Biochemical Basis for the Histo-& Cytochemical Applications of Endogenous Lectins.

1)利用重组DNA技术在大肠杆菌中获得了人14K和鸡16K卵磷脂的重组蛋白。这使得为各种生化研究提供足够数量的蛋白质成为可能。2)对人14K凝集素的表达质粒进行定点诱变，对其氨基酸残基进行替代实验，以评估残基的意义。一些被认为是糖结合活性必需的残基，如色氨酸和几种半胱氨酸，被证明是不必要的。另一方面，鉴定了必需精氨酸和组氨酸残基。(3)利用cDNA验证和特异性抗体，研究了鸡胚发育过程中两种鸡分离素（14K和16K）在不同器官mrna转录和蛋白翻译水平上的表达。虽然结果太复杂，无法给出一个简单的解释，但16K卵磷脂在早期阶段发挥更多作用，而14K卵磷脂在后期阶段发挥更多作用。4)成功克隆了人29K β -半乳糖苷结合卵磷脂cDNA，该卵磷脂是一种人高分子量卵磷脂。完整的一级结构表明29K卵磷脂的c端部分与14K凝集素同源，而n端部分具有不相关的结构。这一结构研究使得讨论脊椎动物β -半乳糖苷结合卵磷脂家族的分子进化成为可能。5)虽然已知某些蛇类的毒液中含有β -半乳糖苷结合卵磷脂，但这些卵磷脂是否与我们广泛研究的不依赖钙的脊椎动物卵磷脂有结构关系尚不清楚。从响尾蛇毒液中纯化的卵磷脂的初级结构清楚地表明它属于另一个动物卵磷脂大家族，即钙依赖性卵磷脂家族。6)采用原位氢化技术，以磺化cDNA为探针，采用酶标法观察14K卵磷脂基因的表达。14K卵磷脂基因主要在表皮中间层表达，在13日龄胚中表达微弱，在表皮分化过程中逐渐增强，在17日龄胚中表达强烈。维生素A可抑制14K卵磷脂基因的表达，诱导表皮粘膜化生。少

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 … More 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