BIOLOGY OF THE HUMAN GLYCOPHORIN BLOOD GROUP ANTIGENS

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Glycophorin A is the major glycoprotein on the human red blood cell surface and contains both N- and O-linked oligosaccharides. Although structurally well characterized, relatively little is known concerning either the biosynthesis of glycophorin A or the role of glycosylation in translocation and surface expression of this molecule. Glycophorin A is important in the practice of transfusion medicine since it carries 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 or mouse monoclonal antibodies to this molecule. Glycophorin A is also of medical importance because it can serve as the red blood cell surface receptor for the invasion of Plasmodium falciparum malaria merozoites. Due to the difficulty in obtaining mutant glycophorin A 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 biology of the human blood group glycophorin antigens by: 1) determining the importance of the N- and O-linked oligosaccharides in intracellular trafficking and surface expression of glycophorin A, 2) examining the fine specificity of the interactions between glycophorin A and human and mouse antibodies, and 3) determining the peptide and carbohydrate portions of glycophorin A which are recognized by human malaria parasites. These goals will be achieved by creating a series of cell lines stably transfected with either wild type or variant glycophorin A cDNA. Mutant glycophorin A cDNAs will be constructed by site-directed mutagenesis using the polymerase chain reaction. By expressing these cDNAs in both normal Chinese hamster ovary fibroblasts and those with defects in glycosylation, it will be possible to create variant glycophorin A molecules which differ in both amino acid and carbohydrate sequence. Both the intact cell lines and glycophorin A proteins purified from the cell lines will then be used to address these three goals.