The leukocyte adhesion molecules, mo1, LFA-1 and p150,95 (Leu-CAM) are three surface membrane heterodimers each with a distinct alpha chain linked to a common beta chain. Leu-CAM mediate vital functions such as phagocytosis, chemotaxis, adhesion to endothelium, aggregation, natural killing and cell-mediated cytotoxicity. Genetic defects in the beta chain (Leu-CAM deficiency) prevent surface expression of the heterodimers, impair cellular inflammation and result in life-threatening bacterial infections in man. On the other hand, massive expression of Mo1 (& p150,95) on phagocytes causes these cells to become hyperadherent resulting in tissue injury as demonstrated in hemodialysis-related pulmonary dysfunction and reperfusion injury during myocardial infarction in dogs. During the current funding period we showed that Leu-CAM deficiency is secondary to defects in the beta chain, cloned the mutant beta chain in one patient, cloned the alpha chain of Mo1 and identified the roles of Leu-CAM in host defense and inflammation. The goals for the next funding period are: 1) Elucidate the structural defects in the naturally occurring beta subunit mutants from selected patients with Leu-CAM deficiency as means to understand the hitherto unknown functions of the beat chain. cDNA cloning techniques will be used for this purpose. 2) Reverse the genetic defects in leukocyte adhesion by transfecting the normal beta gene in naturally occurring mutant cell lines derived from Leu-CAM deficient patients. 3) Achieve a detailed understanding of the structural basis for the adhesion- promoting functions mediated by Mo1. This will be done by co- transfecting normal or mutagenized alpha or beta chains and structural and functional analysis of transfectants. Site-directed mutagenesis will be guided by the results obtained in (1) and knowledge of the primary structure of the alpha and beta chains. 4) Identify the ligand (s) for Mo1 by raising monoclonal antibodies to Leu-CAM deficient cells which inhibit leukocyte adhesion and/or by screening granulocyte cDNA libraries with a cDNA probe for I- CAM, the ligand for LFA-1. The proposed studies will provide a basis for future gene therapy in a disease that is invariably fatal. The structure-function correlation should permit identification of the domains in Mo1 heterodimer involved in promoting cell adhesion. This should allow development of peptides that specifically inhibit leukocyte-mediated inflammatory functions thus producing an acquired Leu-CAM deficiency state. In view of the finding that anti-Mo1 monoclonal antibodies reduce the size of myocardial infarction in dogs by 50%, the chemotherapeutic and immunosuppressive potential of these peptides are obvious.
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