Molecular Cloning And Functional Studies Of Several Lectins From Roughskin Sculpin Trachidermus Fasciatus

Roughskin sculpin Trachidermus fasciatus Heckel (Scorpaeniformes:Cottidae) has ever been widely distributed in Chinese coastal waters and the rivers flowing into this water body. However, wild population of roughskin sculpin has drastically declined since1970s mainly because of overfishing, as well as the broken habitats and spawning sites. Consequently, roughskin sculpin was listed as critically endangered in Category Ⅱ of the National Key Protected Wildlife List. It is crucial that alternatives such as aquaculture be pursued to recover its population in the wild. In fish aquaculture, stressful conditions facilitate the appearance and diffusion of disease. To efficiently manage disease problems and provide knowledge for long-term enhancement in roughskin sculpin aquaculture production, the roughskin sculpin immune response to pathogen exposure should be studied thoroughly. Protein-carbohydrate interactions mediated by lectins have been recognized as key components of innate immunity, not only for recognition of potential pathogens, but also for participating in effector functions, such as agglutination, immobilization, complement activation, opsonization, killing and even regulation of adaptive immune responses. Therefore, the function of various lectins in the immune response contributes not only to quickly recognize and neutralize the microbial challenge, but lead to an effective long term adaptive immunity. As such, the increasing viral, bacterial, fungal and parasitic infections in aquaculture greatly stir up our interest in the role of lectins in roughskin sculpin innate immune responses. In this study, we identified three lectins (F-type. C-type and a galectin) and a MASP (MBL-associated serine protease), and furthurly studied their biological functions in innate immune system. The results are as follows: 1. A galectin with tandem-repeat domain (TfGal-9) is involved in innate immune response of roughskin sculpin Trachidermus fasciatusA galectin-9(TfGal-9) was cloned from roughskin sculpin Trachidermus fasciatus. The cDNA of TfGal-9is1453bp with an open reading frame (ORF) of900bp that encodes a protein of299amino acids. It has tandem repeat carbohydrate-binding domains consisting of the characteristic conserved sequence motif H-NPR and WG-E-R/K, connected by a peptide linker. qRT-PCR analysis revealed that TfGal-9was constitutively expressed in all detected tissues, with a relatively rich amount of mRNA in the intestine followed by the blood, heart and brain. Its expression was up-regulated in the five detected tissues (blood, skin, liver, stomach and heart) challenged with LPS. In the heavy metal exposure experiment. TfGal-9in the brain showed an increased expression. Recombinant TfGal-9protein agglutinated some Gram-positive and Gram-negative bacteria in a calcium-independent manner. The recombinant protein also bound to bacteria in the absence of calcium. The antimicrobial activities of recombinant expressed TfGal-9against Staphylococcus aureus could be detected with cylinder-plate method. The antioxidant experiments in vitro showed that percentage clearance of DPPH was50.38%when TfGal-9concentration reached200μg/ml. These data suggest that TfGal-9might be involved in the immune response to bacteria in roughskin sculpin and exhibited antioxidant activity. This is the first report about antibacterial and antioxidative action of galectin-9in fish.2. Lectin pathway of roughskin sculpin complement:identification, molecular characterization and functional analysis of collectin-11(TfCol-11) and MASPA C-type lectin collectin-11, designated TfCol-11, is reported in roughskin sculpin. TfCol-11encodes a protein of264amino acids. It is composed of three distinct domains:1) a collagenous region characterized by Gly-X-Y repeats;2) neck region; and3) a C-type lectin-like carbohydrate recognition domain (CRD) which contains a Glu-Pro-Asn (EPN) motif predicted binding to mannose specifically. Meanwhile, a993bp-length of partial MASP cDNA with96bp5’untranslated region (UTR) was also cloned from roughskin sculpin, which contained299amino acids consisted of three domains CUB-EGF-CUB. qRT-PCR indicated that TfCol-11and MASP mRNAs were predominately coexpressed in the liver. The temporal expressions of TfCol-11and MASP were both drastically up-regulated in the skin, blood, liver and gill by LPS challenge. It is noticeable that the temporal expressions of TfCol-11and MASP post LPS challenge were strikingly similar. TfCol-11and MASP recombinant proteins were constructed using an E. coli expression system. TfCol-11was able to agglutinate some Gram-positive, Gram-negative bacteria and a yeast in a Ca2+-dependent manner. Further analysis showed that TfCol-11could bind to several kinds of bacteria in a Ca2+-independent manner. In vivo, recombinant protein TfCol-11could significantly reduce mortality of carp Cyprinus carpio infected with Vibrio anguillarum. Pull-down assay showed that the recombinant TfCol-11protein could interact with MASP. It is conceivable that TfCol-11plays a role in activating complement system and in the defense against invading microorganism in roughskin sculpin。3. Molecular characterization and functional study of TfFBL in roughskin sculpin Roughskin sculpin Trachidermus fasciatusIn our study, a F-type lectin gene (TfFBL) was cloned from roughskin sculpin by expression sequence tag (EST) and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of TfFBL contains an open reading frame (ORF) coding for310amino acids. The deduced amino acid sequence of TfFBL contained a signal peptide and two carbohydrate recognition domains (CRDs) arranged in tandem, which both had a carbohydrate-binding site (HX26RX5-6R motif). Results from qRT-PCR in adult tissues indicated that TfFBL mRNA was abundantly expressed in the spawn, moderately expressed in the skin, spleen, kidney, heart and gill, and rarely expressed in other tissues tested. The temporal expression of TfFBL was obviously up-regulated in the blood, liver and skin in time dependent manners by lipopolysaccharide (LPS) challenge. Especially in the liver and blood, expression of TfFBL mRNA was dramatically up-regulated, reaching the highest level (37.4-fold in the blood,35.5-fold in the liver compared with that of the control group) at2h post challenge. Recombinant of TfFBL mature protein was able to agglutinate some bacteria and a yeast in a Ca2+-independent manner, whereas agglutination of TfFBL against microorganism increased greatly in the presence of calcium. Fucose showed inhibition on agglutination, which indicated that TfFBL could bind to fucose. These results suggest that TfFBL might be involved in roughskin sculpin innate response as a PRR.