Functional coupling of glycosyl transfer steps for synthesis of gangliosides in Golgi membranes from neural retina cells.
The synthesis of the oligosaccharide of gangliosides is carried out in the Golgi complex by successive sugar transfers to proper glycolipid acceptors. To examine how the product of one glycosylation step couples with the next transfer step, the endogenous gangliosides of Golgi membranes from 14-day-old chick embryo retina were labeled from CMP-[3H]NeuAc or UDP-[3H]GalNAc or UDP-[3H]Gal in conditions which do not allow vesicular intercompartmental transport. After saturation of the endogenous acceptor capacity, labeling was mostly in the immediate acceptors of the corresponding labeled sugars. However, some labeled intermediates progressed to more glycosylated gangliosides if the membranes were incubated in a second step in the presence of the necessary unlabeled sugar nucleotides. This was particularly evident in the case of membranes incubated with UDP-[3H]Gal, in which most of the [3H]Gal-labeled lactosylceramide synthesized in the first step was converted to GM3 and GD3, or to GM2 or to GD1a in a second incubation step in the presence of unlabeled CMP-NeuAc alone, or together with UDP-GalNAc, or together with UDP-Gal plus UDP-GalNAc, respectively. Conversion was time dependent and dilution-independent. Since prior reports using brefeldin A indicate that transfer steps catalyzed by GalNAc-T, Gal-T2, and Sial-T4 localize in the trans-Golgi network (TGN), our results lead to the following major conclusions: (a) transfer steps catalyzed by GalNAc-T, Gal-T2, and Sial-T4 colocalize and are functionally coupled in the TGN; (b) proximal Golgi Gal-T1, Sial-T1, and Sial-T2, and their corresponding glycolipid acceptors, extend their presence to the TGN, and (c), GalNAc-T and Sial-T2 compete for a common pool of acceptor GM3 in the synthesis of GM2 and GD3.
Maxzúd
M K
MK
Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC (UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina.
Daniotti
J L
JL
Maccioni
H J
HJ
D000818:Animals; D002240:Carbohydrate Sequence; D002642:Chick Embryo; D005732:Gangliosides / Q000096:biosynthesis* / Q000737:chemistry; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry; D012160:Retina / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000378:metabolism
D005732:Gangliosides; D009844:Oligosaccharides; D014536:Uridine Diphosphate N-Acetylgalactosamine; D014532:Uridine Diphosphate Glucose