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.

GD3 and GM2 synthase activities in rat testes during the period of sexual development.

Activities of two key enzymes of gangliosides biosynthesis were determined in rat testes during development. GD3 synthase activity was low and showed small variations with age. GM2 synthase activity increased 10-fold in testes from 10- to 30-d-old animals, showing a maximum activity at 30 d, followed by a small decrease until 45 d and then a constant activity up to adulthood. These developmental changes in the activity of both glycosyltransferases were related to the increasing complexity in the ganglioside pattern observed in rats testes during the period of sexual development.

[Sialyltransferase activity in the spermatic membrane and its relation to human fertility and sterility]. / Actividad de sialiltransferasa en la membrana espermática y su relación con fertilidad y esterilidad humanas.

Sialyltransferase activity was determined on normospermic men sperm cells (greater than 80 x 10(6) sperm/ml and 75% motility) and oligospermic infertile sperm cells (less than 20 x 10(6) sperm/ml and less than 20% motility) and asthenospermic (greater than 40 x 10(6) sperm/ml and less than 10% motility). Sialytransferase activity is quantified by means of the transference of radioactivity of CMP-3H-sialic acid toward the exogenous acceptor (asialofetuin). The enzyme substrate complexes formed in presence of phosphotungstic acid result precipitated insoluble, which was retained on glass fiber filter. The sialyltransferase activity decrease in oligospermic sperm cells 62 +/- 3% and in the asthenospermic decreased 57 +/- 4% with respect normospermic sperm cells. The decrement on sialyltransferase activity in the infertile sperm, permits to assume that this enzyme probably participates as a direct cause of its pathology with detrimental structural and functional integrity of the plasma membrane.