BIOLOGY AND FUNCTION OF LYMPHOCYTE ADHESION MOLECULES

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。短小的 学期目标是阐明细胞表面黏附分子的作用。 CD22在T细胞-B细胞相互作用中的作用CD22是一种B细胞特异性黏附 分子在成熟B细胞上表达为130kD和140kD两种亚型。 编码CD22两种异构体的cDNA已被分离，并预测 氨基酸序列显示含有5个和7个Ig结构域 较小(α)和较大(β)异构体的胞外区 分别进行了分析。CD22α导入COS细胞促进 红细胞和单核细胞的玫瑰花环，而COS细胞 除单核细胞外，CD22β还可结合T和B细胞 红血球。可溶性CD22-Ig融合蛋白(CD22Rg)用于 鉴定CD22的配体，并发现能免疫沉淀多个 来自T细胞的细胞表面糖蛋白。主要物种有115种， 130和180-220kD分子。免疫印迹实验显示 高分子量条带对应于CD45的不同亚型， 白细胞特异性受体连接的磷酸酪氨酸磷酸酶 (PTPase)。CD22Rg与抗CD3抗体的交联性研究 对细胞内钙动员的显著抑制 T细胞单独用抗CD3刺激，而抑制PLC-Gamma1 磷酸化，与连接CD3和CD3的效果密切相关 CD45。我们已经证明CD22与CD45和其他细胞相互作用 表面分子依赖于配体相关唾液酸的存在 α2，6键中的酸表明CD22是唾液酸结合 凝集素。我们最近也获得了支持这一观点的证据 CD22触发的T细胞激活的调制是由于 CD22与T细胞CD45的相互作用因此，CD22似乎是 CD45的第一个功能配体。我们现在建议研究一下 CD22在B细胞信号转导中的表达及评估CD45水平如何 唾液酸化可能会影响CD22-CD45相互作用产生的信号。 其次，我们将分析CD22介导的黏附的调节。我们 将表征一种最近发现的与CD22和CD22结合的糖脂 它似乎调节CD22介导的细胞间相互作用。 最后，我们将确定哪些CD22序列是必需的 配基结合，通过使用定点突变。

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.