title: A novel C1q-domain-containing protein from Zhikong scallop Chlamys farreri with lipopolysaccharide binding activity.
abstract: The C1q-domain-containing (C1qDC) proteins are a family of proteins characterized by a globular C1q (gC1q) domain in their C-terminus. They are involved in various processes of vertebrates and supposed to be an important pattern recognition receptor in innate immunity of invertebrates. In this study, a novel member of C1q-domain-containing protein family was identified from Zhikong scallop Chlamys farreri (designated as CfC1qDC) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfC1qDC was of 777 bp, consisting of a 5'-terminal untranslated region (UTR) of 62 bp and a 3' UTR of 178 bp with a polyadenylation signal sequence AATAAA and a poly (A) tail. The CfC1qDC cDNA encoded a polypeptide of 178 amino acids, including a signal peptide and a C1q-domain of 158 amino acids with the theoretical isoelectric point of 5.19 and the predicted molecular weight of 17.2 kDa. The C1q-domain in CfC1qDC exhibited homology with those in sialic acid binding lectin from mollusks and C1qDC proteins from higher vertebrates. The typical 10 beta-strand jelly-roll folding topology structure of C1q-domain and the residues essential for effective packing of the hydrophobic core were well conserved in CfC1qDC. By fluorescent quantitative real-time PCR, mRNA transcripts of CfC1qDC were mainly detected in kidney, mantle, adductor muscle and gill, and also marginally detectable in hemocytes. In the bacterial challenge experiment, after the scallops were challenged by Listonella anguillarum, there was a significant up-regulation in the relative expression level of CfC1qDC and at 6h post-injection, the mRNA expression reached the maximum level and was 4.55-fold higher than that of control scallops. Similarly, the expression of CfC1qDC mRNA in mixed primary cultures of hemocytes stimulated by lipopolysaccharides (LPS) was up-regulated and reached the maximum level at 6h post-stimulation, and then dropped back to the original level gradually. In order to investigate its function, the cDNA fragment encoding the mature peptide of CfC1qDC was recombined and expressed in Escherichia coli BL21(DE3). The recombinant CfC1qDC protein displayed a significantly strong activity to bind LPS from E. coli, although no obvious antibacterial or agglutinating activity toward Gram-negative bacteria E. coli JM109, L. anguillarum and Gram-positive bacteria Micrococcus luteus was observed. These results suggested that CfC1qDC was absolutely a novel member of the C1qDC protein family and was involved in the recognition of invading microorganisms probably as a pattern recognition molecule in mollusk.
authors.affiliation: Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Zhang
authors.forename: Linsheng
authors.initials: L
authors.lastname: Song
authors.forename: Chenghua
authors.initials: C
authors.lastname: Li
authors.forename: Jianmin
authors.initials: J
authors.lastname: Zhao
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.forename: Limei
authors.initials: L
authors.lastname: Qiu
authors.forename: Duojiao
authors.initials: D
authors.lastname: Ni
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D006434:Hemocytes / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism* / Q000494:pharmacology*; D053526:Listonella / Q000502:physiology; D008969:Molecular Sequence Data; D049897:Pectinidae / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
chemical_list: D000276:Adjuvants, Immunologic; D008070:Lipopolysaccharides; D011506:Proteins; D011994:Recombinant Proteins