BIOLOGY AND FUNCTION OF LYMPHOCYTE ADHESION MOLECULES

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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.