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Carbohydrate binding protein, lectin, is considered to have various functions such as adhesion molecules, signaling molecules, immune related molecules in vertebrates. However, its function in invertebrate has not been elucidated in detail yet. Using manila clam as a model system, we investigated possible roles of lectin in host defense against protozoan and microbial infection. We find Manila clam (Ruditapes philippinarum) synthesizes a lectin termed Manila clam lectin (MCL) only after infection with the protozoan parasite Perkinsus olseni. MCL is synthesized in hemocytes as a ~74 kDa precursor and secreted into hemolymph where it is converted to 30 kDa and 34 kDa polypeptides. The synthesis of MCL in hemocytes is stimulated by a factor(s) in Perkinsus-infected hemolymph, but not directly by Perkinsus itself. MCL can bind to the surfaces of purified hypnospores and zoospores of the parasite and this binding was inhibited by EDTA and GalNAc. Fluorescent beads coated with purified MCL were actively phagocytosed by hemocytes from the clam. Immunohistochemistry showed that secreted MCL localized inside of cyst-like structure. To investigate the patterns of genes expressed in Manila clams (Ruditapes philippinarum) infected with the protozoan parasite Perkinsus olseni, we constructed a cDNA library and generated 1,850 clones (expressed sequence tags). The 1,850 expressed sequence tags (ESTs) were compared to sequences in the GenBank database. Of these, 1,151 clones (62.2%) were unknown and are likely to represent newly described genes, while 699 clones (37.8%) were identified based on matches to sequences in the database. Lectins were the largest group of immune-function ESTs found in the Manila clams (6 different lectin were expressed). The expressions of lectins in the hemocytes were analyzed by RT-PCR using gene specific primers. Hemocyte from Perkinsus-infected clam expressed different set of lectins from that of Vibrio-infected one. Among these lectins we cloned MCL3, MCgalectin, and sialic acid binding lectin and characterized. It showed that has an open reading frame of 918 nucleotide and composed of 309 amino acid residues with a predicted molecular weight of 33.9kDa. Like other galectins, it did not contain signal peptide or transmembrane domain. It contained tandem repeated carbohydrate recognition domains (CRDs), in which the typical motifs important for carbohydrate recognition were conserved. Gemonic DNA analysis revealed MCGal is intronless. The carbohydrate recognition of MCGal analyzed by heptan inhibition of hemagglutination revealed that rMCGal showed a general feature of the galectin family, i.e. significant affinity for galactose and GalNAc. The expression of MCGal mRNA mainly detected in the tissues of heart, mantle, foot, adductor, palp and siphon. Immunohistochemistry using anti-MCGal antibody also conformed expression of MCGal in those tissues and hemocytes. The temporal expression of MCGal mRNA in manila clam challenged by perkinsus or vibrio was both up-regulated compared with non-challenged healthy manila clam. The recombinant MCGal agglutinated Vibrio tapitus and agglutination was inhibited by incubation with α-lactose. The rMCGal also bind surface of Perkinsus. We also find a specific protease associated with manila clam lectin (MCL). Protease was appeared in clams that were infected with Perkinsus sp, whereas uninfected clams did not contain protease. We suggest that in clam, which does not have adaptive immunity, lectins might act as strong defense molecules and their production is very inducible.

저자

• Young Mee Kim [ Department of Medicine, Cheju National University school of Medicine ]
• Jin Young Kim [ Department of Medicine, Cheju National University school of Medicine ]
• Hee-kyoung Lim [ Department of Medicine, Cheju National University school of Medicine ]
• Moshumy Adhya [ Department of Medicine, Cheju National University school of Medicine ]
• Moonjae Cho [ Department of Medicine, Cheju National University school of Medicine ]

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• 간행물명

  한국당과학회 학술대회

• 간기

  연간

• 수록기간

  2006~2022

• 십진분류

  KDC 517 DDC 614