RESUMEN
Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating this technology to clinical practice has been the lack of efficient glycosidase enzymes. Here we report two bacterial glycosidase gene families that provide enzymes capable of efficient removal of A and B antigens at neutral pH with low consumption of recombinant enzymes. The crystal structure of a member of the alpha-N-acetylgalactosaminidase family reveals an unusual catalytic mechanism involving NAD+. The enzymatic conversion processes we describe hold promise for achieving the goal of producing universal RBCs, which would improve the blood supply while enhancing the safety of clinical transfusions.
Asunto(s)
Bacterias/enzimología , Eritrocitos/metabolismo , Glicósido Hidrolasas/metabolismo , Sistema del Grupo Sanguíneo ABO/química , Sitios de Unión , Tipificación y Pruebas Cruzadas Sanguíneas , Catálisis , Cromatografía en Capa Delgada , Citometría de Flujo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Células Procariotas/enzimología , Estructura Secundaria de Proteína , Especificidad por Sustrato , Volumetría , alfa-N-Acetilgalactosaminidasa/químicaAsunto(s)
Química/historia , Glicoconjugados/química , Sistema del Grupo Sanguíneo ABO/química , Sistema del Grupo Sanguíneo ABO/historia , Sistema del Grupo Sanguíneo ABO/metabolismo , Química/métodos , Glicoconjugados/historia , Glicoconjugados/metabolismo , Glucolípidos/química , Glucolípidos/historia , Glucolípidos/metabolismo , Glicómica/historia , Glicómica/métodos , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Estructura Molecular , Neoplasias/metabolismo , Poesía como Asunto , Proteómica/historia , Proteómica/métodos , Estados UnidosRESUMEN
In search of alpha-galactosidases with improved kinetic properties for removal of the immunodominant alpha1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of alpha-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454-464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Galalpha1-3(Fucalpha1-2)Gal, whereas linear oligosaccharides terminated by alpha1,3-linked galactose such as the immunodominant xenotransplantation epitope Galalpha1-3Galbeta1-4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific alpha1,3-galactosidases that act equally well on both branched blood group B and linear alpha1,3Gal structures. We determined by one-dimensional (1)H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known alpha-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant alpha3Gal xenotransplantation epitope.