Down-regulation of the α-Gal Epitope Expression inN-Glycans of Swine Endothelial Cells by Transfection with theN-Acetylglucosaminyltransferase III Gene

Koyota, Souichi; Ikeda, Yoshitaka; Miyagawa, Shuji; Ihara, Hidetoshi; Koma, Masaru; Honke, Koichi; Shirakura, R; Taniguchi, Naoyuki

doi:10.1074/jbc.m102371200

Cited by 41 publications

(25 citation statements)

References 61 publications

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“…The reducing ends of the oligosaccharides were labeled with 2-aminopyridine (PA) as described in ref. 17. PA-oligosaccharides were subjected to HPLC analysis and further to liquid chromatography-electrospray ionization-MS analysis, which was performed on an HCT ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an electrospray source working in positive ion mode.…”

Section: Methodsmentioning

confidence: 99%

Dysregulation of TGF-β1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice

Wang

Inoué

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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The core fucosylation (␣1,6-fucosylation) of glycoproteins is widely distributed in mammalian tissues, and is altered under pathological conditions. To investigate physiological functions of the core fucose, we generated ␣1,6-fucosyltransferase (Fut8)-null mice and found that disruption of Fut8 induces severe growth retardation and death during postnatal development. Histopathological analysis revealed that Fut8 ؊/؊ mice showed emphysema-like changes in the lung, verified by a physiological compliance analysis. Biochemical studies indicated that lungs from Fut8 ؊/؊ mice exhibit a marked overexpression of matrix metalloproteinases (MMPs), such as MMP-12 and MMP-13, highly associated with lung-destructive phenotypes, and a down-regulation of extracellular matrix (ECM) proteins such as elastin, as well as retarded alveolar epithelia cell differentiation. These changes should be consistent with a deficiency in TGF-␤1 signaling, a pleiotropic factor that controls ECM homeostasis by down-regulating MMP expression and inducing ECM protein components. In fact, Fut8 ؊/؊ mice have a marked dysregulation of TGF-␤1 receptor activation and signaling, as assessed by TGF-␤1 binding assays and Smad2 phosphorylation analysis. We also show that these TGF-␤1 receptor defects found in Fut8 ؊/؊ cells can be rescued by reintroducing Fut8 into Fut8 ؊/؊ cells. Furthermore, exogenous TGF-␤1 potentially rescued emphysema-like phenotype and concomitantly reduced MMP expression in Fut8 ؊/؊ lung. We propose that the lack of core fucosylation of TGF-␤1 receptors is crucial for a developmental and progressive͞ destructive emphysema, suggesting that perturbation of this function could underlie certain cases of human emphysema.fucosylation ͉ glycobiology ͉ matrix metalloproteinase

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Section: Methodsmentioning

confidence: 99%

Dysregulation of TGF-β1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice

Wang

Inoué

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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411

347

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“…A lack of a dominant-negative effect was confirmed by overexpressing a cDNA encoding the Mgat3 T37 gene in LEC10 CHO cells, which express GlcNAc-TIII activity (3), due to a gain-of-function mutation that activates transcription of the endogenous Mgat3 gene. 5 Overexpressed full-length and truncated GlcNAc-TIII were readily detected by Western analysis, although endogenous GlcNAc-TIII was not detected under the same conditions in whole cell 5 X. Yang and P. Stanley, unpublished data.…”

Section: Truncated Glcnac-tiii Does Not Act In a Dominant-negative Famentioning

confidence: 99%

“…LEC10 cells are ϳ15-fold more resistant to ricin and ϳ10-fold more sensitive to the toxicity of the erythroagglutinin from Phaseolus vulgaris (E-PHA) compared with wild-type CHO cells, reflecting dramatic changes in binding of these lectins to N-linked Gal residues of cell-surface glycoproteins. Similarly, the ectopic expression of an Mgat3 cDNA reduces the expression of terminal ␣3-Gal residues, a key determinant in xenotransplantation (4,5). The regulated expression of the Mgat3 gene could therefore control the binding of animal lectins like galectins to cell-surface N-glycans with a bisecting GlcNAc and thereby modulate cellular interactions.…”

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confidence: 99%

Truncated, InactiveN-Acetylglucosaminyltransferase III (GlcNAc-TIII) Induces Neurological and Other Traits Absent in Mice That Lack GlcNAc-TIII

Bhattacharyya

Bhaumik

Raju³

et al. 2002

Journal of Biological Chemistry

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N-Acetylglucosaminyltransferase III (GlcNAc-TIII), the product of the Mgat3 gene, transfers the bisecting GlcNAc to the core mannose of complex N-glycans. The addition of this residue is regulated during development and has functional consequences for receptor signaling, cell adhesion, and tumor progression. Mice homozygous for a null mutation at the Mgat3 locus (Mgat3 ⌬ ) or for a targeted mutation in the Mgat3 gene (previously called Mgat3 neo , but herein renamed Mgat3 T37 because the allele generates inactive GlcNAc-TIII of ϳ37 kDa) were found to exhibit retarded progression of liver tumors. Matrix-assisted laser desorption/ionization time-offlight mass spectrometry of neutral N-glycans from kidneys revealed no significant differences, and both mutants showed the expected lack of N-glycan species with an additional GlcNAc. However, the two mutants differed in several biological traits. Mgat3 T37/T37 homozygotes in a mixed or 129SvJ background were retarded in growth rate and exhibited an altered leg clasp reflex, an altered gait, and defective nursing behavior. Pups abandoned by Mgat3 T37/T37 mothers were rescued by wildtype foster mothers. None of these Mgat3 T37/T37 traits were exhibited by Mgat3 ⌬/⌬ mice or by heterozygous mice carrying the Mgat3 T37 mutation. Similarly, no dominant-negative effect was observed in Chinese hamster ovary cells expressing truncated GlcNAc-TIII in the presence of wild-type GlcNAc-TIII. However, compound heterozygotes carrying both the Mgat3 T37 and Mgat3 ⌬ mutations exhibited a marked leg clasp reflex, indicating that in the absence of wild-type GlcNAc-TIII, truncated GlcNAc-TIII causes this phenotype. The Mgat3 gene was expressed in brain at embryonic day 10.5 and thereafter and in neurons of adult cerebellum. The mutant Mgat3 gene was also highly expressed in Mgat3 T37/T37 brain. This may be the basis of the unexpected neurological phenotype induced by truncated, inactive GlcNAc-TIII in the mouse.The N-glycans of mammalian glycoproteins vary widely in structure, but the biological significance of this variation is largely unknown. A well studied example is the bisecting GlcNAc. This residue is transferred to the ␤-linked Man of the core of N-glycans by the glycosyltransferase termed N-acetylglucosaminyltransferase III (GlcNAc-TIII 1 ; EC 2.4.1.144) (1), the product of the Mgat3 gene (2). The presence of the bisecting GlcNAc alters the lectin binding properties of a cell, a fact initially revealed by the gain-of-function Chinese hamster ovary (CHO) glycosylation mutant LEC10, which expresses GlcNAc-TIII (3). LEC10 cells are ϳ15-fold more resistant to ricin and ϳ10-fold more sensitive to the toxicity of the erythroagglutinin from Phaseolus vulgaris (E-PHA) compared with wild-type CHO cells, reflecting dramatic changes in binding of these lectins to N-linked Gal residues of cell-surface glycoproteins. Similarly, the ectopic expression of an Mgat3 cDNA reduces the expression of terminal ␣3-Gal residues, a key determinant in xenotransplantation (4, 5). The regulated expression o...

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“…These researchers found that ␣2,6 sialic acid affected the interactions between N-glycans and the I-like domain, which in turn altered the accessibility of the loop that determines specificity of ligand binding. In fact, the remodeling of N-glycans by GnT-III affects either the branching formations catalyzed by GnT-V and GnT-IV or the sialylation on the terminus of the N-glycans (11,36). Therefore, a possible mechanism by which N-glycans are involved in the ␣␤ interaction or conformational arrangement is that an unknown lectin domain may exist on the ␣ or ␤ subunit.…”

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confidence: 99%

An N-Glycosylation Site on theβ-Propeller Domain of the Integrin α5 Subunit Plays Key Roles in Both Its Function and Site-specific Modification byβ1,4-N-Acetylglucosaminyltransferase III

Sato

Isaji

Tajiri

et al. 2009

Journal of Biological Chemistry

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Recently we reported that N-glycans on the ␤-propeller domain of the integrin ␣5 subunit (S-3,4,5) are essential for ␣5␤1 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type ␣5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the ␣5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type ␣5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the ␣5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.Glycosylation is a crucial post-translational modification of most secreted and cell surface proteins (1). Glycosylation is involved in a variety of physiological and pathological events, including cell growth, migration, differentiation, and tumor invasion. It is well known that glycans play important roles in cell-cell communication, intracellular signal transduction, protein folding, and stability (2, 3).Integrins comprise a family of receptors that are important for cell adhesion. The major function of integrins is to connect cells to the extracellular matrix, activate intracellular signaling pathways, and regulate cytoskeletal formation (4). Integrin ␣5␤1 is well known as a fibronectin (FN) 3 receptor. The interaction between integrin ␣5 and FN is essential for cell migration, cell survival, and development (5-8). In addition, integrins are N-glycan carrier proteins. For example, ␣5␤1 integrin contains 14 and 12 putative N-glycosylation sites on the ␣5 and ␤1 subunits, respectively. Several studies suggest that N-glycosylation is essential for functional integrin ␣5␤1. When human fibroblasts were cultured in the presence of 1-deoxymannojirimycin, which prevents N-linked oligosaccharide processing, immature ␣5␤1 integrin appeared on the cell surface, an...

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Down-regulation of the α-Gal Epitope Expression inN-Glycans of Swine Endothelial Cells by Transfection with theN-Acetylglucosaminyltransferase III Gene

Cited by 41 publications

References 61 publications

Dysregulation of TGF-β1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice

Dysregulation of TGF-β1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice

Truncated, InactiveN-Acetylglucosaminyltransferase III (GlcNAc-TIII) Induces Neurological and Other Traits Absent in Mice That Lack GlcNAc-TIII

An N-Glycosylation Site on theβ-Propeller Domain of the Integrin α5 Subunit Plays Key Roles in Both Its Function and Site-specific Modification byβ1,4-N-Acetylglucosaminyltransferase III

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