Emerging structural insights into glycosyltransferase-mediated synthesis of glycans

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Abstract

Glycans linked to proteins and lipids play key roles in biology; thus, accurate replication of cellular glycans is crucial for maintaining function following cell division. The fact that glycans are not copied from genomic templates suggests that fidelity is provided by the catalytic templates of glycosyltransferases that accurately add sugars to specific locations on growing oligosaccharides. To form new glycosidic bonds, glycosyltransferases bind acceptor substrates and orient a specific hydroxyl group, frequently one of many, for attack of the donor sugar anomeric carbon. Several recent crystal structures of glycosyltransferases with bound acceptor substrates reveal that these enzymes have common core structures that function as scaffolds upon which variable loops are inserted to confer substrate specificity and correctly orient the nucleophilic hydroxyl group. The varied approaches for acceptor binding site assembly suggest an ongoing evolution of these loop regions provides templates for assembly of the diverse glycan structures observed in biology.

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Fig. 1: GT reaction mechanisms.
Fig. 2: Comparison of representative inverting and retaining catalytic mechanisms and enzyme topologies for substrate interactions.
Fig. 3: Structural gallery of eukaryotic GT-A fold inverting enzymes.
Fig. 4: Structural gallery of eukaryotic GT29 sialyltransferases as acceptor-bound complexes.
Fig. 5: Structural gallery of eukaryotic GT-A fold retaining enzymes.
Fig. 6: Glycosyltransferases recognizing linear peptides.
Fig. 7: Domain specific GTs (POFUT1, POFUT2, and POGLUT1) with substrates.

References

  1. 1.

    Cummings, R. D. & Pierce, J. M. The challenge and promise of glycomics. Chem. Biol. 21, 1–15 (2014).

  2. 2.

    Varki, A. Biological roles of glycans. Glycobiology 27, 3–49 (2017).

  3. 3.

    Moremen, K. W., Tiemeyer, M. & Nairn, A. V. Vertebrate protein glycosylation: diversity, synthesis and function. Nat. Rev. Mol. Cell Biol. 13, 448–462 (2012).

  4. 4.

    Moremen, K. W. et al. Expression system for structural and functional studies of human glycosylation enzymes. Nat. Chem. Biol. 14, 156–162 (2018).

  5. 5.

    Lairson, L. L., Henrissat, B., Davies, G. J. & Withers, S. G. Glycosyltransferases: structures, functions, and mechanisms. Annu. Rev. Biochem. 77, 521–555 (2008).

  6. 6.

    Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M. & Henrissat, B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, D490–D495 (2014).

  7. 7.

    Barford, D. & Johnson, L. N. The allosteric transition of glycogen phosphorylase. Nature 340, 609–616 (1989).

  8. 8.

    Ju, T., Aryal, R. P., Kudelka, M. R., Wang, Y. & Cummings, R. D. The Cosmc connection to the Tn antigen in cancer. Cancer Biomark. 14, 63–81 (2014).

  9. 9.

    Rini, J.M. & Esko, J.D. in Essentials of Glycobiology (eds. Varki, A, Cummings, R.D., Esko, J.D. et al.) 65–75 (Cold Spring Harbor, NY, 2015).

  10. 10.

    Levine, Z. G. & Walker, S. The biochemistry of O-GlcNAc transferase: which functions make it essential in mammalian cells? Annu. Rev. Biochem. 85, 631–657 (2016).

  11. 11.

    Bai, L. & Li, H. Cryo-EM is uncovering the mechanism of eukaryotic protein N-glycosylation. FEBS J. 286, 1638–1644 (2019).

  12. 12.

    Fujiwara, R., Yokoi, T. & Nakajima, M. Structure and protein-protein interactions of human UDP-glucuronosyltransferases. Front. Pharmacol. 7, 388 (2016).

  13. 13.

    Ramakrishnan, B. & Qasba, P. K. Crystal structure of lactose synthase reveals a large conformational change in its catalytic component, the beta1,4-galactosyltransferase-I. J. Mol. Biol. 310, 205–218 (2001).

  14. 14.

    Qasba, P. K., Ramakrishnan, B. & Boeggeman, E. Structure and function of β-1,4-galactosyltransferase. Curr. Drug Targets 9, 292–309 (2008).

  15. 15.

    Ramakrishnan, B., Balaji, P. V. & Qasba, P. K. Crystal structure of β1,4-galactosyltransferase complex with UDP-Gal reveals an oligosaccharide acceptor binding site. J. Mol. Biol. 318, 491–502 (2002).

  16. 16.

    Gagnon, S. M. et al. High resolution structures of the human ABO(H) blood group enzymes in complex with donor analogs reveal that the enzymes utilize multiple donor conformations to bind substrates in a stepwise manner. J. Biol. Chem. 290, 27040–27052 (2015).

  17. 17.

    Wagner, G. K., Pesnot, T., Palcic, M. M. & Jørgensen, R. Novel UDP-GalNAc derivative structures provide insight into the donor specificity of human blood group glycosyltransferase. J. Biol. Chem. 290, 31162–31172 (2015).

  18. 18.

    Breton, C., Snajdrová, L., Jeanneau, C., Koca, J. & Imberty, A. Structures and mechanisms of glycosyltransferases. Glycobiology 16, 29R–37R (2006).

  19. 19.

    Lira-Navarrete, E. et al. Structural insights into the mechanism of protein O-fucosylation. PLoS One 6, e25365 (2011).

  20. 20.

    Li, Z. et al. Recognition of EGF-like domains by the Notch-modifying O-fucosyltransferase POFUT1. Nat. Chem. Biol. 13, 757–763 (2017).

  21. 21.

    Urbanowicz, B. R. et al. Structural, mutagenic and in silico studies of xyloglucan fucosylation in Arabidopsis thaliana suggest a water-mediated mechanism. Plant J. 91, 931–949 (2017).

  22. 22.

    Yu, H. et al. Notch-modifying xylosyltransferase structures support an SNi-like retaining mechanism. Nat. Chem. Biol. 11, 847–854 (2015).

  23. 23.

    Lira-Navarrete, E. et al. Substrate-guided front-face reaction revealed by combined structural snapshots and metadynamics for the polypeptide N-acetylgalactosaminyltransferase 2. Angew. Chem. Int. Edn Engl. 53, 8206–8210 (2014).

  24. 24.

    Albesa-Jové, D. et al. A native ternary complex trapped in a crystal reveals the catalytic mechanism of a retaining glycosyltransferase. Angew. Chem. Int. Edn Engl. 54, 9898–9902 (2015).

  25. 25.

    Albesa-Jové, D., Sainz-Polo, M. A., Marina, A. & Guerin, M. E. Structural snapshots of α-1,3-galactosyltransferase with native substrates: insight into the catalytic mechanism of retaining glycosyltransferases. Angew. Chem. Int. Edn Engl. 56, 14853–14857 (2017).

  26. 26.

    Ramakrishnan, B. & Qasba, P. K. Crystal structure of the catalytic domain of Drosophila beta1,4-galactosyltransferase-7. J. Biol. Chem. 285, 15619–15626 (2010).

  27. 27.

    Hurtado-Guerrero, R. Recent structural and mechanistic insights into protein O-GalNAc glycosylation. Biochem. Soc. Trans. 44, 61–67 (2016).

  28. 28.

    Ardèvol, A., Iglesias-Fernández, J., Rojas-Cervellera, V. & Rovira, C. The reaction mechanism of retaining glycosyltransferases. Biochem. Soc. Trans. 44, 51–60 (2016).

  29. 29.

    Gómez, H., Mendoza, F., Lluch, J. M. & Masgrau, L. QM/MM studies reveal how substrate-substrate and enzyme-substrate interactions modulate retaining glycosyltransferases catalysis and mechanism. Adv. Protein Chem. Struct. Biol. 100, 225–254 (2015).

  30. 30.

    Schuman, B., Evans, S. V. & Fyles, T. M. Geometric attributes of retaining glycosyltransferase enzymes favor an orthogonal mechanism. PLoS One 8, e71077 (2013).

  31. 31.

    Kadirvelraj, R. et al. Human N-acetylglucosaminyltransferase II substrate recognition uses a modular architecture that includes a convergent exosite. Proc. Natl Acad. Sci. USA 115, 4637–4642 (2018).

  32. 32.

    Gordon, R. D. et al. X-ray crystal structures of rabbit N-acetylglucosaminyltransferase I (GnT I) in complex with donor substrate analogues. J. Mol. Biol. 360, 67–79 (2006).

  33. 33.

    Pak, J. E. et al. X-ray crystal structure of leukocyte type core 2 beta1,6-N-acetylglucosaminyltransferase. Evidence for a convergence of metal ion-independent glycosyltransferase mechanism. J. Biol. Chem. 281, 26693–26701 (2006).

  34. 34.

    Pak, J. E., Satkunarajah, M., Seetharaman, J. & Rini, J. M. Structural and mechanistic characterization of leukocyte-type core 2 β1,6-N-acetylglucosaminyltransferase: a metal-ion-independent GT-A glycosyltransferase. J. Mol. Biol. 414, 798–811 (2011).

  35. 35.

    Kuhn, B. et al. The structure of human α-2,6-sialyltransferase reveals the binding mode of complex glycans. Acta Crystallogr. D Biol. Crystallogr. 69, 1826–1838 (2013).

  36. 36.

    Meng, L. et al. Enzymatic basis for N-glycan sialylation: structure of rat α2,6-sialyltransferase (ST6GAL1) reveals conserved and unique features for glycan sialylation. J. Biol. Chem. 288, 34680–34698 (2013).

  37. 37.

    Rao, F. V. et al. Structural insight into mammalian sialyltransferases. Nat. Struct. Mol. Biol. 16, 1186–1188 (2009).

  38. 38.

    Volkers, G. et al. Structure of human ST8SiaIII sialyltransferase provides insight into cell-surface polysialylation. Nat. Struct. Mol. Biol. 22, 627–635 (2015).

  39. 39.

    Briggs, D. C. & Hohenester, E. Structural basis for the initiation of glycosaminoglycan biosynthesis by human xylosyltransferase 1. Structure 26, 801–809.e3 (2018).

  40. 40.

    Nagae, M. et al. Structure and mechanism of cancer-associated N-acetylglucosaminyltransferase-V. Nat. Commun. 9, 3380 (2018).

  41. 41.

    Rocha, J. et al. Structure of Arabidopsis thaliana FUT1 reveals a variant of the GT-B class fold and provides insight into xyloglucan fucosylation. Plant Cell 28, 2352–2364 (2016).

  42. 42.

    Valero-González, J. et al. A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2. Nat. Chem. Biol. 12, 240–246 (2016).

  43. 43.

    Li, Z. et al. Structural basis of Notch O-glucosylation and O-xylosylation by mammalian protein-O-glucosyltransferase 1 (POGLUT1). Nat. Commun. 8, 185 (2017).

  44. 44.

    Kuwabara, N. et al. Carbohydrate-binding domain of the POMGnT1 stem region modulates O-mannosylation sites of α-dystroglycan. Proc. Natl Acad. Sci. USA 113, 9280–9285 (2016).

  45. 45.

    Kakuda, S. et al. Structural basis for acceptor substrate recognition of a human glucuronyltransferase, GlcAT-P, an enzyme critical in the biosynthesis of the carbohydrate epitope HNK-1. J. Biol. Chem. 279, 22693–22703 (2004).

  46. 46.

    Pedersen, L. C. et al. Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I. J. Biol. Chem. 275, 34580–34585 (2000).

  47. 47.

    Alfaro, J. A. et al. ABO(H) blood group A and B glycosyltransferases recognize substrate via specific conformational changes. J. Biol. Chem. 283, 10097–10108 (2008).

  48. 48.

    Culbertson, A. T., Ehrlich, J. J., Choe, J. Y., Honzatko, R. B. & Zabotina, O. A. Structure of xyloglucan xylosyltransferase 1 reveals simple steric rules that define biological patterns of xyloglucan polymers. Proc. Natl Acad. Sci. USA 115, 6064–6069 (2018).

  49. 49.

    Pedersen, L. C. et al. Crystal structure of an alpha 1,4-N-acetylhexosaminyltransferase (EXTL2), a member of the exostosin gene family involved in heparan sulfate biosynthesis. J. Biol. Chem. 278, 14420–14428 (2003).

  50. 50.

    Lazarus, M. B. et al. Structural snapshots of the reaction coordinate for O-GlcNAc transferase. Nat. Chem. Biol. 8, 966–968 (2012).

  51. 51.

    Barresi, R. & Campbell, K. P. Dystroglycan: from biosynthesis to pathogenesis of human disease. J. Cell Sci. 119, 199–207 (2006).

  52. 52.

    Yoshida-Moriguchi, T. & Campbell, K. P. Matriglycan: a novel polysaccharide that links dystroglycan to the basement membrane. Glycobiology 25, 702–713 (2015).

  53. 53.

    Sheikh, M. O., Halmo, S. M. & Wells, L. Recent advancements in understanding mammalian O-mannosylation. Glycobiology 27, 806–819 (2017).

  54. 54.

    Shah, N., Kuntz, D. A. & Rose, D. R. Golgi alpha-mannosidase II cleaves two sugars sequentially in the same catalytic site. Proc. Natl Acad. Sci. USA 105, 9570–9575 (2008).

  55. 55.

    Bhide, G. P. & Colley, K. J. Sialylation of N-glycans: mechanism, cellular compartmentalization and function. Histochem. Cell Biol. 147, 149–174 (2017).

  56. 56.

    Chen, X. & Varki, A. Advances in the biology and chemistry of sialic acids. ACS Chem. Biol. 5, 163–176 (2010).

  57. 57.

    Cohen, M. & Varki, A. The sialome—far more than the sum of its parts. OMICS 14, 455–464 (2010).

  58. 58.

    Macauley, M. S., Crocker, P. R. & Paulson, J. C. Siglec-mediated regulation of immune cell function in disease. Nat. Rev. Immunol. 14, 653–666 (2014).

  59. 59.

    Datta, A. K. & Paulson, J. C. The sialyltransferase “sialylmotif” participates in binding the donor substrate CMP-NeuAc. J. Biol. Chem. 270, 1497–1500 (1995).

  60. 60.

    Harduin-Lepers, A., Mollicone, R., Delannoy, P. & Oriol, R. The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach. Glycobiology 15, 805–817 (2005).

  61. 61.

    Harduin-Lepers, A. et al. The human sialyltransferase family. Biochimie 83, 727–737 (2001).

  62. 62.

    Dennis, J. W., Nabi, I. R. & Demetriou, M. Metabolism, cell surface organization, and disease. Cell 139, 1229–1241 (2009).

  63. 63.

    Buckhaults, P., Chen, L., Fregien, N. & Pierce, M. Transcriptional regulation of N-acetylglucosaminyltransferase V by the src oncogene. J. Biol. Chem. 272, 19575–19581 (1997).

  64. 64.

    Kang, R. et al. Transcriptional regulation of the N-acetylglucosaminyltransferase V gene in human bile duct carcinoma cells (HuCC-T1) is mediated by Ets-1. J. Biol. Chem. 271, 26706–26712 (1996).

  65. 65.

    Gu, J. et al. Purification and characterization of UDP-N-acetylglucosamine: alpha-6-d-mannoside beta 1-6N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase V) from a human lung cancer cell line. J. Biochem. 113, 614–619 (1993).

  66. 66.

    Shoreibah, M. G., Hindsgaul, O. & Pierce, M. Purification and characterization of rat kidney UDP-N-acetylglucosamine: α-6-d-mannoside β-1,6-N-acetylglucosaminyltransferase. J. Biol. Chem. 267, 2920–2927 (1992).

  67. 67.

    Boscher, C., Dennis, J. W. & Nabi, I. R. Glycosylation, galectins and cellular signaling. Curr. Opin. Cell Biol. 23, 383–392 (2011).

  68. 68.

    Fritz, T. A., Raman, J. & Tabak, L. A. Dynamic association between the catalytic and lectin domains of human UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase-2. J. Biol. Chem. 281, 8613–8619 (2006).

  69. 69.

    Lira-Navarrete, E. et al. Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation. Nat. Commun. 6, 6937 (2015).

  70. 70.

    de Las Rivas, M. et al. The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences. Nat. Commun. 8, 1959 (2017).

  71. 71.

    Takeuchi, H., Kantharia, J., Sethi, M. K., Bakker, H. & Haltiwanger, R. S. Site-specific O-glucosylation of the epidermal growth factor-like (EGF) repeats of notch: efficiency of glycosylation is affected by proper folding and amino acid sequence of individual EGF repeats. J. Biol. Chem. 287, 33934–33944 (2012).

  72. 72.

    Luo, Y., Nita-Lazar, A. & Haltiwanger, R. S. Two distinct pathways for O-fucosylation of epidermal growth factor-like or thrombospondin type 1 repeats. J. Biol. Chem. 281, 9385–9392 (2006).

  73. 73.

    Wang, Y. & Spellman, M. W. Purification and characterization of a GDP-fucose:polypeptide fucosyltransferase from Chinese hamster ovary cells. J. Biol. Chem. 273, 8112–8118 (1998).

  74. 74.

    Varshney, S. & Stanley, P. Multiple roles for O-glycans in Notch signalling. FEBS Lett. 592, 3819–3834 (2018).

  75. 75.

    Harvey, B. M. & Haltiwanger, R. S. Regulation of Notch function by O-glycosylation. Adv. Exp. Med. Biol. 1066, 59–78 (2018).

  76. 76.

    Holdener, B. C. & Haltiwanger, R. S. Protein O-fucosylation: structure and function. Curr. Opin. Struct. Biol. 56, 78–86 (2019).

  77. 77.

    Yu, H. & Takeuchi, H. Protein O-glucosylation: another essential role of glucose in biology. Curr. Opin. Struct. Biol. 56, 64–71 (2019).

  78. 78.

    Schneider, M., Al-Shareffi, E. & Haltiwanger, R. S. Biological functions of fucose in mammals. Glycobiology 27, 601–618 (2017).

  79. 79.

    Benz, B. A. et al. Genetic and biochemical evidence that gastrulation defects in Pofut2 mutants result from defects in ADAMTS9 secretion. Dev. Biol. 416, 111–122 (2016).

  80. 80.

    Du, J. et al. O-fucosylation of thrombospondin type 1 repeats restricts epithelial to mesenchymal transition (EMT) and maintains epiblast pluripotency during mouse gastrulation. Dev. Biol. 346, 25–38 (2010).

  81. 81.

    Yu, H. et al. Structural analysis of Notch-regulating Rumi reveals basis for pathogenic mutations. Nat. Chem. Biol. 12, 735–740 (2016).

  82. 82.

    Takeuchi, H. et al. O-Glycosylation modulates the stability of epidermal growth factor-like repeats and thereby regulates Notch trafficking. J. Biol. Chem. 292, 15964–15973 (2017).

  83. 83.

    Vasudevan, D., Takeuchi, H., Johar, S. S., Majerus, E. & Haltiwanger, R. S. Peters plus syndrome mutations disrupt a noncanonical ER quality-control mechanism. Curr. Biol. 25, 286–295 (2015).

  84. 84.

    Lira-Navarrete, E. & Hurtado-Guerrero, R. A perspective on structural and mechanistic aspects of protein O-fucosylation. Acta Crystallogr. F Struct. Biol. Commun. 74, 443–450 (2018).

  85. 85.

    Wild, R. et al. Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation. Science 359, 545–550 (2018).

  86. 86.

    Bai, L., Wang, T., Zhao, G., Kovach, A. & Li, H. The atomic structure of a eukaryotic oligosaccharyltransferase complex. Nature 555, 328–333 (2018).

  87. 87.

    Braunger, K. et al. Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum. Science 360, 215–219 (2018).

  88. 88.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

  89. 89.

    Thiyagarajan, N. et al. Structure of a metal-independent bacterial glycosyltransferase that catalyzes the synthesis of histo-blood group A antigen. Sci. Rep. 2, 940 (2012).

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Acknowledgements

The authors would like to thank M. Tiemeyer and H. Takeuchi for providing useful comments. Original work in the author’s laboratories is supported by NIH grants P01GM107012, P41GM103390, U01GM120408, R01GM130915 (K.W.M.) and GM061126, HD090156, HD096030 (R.S.H.).

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Correspondence to Kelley W. Moremen or Robert S. Haltiwanger.

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Competing interests

K.W.M. acknowledges ownership interest and roles as President and CEO of Glyco Expression Technologies, Inc., a biotechnology spinout commercializing recombinant glycosyltransferases, and may conceivably profit from the results described herein.

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Supplementary information

Supplementary Figure 1

Timeline of published GT structures. The GT structures in PDB from all domains of life were collated in Supplementary Dataset 1.

Supplementary Dataset 1

Summary of glycosyltransferase structures in PDB (January 2019).

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