Glycophorins A is the major glycoprotein on the human red blood cell surface and contains N-glycans and O-glycans. Glycophorin B, a highly homologous glycoprotein on human red blood cells, contains only O-glycans. Although appropriate glycosylation is crucial for cell surface expression of glycophorin A, the mechanism responsible for this effect is not understood. Glycophorin A and B are important in the practice of transfusion medicine since they carry several different human blood group antigens, and antibodies to these antigens can cause hemolytic transfusion reactions, hemolytic disease of the newborn, and autoimmune hemolytic anemia. However, there have been few studies examining the fine specificity of binding of human polyclonal and mouse monoclonal antibodies to these molecules. Glycophorins A and B are also of medical importance because they can serve as red blood cell surface receptors for the invasion of Plasmodium falciparum malaria merozoites. Due to the difficulty in obtaining mutant glycophorin molecules with defined variations in amino acid sequence and oligosaccharide structure, a detailed understanding of this red blood cell-parasite interaction is not yet available. The goals of the current proposal are to study the cell biology, immunology, and receptor function of the human blood group glycophorin antigens by: 1) determining the role that the N-glycans and O-glycans play in intracellular transport of glycophorin A, 2) using recombinant DNA approaches to examine the murine and human immune response to glycophorins A and B, and 3) determining the peptide and carbohydrate portions of glycophorins A and B which are recognized by human malaria parasites. These, goals will be achieved by using a series of stably transfected cell lines expressing cDNA of wild type or variant glycophorin A or B. New mutant glycophorin A and B cDNAs will be constructed by site-directed mutagenesis. By expressing the cDNAs in both normal Chinese hamster ovary fibroblsts and those with defects in glycosylation, it is possible to create variant glycophorins which differ in both amino acid and carbohydrate sequence. Phage display libraries will be constructed to examine the murine immune response to glycophorin A. This will determine whether the immune response is restricted and will also result in the construction of new clinically, useful serological reagents. In summary, these studies will result in greater understanding of the cell biology, immunology, and receptor function of glycophorins A and B.
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