Regulation of ganglioside biosynthesis in the nervous system
2004; Elsevier BV; Volume: 45; Issue: 5 Linguagem: Inglês
10.1194/jlr.r300020-jlr200
ISSN1539-7262
AutoresRobert K. Yu, Erhard Bieberich, Tian Xia, Guichao Zeng,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoGanglioside biosynthesis is strictly regulated by the activities of glycosyltransferases and is necessarily controlled at the levels of gene transcription and posttranslational modification. Cells can switch between expressing simple and complex gangliosides or between different series within these two groups during brain development. The sequential biosynthesis of gangliosides in parallel enzymatic pathways, however, requires fine-tuned subcellular sequestration and orchestration of glycosyltransferases. A popular model predicts that this regulation is achieved by the vectorial organization of ganglioside biosynthesis: sequential biosynthetic steps occur with the traffic of ganglioside intermediates through subsequent subcellular compartments. Here, we review current models for the subcellular distribution of glycosyltransferases and discuss results that suggest a critical role of N-glycosylation for the processing, transport, and complex formation of these enzymes. In this context, we attempt to illustrate the regulation of ganglioside biosynthesis as well as the biological significance of N-glycosylation as a posttranslational regulatory mechanism.We also review the results of analyses of the 5′ regulatory sequences of several glycosyltransferases in ganglioside biosynthesis and provide insights into how their synthesis can be regulated at the level of transcription. Ganglioside biosynthesis is strictly regulated by the activities of glycosyltransferases and is necessarily controlled at the levels of gene transcription and posttranslational modification. Cells can switch between expressing simple and complex gangliosides or between different series within these two groups during brain development. The sequential biosynthesis of gangliosides in parallel enzymatic pathways, however, requires fine-tuned subcellular sequestration and orchestration of glycosyltransferases. A popular model predicts that this regulation is achieved by the vectorial organization of ganglioside biosynthesis: sequential biosynthetic steps occur with the traffic of ganglioside intermediates through subsequent subcellular compartments. Here, we review current models for the subcellular distribution of glycosyltransferases and discuss results that suggest a critical role of N-glycosylation for the processing, transport, and complex formation of these enzymes. In this context, we attempt to illustrate the regulation of ganglioside biosynthesis as well as the biological significance of N-glycosylation as a posttranslational regulatory mechanism. We also review the results of analyses of the 5′ regulatory sequences of several glycosyltransferases in ganglioside biosynthesis and provide insights into how their synthesis can be regulated at the level of transcription. ErrataJournal of Lipid ResearchVol. 46Issue 6PreviewIn the article "Substrate reduction reduces gangliosides in postnatal cerebrum-brainstem and cerebellum in GM1 gangliosidosis mice" by Kasperzyk et al., published in the April 2004 issue of the Journal of Lipid Research (Volume 46, pages 744–751), the affiliations should read as follows: Full-Text PDF Open Access Gangliosides are important constituents of cells and play a variety of biological functions, including cellular recognition and adhesion as well as signaling. The expression of gangliosides is not only cell specific and developmentally regulated but also closely related to the differentiation state of the cell (1Yu R.K. Macala L.J. Taki T. Weinfeld H.M. Yu F.S. Developmental changes in ganglioside composition and synthesis in embryonic rat brain.J. Neurochem. 1988; 50: 1825-1829Google Scholar, 2Yu R.K. Developmental regulation of ganglioside metabolism.Prog. Brain Res. 1994; 101: 31-44Google Scholar). Numerous studies have indicated that ganglioside changes during cellular differentiation are closely related to their metabolism, particularly in their biosynthesis. Thus, it is possible to control the expression of gangliosides by regulating glycosyltransferase (GT) activities that are responsible for ganglioside biosynthesis. The activities of GTs can be regulated at several levels, including transcriptional and posttranslational controls. At least two basic mechanisms of posttranslational control can be affected, phosphorylation and N-glycosylation. Phosphorylation/dephosphorylation offers an important "fast" regulatory mechanism in response to physiological demands, and its implications have recently been reviewed (3Yu R.K. Bieberich E. Regulation of glycosyltransferases in ganglioside biosynthesis by phosphorylation and dephosphorylation.Mol. Cell. Endocrinol. 2001; 177: 19-24Google Scholar). Here, we focus on the N-glycosylation of GTs, an important step in regulating their activities, half-life, and intracellular transport and localization. In addition, in recent years, many cDNAs encoding GTs for ganglioside biosynthesis have been cloned by several investigators, including us. The genes of these enzymes can also be regulated at the transcriptional level during brain development. Analysis of the 5′ flanking region of the GT genes revealed a number of common features and potential sites for known transcription factors. Here, we review studies of the promotor region to gain insights into these regulatory mechanisms. An understanding of the regulation of GTs should provide powerful means for investigating the functional roles of gangliosides in cellular differentiation and proliferation. The use of radiolabeled sphingolipid precursors has shown that in ganglioside biosynthesis, subsequent reactions are catalyzed by GTs that act in parallel pathways. As shown in Fig. 1, these pathways give rise to different series of simple and complex gangliosides. In general, ganglioside biosynthesis starts with ceramide, the common precursor for acidic and nonacidic glycosphingolipids. Ceramide is first converted to glucosylceremide by UDP-glucose:ceramide β-glucosyltransferase, which is then followed by the addition of galactose, yielding lactosylceramide. The addition of the first sialic acid residue converts lactosylceramide to GM3, the precursor of most of the complex brain gangliosides, which is catalyzed by sialyltransferase I (ST1) or GM3-synthase. Further addition of sialic acid residues generates GD3 (catalyzed by ST2 or GD3-synthase) and GT3 (catalyzed by ST3 or GT3-synthase). ST1, ST2, and ST3 are all distinct enzyme entities arising from different genes. GM3, GD3, and GT3 represent the entry substrates for the biosynthesis of complex-type gangliosides in the a-, b-, and c-series pathways, respectively (Fig. 1). Further synthesis of the complex hexosamine-containing gangliosides is unique in that several identical GTs may participate in catalyzing the addition of various sugar residues to different ganglioside acceptor substrates. These enzymes include N-acetylgalactosaminyltransferase I or GM2/GD2-synthase, galactosyltransferase II (GalT2) or GM1/GD1b-synthase, ST4 or GD1a/GT1b-synthase, and ST5 or GT1a/GQ1b-synthase. Most of the basic steps have been characterized using rather crude cell-free systems (4Basu S. Kauman B. Roseman S. Conversion of Tay-Sachs ganglioside to monosialoganglioside by brain uridine diphosphate D-galactose:glycolipid galactosyltransferase.J. Biol. Chem. 1965; 240: 4115-4117Google Scholar, 5Kaufman B. Basu S. Roseman S. Enzymatic synthesis of disialogangliosides from monosialogangliosides by sialyltransferases from embryonic chicken brain.J. Biol. Chem. 1968; 243: 5804-5807Google Scholar, 6Panzetta P. Maccioni H.J.F. Caputto R. Synthesis of retinal gangliosides during chick embryonic development.J. Neurochem. 1970; 35: 100-108Google Scholar, 7Maccioni H.J.F. Arce A. Caputto R. The biosynthesis of gangliosides, labelling of rat brain gangliosides in vivo.Biochem. J. 1971; 125: 1131-1137Google Scholar, 8Fishman P.H. Brady R.O. Biosynthesis and function of gangliosides.Science. 1976; 194: 906-914Google Scholar, 9Yu R.K. Lee S.H. In vitro biosynthesis of sialosylgalactosylceramide (G7) by mouse brain microsomes.J. Biol. Chem. 1976; 251: 198-203Google Scholar). The development of parallel pathways was achieved by Yu and Ando (10Yu R.K. Ando S. Structures of new complex gangliosides of fish brain.Adv. Exp. Med. Biol. 1980; 125: 33-45Google Scholar, 11Ando S. Yu R.K. Isolation and characterization of two isomers of brain tetrasialogangliosides.J. Biol. Chem. 1979; 254: 12224-12229Google Scholar) after the structural characterization of the c-series gangliosides, including GT3, GT2, GT1c, and GQ1c (Fig. 1). They also proposed the c-series pathway of ganglioside biosynthesis. The addition of the a- and b-series pathways is a logical extension of the proposed pathways based on the original work in Roseman's laboratory (4Basu S. Kauman B. Roseman S. Conversion of Tay-Sachs ganglioside to monosialoganglioside by brain uridine diphosphate D-galactose:glycolipid galactosyltransferase.J. Biol. Chem. 1965; 240: 4115-4117Google Scholar, 5Kaufman B. Basu S. Roseman S. Enzymatic synthesis of disialogangliosides from monosialogangliosides by sialyltransferases from embryonic chicken brain.J. Biol. Chem. 1968; 243: 5804-5807Google Scholar). Subsequently, the so-called α-series pathway (12Irie F. Hidari K. I-P. J. Tai T. Li Y-T. Seyama Y. Hirabayashi Y. Biosynthetic pathway for a new series of gangliosides, GT1aα and GQ1aα.FEBS Lett. 1994; 351: 291-294Google Scholar) was added. Evidence has been presented that some of the GTs may also catalyze the biosynthesis of the asialo-series of gangliosides, indicating a low degree of substrate specificity (13Seyfried T.N. Novikov A.M. Irwin R.A. Brigande J.V. Ganglioside biosynthesis in mouse embryos: sialyltransferase IV and the asialo pathway.J. Lipid Res. 1994; 35: 993-1001Google Scholar). The validity and completion of most of the steps have also been established by many investigators, including one of us (9Yu R.K. Lee S.H. In vitro biosynthesis of sialosylgalactosylceramide (G7) by mouse brain microsomes.J. Biol. Chem. 1976; 251: 198-203Google Scholar, 10Yu R.K. Ando S. Structures of new complex gangliosides of fish brain.Adv. Exp. Med. Biol. 1980; 125: 33-45Google Scholar, 11Ando S. Yu R.K. Isolation and characterization of two isomers of brain tetrasialogangliosides.J. Biol. Chem. 1979; 254: 12224-12229Google Scholar, 12Irie F. Hidari K. I-P. J. Tai T. Li Y-T. Seyama Y. Hirabayashi Y. Biosynthetic pathway for a new series of gangliosides, GT1aα and GQ1aα.FEBS Lett. 1994; 351: 291-294Google Scholar, 13Seyfried T.N. Novikov A.M. Irwin R.A. Brigande J.V. Ganglioside biosynthesis in mouse embryos: sialyltransferase IV and the asialo pathway.J. Lipid Res. 1994; 35: 993-1001Google Scholar, 14Yohe H.C. Yu R.K. In vitro biosynthesis of an isomer of trisialoganglioside, GT1a.J. Biol. Chem. 1980; 255: 608-613Google Scholar, 15Yohe H.C. Macala L.J. Yu R.K. In vitro biosynthesis of a tetrasialoganglioside.J. Biol. Chem. 1982; 257: 249-252Google Scholar, 16Freischütz B. Saito M. Rahmann H. Yu R.K. Activities of five different sialyltransferases in fish and rat brains.J. Neurochem. 1994; 62: 1965-1973Google Scholar, 17Freischütz B. Saito M. Rahmann H. Yu R.K. Characterization of sialyltransferase-IV activity and its involvement in the c-pathway of brain ganglioside metabolism.J. Neurochem. 1995; 64: 385-393Google Scholar). In 1988, Sandhoff's group (18Pohlentz G. Klein D. Schwarzmann G. Schmitz D. Sandhoff K. Both GA2, GM2, and GD2 synthases and GM1b, GD1a, and GT1b synthases are single enzymes in Golgi vesicles from rat liver.Proc. Natl. Acad. Sci. USA. 1988; 85: 7044-7048Google Scholar) demonstrated that equivalent steps in the parallel pathways are catalyzed by identical enzymes and that the biosynthesis of complex gangliosides is controlled by the entry reaction for each pathway. The validity of this observation was confirmed by us (17Freischütz B. Saito M. Rahmann H. Yu R.K. Characterization of sialyltransferase-IV activity and its involvement in the c-pathway of brain ganglioside metabolism.J. Neurochem. 1995; 64: 385-393Google Scholar). We further demonstrated that GT3-synthase is the rate-limiting step for the c-series ganglioside biosynthesis (17Freischütz B. Saito M. Rahmann H. Yu R.K. Characterization of sialyltransferase-IV activity and its involvement in the c-pathway of brain ganglioside metabolism.J. Neurochem. 1995; 64: 385-393Google Scholar). There is evidence that deviation from strict substrate specificity may be contributed, at least in part, by the cell- or tissue-specific expression of isoenzymes (19Kono M. Yoshida Y. Kojima N. Tsuji S. Molecular cloning and expression of a fifth type of α2,8-sialyltransferase (ST8Sia V). Its substrate specificity is similar to that of SAT-V/III, which synthesize GD1c, GT1a, GQ1b, and GT3.J. Biol. Chem. 1996; 271: 29366-29371Google Scholar, 20Nara K. Watanabe Y. Kawashima I. Kai T. Nagai Y. Sanai Y. Acceptor substrate specificity of a cloned GD3 synthase that catalyzes the biosynthesis of both GD3 and GD1c/GT1a/GQ1b.Eur. J. Biochem. 1996; 238: 647-652Google Scholar). Although glycosidases are clearly involved in the catabolism of gangliosides, the expression of cellular gangliosides has long been believed to rely to a large extent on the biosynthetic steps that provide the product gangliosides. In other words, to increase a particular ganglioside, the cell has to increase the activity of the enzyme that catalyzes its biosynthesis. Although this conclusion comes naturally, it is clear that the mechanism by which cells regulate the composition of gangliosides is not that simple. Other factors must also be considered. For this reason, we have proposed an enzyme kinetic model that predicts a steady-state concentration or continuous "flow" of gangliosides in each of the pathways (21Bieberich E. Yu R.K. Multi-enzyme kinetic analysis of glycolipid biosynthesis.Biochim. Biophys. Acta. 1999; 1432: 113-124Google Scholar). Assuming that the concentration of the other substrate, the sugar nucleotide, is in a constant supply, the steady state is dependent on the enzyme that consumes a ganglioside intermediate and not on the enzyme that catalyzes its biosynthesis. In other words, to increase a particular ganglioside, the cell has to decrease the activity of the enzyme that uses this ganglioside as its substrate. This model implies two consequences that distinguish it from the first assumption of regulating the biosynthesis of a product ganglioside. As the first consequence, our model allows for the simultaneous calculation of all enzyme activities from the ganglioside composition in any of the parallel pathways. This has led to a simplified kinetic analysis [multienzyme kinetic analysis (MEKA)] of ganglioside biosynthesis by matrix calculation, which shows a remarkable consistency with the actual measured concentrations and enzyme activities (21Bieberich E. Yu R.K. Multi-enzyme kinetic analysis of glycolipid biosynthesis.Biochim. Biophys. Acta. 1999; 1432: 113-124Google Scholar). As a second consequence, our model predicts the existence of distinct subcellular ganglioside pools that can be distinguished by the concrete values of the kinetic analysis. The existence of distinct subcellular ganglioside pools directing the biosynthetic flow and traffic of ganglioside intermediates has been suggested in several previous studies (22Miller-Podraza H. Fishman P.H. Translocation of newly synthesized gangliosides to the cell surface.Biochemistry. 1982; 21: 3265-3270Google Scholar, 23Caputto R. Maccioni H.J. Arce A. Cumar B.L. Biosynthesis of brain gangliosides.Adv. Exp. Med. Biol. 1976; 71: 27-44Google Scholar, 24Maccioni H.J. Landa C. Arce R. Caputto R. The biosynthesis of brain gangliosides—evidence for a "transient pool" and an "end product" pool of gangliosides.Adv. Exp. Med. Biol. 1977; 83: 267-281Google Scholar). However, only MEKA has been able to correlate quantitatively the ganglioside concentration in each pool with the localization of GTs that are required for its biosynthesis. In a model of vectorial ganglioside biosynthesis, the traffic of ganglioside intermediates moves along with the subcellular localization and/or transport of GTs throughout the endoplasmic reticulum (ER)-to-Golgi axis. In cases in which the transport of the product ganglioside is slow, it will be enriched in a common pool with the enzyme that catalyzes its biosynthesis. However, if the transport of the product ganglioside is fast but its consumption is slow, the ganglioside will become enriched in the subcellular compartment where the enzyme that uses this ganglioside as its substrate is located (21Bieberich E. Yu R.K. Multi-enzyme kinetic analysis of glycolipid biosynthesis.Biochim. Biophys. Acta. 1999; 1432: 113-124Google Scholar). In the last two decades, tremendous progress has been achieved in localizing gangliosides and their respective GTs. Initial efforts used exclusively subcellular fractionation, probably because of the lack of antibodies that can be used for localization studies by immunofluorescence or electron microscopy (25Trinchera M. Pirovano B. Ghidoni R. Sub-Golgi distribution in rat liver of CMP-NeuAc:GM3 and CMP-NeuAc:GT1b alpha 2-8sialyltransferases and comparison with the distribution of the other glycosyltransferase activities involved in ganglioside biosynthesis.J. Biol. Chem. 1990; 265: 18242-18247Google Scholar, 26Maccioni H.J.F. Daniotti J.L. Martina J.A. Organization of ganglioside synthesis in the Golgi apparatus.Biochim. Biophys. Acta. 1999; 1437: 101-118Google Scholar, 27Trinchera M. Ghidoni R. Two glycosphingolipid sialyltransferases are localized in different sub-Golgi compartments in rat liver.J. Biol. Chem. 1989; 264: 15766-15769Google Scholar, 28Iber H. van Echten G. Sandhoff K. Fractionation of primary cultured cerebellar neurons: distribution of sialyltransferases involved in ganglioside biosynthesis.J. Neurochem. 1992; 58: 1533-1537Google Scholar). Meanwhile, specific antibodies against different gangliosides and epitope-tagged or fluorescent protein-linked GTs are available that have contributed significantly to the analysis of subcellular ganglioside and GT transport and localization (29Martina J.A. Daniotti J.L. Maccioni H.J. Influence of N-glycosylation and N-glycan trimming on the activity and intracellular traffic of GD3 synthase.J. Biol. Chem. 1998; 273: 3725-3731Google Scholar, 30Bieberich E. Tencomnao T. Kapitonov D. Yu R.K. Effect of N-glycosylation on turnover and subcellular distribution of N-acetylgalactosaminyltransferase I and sialyltransferase II in neuroblastoma cells.J. Neurochem. 2000; 74: 2359-2364Google Scholar, 31Giraudo C.G. Daniotti J.L. Maccioni H.J. Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferase in the Golgi apparatus.Proc. Natl. Acad. Sci. USA. 2001; 98: 1625-1630Google Scholar, 32Giraudo C.G. Maccioni H.J.F. Ganglioside glycosyltransferases organize in distinct multienzyme complexes in CHO-K1 cells.J. Biol. Chem. 2003; 278: 40262-40271Google Scholar, 33Bieberich E. MacKinnon S. Silva J. Li D.D. Tencomnao T. Irwin L. Kapitonov D. Yu R.K. Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltansferases.Biochemistry. 2002; 41: 11479-11487Google Scholar). Most of these reports agree that the initial steps of ceramide and neutral glycosphingolipid biosynthesis occur in the ER (34Maccioni H.J.F. Giraudo C.G. Daniotti J.L. Understanding the stepwise synthesis of glycolipids.Neurochem. Res. 2002; 27: 629-636Google Scholar). Interestingly, ceramide is first synthesized on the cytosolic face of the ER membrane and then glucosylated. Glucosylceramide flips to the luminal side and is converted to lactosylceramide by Golgi-resident galactosyltransferase I (Fig. 1). There is an ongoing debate, however, about the site of GM3 biosynthesis. Early studies reported that GM3 is synthesized in the Golgi apparatus, most likely in its cis subcompartment (34Maccioni H.J.F. Giraudo C.G. Daniotti J.L. Understanding the stepwise synthesis of glycolipids.Neurochem. Res. 2002; 27: 629-636Google Scholar, 35Stern C.A. Braverman T.R. Tiemeyer M. Molecular identification, tissue distribution and subcellular localization of mST3GalV/GM3 synthase.Glycobiology. 2000; 10: 365-374Google Scholar, 36Kapitonov D. Bieberich E. Yu R.K. Combinatorial PCR approach to homology-based cloning: cloning and expression of mouse and human GM3 synthase.Glycoconj. J. 1999; 16: 337-350Google Scholar). Later studies, in particular from our laboratory, have shown that GM3 may already be present in the ER (33Bieberich E. MacKinnon S. Silva J. Li D.D. Tencomnao T. Irwin L. Kapitonov D. Yu R.K. Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltansferases.Biochemistry. 2002; 41: 11479-11487Google Scholar). These results were based on subcellular localization studies with GM3-synthase that was tagged with a fluorescent protein tag. At present, it is fair to conclude that the localization of GM3-synthase is most likely cell or tissue specific and may encompass the ER as well as Golgi compartments. There has also been considerable discussion of the subcellular localization of other GTs, in particular GM2/GD2-synthase and GD3-synthase. Subcellular localization studies reported in the literature indicate a Golgi localization for both enzymes (29Martina J.A. Daniotti J.L. Maccioni H.J. Influence of N-glycosylation and N-glycan trimming on the activity and intracellular traffic of GD3 synthase.J. Biol. Chem. 1998; 273: 3725-3731Google Scholar, 31Giraudo C.G. Daniotti J.L. Maccioni H.J. Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferase in the Golgi apparatus.Proc. Natl. Acad. Sci. USA. 2001; 98: 1625-1630Google Scholar). Our own studies, however, could only confirm a strict Golgi localization for GD3-synthase, whereas GM2/GD2-synthase appeared to be broadly distributed in the ER and Golgi (30Bieberich E. Tencomnao T. Kapitonov D. Yu R.K. Effect of N-glycosylation on turnover and subcellular distribution of N-acetylgalactosaminyltransferase I and sialyltransferase II in neuroblastoma cells.J. Neurochem. 2000; 74: 2359-2364Google Scholar, 33Bieberich E. MacKinnon S. Silva J. Li D.D. Tencomnao T. Irwin L. Kapitonov D. Yu R.K. Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltansferases.Biochemistry. 2002; 41: 11479-11487Google Scholar). To date, we can only conclude from these results that GM3- and GM2/GD2-synthases are localized in the ER and/or Golgi, whereas GD3-synthase is distributed to the Golgi. These results are not trivial, because the subcellular localization of GTs may determine the sequence in which gangliosides are synthesized. For example, if GM3 encounters GM2/GD2-synthase before GD3-synthase, it will most likely be converted to a-series complex gangliosides; in the opposite case, b-series complex gangliosides will prevail. At present, however, the possibility cannot be excluded that some of the results obtained with epitope- or fluorescent protein-tagged GTs are flawed by overexpression or tagging of the recombinant protein. Hence, we will have to wait for the availability of antibodies against the natural enzymes to clearly determine the subcellular localization of GTs under physiological conditions. Within the last decade, most of the cDNAs for GTs in the ganglioside biosynthetic pathways have been cloned from and expressed in various mammalian tissues and cells (37Tsuji S. Molecular cloning and functional analysis of sialyltransferases.J. Biochem. 1996; 120: 1-13Google Scholar, 38Nagata Y. Yamahiro S. Yodoi J. Lloyd K.O. Shiku H. Furukawa K. Expression cloning of β1,4-N-acetylgalactosaminyltransferase cDNAs that determine the expression of GM2 and GD2 gangliosides.J. Biol. Chem. 1992; 267: 12058-12089Google Scholar, 39Haraguchi M. Yamashiro S. Yamamoto A. Furukawa K. Takamiya K. Lloyd K.O. Shiku H. Furukawa K. Isolation of GD3 synthase gene by expression cloning of GM3 α-2,8-sialyltransferase cDNA using anti-GD2 monoclonal antibody.Proc. Natl. Acad. Sci. USA. 1994; 91: 10455-10459Google Scholar, 40Sasaki K. Kurata K. Kojima N. Kurosawa N. Ohta S. Hanai N. Tsuji S. Nishi T. Expression cloning of a GM3-specific α2,8-sialyltransferase (GD3 synthase).J. Biol. Chem. 1994; 269: 15950-15956Google Scholar, 41Zeng G. Gao L. Ariga T. Yu R.K. Molecular cloning of cDNA for rat brain GD3-synthase.Biochem. Biophys. Res. Commun. 1996; 226: 319-323Google Scholar). Common to all of the GTs is the presence of three or four N-glycosylation sites and a transmembrane II topology that predicts a short cytoplasmic N-terminal tail, a single hydrophobic transmembrane domain, and a larger C-terminal, catalytically active domain that is sequestered in the ER lumen (42Kapitonov D. Yu R.K. Conserved domains of glycosyltransferases.Glycobiology. 1999; 9: 961-978Google Scholar, 43Paulson J.C. Colley K.J. Glycosyltransferases. Structure, localization, and control of cell type-specific glycosylation.J. Biol. Chem. 1989; 264: 17615-17618Google Scholar). Maccioni's group and our group (29Martina J.A. Daniotti J.L. Maccioni H.J. Influence of N-glycosylation and N-glycan trimming on the activity and intracellular traffic of GD3 synthase.J. Biol. Chem. 1998; 273: 3725-3731Google Scholar, 30Bieberich E. Tencomnao T. Kapitonov D. Yu R.K. Effect of N-glycosylation on turnover and subcellular distribution of N-acetylgalactosaminyltransferase I and sialyltransferase II in neuroblastoma cells.J. Neurochem. 2000; 74: 2359-2364Google Scholar, 31Giraudo C.G. Daniotti J.L. Maccioni H.J. Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferase in the Golgi apparatus.Proc. Natl. Acad. Sci. USA. 2001; 98: 1625-1630Google Scholar, 32Giraudo C.G. Maccioni H.J.F. Ganglioside glycosyltransferases organize in distinct multienzyme complexes in CHO-K1 cells.J. Biol. Chem. 2003; 278: 40262-40271Google Scholar, 33Bieberich E. MacKinnon S. Silva J. Li D.D. Tencomnao T. Irwin L. Kapitonov D. Yu R.K. Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltansferases.Biochemistry. 2002; 41: 11479-11487Google Scholar, 44Martina J.A. Daniotti J.L. Maccioni H.J. GM1 synthase depends on N-glycosylation for enzyme activity and trafficking to the Golgi complex.Neurochem. Res. 2000; 25: 725-731Google Scholar) have clearly shown that GD3-synthase, GM2/GD2-synthase, and also GM3-synthase are expressed as N-glycosylated proteins with transmembrane II topology. Our group has shown that these three proteins undergo N-glycoprotein processing catalyzed by ER- and Golgi-resident glycosidases but do not acquire a complex N-glycan or N-linked oligosaccharide structure (30Bieberich E. Tencomnao T. Kapitonov D. Yu R.K. Effect of N-glycosylation on turnover and subcellular distribution of N-acetylgalactosaminyltransferase I and sialyltransferase II in neuroblastoma cells.J. Neurochem. 2000; 74: 2359-2364Google Scholar). At this point, it is necessary to briefly recapitulate the sequence of reactions that occur during N-glycosylation and N-glycoprotein processing (45Kornfeld R. Kornfeld S. Assembly of asparagine-linked oligosaccharides.Annu. Rev. Biochem. 1985; 54: 631-664Google Scholar, 46Helenius A. Aebi M. Intracellular functions of N-linked glycans.Science. 2001; 291: 2364-2369Google Scholar). Interestingly, N-glycosylation begins with an ER membrane-bound carrier lipid termed dolichol pyrophosphate. At the cytosolic face of the ER membrane, a mannose(9)-N-acetylglucosamine(2) (Man9GlcNAc2) oligosaccharide is synthesized by the sequential addition of sugar residues onto dolichol pyrophosphate. This oligosaccharide flips into the ER lumen, where it is further glucosylated to a Glc3Man9GlcNAc2 precursor. The dolichol-linked precursor oligosaccharide serves as a donor substrate for the N-glycosylation of a particular acceptor sequence of the type Asn-X-Ser/Thr within the nascent polypeptide of the presumptive N-glycoprotein. This reaction is catalyzed by oligosaccharyltransferase, which transfers the entire Glc3Man9GlcNAc2 precursor from dolichol pyrophosphate onto the Asn residue within the acceptor sequence (47Geetha-Habib M. Noiva R. Kaplan H.A. Lennarz W.J. Glycosylation site binding protein, a component of oligosaccharyl transferase, is highly similar to three other 57 kD luminal proteins of the ER.Cell. 1988; 54: 1053-1060Google Scholar, 48Breuer W. Bause E. Oligosaccharyl transferase is a constitutive component of an oligomeric protein complex from pig liver endoplasmic reticulum.Eur. J. Biochem. 1995; 228: 689-696Google Scholar). It is important to note that this en bloc transfer occurs cotranslationally, before the N-glycoprotein is completely folded and has acquired its native conformation. After the en bloc transfer of the precursor oligosaccharide, ER- and Golgi-resident glycosidases trim down the Glc3Man9GlcNAc2 structure by sequential hydrolytic removal of terminal sugar residues from the N-linked oligosaccharide (49Kalz-Füller B. Bieberich E. Bause E. Cloning and expression of glucosidase I from human hippocampus.Eur. J. Biochem. 1995; 231: 344-351Google Scholar, 50Bieberich E. Bause E. Man9-mannosidase from human kidney is expressed in COS-cells as a Golgi resident type II transmembrane N-glycoprotein.Eur. J. Biochem. 1995; 233: 644-649Google Scholar). In the trans-Golgi subcompartment, the residual core structure of Man3–5GlcNAc2 will then be reglycosylated by the addition of GalNAc, galactose, and sialic acid residues, giving rise to complex N-glycoproteins (47Geetha-Habib M. Noiva R. Kaplan H.A. Lennarz W.J. Glycosylation site binding protein, a component of oligosaccharyl transferase, is highly similar to three other 57 kD luminal proteins of the ER.Cell. 1988; 54: 1053-1060Google Scholar). We have analyzed the
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