RESUMEN
Recent studies demonstrated that mutations in B3GNT1, an enzyme proposed to be involved in poly-N-acetyllactosamine synthesis, were causal for congenital muscular dystrophy with hypoglycosylation of α-dystroglycan (secondary dystroglycanopathies). Since defects in the O-mannosylation protein glycosylation pathway are primarily responsible for dystroglycanopathies and with no established O-mannose initiated structures containing a ß3 linked GlcNAc known, we biochemically interrogated this human enzyme. Here we report this enzyme is not a ß-1,3-N-acetylglucosaminyltransferase with catalytic activity towards ß-galactose but rather a ß-1,4-glucuronyltransferase, designated B4GAT1, towards both α- and ß-anomers of xylose. The dual-activity LARGE enzyme is capable of extending products of B4GAT1 and we provide experimental evidence that B4GAT1 is the priming enzyme for LARGE. Our results further define the functional O-mannosylated glycan structure and indicate that B4GAT1 is involved in the initiation of the LARGE-dependent repeating disaccharide that is necessary for extracellular matrix protein binding to O-mannosylated α-dystroglycan that is lacking in secondary dystroglycanopathies.
Asunto(s)
Distroglicanos/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Secuencia de Aminoácidos , Biocatálisis , Disacáridos/metabolismo , Glicosilación , Células HEK293 , Humanos , Cinética , Modelos Biológicos , Datos de Secuencia Molecular , N-Acetilglucosaminiltransferasas/química , Pentosiltransferasa/metabolismo , Solubilidad , Estereoisomerismo , Especificidad por Sustrato , Trisacáridos/metabolismo , Uridina Difosfato Ácido Glucurónico/metabolismo , Xilosa/química , Xilosa/metabolismo , UDP Xilosa Proteína XilosiltransferasaRESUMEN
Dystroglycan is a cell membrane receptor that organizes the basement membrane by binding ligands in the extracellular matrix. Proper glycosylation of the α-dystroglycan (α-DG) subunit is essential for these activities, and lack thereof results in neuromuscular disease. Currently, neither the glycan synthesis pathway nor the roles of many known or putative glycosyltransferases that are essential for this process are well understood. Here we show that FKRP, FKTN, TMEM5 and B4GAT1 (formerly known as B3GNT1) localize to the Golgi and contribute to the O-mannosyl post-phosphorylation modification of α-DG. Moreover, we assigned B4GAT1 a function as a xylose ß1,4-glucuronyltransferase. Nuclear magnetic resonance studies confirmed that a glucuronic acid ß1,4-xylose disaccharide synthesized by B4GAT1 acts as an acceptor primer that can be elongated by LARGE with the ligand-binding heteropolysaccharide. Our findings greatly broaden the understanding of α-DG glycosylation and provide mechanistic insight into why mutations in B4GAT1 disrupt dystroglycan function and cause disease.