This proposal deals with structural, biosynthetic and developmental aspects of the major sialoglycoprotein of the human erythrocyte, glycophorin A, whose carbohydrate and peptide primary structures are well understood. The protein is a product of the polymorphic M-N gene locus, and this polymorphism is expressed by amino acid substitutions within the amino terminal pentapeptide. The protein also exhibits single-site carbohydrate polymorphism and this renders it a potentially useful marker of the influence of peptide substitution on co-translational and post-translational events. Carbohydrate and peptide variants of the M-N locus and related other loci will be identified by serological screening, the glycoproteins isolated and their carbohydrate and polypeptide structures determined. A cDNA glycophorin A probe will be prepared and used to assess the organization and expression of the M-N locus in erythroid cells from M-N and variant individuals. Because glycophorin A is extensively glycosylated, and because it resides on the surface of a terminally differentiated cell, we propose to investigate whether carbohydrate changes may precede or be a consequence of cell differentiation, and whether glycosylation is critical to both translocation to and permanent residence on the cell surface. We will continue the biosynthetic and structural studies of glycophorin A in the K562 leukemic human cell line that appears arrested at some static stage of erythroid differentiation. We have isolated its major glycoprotein species, three of which are immunoreactive with anti-glycophorin A; using microscale procedures, we will examine their structures and assess how these molecules relate to each other, to erythrocyte glycophorin A, as well as erythrocyte band 3. We will evaluate whether or not the carbohydrate units differ structurally from those of erythrocytes. We will study the sequence of temporal and metabolic events leading to the assembly of O- and N- glycosidic units and assess their role in the insertion of the protein into the surface membrane. We propose to use the cDNA probe as a tool to relate the expression of the glycophorin gene to the processes of glycosylation and membrane targeting. Ultimately we propose to ask similar questions in primary cells undergoing dynamic erythroid differentiation. However, because semi-solid cultures of peripheral blood erythroid cells, that we now use, provide limited numbers of erythroblasts for some of the studies, we propose to develop multipotent hemopoietic cell lines that respond to erythropoietin and other hemopoietic growth factors.
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