The Critical Role of the Stem Region as a Functional Domain Responsible for the Oligomerization and Golgi Localization of N-Acetylglucosaminyltransferase V

Sasai, Ken; Ikeda, Yoshitaka; Tsuda, Toshihide; Ihara, Hidetoshi; Korekane, Hiroaki; Shiota, Kunio; Taniguchi, Naoyuki

doi:10.1074/jbc.m004972200

Cited by 49 publications

(34 citation statements)

References 40 publications

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“…Similar results have been reported for GnT-V, a Golgi-resident protein whose stem region is not required for its enzymatic activity but is responsible for its intracellular localization (41). However, it is not yet known whether this is also valid for XT-II, and we will have to investigate this after identification of the enzymatic function of this protein.…”

Section: Discussionsupporting

confidence: 48%

“…The way in which our XT-I and XT-II data can be reconciled with the kin recognition theory should be investigated in further studies. The present investigations reported that for some glycoyltransferases, oligomerization of the stem region was responsible for their Golgi retention (41), whereas others still showed Golgi retention after the disruption of the protein-protein interactions (42,43). We could only hypothesize that the truncated stem region of XT-I leads to decreased formation of aggregates and therefore to impaired Golgi retention.…”

Section: Discussionmentioning

confidence: 75%

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Cloning and Recombinant Expression of Active Full-length Xylosyltransferase I (XT-I) and Characterization of Subcellular Localization of XT-I and XT-II

Schön

Prante

Bahr

et al. 2006

Journal of Biological Chemistry

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Xylosyltransferase I (XT-I) catalyzes the transfer of xylose from UDP-xylose to serine residues in proteoglycan core proteins. This is the first and apparently rate-limiting step in the biosynthesis of the tetrasaccharide linkage region in glycosaminoglycan-containing proteoglycans. The XYLT-II gene codes for a highly homologous protein, but its physiological function is not yet known. Here we present for the first time the construction of a vector encoding the full-length GFP-tagged human XT-I and the recombinant expression of the active enzyme in mammalian cells. We expressed XT-I-GFP and various GFP-tagged XT-I and XT-II mutants with C-terminal truncations and deletions in HEK-293 and SaOS-2 cells in order to investigate the intracellular localization of XT-I and XT-II. Immunofluorescence analysis showed a distinct perinuclear pattern of XT-I-GFP and XT-II-GFP similar to that of ␣-mannosidase II, which is a known enzyme of the Golgi cisternae. Furthermore, a co-localization of native human XT-I and ␣-mannosidase II could also be demonstrated in untransfected cells. Using brefeldin A, we could also show that both xylosyltransferases are resident in the early cisternae of the Golgi apparatus. For its complete Golgi retention, XT-I requires the N-terminal 214 amino acids. Unlike XT-I, for XT-II, the first 45 amino acids are sufficient to target and retain the GFP reporter in the Golgi compartment. Here we show evidence that the stem regions were indispensable for Golgi localization of XT-I and XT-II.

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Section: Discussionsupporting

confidence: 48%

Section: Discussionmentioning

confidence: 75%

Cloning and Recombinant Expression of Active Full-length Xylosyltransferase I (XT-I) and Characterization of Subcellular Localization of XT-I and XT-II

Schön

Prante

Bahr

et al. 2006

Journal of Biological Chemistry

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“…Neither model completely explains the observations in the literature. The stem region or cytoplasmic tail also may influence Golgi retention of some glycosyltransferases (Milland et al, 2001;Sasai et al, 2001). In any case, the Golgi retention mechanism must be altered or overridden for GalT-I to move to the plasma membrane.…”

Section: B Receptors For Zona Pellucida Proteinsmentioning

confidence: 99%

Molecular Basis of Mammalian Gamete Binding

Miller

Shi

Burkin

2002

Recent Progress in Hormone Research

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Despite the importance of fertilization for controlling human reproduction, regulating animal production, and promoting preservation of endangered species, the molecular basis underlying gamete binding and fertilization has been perplexing. More progress has been made in the mouse than in other mammals and, recently, targeted deletion of specific genes in the mouse has yielded intriguing results. This review will emphasize research performed by our laboratory and others done primarily with mouse gametes but will include some interesting observations from other mammals. Studies of murine fertilization indicate that oligosaccharides on the egg coat glycoprotein ZP3 bind sperm. The precise oligosaccharides that bind sperm are the subject of considerable debate. ZP3 also induces exocytosis of the sperm acrosome, allowing sperm to penetrate through the egg coat (zona pellucida). A number of candidate ZP3 receptors have been proposed and studies of ␤1,4galacto-syltransferase-I (GalT-I) are reviewed here in the most detail. Sperm from mice with a targeted deletion of GalT-I still are able to bind the zona pellucida but are unable to acrosome react and penetrate through the zona. Therefore, the unique role of GalT-I appears to be in signal transduction. GalT-I forms a complex with heterotrimeric G proteins and activates signaling, leading to exocytosis in sperm and in heterologous cells expressing GalT-I. Other signaling steps triggered by GalT-I are under active investigation; this receptor forms a complex with a protein kinase anchoring protein.After exocytosis of the acrosome, sperm penetrate the zona pellucida and fuse with the oocyte plasma membrane using ADAM family members on sperm and integrins on oocytes. These proteins, along with the tetraspanins on oocytes, may form a complex web at gamete fusion. Targeted deletion of specific genes in this putative complex has provided important information about their redundancy. After the oocyte is fertilized, the binding site for GalT-I is lost from ZP3, preventing additional sperm from binding to the zona pellucida. New technical advances and creative ideas offer the opportunity to make important advances and to solve the conundrum of fertilization.

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“…Because deletion mutants, which contain CTS regions alone, could be localized at the Golgi apparatus (31)(32)(33), the CTS regions function as Golgi retention signals for various glycosyltransferases (34). Furthermore, because the substitution of CTS regions for those of other glycosyltransferases resulted in different ultimate oligosaccharide structures because of an alteration in subcompartment distribution (8), the CTS regions of glycosyltransferases play important roles as intrinsic signals for regulating their distribution in the Golgi subcompartment.…”

Section: Discussionmentioning

confidence: 99%

Caveolin-1 Regulates the Functional Localization of N-Acetylglucosaminyltransferase III within the Golgi Apparatus

Sasai¹,

Ikeda

Ihara

et al. 2003

Journal of Biological Chemistry

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In an investigation of the mechanism underlying the functional sublocalization of glycosyltransferases within the Golgi apparatus, caveolin-1 was identified as a possible cellular factor. Caveolin-1 appears to regulate the localization of N-acetylglucosaminyltransferase III (GnT-III) in the intra-Golgi subcompartment. Structural analyses of total cellular N-glycans indicated that the overexpression of GnT-III in human hepatoma cells, in which caveolin-1 is not expressed, failed to reduce branch formation, whereas expression of caveolin-1 led to a dramatic decrease in the extent of branching with no enhancement in GnT-III activity. Because the addition of a bisecting GlcNAc by GnT-III to the core ␤-Man in N-glycans prevents the action of GnT-IV and GnT-V, both of which are involved in branch formation, this result suggests that caveolin-1 facilitates the prior action of GnT-III, relative to the other GnTs, on the nascent sugar chains in the Golgi apparatus and that GnT-III is redistributed in the earlier Golgi subcompartment by caveolin-1. Indeed, when caveolin-1 was expressed in human hepatoma cells, it was found to be co-localized with GnT-III, as evidenced by the fractionation of Triton X-100-insoluble cellular membranes by density gradient ultracentrifugation. Caveolin-1 may modify the biosynthetic pathway of sugar chains via the regulation of the intra-Golgi subcompartment localization of this key glycosyltransferase. N-Acetylglucosaminyltransferase III (GnT-III)1 is one of the Golgi-resident glycosyltransferases that catalyzes the transfer of GlcNAc from an UDP-GlcNAc to a core ␤-mannosyl residue of asparagine-linked glycans (N-glycans) via a ␤1,4-linkage, resulting in the formation of a unique structure, a bisecting GlcNAc (1). Oligosaccharides containing a bisecting GlcNAc (bisected oligosaccharides) do not serve as substrates for GnT-IV and GnT-V, which catalyze the formation of a ␤1,4-branch at an ␣1,3Man and a ␤1,6-branch at an ␣1,6Man, respectively (2, 3). We previously reported that the action of GnT-V was strictly inhibited by the addition of a bisecting GlcNAc at the catalytic step (4). Therefore, the action of GnT-III prior to these enzymes in the Golgi leads to an inhibition of the biosynthesis of multiantennary oligosaccharides. Indeed, the ectopic expression of GnT-III caused an effective reduction in multiantennary oligosaccharides in various types of cells (5, 6). For example, in the case of swine endothelial cells, the expression of GnT-III led to a dramatic decrease in multiantennary oligosaccharides, accompanied by a marked increase in bisected biantennary ones (6). However, there are exceptions, in which a high GnT-III activity did not lead to a decrease in branching. In the case of human hepatoma cells, the overexpression of GnT-III resulted in a dramatic increase in bisected oligosaccharides but did not cause a decrease of the number of antenna (7). It would be expected that GnT-IV and GnT-V act prior to GnT-III in Huh6 and HepG2 cells, because the activities of GnT-III, GnT-IV, and Gn...

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The Critical Role of the Stem Region as a Functional Domain Responsible for the Oligomerization and Golgi Localization of N-Acetylglucosaminyltransferase V

Cited by 49 publications

References 40 publications

Cloning and Recombinant Expression of Active Full-length Xylosyltransferase I (XT-I) and Characterization of Subcellular Localization of XT-I and XT-II

Cloning and Recombinant Expression of Active Full-length Xylosyltransferase I (XT-I) and Characterization of Subcellular Localization of XT-I and XT-II

Molecular Basis of Mammalian Gamete Binding

Caveolin-1 Regulates the Functional Localization of N-Acetylglucosaminyltransferase III within the Golgi Apparatus

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