Hisao Kamiya

Galectin containing cells in the skin and mucosal tissues in Japanese conger eel, Conger myriaster: an immunohistochemical study

Congerin is a beta-galactoside binding lectin (galectin) purified from the skin mucus of the Japanese conger, Conger myriaster. To clarify its tissue distribution and productive cells, several tissue samples including skin, buccal cavity wall, tang, pharynx, gills, esophagus, stomach, intestine, liver, kidney, spleen and ovary of conger were stained immunohistochemically using polyclonal rabbit anti-congerin serum. In the epidermis, a number of club cells were strongly stained. Because no agglutinating activity was detected in plasma, it appears evident that congerin is produced and secreted into mucus by those cells. In addition, congerin-positive club cells were distributed in the mucosal epithelium lining the digestive tract preceding the stomach and in the gills. These findings suggest that congerin participates in innate immunity on the intra- and the extra-body surface of the conger. The putative functions of club cells in fish and their contained lectin are discussed.

Rhamnose-binding lectins from steelhead trout (Oncorhynchus mykiss) eggs recognize bacterial lipopolysaccharides and lipoteichoic acid.

The interaction between bacteria and three L-rhamnose-binding lectins, named STL1, STL2, and STL3, from steelhead trout (Oncorhynchus mykiss) eggs was investigated. Although STLs bound to most Gram-negative and Gram-positive bacteria, they agglutinated only Escherichia coli K-12 and Bacillus subtilis among the bacteria tested. The binding was inhibited by L-rhamnose. STLs bound to distinct serotypes of lipopolysaccharides (LPSs), and showed much higher binding activities to smooth-type LPSs of Escherichia coli K-12 and Shigella flexneri 1A than to their corresponding rough-type LPSs. STLs also bound to lipoteichoic acid (LTA) of Bacillus subtilis. These results indicate that STLs bound to bacteria by recognizing LPSs or LTA on the cell surfaces.

Immunohistochemical localization of rhamnose-binding lectins in the steelhead trout (Oncorhynchus mykiss)

The localization of three -rhamnose-binding lectins named STL1, STL2, and STL3 from eggs of steelhead trout (Oncorhynchus mykiss) was analyzed by indirect immunohistochemical staining with specific antisera against individual lectins. In early previtellogenic oocyte, STL1 was localized not only in the cortical vesicles, but also in the plasma membrane and germinal vesicle. On the other hand, STL2 and STL3 were localized only in the cortical vesicles. In pre-fertilization mature egg, STLs were localized in a thin layer of cortical granules at the cytoplasmic side of the plasma membrane. STLs were accumulated on the surface of cytoplasm and inner membrane 30 min after fertilization. The strong staining with anti-STL1 antiserum was observed in several tissues and cells of the steelhead trout, such as spleen, thrombocytes, and blood leukocytes, but not erythrocytes. STL1 was also identified in exocrine cells, such as goblet cells of intestine and mucous cells of gill. These results indicate that the multiple lectins found in eggs of the steelhead trout play physiological roles not only in eggs, but also in various cells related to the innate immunity.

A Novel Rhamnose-binding Lectin Family from Eggs of Steelhead Trout (Oncorhynchus mykiss) with Different Structures and Tissue Distribution

An L-rhamnose-binding isolectin named STL3 (subunit Mr, 21.5 k) was isolated from eggs of the steelhead trout (Oncorhynchus mykiss) in addition to STL1 (subunit Mr, 31.4 k) and STL2 (subunit Mr, 21.3 k) that had been already isolated. STLs were composed of non-covalently linked subunits. The primary structures of STL1 and STL3 were analyzed by the combined use of protein sequencing and cDNA sequencing. A cDNA encoding STL2, of which the protein sequence had been previously studied, was also analyzed. The STL1 subunit (289 amino acid residues) had different structural properties compared to those of the STL2 subunit (195 amino acid residues) and the STL3 subunit (195 amino acid residues); e.g., the number of repeated domain (three for STL1, and two for STL2 and STL3), although all of them were composed of tandemly repeated homologous domains (40 to 53% identities). The lectin levels in various tissues and during the embryonic development showed that STL1 had different distribution and expression profiles from those of STL2 and STL3. Although STL1 could be detected in several tissues and serum of both male and female steelhead trout, STL2 and STL3 were only abundant in the ovary. STL2 and STL3 levels dramatically decreased just after hatching, however, the STL1 level increased temporarily. These results indicate that the multiple lectins from eggs of the steelhead trout form a novel rhamnose-binding lectin family with different structures and tissue distribution to share distinct functions in eggs.

Tissue-specific expression of rhamnose-binding lectins in the steelhead trout (Oncorhynchus mykiss)

Tissue-specific expression of three L-rhamnose-binding lectins, named STL1, STL2, and STL3, in the steelhead trout (Oncorhynchus mykiss) was investigated. STL2 and STL3 mRNAs were restricted in the oocytes. In contrast, STL1 mRNA was detected only in the liver. The transcription of STL2 and STL3 started in previtellogenic oocytes. These results showed distinct expression profiles of rhamnose-binding lectins in the fish.

High-level expression and characterization of fully active recombinant conger eel galectins in Eschericia coli.

An expression system for recombinant conger eel galectins, congerins I and II, were constructed using the pTV 118N plasmid vector and Escherichia coli. Recombinant congerins I and II could be obtained in the soluble active form with high quantitative yield. Mutation of codons for Val and Leu located in the N-terminal region of Con I increased the expression efficiency. Purification of recombinant proteins were done by only two chromatographical steps from E. coli extract. The purified recombinant congerins were found to be almost the same as the native ones except for the acetyl group at the N-terminus; that is, they showed the same structures and carbohydrate binding activities, suggesting that N-terminal acetyl groups of congerins were not significant for activity.