NOVEL GLYCOCONJUGATE-BINDING PROTEINS OF LEUCOCYTES

膜相关的碳水化合物结合分子似乎 参与各种生理上重要的相互作用。 半乳糖肽，一类内源性的高度保守的β-半乳糖肽， 半乳糖苷结合凝集素，似乎在生长中发挥作用， 控制、细胞粘附和转移现象以及免疫 调变 半乳糖肽似乎在人体组织中普遍存在 但在人白细胞中差异表达。 是 该项目的目的是进行物理和 半乳糖肽免疫化学表征 人血沉棕黄层细胞。 半乳糖肽将从正常 人外周血白细胞去唾液酸胎球蛋白-琼脂糖亲和法 层析 纯度、分子量和亚基 将通过单向和二维电泳分析组成 方法和Western印迹法，然后进行免疫检测。 将通过FPLC凝胶过滤分析天然半乳糖肽。 物理- 半乳糖肽的化学表征将包括氨基酸 组成和氨基酸序列分析。 组成 将在沃茨皮可标签系统上进行研究。 将对半乳糖肽降解进行序列研究 用Applied Biosystems 470 A测序仪分析片段。 的 将测定抗半乳糖肽兔血清在手部的特异性。 将表征半乳糖肽碳水化合物结合特异性 利用ELISA和亲和珠方法。 结合位点 探针将包括良好表征的糖缀合物及其 降解产物以及一个大电池的完全 以合成碳水化合物为特征。 的 半乳糖肽在白细胞亚型中的分布将是 通过免疫组织化学程序，利用我们的抗- 半乳糖肽血清 本申请中描述的研究将导致 碳水化合物结合的物理化学表征 人外周血白细胞中存在的蛋白质及其鉴定 细胞的起源。 这些研究将奠定必要的基础 进一步阐明碳水化合物结合蛋白的功能 以及功能扰动如何影响细胞行为。

Membrane-associated carbohydrate-binding molecules appear to be involved in a variety of physiologically significant interactions. Galaptins, a class of endogenous well-conserved, beta- galactoside-binding lectins, appear to play a role in growth control, cell adhesion and metastatic phenomena and immune modulation. Galaptin appears to be ubiquitous in human tissues but is differentially expressed in human leucocytes. It is the purpose of this project to carry out the physico- and immunochemical characterization of galaptin isolated from human buffy coat cells. Galaptin will be isolated from normal human peripheral leucocytes by asialofetuin-Sepharose affinity chromatography. The purity, molecular weight and subunit composition will be analyzed by single and 2-D electrophoretic procedures and by Western blotting followed by immunodetection. Native galaptin will be analyzed by FPLC gel filtration. Physico- chemical characterization of galaptin will include amino acid composition and amino acid sequence analyses. Compositional studies will be carried out on a Waters Pico-Tag system. Sequence studies will be carried out on galaptin degradation fragments with an Applied Biosystems 470A sequenator. The specificity of anti-galaptin rabbit sera in hand will be determined. Galaptin carbohydrate-binding specificity will be characterize utilizing ELISA and affinity bead methodologies. Binding-site probes will include well-characterize glycoconjugates and their degradation products as well as a large battery of completely characterized synthetic carbohydrate compounds. The distribution of galaptin among leucocyte subtypes will be determined by immunohistochemical procedures utilizing our anti- galaptin sera. The research described in this application will lead to the physico-chemical characterization of a carbohydrate-biding protein present in human peripheral leucocytes and identification of cells of origin. These studies will lay the necessary foundation to pursue the elucidation of carbohydrate-binding protein function and how pertubation of function affects cell behavior.