The activation of B-lymphocytes (B-cells) leads to increased synthesis of membrane-bound and secretory glycoproteins. This application proposes studies on the regulation of N-glycosylation activity when membrane glycoprotein synthesis is stimulated in resting B-cells induced to proliferate by bacterial lipopolysaccharide (LPS). The control of N-glycosylation will also be investigated in the human B lymphoblastoid cell line (CESS) stimulated to differentiate and secrete large amounts of immunoglobulins by exposure to B-cell differentiation factor (BCDF). Changes in N-glycosylation activity in response to the induction of proliferation or differentiation of the B-cells will be correlated with changes in several enzymes involved in dolichyl phosphate (Do1-P) metabolism and the synthesis of dolichol-linked oligosaccharides. Dolichol kinase and Dol-P phosphatase activity will be assayed in vitro to determine if Dol-P levels could be altered by shifting the balance in a phosphorylation- dephosphorylation scheme during the proliferation of B-cells or the differentiation of the CESS line. The requirement for de novo synthesis of dolichol or Dol-P during the proliferative and differentiative changes will be explored by metabolic labeling experiments with (3H)mevalonate, and studies with exogenously supplied dolichol and capactin, the competitive inhibitor of HMG- CoA reductase. The possible involvement of Ca++-mobilization and protein kinase C in the activation processes will be explored by cellular studies with ionomycin and phorbol diesters. The proposed experiments could shed new light on the control of the N-glycosylation apparatus in B-cells, and extend the current knowledge of the biochemical events occurring during the proliferation and differentiation of B-lymphocytes.
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