The long term objective is to determine how cobalamin (Cbl) functions in the synthesis and release of immunoglobulins by human lymphocytes. The postulate is that methionine (Met) synthesis via theCbl dependent methionine synthesis (MS) reaction makes available S-adenosylmethionine (AdoMet) which serves as a methyl donor to intracellular macromolecules, e.g. t RNA. The first immediate aim is to characterize, identify the mechanisms, and determine the consequences of the Met auxotrophy of human lymphocytes. The models, as in the past, will be cultured human peripheral lymphocytes and an established line of human B cells. The studies will include examination of synthesis of Met and AdoMet, studying the effects of several stimulations of blastogenesis, studying the effects of transcobalamin II (TC II) in other auxotropies of Met and examining cell methylation in the presence and absence of Met.
A second aim will be to determine why the transport protein of Cbl TC II influences the intracellular function of Cbl, i.e. in circumventing Met auxotrohy. The basic approach will be to compare the fate of TC II-Cbl and free Cbl in the effects on MS activity, synthesis of Met and AdoMet, and conversion to coenzymes of Cbl.
A third aim will be to correlate synthesis and release of total protein and IgG with MS activity (including blocking studies), synthesis of Met and AdoMet and methylation of t RNA. Finally the effects of Cbl depletion on blood cells will be studied in both in vitro models and after depletion in vivo in order to apply some of the data obtained here in clinical diagnosis. It is already known that Cbl deficiency affects adversely responses to infection and the thrust of the present study is to identify consequences and mechanisms with expectations for better management.
