Bisecting GlcNAc Is a General Suppressor of Terminal Modification of N-glycan*[S]
2019; Elsevier BV; Volume: 18; Issue: 10 Linguagem: Inglês
10.1074/mcp.ra119.001534
ISSN1535-9484
AutoresMiyako Nakano, Sushil Kumar Mishra, Yuko Tokoro, Keiko Sato, Kazuki Nakajima, Yoshiki Yamaguchi, Naoyuki Taniguchi, Yasuhiko Kizuka,
Tópico(s)Galectins and Cancer Biology
ResumoGlycoproteins are decorated with complex glycans for protein functions. However, regulation mechanisms of complex glycan biosynthesis are largely unclear. Here we found that bisecting GlcNAc, a branching sugar residue in N-glycan, suppresses the biosynthesis of various types of terminal epitopes in N-glycans, including fucose, sialic acid and human natural killer-1. Expression of these epitopes in N-glycan was elevated in mice lacking the biosynthetic enzyme of bisecting GlcNAc, GnT-III, and was conversely suppressed by GnT-III overexpression in cells. Many glycosyltransferases for N-glycan terminals were revealed to prefer a nonbisected N-glycan as a substrate to its bisected counterpart, whereas no up-regulation of their mRNAs was found. This indicates that the elevated expression of the terminal N-glycan epitopes in GnT-III-deficient mice is attributed to the substrate specificity of the biosynthetic enzymes. Molecular dynamics simulations further confirmed that nonbisected glycans were preferentially accepted by those glycosyltransferases. These findings unveil a new regulation mechanism of protein N-glycosylation. Glycoproteins are decorated with complex glycans for protein functions. However, regulation mechanisms of complex glycan biosynthesis are largely unclear. Here we found that bisecting GlcNAc, a branching sugar residue in N-glycan, suppresses the biosynthesis of various types of terminal epitopes in N-glycans, including fucose, sialic acid and human natural killer-1. Expression of these epitopes in N-glycan was elevated in mice lacking the biosynthetic enzyme of bisecting GlcNAc, GnT-III, and was conversely suppressed by GnT-III overexpression in cells. Many glycosyltransferases for N-glycan terminals were revealed to prefer a nonbisected N-glycan as a substrate to its bisected counterpart, whereas no up-regulation of their mRNAs was found. This indicates that the elevated expression of the terminal N-glycan epitopes in GnT-III-deficient mice is attributed to the substrate specificity of the biosynthetic enzymes. Molecular dynamics simulations further confirmed that nonbisected glycans were preferentially accepted by those glycosyltransferases. These findings unveil a new regulation mechanism of protein N-glycosylation. Protein glycosylation is the most abundant post-translational modification (1Varki A. Biological roles of glycans.Glycobiology. 2017; 27: 3-49Crossref PubMed Scopus (1209) Google Scholar). Glycan structures even on a single glycoprotein are diverse, and protein functions are dynamically regulated by their specific glycosylation states (2Moremen K.W. Tiemeyer M. Nairn A.V. Vertebrate protein glycosylation: diversity, synthesis and function.Nat. Rev. Mol. Cell Biol. 2012; 13: 448-462Crossref PubMed Scopus (1111) Google Scholar, 3Ohtsubo K. Marth J.D. Glycosylation in cellular mechanisms of health and disease.Cell. 2006; 126: 855-867Abstract Full Text Full Text PDF PubMed Scopus (2087) Google Scholar). Indeed, alteration of or dynamic changes to the glycan structures on proteins affect various physiological processes, such as protein folding, stability, trafficking and activity (2Moremen K.W. Tiemeyer M. Nairn A.V. Vertebrate protein glycosylation: diversity, synthesis and function.Nat. Rev. Mol. Cell Biol. 2012; 13: 448-462Crossref PubMed Scopus (1111) Google Scholar, 3Ohtsubo K. Marth J.D. Glycosylation in cellular mechanisms of health and disease.Cell. 2006; 126: 855-867Abstract Full Text Full Text PDF PubMed Scopus (2087) Google Scholar). Further, genetic deletion of glycans in mice improved or accelerated the pathology of various diseases including cancer, Alzheimer's disease (AD) 1The abbreviations used are:ADAlzheimer's diseaseERendoplasmic reticulumAALAleuria aurantia lectinE4-PHAerythroagglutinating phytohemagglutininFucfucoseGalgalactoseGlcAglucuronic acidGlcNAcN-acetylglucosamineGluA2AMPA-type glutamate receptor-2′GGnGGnbiGal-terminated biantennaryGnGnbiGlcNAc-terminated biantennaryGnTN-acetylglucosaminyltransferaseHexhexoseHNK-1human natural killer-1LacNAcN-acetyllactosamineLeLewisMDmolecular dynamicsManmannoseMAMMaakia amurensis lectinNCAMneural cell adhesion moleculePApyridylaminePhoSLPholiota squarrosa lectinRMSFroot mean square fluctuationsSiasialic acidSSASambucus sieboldiana lectin. 1The abbreviations used are:ADAlzheimer's diseaseERendoplasmic reticulumAALAleuria aurantia lectinE4-PHAerythroagglutinating phytohemagglutininFucfucoseGalgalactoseGlcAglucuronic acidGlcNAcN-acetylglucosamineGluA2AMPA-type glutamate receptor-2′GGnGGnbiGal-terminated biantennaryGnGnbiGlcNAc-terminated biantennaryGnTN-acetylglucosaminyltransferaseHexhexoseHNK-1human natural killer-1LacNAcN-acetyllactosamineLeLewisMDmolecular dynamicsManmannoseMAMMaakia amurensis lectinNCAMneural cell adhesion moleculePApyridylaminePhoSLPholiota squarrosa lectinRMSFroot mean square fluctuationsSiasialic acidSSASambucus sieboldiana lectin., diabetes and muscle dystrophy (4Kizuka Y. 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Alzheimer's disease endoplasmic reticulum Aleuria aurantia lectin erythroagglutinating phytohemagglutinin fucose galactose glucuronic acid N-acetylglucosamine AMPA-type glutamate receptor-2′ Gal-terminated biantennary GlcNAc-terminated biantennary N-acetylglucosaminyltransferase hexose human natural killer-1 N-acetyllactosamine Lewis molecular dynamics mannose Maakia amurensis lectin neural cell adhesion molecule pyridylamine Pholiota squarrosa lectin root mean square fluctuations sialic acid Sambucus sieboldiana lectin. N-glycans are highly conserved and abundant, and expressed on most proteins that pass through the secretory pathway (10Aebi M. N-linked protein glycosylation in the ER.Biochim. Biophys. Acta. 2013; 1833: 2430-2437Crossref PubMed Scopus (458) Google Scholar). Although the early biosynthetic system of N-glycans that occurs in endoplasmic reticulum (ER) is highly conserved among all eukaryotic cells (10Aebi M. N-linked protein glycosylation in the ER.Biochim. Biophys. 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Cold Spring Harbor Lab Press, Cold Spring Harbor, NY2015: 99-111Google Scholar). Biochemical studies showed that branch formation basically precedes the biosynthesis of terminal modifications (11Stanley P. Taniguchi N. Aebi M. N-Glycans.in: Varki A. Cummings R.D. Esko J.D. Stanley P. Hart G.W. Aebi M. Darvill A.G. Kinoshita T. Packer N.H. Prestegard J.H. Schnaar R.L. Seeberger P.H. Essentials of Glycobiology. Cold Spring Harbor Lab Press, Cold Spring Harbor, NY2015: 99-111Google Scholar). Final N-glycan structures on each glycoprotein or even a single N-glycosylation site are biosynthesized by concerted and competitive actions of various glycosyltransferases in the Golgi, but how the actions of these glycosyltransferases are regulated in the Golgi remains to be elucidated.Fig. 2Increase in the HNK-1 glycan level in Mgat3−/− brain. A, A schematic model describing the biosynthesis of bisecting GlcNAc, HNK-1 and polySia in N-glycans. B, Proteins of brain membranes from 7-week-old Mgat3+/+ and Mgat3−/− mice were stained with the anti-HNK-1, anti-NCAM, anti-GluA2 and anti-VDAC1 Ab. The data of two representative mice for each genotype are shown. C, Brain sections from 20-week-old Mgat3+/+ and Mgat3−/− mice were immunostained with the anti-HNK-1 mAb. Hippocampus area is shown. Bar, 300 μm. D, Proteins of brain homogenates from P0 Mgat3+/+ and Mgat3−/− mice (left) or of DIV15 cultured cerebral neurons from Mgat3+/+ and Mgat3−/− embryos (right) were stained with the anti-polySia, anti-actin, or anti-βIII-tubulin (neuron marker) Ab. E, Brain sections from 20-week-old Mgat3+/+ and Mgat3−/− mice were immunostained with the anti-polySia mAb. Hippocampus area is shown. Bar, 300 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Bisecting GlcNAc, the central branch of N-glycan expressed highly in brain and kidney, is biosynthesized by a glycosyltransferase GnT-III (encoded by the MGAT3 gene) (Fig. 1A) (12Nishikawa A. Ihara Y. Hatakeyama M. Kangawa K. Taniguchi N. Purification, cDNA cloning, and expression of UDP-N-acetylglucosamine: beta-D-mannoside beta-1,4N-acetylglucosaminyltransferase III from rat kidney.J. Biol. Chem. 1992; 267: 18199-18204Abstract Full Text PDF PubMed Google Scholar) and was reported to be associated with several diseases. Mgat3-deficient mice showed improved AD pathology with reduced amyloid-plaque formation in brain (4Kizuka Y. Kitazume S. Fujinawa R. Saito T. Iwata N. Saido T.C. Nakano M. Yamaguchi Y. Hashimoto Y. Staufenbiel M. Hatsuta H. Murayama S. Manya H. Endo T. Taniguchi N. 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In this study, to understand whether bisecting GlcNAc regulates whole N-glycan structures, we carried out N-glycomic and biochemical analysis of Mgat3-deficient mouse brain. We found significant increases in various terminal modifications of N-glycans, including Le-type fucose, sialic acid and the HNK-1 epitope. We also reveal that most glycosyltransferases acting on N-glycan terminals have lower preference toward bisected glycans as substrates over nonbisected glycans, which was further supported by our docking models and molecular dynamics (MD) simulations. These data indicate that bisecting GlcNAc serves as a general suppressor of various terminal modifications of N-glycans, highlighting its importance for fine regulation of glycan structures on proteins. Commercially available antibodies used were as follows: anti-ST6GAL1 (M2, 28047) was from Immuno-Biological Laboratories (Gunma, Japan), anti-VDAC1 (ab14734) from Abcam, Cambridge, UK, anti-actin (A4700) from Sigma, St Louis, MO, anti-GAPDH (MAB374) and anti-myc (4A6, 05–724) from Millipore, Darmstadt, Germany, anti-polysialic acid (12E3, 14–9118-80) from Thermo Fisher Scientific, Waltham, MA, anti-HNK-1 (559048) from BD Biosciences, Franklin Lakes, NJ, anti-beta-III-tubulin (tuj1, MMS-435P) from Covance and anti-myc, Princeton, NJ (9E10, BML SA-294) from Enzo Life Sciences, Farmingdale, NY. Biotinylated Aleuria Aurantia Lectin (AAL) (J201-R), erythroagglutinating phytohemagglutinin (E4-PHA) lectin (J211), Sambucus sieboldiana (SSA) (J218), Maackia amurensis (MAM) (J210) were from J-Chemical, Tokyo, Japan. The biotinylated Pholiota squarrosa lectin (PhoSL) was provided by J-Chemical. The anti-GnT-III (clone 33A8) was provided by Dr. Eiji Miyoshi (Osaka University). The anti-GlcAT-P (GP2) was provided by Dr. Shogo Oka (Kyoto University). The anti-Sialyl Lex (F2) was provided by Dr. Hiroto Kawashima (Chiba University). The generation of the Mgat3-deficient mice has been described previously (27Priatel J.J. Sarkar M. Schachter H. Marth J.D. Isolation, characterization and inactivation of the mouse Mgat3 gene: the bisecting N-acetylglucosamine in asparagine-linked oligosaccharides appears dispensable for viability and reproduction.Glycobiology. 1997; 7: 45-56Crossref PubMed Scopus (94) Google Scholar). All mice were from a C57BL/6 genetic background. Mgat3-deficient mice were generously provided by Dr. Jamey D. Marth (University of California-Santa Barbara). Mice were housed (three or fewer mice per cage) at 23 ± 3 °C and 55 ± 10% humidity. The light condition was 14 h : 10 h (lights on at 7:00). All animal experiments were approved by the Animal Experiment Committee of RIKEN and Gifu University. The construction of pcDNA6-mycHisA/mouse FUT1, FUT2, FUT4 and FUT9 to express C-terminally myc-tagged full-length fucosyltransferases was described previously (28Kizuka Y. Funayama S. Shogomori H. Nakano M. Nakajima K. Oka R. Kitazume S. Yamaguchi Y. Sano M. Korekane H. Hsu T.L. Lee H.Y. Wong C.H. Taniguchi N. High-Sensitivity and low-toxicity fucose probe for glycan imaging and biomarker discovery.Cell Chem. Biol. 2016; 23: 782-792Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Mouse FUT7 cDNA was amplified by PCR by using a mouse liver cDNA library and then ligated into pCR-Blunt (ThermoFisher Scientific). By using pCR-Blunt/mouse FUT7 as a template, FUT7 cDNA was amplified and subcloned into the EcoRI/XhoI site of pcDNA6-mycHisA to express C-terminally myc-tagged FUT7. Human GnT-III (MGAT3) cDNA was amplified by using pCXN2/human GnT-III (29Kitada T. Miyoshi E. Noda K. Higashiyama S. Ihara H. Matsuura N. Hayashi N. Kawata S. Matsuzawa Y. Taniguchi N. 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An alkynyl-fucose halts hepatoma cell migration and invasion by inhibiting GDP-fucose-synthesizing enzyme FX, TSTA3.Cell Chem. Biol. 2017; 24: 1467-1478Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 36Nakano M. Nakagawa T. Ito T. Kitada T. Hijioka T. Kasahara A. Tajiri M. Wada Y. Taniguchi N. Miyoshi E. Site-specific analysis of N-glycans on haptoglobin in sera of patients with pancreatic cancer: a novel approach for the development of tumor markers.Int. J. Cancer. 2008; 122: 2301-2309Crossref PubMed Scopus (123) Google Scholar) with modifications as follows. Mouse brains (30 mg) were crushed in 2 ml of homogenization buffer (50 mm Tris-HCl, pH 7.4, 0.1 m NaCl, 1 mm EDTA and protease inhibitor mixture (Roche, Basel, Switzerland)) using a Dounce tissue grinder and then homogenized using a polytron homogenizer, followed by centrifugation at 760 × g for 20 min at 4 °C to remove nuclei and unbroken cells. The supernatant was diluted with 2 ml of Tris-buffer (50 mm Tris-HCl, pH 7.4, 0.1 m NaCl) and then ultracentrifuged at 120,000 × g for 80 min at 4 °C. The membrane pellet was suspended in 100 μl of the Tris-buffer, followed by addition of 400 μl of the Tris-buffer containing 1% Triton X-114 with pipetting. The lysate was incubated on ice for 10 min and then at 37 °C for 20 min, followed by phase partitioning by centrifugation at 1940 × g for 2 min. The upper aqueous phase was removed, and the lower detergent phase was mixed with 1 ml of ice-cold acetone and kept at −25 °C overnight. After centrifugation at 1940 × g for 2 min, the precipitated membrane proteins were dissolved with 11 μl of 8 m Urea and spotted (2.5 μl × 4 times) onto an ethanol-pretreated PVDF membrane. After drying at room temperature for > 4 h, the membrane was washed with ethanol for 1 min once and then with water for 1 min three times. The protein on the membrane was stained for 5 min with Direct Blue 71 (Sigma Aldrich) (800 μl solution A (0.1% Direct Blue 71) in 10 ml solution B (acetic acid/ethanol/water = 1:4:5)). After destaining with solution B for 1 min, the membrane was dried at room temperature for > 3 h. The protein spots were excised from the membrane and placed into a well of a 96-well plate. The spots were covered with 100 μl of 1% (w/v) poly(vinylpyrrolidone) 40,000 in 50% (v/v) methanol, agitated for 20 min and washed with water (100 μl × 5 times). PNGase F (2U in 10 μl of 20 mm phosphate buffer, pH 7.3, Roche) was added to the well and the spots were incubated at 37 °C for 15 min, followed by the addition of 10 μl of water and incubated at 37 °C overnight. The samples were sonicated (in the 96-well plate) for 10 min and the released N-glycans (20 μl) were transferred to 1.5-ml polypropylene tubes. The well was washed with water (50 μl twice), and the washings were combined and evaporated. The dried N-glycans were reacted with aminoxyTMT6 reagent (Thermo Fisher Scientific, 0.02 mg in 200 μl of 95% methanol, 0.1% acetic acid solution) by continuous shaking for 15 min at room temperature. After evaporating the reaction solution, 200 μl of 95% methanol was added to the samples, followed by further shaking for 15 min. After evaporating the samples, 100 μl of 10% acetone solution was added to the samples, followed by incubation at room temperature for 15 min with continuous shaking. The sample was evaporated, and excess reagent was removed using Sepharose CL4B. The samples were dried, dissolved with 20 μl of 10 mm ammonium bicarbonate and analyzed by LC-ESI MS/MS. N-Glycans labeled with aminoxyTMT6 were separated on a carbon column (5 μm HyperCarb, 1 mm I.D. × 100 mm, Thermo Fisher Scientific) using an Accela HPLC pump (flow rate: 50 μl/min) under the following gradient conditions; a sequence of isocratic and two segmented linear gradients: 0–8 min, 10 mm NH4HCO3; 8–38 min, 9–22.5% (v/v) CH3CN in 10 mm NH4HCO3; 38–73 min, 22.5–51.75% (v/v) CH3CN
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