GM1 Synthase Depends on N-Glycosylation for Enzyme Activity and Trafficking to the Golgi Complex
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Glycosyltransferase cDNAs contain a variable number of potential N-glycosylation sites. Here we examined the occupancy and relevance for the activity and intracellular trafficking of the only potential N-glycosylation site of the mouse β1,3galactosyltransferase (Gal-T2 or GA1/GM1/GD1b synthase) in Gal-T2 cDNA transfected CHO-K1 cells. Transfected cells synthesize a Golgi located active enzyme of 43 kDa whose N-glycan was metabolically labeled from [3H]mannose and was Endo-H sensitive. Inhibition of N-glycosylation by Tunicamycin or by point mutation of the N-glycosylation site resulted in the synthesis of a polypeptide of 40 kDa which lacked enzyme activity and was concentrated in the endoplasmic reticulum (ER). Inhibition of ER glucosidases by Castanospermine impaired the exit of a form of Gal-T2 having reduced enzyme activity from the ER. The N-terminal Gal-T2 domain (aa 1–52) was able to direct and to retain the green fluorescence protein in the Golgi complex. Taken together, these results indicate that Gal-T2 depends on N-glycosylation for its activity and for proper trafficking to, but not its retention in, the Golgi complex.
- van Echten-Deckert, G. and Sandhoff, K. 1998. Organization and Topology of Sphingolipid Metabolism, Chapter 5, in Pinto, B. M. (ed.). Comprehensive Natural Products Chemistry, Vol. 3, Pergamon Elsevier Science, New York.
- Panzetta, P., Maccioni, H. J. F., and Caputto, R. 1980. Synthesis of retinal gangliosides during chick embryonic development. J. Neurochem. 35:100-108.
- Yu, R. K., Macala, L. J., Taki, T., Weinfield, H. M., and Yu, F. S. 1988. Developmental changes in ganglioside composition and synthesis in embryonic rat brain. J. Neurochem. 50:1825-1829.
- Ruan, S., and Lloyd, K. O. 1992. Glycosylation pathways in the biosynthesis of gangliosides in melanoma and neuroblastoma cells: relative glycosyltransferase levels determine ganglioside patterns. Cancer Res. 52:5725-5731.
- Zeng, G., Ariga, T., Gu, X. B., and Yu, R. K. 1995. Regulation of glycolipid synthesis in HL-60 cells by antisense oligodeoxynucleotides to glycosyltransferase sequences: effect on cellular differentiation. Proc. Natl. Acad. Sci. U S A, 92:8670-8674.
- Jaskiewicz, E., Zhu, G., Taatjes, D. J., Darling, D. S., Zwanzig, G. E. Jr, and Young, W. W. Jr 1996. Cloned beta 1,4N-acetylgalactosaminyltransferase: subcellular localization and formation of disulfide bonded species. Glycoconjugate J. 13:213-223.
- Rosales Fritz, V. M., Daniotti, J. L., and Maccioni, H. J. F. 1997. Chinese hamster ovary cells lacking GM1 and GD1a synthesize gangliosides upon transfection with human GM2 synthase. Biochim. Biophys. Acta 1354:153-158.
- Maccioni, H. J. F., Daniotti, J. L., and Martina, J. A. 1999. Organisation of ganglioside synthesis in the Golgi apparatus. Biochim. Biophys. Acta 1437, 101-118.
- Bieberich, E., and Yu, R. K. 1999. Multi-enzyme kinetic analysis of glycolipid biosynthesis. Biochim. Biophys. Acta 1432: 113-124.
- Field, M. C., and Wainwright, L. J. 1995. Molecular cloning of eukaryotic glycoprotein and glycolipid glycosyltransferases: a survey. Glycobiology 5:463-472.
- Haraguchi, M., Yamashiro, S., Furukawa, K., Takamiya, K., Shiku, H., and Furukawa, K. 1995. The effects of the site-directed removal of N-glycosylation sites from beta-1,4-N-acetylgalactosaminyltransferase on its function. Biochem. J. 312: 273-280.
- Nagai, K., Ihara, Y., Wada, Y., and Taniguchi, N. 1997. Nglycosylation is requisite for the enzyme activity and Golgi retention of N-acetylglucosaminyltransferase III. Glycobiology 7:769-76.
- Martina, J. A., Daniotti, J. L., and Maccioni, H. J. 1998. Influence of N-glycosylation and N-glycan trimming on the activity and intracellular traffic of GD3 synthase J. Biol. Chem. 273: 3725-3731.
- Daniotti, J. L., Martina, J. A., Zurita, A., and Maccioni, H. J. F. 1999. Mouse beta 1,3-Galatosyltransferase (GA1/GM1/GD1b Synthase): protein characterization, tissue expression, and developmental regulation in neural retina. J. Neurosc. Res. 56: 318-327.
- Sells, M. A., and Chernoff, J. 1995. Epitope tag vectors for eukaryotic protein production. Gene 152:187-189.
- Laemmli, U. K. 1970. Cleave and structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
- Towbin, H., Staehelin, T., and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354.
- Martina, J. A., Daniotti, J. L., and Maccioni, H. J. F. 1995. A UDP-GalNAc pyrophosphatase activity is developmentally regulated in the rat retina. J. Neurochem. 64:1274-1280.
- Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 7:248-254.
- Cole, N. B., Smith, C. L., Sciaky, N., Terasaki, M., Edidin, M., and Lippincott-Schwartz, J. 1996. Diffusional mobility of Golgi proteins in membranes of living cells. Science 273:797-801.
- Donaldson, J. G., and Klausner, R. D. 1994. ARF: a key regulatory switch in membrane traffic and organelle structure. Curr. Opin. Cell Biol. 6:527-532.
- Haraguchi, M., Yamashiro, S., Furukawa, K., Takamiya, K., Shiku, H., and Furukawa, K. 1995. The effects of the site-directed removal of N-glycosylation sites from beta-l,4-N-acetylgalactosaminyltransferase on its function. Biochem. J. 312:273-280.
- Colley, K. J. 1997. Golgi localization of glycosyltransferases: more question than answers. Glycobiology 7:1-13.
- Trombetta, E. S., and Helenius, A. 1998. Lectins as chaperones in glycoprotein folding. Curr. Opin. Struct. Biol. 5:587-592.
- Parodi, A. J. 1999. Reglucosylation of glycoproteins and quality control of glycoprotein folding in the endoplasmic reticulum of yeast cells. Biochim. Biophys. Acta 1426, 287-295.
- GM1 Synthase Depends on N-Glycosylation for Enzyme Activity and Trafficking to the Golgi Complex
Volume 25, Issue 5 , pp 725-731
- Cover Date
- Print ISSN
- Online ISSN
- Kluwer Academic Publishers-Plenum Publishers
- Additional Links
- GM1 synthase
- glycolipid expression
- Golgi complex
- N-glycan trimming
- Industry Sectors
- Author Affiliations
- 1. Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC (UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, 5000, Córdoba, Argentina
- 3. Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC (UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, 5000, Córdoba, Argentina