RESUMEN
A/B-Transferase is a glycosyltransferase that transfers a sugar substrate onto H-antigen, which is responsible for the synthesis of glycoprotein- and glycolipid-conjugates termed A/B-antigens. One polymorphism that causes the Pro234Ser substitution in B-transferase was recently found in a genotyping study, and might be cis-AB. In the present study, we analyzed the phenotypes arising from the enzymatic specificity of B-transferase with the Pro234Ser mutation. To evaluate the effect of the P234S mutation on enzymatic specificity, we generated an expression plasmid for B-transferase with Pro234Ser as well as A-transferase with Leu266Met, which is frequently found in cis-ABs. Transfection of B-transferase/P234S or A-transferase/L266M cDNA into HeLa cells, an O-blood group cell line, resulted in an AB-phenotype by absorption-elution testing and immunostaining, whereas A- and B-transferase-expressing HeLa cells exhibited only their own activity. Molecular simulation indicated that the P234S mutation causes a conformational change in the substrate pocket making it suitable for N-acetylgalactosamine.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/genética , Acetilgalactosamina/metabolismo , Mutación , N-Acetilgalactosaminiltransferasas/genética , Polimorfismo Genético/inmunología , Sistema del Grupo Sanguíneo ABO/inmunología , Sistema del Grupo Sanguíneo ABO/metabolismo , Cromatografía de Afinidad , Femenino , Genotipo , Células HeLa , Humanos , Inmunohistoquímica , Simulación de Dinámica Molecular , N-Acetilgalactosaminiltransferasas/inmunología , N-Acetilgalactosaminiltransferasas/metabolismo , Fenotipo , Plásmidos , Estructura Terciaria de Proteína , Especificidad por Sustrato , TransfecciónRESUMEN
A/B-transferase is a glycosyltransferase that transfers a sugar substrate onto H-antigen resulting in the synthesis of glycoproteins and glycolipids termed A/B-antigens. The ABO blood group (ABO) gene encoding A/B-transferase possesses numerous polymorphisms affecting the specificity and/or activity of the enzyme. The relationship between genotype and phenotype is very complicated, except for those of some critical polymorphisms. In order to establish a system for evaluating the effect of each polymorphism on the transferase function, an A- or B-transferase cDNA expressing vector was introduced into HeLa cells, a cell line that do not possess endogenous A/B-transferase activity. We successfully detected substrate-specific transferase activity in the cells and in the culture medium. Furthermore, in three different assays, each corresponding A- or B-antigen was detected in the transfectants with high sensitivity. Accordingly, the present study demonstrates a possibility that A/B-transferase variants may be characterized by using this method.
