Biology:OGT (gene)

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UDP-N-acetylglucosamine—peptide N-acetylglucosaminyltransferase (EC 2.4.1.255), also known as O-linked β-N-acetylglucosamine transferase and O-GlcNAc transferase, OGT is an enzyme that in humans is encoded by the OGT gene.[1][2]

Function

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) catalyzes the addition of a single N-acetylglucosamine through an O-glycosidic linkage to serine or threonine and an S-glycosidic linkage to cysteine[3][4] residues of intracellular proteins. Since both phosphorylation and O-GlcNAcylation compete for similar serine or threonine residues, the two processes may compete for sites, or they may alter the substrate specificity of nearby sites by steric or electrostatic effects. The protein contains 9 or 14 tetratricopeptide repeats, depending on the splice variant, and a putative bipartite nuclear localization signal. Two alternatively spliced transcript variants encoding distinct isoforms (nucleocytoplasmic and mitochondrial) have been found for this gene.[2] OGT glycosylates many proteins including: Histone H2B,[5] AKT1,[6] PFKL,[7] KMT2E/MLL5,[7] MAPT/TAU,[8] Host cell factor C1,[9] and SIN3A.[10]

O-GlcNAc transferase is a part of a host of biological functions within the human body. OGT is involved in the resistance of insulin in muscle cells and adipocytes by inhibiting the Threonine 308 phosphorylation of AKT1, increasing the rate of IRS1 phosphorylation (at Serine 307 and Serine 632/635), reducing insulin signaling, and glycosylating components of insulin signals.[11] Additionally, O-GlcNAc transferase catalyzes intracellular glycosylation of serine and threonine residues with the addition of N-acetylglucosamine. Studies show that OGT alleles are vital for embryogenesis, and that OGT is necessary for intracellular glycosylation and embryonic stem cell vitality.[12] O-GlcNAc transferase also catalyzes the posttranslational modification that modifies transcription factors and RNA polymerase II, however the specific function of this modification is mostly unknown.[13]

OGT cleaves Host Cell Factor C1, at one of the 6 repeat sequences. The TPR repeat domain of OGT binds to the carboxyl terminal portion of an HCF1 proteolytic repeat so that the cleavage region is in the glycosyltransferase active site above uridine-diphosphate-GlcNAc [14] The large proportion of OGT complexed with HCF1 is necessary for HCF1 cleavage, and HCFC1 is required for OGT stabilization in the nucleus. HCF1 regulates OGT stability using a post-transcriptional mechanism, however the mechanism of the interaction with HCFC1 is still unknown.[15]

Structure

The human OGT gene has 1046 amino acid residues, and is a heterotrimer consisting of two 110 kDa subunits and one 78 kDa subunit.[16] The 110 kDa subunit contains 13 tetratricopeptide (TPR) repeats; the 13th repeat is truncated. These subunits are dimerized by TPR repeats 6 and 7. OGT is highly expressed in the pancreas and also expressed in the heart, brain, skeletal muscle, and the placenta. There have been trace amounts found in the lung and the liver.[1] The binding sites have been determined for the 110 kDa subunit. It has 3 binding sites at amino acid residues 849, 852, and 935. The probable active site is residue 508.[7]

The crystal structure of O-GlcNAc transferase has not been well studied, but the structure of a binary complex with UDP and a ternary complex with UDP and a peptide substrate has been researched.[14] The OGT-UDP complex contains three domains in its catalytic region, the amino (N)-terminal domain, the carboxy (C)-terminal domain, and the intervening domain (Int-D). The catalytic region is linked to TPR repeats by a translational helix (H3), which loops from the C-cat domain to the N-Cat domain along the upper surface of the catalytic region.[14] The OGT-UDP-peptide complex has a larger space between the TPR domain and the catalytic region than the OGT-UDP complex. The CKII peptide, which contains three serine residues and a threonine residue, binds in this space. This structure supports an ordered sequential bi-bi mechanism that matches the fact that “at saturating peptide concentrations, a competitive inhibition pattern was obtained for UDP with respect to UDP-GlcNAc.”[14]

Mechanism of catalysis

The molecular mechanism of O-linked N-acetylglucosamine transferase has not been extensively studied either, since there is not a confirmed crystal structure of the enzyme. A proposed mechanism supported by product inhibition patterns by UDP at saturating peptide conditions proceeds with starting materials Uridine diphosphate N-acetylglucosamine, and a peptide chain with a reactive serine or threonine hydroxyl group. The proposed reaction is an ordered sequential bi-bi mechanism.[14]

Figure 2: The proposed catalytic mechanism of O-GlcNAc transferase. It is an ordered sequential bi-bi mechanism with only one step, not including a proton transfer. The peptide is represented by a serine residue with a reactive hydroxyl group.[14]

The chemical reaction can be written as:

(1) UDP-N-acetyl-D-glucosamine + [protein]-L-serine → UDP + [protein]-3-O-(N-acetyl-D-glucosaminyl)-L-serine

(2) UDP-N-acetyl-D-glucosamine + [protein]-L-threonine → UDP + [protein]-3-O-(N-acetyl-D-glucosaminyl)-L-threonine

First, the hydroxyl group of serine is deprotonated by Histidine 498, a catalytic base in this proposed reaction. Lysine 842 is also present to stabilize the UDP moiety. The oxygen ion then attacks the sugar-phosphate bond between the glucosamine and UDP. This results in the splitting of UDP-N-acetylglucosamine into N-acetylglucosamine – Peptide and UDP. Proton transfers take place at the phosphate and Histidine 498. This mechanism is spurred by OGT gene containing O-linked N-acetylglucosamine transferase. Aside from proton transfers the reaction proceeds in one step, as shown in Figure 2.[14] Figure 2 uses a lone serine residue as a representative of the peptide with a reactive hydroxyl group. Threonine could have also been used in the mechanism.

Regulation

File:Competition Between Glycosylation and Phosphorylation of Proteins.tiff

O-GlcNAc transferase is part of a dynamic competition for a serine or threonine hydroxyl functional group in a peptide unit. Figure 3 shows an example of both reciprocal same-site occupancy and adjacent-site occupancy. For the same-site occupancy, OGT competes with kinase to catalyze the glycosylation of the protein instead of phosphorylation. The adjacent-site occupancy example shows the naked protein catalyzed by OGT converted to a glycoprotein, which can increase the turnover of proteins such as the tumor repressor p53.[17]

The post-translational modification of proteins by O-GlcNAc is spurred by glucose flux through the hexosamine biosynthetic pathway. OGT catalyzes attachment of the O-GlcNAc group to serine and threonine, while O-GlcNAcase spurs sugar removal.[18][19]

This regulation is important for multiple cellular processes including transcription, signal transduction, and proteasomal degradation. Also, there is competitive regulation between OGT and kinase for the protein to attach to a phosphate group or O-GlcNAc, which can alter the function of proteins in the body through downstream effects.[7][18] OGT inhibits the activity of 6-phosophofructosekinase PFKL by mediating the glycosylation process. This then acts as a part of glycolysis regulation. O-GlcNAc has been defined as a negative transcription regulator in response to steroid hormone signaling.[11]

Studies show that O-GlcNAc transferase interacts directly with the Ten eleven translocation 2 (TET2) enzyme, which converts 5-methylcytosine to 5-hydroxymethylcytosine and regulates gene transcription.[20] Additionally, increasing levels of OGT for O-GlcNAcylation may have therapeutic effects for Alzheimer's disease patients. Brain glucose metabolism is impaired in Alzheimer's disease, and a study suggests that this leads to hyperphosphorylation of tau and degerenation of tau O-GlcNCAcylation. Replenishing tau O-GlcNacylation in the brain along with protein phosphatase could deter this process and improve brain glucose metabolism.[8]

References

  1. ↑ 1.0 1.1 "O-Linked GlcNAc transferase is a conserved nucleocytoplasmic protein containing tetratricopeptide repeats". The Journal of Biological Chemistry 272 (14): 9316–24. April 1997. doi:10.1074/jbc.272.14.9316. PMID 9083068. 
  2. ↑ 2.0 2.1 "Entrez Gene: OGT O-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase)". https://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=8473. 
  3. ↑ "Cysteine S-linked N-acetylglucosamine (S-GlcNAcylation), A New Post-translational Modification in Mammals". Molecular & Cellular Proteomics 15 (11): 3405–3411. November 2016. doi:10.1074/mcp.M116.061549. PMID 27558639. 
  4. ↑ "Genetic recoding to dissect the roles of site-specific protein O-GlcNAcylation". Nature Structural & Molecular Biology 26 (11): 1071–1077. November 2019. doi:10.1038/s41594-019-0325-8. PMID 31695185. 
  5. ↑ "GlcNAcylation of histone H2B facilitates its monoubiquitination". Nature 480 (7378): 557–60. November 2011. doi:10.1038/nature10656. PMID 22121020. 
  6. ↑ "Regulation of insulin receptor substrate 1 (IRS-1)/AKT kinase-mediated insulin signaling by O-Linked beta-N-acetylglucosamine in 3T3-L1 adipocytes". The Journal of Biological Chemistry 285 (8): 5204–11. February 2010. doi:10.1074/jbc.M109.077818. PMID 20018868. 
  7. ↑ 7.0 7.1 7.2 7.3 "O15294 (OGT1_HUMAN) Reviewed, UniProtKB/Swiss-Prot". UniProt. https://www.uniprot.org/uniprot/O15294#ref11. 
  8. ↑ 8.0 8.1 "Reduced O-GlcNAcylation links lower brain glucose metabolism and tau pathology in Alzheimer's disease". Brain 132 (Pt 7): 1820–32. July 2009. doi:10.1093/brain/awp099. PMID 19451179. 
  9. ↑ "Human Sin3 deacetylase and trithorax-related Set1/Ash2 histone H3-K4 methyltransferase are tethered together selectively by the cell-proliferation factor HCF-1". Genes & Development 17 (7): 896–911. April 2003. doi:10.1101/gad.252103. PMID 12670868. 
  10. ↑ "Recruitment of O-GlcNAc transferase to promoters by corepressor mSin3A: coupling protein O-GlcNAcylation to transcriptional repression". Cell 110 (1): 69–80. July 2002. doi:10.1016/S0092-8674(02)00810-3. PMID 12150998. 
  11. ↑ 11.0 11.1 "Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance". Nature 451 (7181): 964–9. February 2008. doi:10.1038/nature06668. PMID 18288188. 
  12. ↑ "The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny". Proceedings of the National Academy of Sciences of the United States of America 97 (11): 5735–9. May 2000. doi:10.1073/pnas.100471497. PMID 10801981. 
  13. ↑ "TET2 promotes histone O-GlcNAcylation during gene transcription". Nature 493 (7433): 561–4. January 2013. doi:10.1038/nature11742. PMID 23222540. 
  14. ↑ 14.0 14.1 14.2 14.3 14.4 14.5 14.6 "Structure of human O-GlcNAc transferase and its complex with a peptide substrate". Nature 469 (7331): 564–7. January 2011. doi:10.1038/nature09638. PMID 21240259. 
  15. ↑ "Crosstalk between O-GlcNAcylation and proteolytic cleavage regulates the host cell factor-1 maturation pathway". Proceedings of the National Academy of Sciences of the United States of America 108 (7): 2747–52. February 2011. doi:10.1073/pnas.1013822108. PMID 21285374. 
  16. ↑ "Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase". The Journal of Biological Chemistry 267 (13): 9005–13. May 1992. PMID 1533623. 
  17. ↑ 17.0 17.1 "Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins". Nature 446 (7139): 1017–22. April 2007. doi:10.1038/nature05815. PMID 17460662. 
  18. ↑ 18.0 18.1 "O-linkage of N-acetylglucosamine to Sp1 activation domain inhibits its transcriptional capability". Proceedings of the National Academy of Sciences of the United States of America 98 (12): 6611–6. June 2001. doi:10.1073/pnas.111099998. PMID 11371615. 
  19. ↑ "The hexosamine signaling pathway: deciphering the "O-GlcNAc code"". Science's STKE 2005 (312): re13. November 2005. doi:10.1126/stke.3122005re13. PMID 16317114. 
  20. ↑ "Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1". Science 324 (5929): 930–5. May 2009. doi:10.1126/science.1170116. PMID 19372391. 

Further reading

  • "The role of O-linked protein glycosylation in beta-cell dysfunction". International Journal of Molecular Medicine 10 (5): 535–9. November 2002. doi:10.3892/ijmm.10.5.535. PMID 12373287. 
  • "Localization of O-GlcNAc modification on the serum response transcription factor". The Journal of Biological Chemistry 267 (24): 16911–21. August 1992. PMID 1512232. 
  • "Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase". The Journal of Biological Chemistry 267 (13): 9005–13. May 1992. PMID 1533623. 
  • "Vertebrate lens alpha-crystallins are modified by O-linked N-acetylglucosamine". The Journal of Biological Chemistry 267 (1): 555–63. January 1992. PMID 1730617. 
  • "c-Myc is glycosylated at threonine 58, a known phosphorylation site and a mutational hot spot in lymphomas". The Journal of Biological Chemistry 270 (32): 18961–5. August 1995. doi:10.1074/jbc.270.32.18961. PMID 7642555. 
  • "Clathrin assembly protein AP-3 is phosphorylated and glycosylated on the 50-kDa structural domain". The Journal of Biological Chemistry 269 (33): 21346–52. August 1994. PMID 8063760. 
  • "The addition of 5'-coding information to a 3'-directed cDNA library improves analysis of gene expression". Gene 146 (2): 199–207. September 1994. doi:10.1016/0378-1119(94)90293-3. PMID 8076819. 
  • "Glycosylation of mammalian neurofilaments. Localization of multiple O-linked N-acetylglucosamine moieties on neurofilament polypeptides L and M". The Journal of Biological Chemistry 268 (22): 16679–87. August 1993. PMID 8344946. 
  • "A "double adaptor" method for improved shotgun library construction". Analytical Biochemistry 236 (1): 107–13. April 1996. doi:10.1006/abio.1996.0138. PMID 8619474. 
  • "Dynamic O-GlcNAcylation of the small heat shock protein alpha B-crystallin". Biochemistry 35 (11): 3578–86. March 1996. doi:10.1021/bi951918j. PMID 8639509. 
  • "Cytoplasmic O-GlcNAc modification of the head domain and the KSP repeat motif of the neurofilament protein neurofilament-H". The Journal of Biological Chemistry 271 (34): 20845–52. August 1996. doi:10.1074/jbc.271.34.20845. PMID 8702840. 
  • "The microtubule-associated protein tau is extensively modified with O-linked N-acetylglucosamine". The Journal of Biological Chemistry 271 (46): 28741–4. November 1996. doi:10.1074/jbc.271.46.28741. PMID 8910513. 
  • "Dynamic glycosylation of nuclear and cytosolic proteins. Cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeats". The Journal of Biological Chemistry 272 (14): 9308–15. April 1997. doi:10.1074/jbc.272.14.9308. PMID 9083067. 
  • "Large-scale concatenation cDNA sequencing". Genome Research 7 (4): 353–8. April 1997. doi:10.1101/gr.7.4.353. PMID 9110174. 
  • "O glycosylation of an Sp1-derived peptide blocks known Sp1 protein interactions". Molecular and Cellular Biology 17 (11): 6472–80. November 1997. doi:10.1128/mcb.17.11.6472. PMID 9343410. 
  • "SV40 large T antigen is modified with O-linked N-acetylglucosamine but not with other forms of glycosylation". Glycobiology 8 (4): 383–91. April 1998. doi:10.1093/glycob/8.4.383. PMID 9499386. 
  • "Glycosylation sites flank phosphorylation sites on synapsin I: O-linked N-acetylglucosamine residues are localized within domains mediating synapsin I interactions". Journal of Neurochemistry 73 (1): 418–28. July 1999. doi:10.1046/j.1471-4159.1999.0730418.x. PMID 10386995. 
  • "Localization of the O-linked N-acetylglucosamine transferase in rat pancreas". Diabetes 48 (12): 2407–13. December 1999. doi:10.2337/diabetes.48.12.2407. PMID 10580430. 
  • "Functional expression of O-linked GlcNAc transferase. Domain structure and substrate specificity". The Journal of Biological Chemistry 275 (15): 10983–8. April 2000. doi:10.1074/jbc.275.15.10983. PMID 10753899. 




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