BIOLOGY AND FUNCTION OF LYMPHOCYTE ADHESION MOLECULES

淋巴细胞粘附分子的生物学和功能

• 批准号: 2186113
• 负责人: Ivan Stamenkovic
• 金额: $ 21.58万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2186113
• 关键词: B lymphocyte; CD antigens; T lymphocyte; biological signal transduction; cell adhesion; cell cell interaction; glycolipids; human tissue; leukocyte adhesion molecules; protein sequence; sialate; site directed mutagenesis; tissue /cell culture; transfection

The long term objectives of the present proposal are to understand the molecular mechanisms which regulate lymphocyte interactions. The short term goal is to elucidate the role of the cell surface adhesion molecule CD22 in T cell-B cell interactions. CD22 is a B cell-specific adhesion molecule expressed on mature B cells as two isoforms of 130 and 140 kD. cDNAs encoding two isoforms of CD22 have been isolated, and the predicted amino acid sequence shown to contain 5 and 7 Ig domains in the extracellular region of the smaller (alpha) and larger (beta) isoform respectively. Introduction of CD22alpha into COS cells promotes rosetting of erythrocytes and monocytes, whereas COS cells transfected with CD22beta bind T and B cells in addition to monocytes and erythrocytes. A soluble CD22-Ig fusion protein (CD22Rg) was used to identify ligands of CD22, and was found to immunoprecipitate multiple cell surface glycoproteins from T cells. the major species being 115, 130 and 180-220 kD molecules. Immunoblotting experiments revealed that the high molecular weight bands correspond to different isoforms of CD45, the leukocyte specific receptor-linked phosphotyrosine phosphatase (PTPase). Cross-linking of CD22Rg with anti-CD3 antibody induced a dramatic inhibition of intracellular calcium mobilization produced when T cells are stimulated with anti-CD3 alone, and inhibited PLCgamma1 phosphorylation, closely reminiscent of the effect of coligating CD3 and CD45. We have shown that CD22 interaction with CD45 and other cell surface molecules dependent upon the presence of ligand-associated sialic acid in alpha2,6 linkage indicating that CD22 is a sialic acid binding lectin. We have also recently obtained evidence supporting the notion that CD22-triggered modulation of T cell activation is due to the interaction between CD22 and T cell CD45. CD22 therefore appears to be the first functional ligand of CD45. We now propose to study the role of CD22 expression in B cell signaling and to assess how the level of CD45 sialylation may influence signals resulting from CD22-CD45 interaction. Secondly, we will analyse the regulation of CD22-mediated adhesion. We will characterize a recently identified glycolipid which binds CD22 and which appears to regulate CD22-mediated cell - cell interactions. Finally, we will determine which sequences of CD22 are required for ligand binding, by using site specific mutagenesis.

本提案的长期目标是了解 调节淋巴细胞相互作用的分子机制。短 一个术语的目的是阐明细胞表面粘附分子的作用， CD 22与T细胞-B细胞相互作用CD 22是一种B细胞特异性粘附分子， 在成熟B细胞上表达为130和140 kD两种亚型的分子。 已经分离了编码两种CD 22亚型的cDNA，并且预测了CD 22亚型的表达。 显示的氨基酸序列中含有5和7个IG结构域， 较小（α）和较大（β）亚型的细胞外区域 分别 将CD 22 α导入COS细胞促进 红细胞和单核细胞的玫瑰花结，而COS细胞转染 CD 22 β结合T和B细胞以及单核细胞， 红细胞 使用可溶性CD 22-IG融合蛋白（CD 22 Rg）， 鉴定CD 22配体，并发现免疫沉淀多种 细胞表面糖蛋白。 主要种类有115种， 130和180-220 kD分子。免疫印迹实验显示， 高分子量条带对应于CD 45的不同同种型， 白细胞特异性受体连接磷酸酪氨酸磷酸酶 （PTD）. CD 22 Rg与抗-CD 3抗体的交联诱导了一种新的抗-CD 3抗体的产生。 细胞内钙动员的显著抑制产生时， 单独用抗CD 3刺激T细胞，并抑制PLC γ 1 磷酸化，密切联想到协同连接CD 3的作用， CD45。我们已经表明，CD 22与CD 45和其他细胞的相互作用， 表面分子依赖于配体相关唾液酸的存在 α 2，6键中的酸性，表明CD 22是唾液酸结合的 凝集素。我们最近也获得了支持这一观点的证据 CD 22触发的T细胞活化调节是由于 CD 22和T细胞CD 45之间的相互作用。因此，CD 22似乎是 CD 45的第一个功能性配体。我们现在建议研究 CD 22在B细胞信号传导中的表达并评估CD 45的水平 唾液酸化可影响由CD 22-CD 45相互作用产生的信号。 其次，我们将分析CD 22介导的粘附的调节。我们 将表征最近鉴定的结合CD 22的糖脂， 其似乎调节CD 22介导的细胞与细胞相互作用。 最后，我们将确定哪些CD 22序列是需要的， 通过使用位点特异性诱变进行配体结合。