| Research Abstract |
1) Production of recombinant proteins of both human 14K and chick 16K lecithins in E. coli has been attained by recombinant DNA techniques. This made it possible to provide sufficient amount of proteins required for a variety of biochemical studies.
2) Substitution experiments of amino acid residues in human 14K lectin by site-directed mutagenesis of its expression plasmid which enables to assess significance of residues was done. Some residues which had been considered essential for sugar-binding activity, such as tryptophan and several cysteins, proved to be unessential. On the other hand, essential arginine and histidine residues were identified.
(3) Expression of two chick isolectins (14K and 16K) in various organs during development of embryo was studied at the levels of both transcription of their mRNAs and translation to proteins by using cDNA proves and specific antibodies. Though results were too complicated to give a simple explanation, a tendency that 16K lecithin plays roles
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at earlier stages while 14K lecithin at later stages.
4) We succeeded in cloning of cDNA for human 29K beta-galactoside-binding lecithin which is a human high-molecular weight lecithin. Revealed complete primary structure indicated that the C-terminal half of 29K lecithin is homologous to 14K lectin while the N-terminal half has an unrelated structure. This structural studies made it possible to discuss molecular evolution of vertebrate beta-galactoside-binding lecithin family.
5) Though venoms of some species of snake were known to contain beta-galactoside-binding lecithins, it had not been clarified whether these lecithins have any structural relationship with Ca-independent vertebrate lectins which we have been studying extensively. Primary structure of a lecithin purified from rattlesnake venom clearly showed that it belongs to another big family of animal lecithin, that is, Ca-dependent lecithin family.
6) The 14K lecithin gene expression was visualized by HRP-labeling method using in situ hydridization techniques, in which sulfonated cDNA was employed as a probe. The 14K lecithin gene expression was detected mainly in the intermediate layer of the epidermis : faintly in 13-day-old embryo, graduallly increased in intensity during epidermal differentiation, and intensely positive in 17-day-old embryo. The expression of the 14K lecithin gene was supressed by vitamin A which induced the mucous metaplasia of the epidermis. Less
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