ANIMAL LECTINS AS RECOGNITION MOLECULES

无脊椎动物、低等脊索动物和高等胚胎发育阶段 脊椎动物具有精确辨别自我的能力 和非自我，尽管它们缺乏经典的免疫球蛋白介导的 识别系统。由于凝集素(特定的糖结合分子) 存在于不同物种和发育阶段的血清和细胞中 缺乏免疫球蛋白，很可能这些特定的分子 可能直接参与了与发育相关的认知和 最早的防御阶段，尤其是对微生物的防御。这个 这项建议的主要目的是分离、纯化和确定 原脊索动物(被膜动物)和原脊索动物凝集素的分子结构 一种低等脊椎动物，海鳗(环口)。在…的基础上 10种有代表性的尿腹水蚤的筛选 凝集素和这些分子对特定糖类的专一性，我们有 选择了半乳糖结合的白花毛竹凝集素，并 被囊藻唾液酸偶联物的两种凝集素 梨状菌和皱瘤海鞘作为血清学和免疫学检测的适宜分子 计划进行生化研究。大鼠唾液酸偶联物结合凝集素的研究 七鳃鳗(Petromyzon Marinus)的血清和卵将用作 与相应的非免疫球蛋白分子直接比较 具体细节。用亲和层析法分离凝集素 使用固定化的纯化糖和糖蛋白。一种纯净的 分离的凝集素将通过血清学和分析方法进行评估 钠溶液中的聚丙烯酰胺凝胶电泳法 含十二烷基硫酸盐的缓冲液和等电聚焦。这个 将从分子的角度对分子进行物理化学表征 体重分析，碳水化合物和氨基酸组成分析， 结合位点数和亲和力的测定及研究 使用光学旋转色散和圆形的二级结构 二向色性。我们已经能够使Didemnum的镁含量 凝集素，并启动了N-末端氨基酸序列分析。我们计划 用已定义的蛋白水解酶从凝集素中制备多肽 特异性和化学试剂，如CNBR和凝胶拆分肽 高效液相色谱过滤层析和 采用反相多肽层析法。这些多肽将被测序 利用Beckman自动测序仪对衍生氨基酸进行测序 用高效液相色谱法测定，目的是获得完整的氨基酸 序列，以便与已知识别分子进行详细比较 被创造出来。这些研究将对这一假设进行关键检验 动物凝集素构成了一个多基因识别分子家族。

Invertebrates, lower chordates and developing embryonic stages of higher vertebrates possess the capacity for precise discrimination between self and non-self even though they lack the classical immunoglobulin-mediated recognition system. Since lectins (specific saccharide-binding molecules) occur in the sera and on the cells of species and developmental stages which lack immunoglobulins, it is possible that these specific molecules might be directly involved in developmentally related recognition and in the earliest phases of defense, particularly against microorganisms. The chief aim of this proposal is to isolate, purify and determine the molecular structure of defined lectins from protochordates (tunicates) and a lower vertebrate, the sea lamprey (a cyclostome). On the basis of screening of 10 representative species of urocordates for the presence of lectins and the specificity of these molecules for defined sugars, we have selected a galactose-binding lectin of the tunicate Didemnum candidum and two lectins specific for sialoconjugates from the tunicates Halocynthia pyriformis and Styela plicata as suitable molecules for the serological and biochemical studies planned. Study of sialoconjugate-binding lectins from the serum and eggs of the sea lamprey (Petromyzon marinus) will serve as a direct comparison with non-immunoglobulin molecules of corresponding specificities. The lectins will be isolated by affinity chromatography using immobilized purified sugars and glycoproteins. The purity of the isolated lectins will be assessed serologically and by analytical techniques including polyacrylamide gel electrophoresis in sodium dodecylsulfate-containing buffers and by isoelectric focusing. The molecules will be characterized physicochemically in terms of molecular weight analyses, carbohydrate and amino acid composition analyses, determination of the number and affinity of binding sites and investigation of secondary structures using optical rotatory dispersion and circular dichroism. We have been able to silate mg quantitites of the Didemnum lectin and have initiated N-terminal amino acid sequence analysis. We plan to prepare peptides from the lectins using proteolytic enzymes of defined specificity and chemical agents such as CNBR and to resolve peptides by gel filtration chromatography using high performance liquid chromatography and by reverse phase peptide chromatography. The peptides will be sequenced using the Beckman automatic sequencer with derivatized amino acids determined using HPLC with the goal of obtaining complete amino acid sequences so that detailed comparisons with known recognition molecules can be made. These studies will provide a critical test of the hypothesis that animal lectins constitute a multigenic family of recognition molecules.