Kimmo K. Karhi

Identification of blood group A-active glycoproteins in the human erythrocyte membrane

Normal human erythrocytes of blood groups A1, A2, B and O, and En (a-) erythrocytes lacking glycophorin A, but with A1B-activity, were surface-labeled with tritiated sodium borohydride after oxidation of terminal galactosyl and N-acetylgalactosaminyl residues with galactose oxidase. A1 cells were also labeled by lactoperoxidase catalyzed iodination. After solubilization in Triton X-100, the blood group A-active glycoconjugates were isolated using the A-specific lectin from Vicia cracca coupled to Sepharose. No radioactivity was bound from erythrocytes of B and O blood groups. The glycoconjugates from A cell membranes which bound to the lectin and were eluted with 0.01 M N-acetyl-D-galactosamine were analyzed using cylindrical or slab gel electrophoresis in the presence of sodium dodecyl sulfate. The A-active glycoproteins included the major integral glycoprotein, band 3, and many minor, previously poorly defined components. Glycophorins A and B did not contain A-activity.

Glycophorin A as an Erythroid Marker in Normal and Malignant Hematopoiesis

Glycophorin A (GpA), which is the major sialoglycoprotein on human red cells, is one of the best characterized mammalian integral membrane proteins (Marchesi et al. 1972; Tomita and Marchesi 1975). Its amino acid sequence is known. The protein molecule contains three distinct domains. A large hydrophilic portion, carrying the NH2-terminal, is located on the external surface of the red cell, and the COOH-terminal is located in the cytoplasm (Bretscher 1975) and probably interacts with peripheral proteins on the inner aspect of the membrane. These two hydrophilic sequences are connected by a hydrophobic segment of 23 amino acids which must be embedded within the lipid bilayer.

Isolation and characterization of the blood group A-specific lectin from Vicia cracca

We have isolated the blood group A-specific lectin from Vicia cracca by affinity chromatography on immobilized porcine blood group A/H substance. A molecular weight of 100 000 was obtained by gel filtration and analytical ultracentrifugation. The subunit size when determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 29 000. The lectin contains no half-cystine or methionine. It is a glycoprotein apparently with one oligosaccharide per subunit. The oligosaccharide contains mainly D-mannose and D-glucosamine but no D-galactose or sialic acid.

[22] Glycophorin A: In vitro biogenesis and processing

Publisher Summary This chapter emphasizes on the synthesis of the glycophorin A polypeptide in the K562 cell line and its intriguing posttranslational modifications, including N- and O-glycosylation, phosphorylation, and sulfation. The chapter also discusses its cell-free synthesis using glycophorin A mRNA obtained from K562 cells. The methods involve radioactive cell surface labeling of erythrocytes, radioactive metabolic labeling of K562 Cells, isolation of mRNA from K562 Cells, translation of mRNA in vitro, preparation of Lectin-Sepharose columns, preparation of anti-glycophorin A antiserum, Lectin-Sepharose affinity chromatography and immune precipitations, treatment of glycophorin A with endoglycosidase H, and polyacrylamide slab gel electrophoresis. The illustrative data and interpretation shows that tunicamycin inhibits N-glycosylation of proteins, and endoglycosidase H has no effect on either the lentil lectin- or wheat germ lectin-adsorbed molecules. The tunicamycin experiments clearly shows that the absence of the N-glycosidic oligosaccharide does not affect the intracellular migration of glycophorin A.