GM3 alpha2,8-sialyltransferase (GD3 synthase): protein characterization and sub-golgi location in CHO-K1 cells.
GD3 synthase (Sial-T2) is a key enzyme of ganglioside synthesis that, in concert with GM2 synthase (GalNAc-T), regulates the ratio of a- and b-pathway gangliosides. In this work, we study the sub-Golgi location of an epitope-tagged version of chicken Sial-T2 transfected to CHO-K1 cells. The expressed protein was enzymatically active both in vitro and in vivo and showed a molecular mass of approximately 47 or approximately 95 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of, respectively, beta-mercaptoethanol. The 95-kDa form of Sial-T2 was also detected if the protein was retained in the endoplasmic reticulum (ER) due to impaired glycosylation, indicating that it was formed in the ER. Confocal immunofluorescence microscopy showed Sial-T2 localized to the Golgi complex and, within the organelle, partially co-localizing with the mannose-6-phosphate receptor, a marker of the trans-Golgi network (TGN). In cells treated with brefeldin A, a major fraction of Sial-T2 redistributed to the ER, even under controlled expression to control for mislocalization due to protein overloading. In experiments of incorporation of sugars into endogenous acceptors of Golgi membranes in vitro, GD3 molecules formed by incubation with CMP-NeuAc were converted to GD2 upon incubation with UDP-GalNAc. These results indicate that Sial-T2 localizes mainly to the proximal Golgi, although a fraction is located in the TGN functionally coupled to GalNAc-T. Consistent with this, most of the enzyme was in an endoglycosidase H (Endo-H)-sensitive, neuraminidase (NANase)-insensitive form. A minor secreted form lacking approximately 40 amino acids was Endo-H-resistant and NANase-sensitive, indicating that the cells were able to process N-glycans to Endo-H-resistant forms. Taken together, the results of these biochemical and immunocytochemical experiments indicate that in CHO-K1 cells, most Sial-T2 localizes in the proximal Golgi and that a functional fraction is also present in the TGN.
Daniotti
J L
JL
Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC (UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, Argentina.
Martina
J A
JA
Giraudo
C G
CG
Zurita
A R
AR
Maccioni
H J
HJ
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002645:Chickens; D006224:Cricetinae; D018076:DNA, Complementary; D004441:Ecdysterone / Q000031:analogs & derivatives / Q000494:pharmacology; D000939:Epitopes / Q000235:genetics; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic; D006031:Glycosylation; D006056:Golgi Apparatus / Q000187:drug effects / Q000201:enzymology*; D008360:Mannosephosphates / Q000235:genetics; D017350:N-Acetylgalactosaminyltransferases / Q000032:analysis / Q000378:metabolism; D012799:Sialyltransferases / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D018076:DNA, Complementary; D004441:Ecdysterone; D000939:Epitopes; D008360:Mannosephosphates; D017350:N-Acetylgalactosaminyltransferases; D012799:Sialyltransferases; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C027693:mannose-6-phosphate; C008999:ponasterone A; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase; C043662:alpha-N-acetylneuraminate alpha-2,8-sialyltransferase