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. 2010 Sep 10;285(37):28777-86.
doi: 10.1074/jbc.M110.131318. Epub 2010 Jul 8.

Characterization of C1q in Teleosts: Insight Into the Molecular and Functional Evolution of C1q Family and Classical Pathway

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Free PMC article

Characterization of C1q in Teleosts: Insight Into the Molecular and Functional Evolution of C1q Family and Classical Pathway

Yu-Lan Hu et al. J Biol Chem. .
Free PMC article

Abstract

C1qs are key components of the classical complement pathway. They have been well documented in human and mammals, but little is known about their molecular and functional characteristics in fish. In the present study, full-length cDNAs of c1qA, c1qB, and c1qC from zebrafish (Danio rerio) were cloned, revealing the conservation of their chromosomal synteny and organization between zebrafish and other species. For functional analysis, the globular heads of C1qA (ghA), C1qB (ghB), and C1qC (ghC) were expressed in Escherichia coli as soluble proteins. Hemolytic inhibitory assays showed that hemolytic activity in carp serum can be inhibited significantly by anti-C1qA, -C1qB, and -C1qC of zebrafish, respectively, indicating that C1qA, C1qB, and C1qC are involved in the classical pathway and are conserved functionally from fish to human. Zebrafish C1qs also could specifically bind to heat-aggregated zebrafish IgM, human IgG, and IgM. The involvement of globular head modules in the C1q-dependent classical pathway demonstrates the structural and functional conservation of these molecules in the classical pathway and their IgM or IgG binding sites during evolution. Phylogenetic analysis revealed that c1qA, c1qB, and c1qC may be formed by duplications of a single copy of c1qB and that the C1q family is, evolutionarily, closely related to the Emu family. This study improves current understanding of the evolutionary history of the C1q family and C1q-mediated immunity.

Figures

FIGURE 1.
FIGURE 1.
Full-length nucleotide sequence with predicted amino acid sequence schematic structure of zebrafish c1qA (accession no. FJ713133.1) (A), c1qB (accession no. FJ713134.1) (B), and c1qC (accession no. FJ713135.1) (C). The start and stop codons are in boldface type. The polyadenylation signal (AATAAA) is in boldface and italic type. The putative signal peptide is underlined. The collagen-like region (CLR) is in boldface type and underlined, and the C1q domain is highlighted. The cysteines are boxed. C1q protein consists of a signal peptide (SP), a collagen-like region, and a C-terminal C1q domain.
FIGURE 2.
FIGURE 2.
A, comparison of gene locations of c1qA, c1qB, and c1qC among human, mouse, and zebrafish. Arrows indicate gene orientation. Numbers below gene names indicate gene positions. B, comparison of genomic organizations of c1qA, c1qB, and c1qC genes among human, mouse, chicken, and zebrafish. Rectangles represent the exons, whereas the lines between them indicate the introns. Exons are indicated by black boxes, and untranslated regions are indicated by white boxes. The sizes of exons and introns are indicated above them.
FIGURE 3.
FIGURE 3.
Tissue distributions of zebrafish c1qA, c1qB, and c1qC. A, detection of c1qA, c1qB, and c1qC transcripts in different tissues by RT-PCR. B, semiquantitative analysis of the expression of zebrafish c1qA, c1qB, and c1qC. Expression levels are expressed as a ratio to β-actin mRNA levels after densitometric scanning of gels stained with ethidium bromide. Values are mean ± S.D. The relative expression values were averaged from three fish.
FIGURE 4.
FIGURE 4.
Western blot analysis of C1q in carp serum (lane M: molecular marker). Carp serum was electrophoresed on 12% SDS-PAGE gel using Laemmli buffer. The proteins separated were blotted on nitrocellulose membrane and immunostained with rabbit anti-fish ghA, ghB, or ghC antibodies, followed by staining with HRP-labeled anti-rabbit IgG.
FIGURE 5.
FIGURE 5.
Hemolytic study of carp serum using SRBC sensitized with carp anti-SRBC antibody. 100 μl of serum was incubated with an equal amount of sensitized SRBC at 37 °C for 30 min. After centrifugation, the sera were tested for hemolytic activity by measuring the A405 values. The guinea pig serum (1:20 diluted) was used as a positive control. Vertical bars represent the mean ± S.D. (n = 4). Significant differences are indicated with asterisks at p < 0.05.
FIGURE 6.
FIGURE 6.
Inhibition by Trx-ghA, Trx-ghB, or Trx-ghC of C1q-dependent hemolysis of SRBC sensitized with rabbit anti-SRBC antibody (IgG) (A), mouse anti-SRBC antibody (IgM) (B), and carp anti-SRBC antibody (C). In A and B, SRBC sensitized with IgG (SRBCIgG) or IgM (SRBCIgM) were pretreated with various concentrations of Trx-ghA, Trx-ghB, or Trx-ghC for 1 h at 37 °C. 1:20 diluted guinea pig serum was then added to pretreated SRBC to initiate hemolysis. In C, nonimmunized zebrafish serum diluted 1:3 was alternatively used; nonimmunized zebrafish serum has been proven previously to not lead to hemolysis). The unlysed cells were centrifuged, and A405 values of the supernatants were measured. The percentage inhibited was determined relative to the untreated sample. The means of three experiments are presented for each set of experiments.
FIGURE 7.
FIGURE 7.
Binding of heat-aggregated IgG and IgM to Trx-ghA, Trx-ghB, and Trx-ghC. Various concentrations of Trx-ghA, Trx-ghB, or Trx-ghC were coated to the microtiter wells and a fixed concentration of either heat-aggregated human IgG (10 μg/well) (A) or heat-aggregated human IgM (20 μg/well) (B) was added and probed with HRP-conjugated anti-human IgG (A) or anti-human IgM (B), respectively. Color was developed using p-nitrophenyl phosphate, and A450 values were measured. All experiments were performed in duplicate, and Trx was used as a negative control.
FIGURE 8.
FIGURE 8.
The evolution of the C1q and Emu families. □, a gene duplication event accompanied by loss of some gene segment. Finally, five subsets were formed in the two families. The topmost subset is the ancestor containing the three domains.

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