Functional Study Of The Spiny Lectin And C1qDC Protein In Innate Immunity

Molluscs are important aquaculture creatures, and can creat great economic value each year. However, in recent years, aquaculture of aquatic diseases caused massive economic losses. In order to prevent and control the occurrence of molluscs aquaculture diseases, the study on immune defense mechanism, especially the mechanism of pathogen identification is needed. C-type lectins and C1qDC proteins are expressed in molluscs with great abundance. However, the functions of them are not clear. To clarify the roles of them in pathogen resisting and to find whether they have specificity, we studied the accurate functions of several molluscs. C-type lectins and C1qDC proteins are from Hyriopsis cumingii. Besides, we also investigated the role of the C-type lectin and C1qDC proteins from Hyriopsis cumingii in the immune system.1. HcLec4 from Hyriopsis cumingii functions as a pattern recognition receptor to participate in anti-bacterial immune responseC-type lectins (CTLs) are found in a wide number of invertebrates, and have been reported to participate in immune responses, such as the activation of prophenoloxidase, cell adhesion, bacterial clearance and phagocytosis. Previous studies on CTLs focused on the function of their carbohydrate recognition domains(CRDs). Currently, studies on the function of the CTLs with multi-CRDs are limited in Hyriopsis cumingii. In this study, a lectin with four CRDs was cloned from Hyriopsis cumingii, and called HcLec4. Four CRD domains are named CRD1,CRD2, CRD3 and CRD4 respectively. The function of HcLec4 in innate immunity was further studied. The full length of the cDNA sequence of the HcLec4 was 2218bp,which was obtained by Race, containing a 1860bp open reading frame (ORF),encoding a protein of 620 amino acids. Multiple alignments and phylogenetic tree analysis revealed that the four CRD proteins highly differ from one another. At the mRNA level,HcLec4 was widely distributed in several tissues, including hemocytes,hepatopancreas, gill, mantle and foot, and had a relatively high expression level in hepatopancreas. HcLec4 was significantly down-regulated at the early stage (2 h) of infection with S. aureus, B. subtilis, E. coli and V. parahaemolyticus. We further analyzed the bacteria and carbohydrate binding activities of four CRD domains of HcLec4. The results showed that four CRD domains could bind to several bacteria,lipopolysaccharide (LPS) and peptidoglycan (PGN). In HcLec4 knockdown mussels,the bacterial clearance rate was increased, and the expression level of anti-microbial peptides (AMPs) was up-regulated. This study reveals that HcLec4 exerts its anti-bacterial effect by regulating the expression of AMPs at the early stage of bacterial infection.2. ClqDC proteins from Hyriopsis cumingii functions as pattern recognition receptor are involved in anti-bacterial immune responseClq is a key subcomponent of the complement Cl complex. This subcomponent contains a globular Clq (gClq) domain with remarkable ligand binding properties.Clq domain-containing (ClqDC) proteins are composed of all proteins with a gClq domain. ClqDC proteins exist in many invertebrates and recognize non-self-ligands.In our study, four ClqDC proteins, namely, HcClqDCl-HcClqDC4, were identified from Hyriopsis cumingii. HcClgDCl-HcClqDC4 encode a protein of 224, 204, 305,and 332 amino acids, respectively. HcClqDCl-HcClqDC3 consist of a gClq domain at the C terminal, in addition to the gClq domain, a coiled-coil region is found in HcClqDC4. Multiple alignments and phylogenetic tree analysis revealed that the ClqDC proteins highly differ from one another. Tissue distribution analysis demonstrated that HcClqDCl-HcClqDC4 are widely distributed in hemocytes,hepatopancreas, gills, mantle and foot. The bacteria binding showed that four recombinant ClqDC proteins exhibit a binding activity against different bacterial species. Our results may suggest that ClqDC proteins may act as a pattern recognition receptor in anti-bacterial immune defense.