of carp complement was purified to homogeneity by a four-step purification procedure: 1) affinity chromatography on Blue-Cellulofine, 2) QAE-Sephadex A-SO chromatography, 3) gel filtration on Sepharose CL-6B, and 4) CM-Sephadex C-50 chromatography. Hemolytic activity of carp C3 was assayed using intermediate cells, EAC14, and hydrazine-treated carp serum.The final recovery of C3 activity was 17%. Carp C3 showed an electrophoretic mobility of β-globulin with a molecular weight of 194, 000. The protein consisted of two carbohydrate-containing subunits of 120, 000 (α-chain) and 74, 000 β-chain) which were linked by disulfide bonds. In agreement with mammalian C3, carp C3 was inactivated by nucleophilic attack by amines such as hydrazine, ammonia, hydroxylamine and methylamine, but it showed considerable resistance against chaotropic reagents such as KBr and KSCN. In addition, it was shown that carp C3 attached covalently to a zymosan particle as a result of the activation of the alternative complement pathway.

The complement system of carp Cyprinus carpio-IV. Formation and characteristics of an intermediate complex EAC1,4,2 in immune hemolysis by carp complement.

A hemolytic intermediate complex EAC1, 4, 2 was prepared by incubating carp EAC1, 4 with hydrazine-treated carp serum, which is deficient in C4 and C3, at 0°C for 20 min. The difference between EAC1, 4, 2 and EAC1, 4 was shown by tests with inactivated carp sera. The former was hemolyzed by carrageenan-treated serum (Cn), ammonia-treated serum (Am) and heat-treated serum (H), whereas the latter was hemolyzed by Cn and Am, but not by H. EAC1, 4, 2 was unstable. Its half-life was about 15 min at 20°C and about 2.5h at 0°C. Addition of Ca2+ to the buffer was not effective in preventing the deterioration of this complex. The reaction between EAC1, 4 and Cy' (a mixture of complement components other than C1 and C4) proceeded only in the presence of Mg2+, whereas the reaction of EAC1, 4, 2 with Cy'proceeded without Mg2+. This indicates that the action of C2 requires Mg2+ while C3 acts without Mg2+.