GA2/GM2/GD2 synthase localizes to the trans-golgi network of CHO-K1 cells.

UDP-GalNAc:lactosylceramide/GM3/GD3 beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T) transforms its acceptors into the gangliosides GA2, GM2 and GD2. It is well established that it is a Golgi-located glycosyltransferase, but its sub-Golgi localization is still unclear. We addressed this question in Chinese hamster ovary K1 cell clones stably transfected with a c-myc-tagged version of GalNAc-T which express the enzyme at different levels of activity. In these cell clones we examined the effect of brefeldin A (BFA) on the synthesis of glycolipids (in metabolic-labelling experiments) and on the sub-Golgi localization of the GalNAc-T (by immunocytochemistry). We found that in cell clones expressing moderate levels of activity, GalNAc-T immunoreactivity behaved as the trans-Golgi network (TGN) marker mannose-6-P receptor (M6PR) both in BFA-treated and untreated cells, and that BFA completely blocked the synthesis of GM2, GM1 and GD1a. On the other hand, in cell clones expressing high levels of activity and treated with BFA, most GalNAc-T immunoreactivity redistributed to the endoplasmic reticulum, as did the medial-Golgi marker mannosidase II, and the synthesis of GM2, GM1 and GD1a was not completely blocked. These results indicate that GalNAc-T is a TGN-located enzyme and that the mechanism that localizes it to this compartment involves steps that, when saturated, lead to its mislocalization to the cis-, medial- or trans-Golgi. Changes of Golgi membrane properties by modification of local glycolipid composition due to the activity of the expressed enzyme were not the main cause of mislocalization, since it persists when glycolipid synthesis is inhibited with d, l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCl.

Identification of an endogenous inhibitor of the UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyl transferase as apolipoprotein A1.

A previously described inhibitor of the UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyltransferase (GalNAc-T) (Quiroga et al., 1, 2), was purified from chicken blood serum by a new procedure. When subjected to SDS-PAGE, two major polypeptides of 27 and 70 kDa were observed. When tested in vitro, only the 27 kDa polypeptide inhibited the GalNAc-T. When added to chick cerebral embryonic neurons in culture, both polypeptides inhibited neuritogenesis. Both the 27 kDa and the 70 kDa fractions were present in the cells at 3 h following their addition to the cultures; both polypeptides had aneuritogenic activity and both inhibited the incorporation of [3H]-galactose into the cell gangliosides modifying their labeling pattern to a similar extent. Sequencing of the amino terminal end of the polypeptides showed that 18 and 9 amino acids from, respectively, the 27 and the 70 kDa polypeptides, were 100% homologues with the corresponding region of chick apolipoprotein Al (apo Al). After addition to cells in culture, no interconversion between the two polypeptides was detected after up to 20 h in culture. A monoclonal antibody that recognizes only the 70 kDa polypeptide, blocks its aneuritogenic effect without modifying that of the 27 kDa fraction. It is concluded that the endogenous inhibitor of GalNAc-T is apo Al.

High-density lipoprotein inhibits UDP-N-acetylgalactosamine:GM3, N-acetylgalactosaminyltransferase and differentiation of cultured cerebral cells: comparison with a formerly described inhibitor of this enzyme.

We have previously described a thermostable inhibitor of the UDP-N-acetylgalactosamine:GM3,N-acetylgalactosaminyltransferase (GM2 synthase) purified from chicken blood serum. Some properties of the GM2 synthase inhibitory preparation (IP) resemble those of high-density lipoprotein (HDL), i.e., both have a MW of 200,000 in native conditions and are resistant to denaturation by heat. These and other facts prompted us to test the possibility that lipoproteins regulate ganglioside biosynthesis in the CNS. For this purpose, serum lipoprotein fractions were isolated from chicken serum by flotation and were assayed as inhibitors of GM2 synthase activity and of neuron differentiation in culture. HDL (in contrast to fractions containing very low-density or low-density lipoprotein) inhibited GM2 synthase with the same specific activity as IP and inhibited neuron cell differentiation in culture in a similar way. Furthermore, these two preparations also share several other characteristics; i.e., both have the same cholesterol content, the same floating behavior on KBr gradients, and the same polypeptide pattern as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with Coomassie Blue, or after western blot and revealing with an antibody prepared against IP, which is able to diminish the inhibitory effect of this preparation. The results described indicate identity between HDL and IP and suggest that HDL (particularly apolipoprotein A) could play an important role on ganglioside biosynthesis modulation during CNS development. The antineuritogenic effect of HDL described in this study could be of physiological relevance during CNS development and response to injury.

Effects of the N-acetylgalactosaminyltransferase inhibitor on cultured cerebral cells.

The inhibitor preparation of the UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyltransferase (EC 2.4.1.92) (GalNAc-T) produces effects on the neurons and the glial (astrocytes) cells of the cerebrum in culture. The effect in culture is evidenced by aneuritogenesis, deficiency in the GalNAc-T activity, and decrease in the content of gangliosides, proteins, and lipids. In isolated glial cells the effect is evidenced by cytoplasm vesiculation and premature cessation of proliferation compared with control culture. The pattern of gangliosides in the inhibited culture shows a decrease in the amount of GD1a with respect to GD3; this is compatible with the notion that the effect is due to an inhibitor of the GM2 synthase. The inhibitor effects are reverted when it is eliminated after 24 or 48 hr in the culture medium.