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Selective β-N-acetylhexosaminidase from Aspergillus versicolor—a tool for producing bioactive carbohydrates

  • Pavla BojarováEmail author
  • Natallia Kulik
  • Kristýna Slámová
  • Martin Hubálek
  • Michael Kotik
  • Josef Cvačka
  • Helena Pelantová
  • Vladimír Křen
Biotechnologically relevant enzymes and proteins
  • 55 Downloads

Abstract

β-N-Acetylhexosaminidases (EC 3.2.1.52) are typical of their dual activity encompassing both N-acetylglucosamine and N-acetylgalactosamine substrates. Here we present the isolation and characterization of a selective β-N-acetylhexosaminidase from the fungal strain of Aspergillus versicolor. The enzyme was recombinantly expressed in Pichia pastoris KM71H in a high yield and purified in a single step using anion-exchange chromatography. Homologous molecular modeling of this enzyme identified crucial differences in the enzyme active site that may be responsible for its high selectivity for N-acetylglucosamine substrates compared to fungal β-N-acetylhexosaminidases from other sources. The enzyme was used in a sequential reaction together with a mutant β-N-acetylhexosaminidase from Talaromyces flavus with an enhanced synthetic capability, affording a bioactive disaccharide bearing an azido functional group. The azido function enabled an elegant multivalent presentation of this disaccharide on an aromatic carrier. The resulting model glycoconjugate is applicable as a selective ligand of galectin-3 — a biomedically attractive human lectin. These results highlight the importance of a general availability of robust and well-defined carbohydrate-active enzymes with tailored catalytic properties for biotechnological and biomedical applications.

Keywords

Aspergillus versicolor β-N-Acetylhexosaminidase Glycosidase Homology modeling Heterologous expression Pichia pastoris 

Notes

Acknowledgments

P. B. and V. K. acknowledge support by mobility projects no. LTC18038 and LTC18041 (MEYS, the Ministry of Education, Youth and Sports of the Czech Republic). N. K. acknowledges access to the computing and storage facilities provided by the CESNET LM2015042 and the CERIT Scientific Cloud LM2015085 under the program “Projects of Large Research, Development, and Innovations Infrastructures.”

Funding

This study was funded by the Ministry of Education, Youth, and Sports of the Czech Republic mobility projects nos. LTC18038 and LTC18041, by CESNET (LM2015042), and by CERIT Scientific Cloud (LM2015085) under the program “Projects of Large Research, Development, and Innovations Infrastructures.”

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2018_9534_MOESM1_ESM.pdf (1.5 mb)
ESM 1 (PDF 1485 kb)

References

  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410.  https://doi.org/10.1016/S0022-2836(05)80360-2 CrossRefGoogle Scholar
  2. Bohne-Lang A, von der Lieth CW (2005) GlyProt: in silico glycosylation of proteins. Nucleic Acids Res 33:W214–W219.  https://doi.org/10.1093/nar/gki385 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bojarová P, Křen V (2009) Glycosidases: a key to tailored carbohydrates. Trends Biotechnol 27:199–209.  https://doi.org/10.1016/j.tibtech.2008.12.003 CrossRefPubMedGoogle Scholar
  4. Bojarová P, Křen V (2011) Glycosidases in carbohydrate synthesis: when organic chemistry falls short. Chimia 65:65–70.  https://doi.org/10.2533/chimia.2011.65 CrossRefPubMedGoogle Scholar
  5. Bojarová P, Křen V (2016) Sugared biomaterial binding lectins: achievements and perspectives. Biomat Sci 4:1142–1160.  https://doi.org/10.1039/c6bm00088f CrossRefGoogle Scholar
  6. Bojarová P, Petrásková L, Ferrandi E, Monti D, Pelantová H, Kuzma M, Simerská P, Křen V (2007) Glycosyl azides – an alternative way to disaccharides. Adv Synth Catal 349:1514–1520CrossRefGoogle Scholar
  7. Bojarová P, Křenek K, Kuzma M, Petrásková L, Bezouška K, Namdjou D-J, Elling L, Křen V (2008) N-Acetylhexosamine triad in one molecule: chemoenzymatic introduction of 2-acetamido-2-deoxy-β-d-galactopyranosyluronic acid residue into a complex oligosaccharide. J Mol Catal B Enzym 50:69–73.  https://doi.org/10.1016/j.molcatb.2007.09.002 CrossRefGoogle Scholar
  8. Bojarová P, Slámová K, Křenek K, Gažák R, Kulik N, Ettrich R, Pelantová H, Kuzma M, Riva S, Adámek D, Bezouška K, Křen V (2011) Charged hexosaminides as new substrates for β-N-scetylhexosaminidase-catalyzed synthesis of immunomodulatory disaccharides. Adv Synth Catal 353:2409–2420.  https://doi.org/10.1002/adsc.201100371 CrossRefGoogle Scholar
  9. Bojarová P, Rosencrantz RR, Elling L, Křen V (2013) Enzymatic glycosylation of multivalent scaffolds. Chem Soc Rev 42:4774–4797.  https://doi.org/10.1039/c2cs35395d CrossRefPubMedGoogle Scholar
  10. Bojarová P, Chytil P, Mikulová B, Bumba L, Konefal R, Pelantová H, Krejzová J, Slámová K, Petrásková L, Kotrchová L, Cvačka J, Etrych T, Křen V (2017) Glycan-decorated HPMA copolymers as high-affinity lectin ligands. Polym Chem 8:2647–2658.  https://doi.org/10.1039/C7PY00271H CrossRefGoogle Scholar
  11. Bojarová P, Tavares MR, Laaf D, Bumba L, Petrásková L, Konefał R, Bláhová M, Pelantová H, Elling L, Etrych T, Chytil P, Křen V (2018) Biocompatible glyconanomaterials based on HPMA-copolymer for specific targeting of galectin-3. J Nanobiotechnol 16:73.  https://doi.org/10.1186/s12951-018-0399-1 CrossRefGoogle Scholar
  12. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.  https://doi.org/10.1016/0003-2697(76)90527-3 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Bumba L, Laaf D, Spiwok V, Elling L, Křen V, Bojarová P (2018) Poly-N-acetyllactosamine neo-glycoproteins as nanomolar ligands of human galectin-3: binding kinetics and modeling. Int J Mol Sci 19:372.  https://doi.org/10.3390/ijms19020372 CrossRefPubMedCentralGoogle Scholar
  14. Chuang G-Y, Boyington JC, Joyce MG, Zhu J, Nabel GJ, Kwong PD, Georgiev I (2012) Computational prediction of N-linked glycosylation incorporating structural properties and patterns. Bioinformatics 28:2249–2255.  https://doi.org/10.1093/bioinformatics/bts426 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Clamp M, Cuff J, Searle SM, Barton GJ (2004) The Jalview Java alignment editor. Bioinformatics 20:426–427.  https://doi.org/10.1093/bioinformatics/btg430 CrossRefPubMedGoogle Scholar
  16. Davis IW, LeaverFay A, Chen VB, Block JN, Kapral GJ, Wang X, Murray LW, Arendall WB 3rd, Snoeyink J, Richardson JS, Richardson DC (2007) MolProbity: allatom contacts and structure validation. Nucleic Acids Res 35:W375–W383.  https://doi.org/10.1093/nar/gkm216. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Drozdová A, Bojarová P, Křenek K, Weignerová L, Henßen B, Elling L, Christensen H, Jensen HH, Pelantová H, Kuzma M, Bezouška K, Krupová M, Adámek D, Slámová K, Křen V (2011) Enzymatic synthesis of dimeric glycomimetic ligands of NK cell activation receptors. Carbohydr Res 346:1599–1609.  https://doi.org/10.1016/j.carres.2011.04.043 CrossRefPubMedGoogle Scholar
  18. Ettrich R, Kopecký V Jr, Hofbauerová K, Baumruk V, Novák P, Pompach P, Man P, Plíhal O, Kutý M, Kulik N, Sklenář J, Ryšlavá H, Křen V, Bezouška K (2007) Structure of the dimeric N-glycosylated form of fungal β-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies. BMC Struct Biol 7:32–45.  https://doi.org/10.1186/1472-6807-7-32 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Fialová P, Weignerová L, Rauvolfová J, Přikrylová V, Pišvejcová A, Ettrich R, Kuzma M, Sedmera P, Křen V (2004) Hydrolytic and transglycosylation reactions of N-acyl modified substrates catalysed by β-N-acetylhexosaminidases. Tetrahedron 60:693–701.  https://doi.org/10.1016/j.tet.2003.10.111 CrossRefGoogle Scholar
  20. Fialová P, Carmona AT, Robina I, Ettrich R, Sedmera P, Přikrylová V, Petrásková-Hušáková L, Křen V (2005) Glycosyl azide - a novel substrate for enzymatic transglycosylations. Tetrahedron Lett 46:8715–8718.  https://doi.org/10.1016/j.tetlet.2005.10.040 CrossRefGoogle Scholar
  21. Friesner RA, Murphy RB, Repasky MP, Frye LL, Greenwood JR, Halgren TA, Sanschagrin PC, Mainz DT (2006) Extra precision Glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J Med Chem 49:6177–6196.  https://doi.org/10.1021/jm051256o CrossRefPubMedGoogle Scholar
  22. Gloster TM, Vocadlo DJ (2010) Mechanism, structure, and inhibition of O-GlcNAc processing enzymes. Curr Signal Transduct Ther 5:74–91.  https://doi.org/10.2174/157436210790226537 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Huňková Z, Křen V, Ščigelová M, Weignerová L, Scheel O, Thiem J (1996) Induction of β-N-acetylhexosaminidase in Aspergillus oryzae. Biotechnol Lett 18:725–730.  https://doi.org/10.1007/BF00130773 CrossRefGoogle Scholar
  24. Jakalian A, Jack DB, Bayly CI (2002) Fast, efficient generation of high quality atomic charges. AM1BCC model: II. Parameterization and validation. J Comput Chem 23:1623–1641.  https://doi.org/10.1002/jcc.10128 CrossRefPubMedGoogle Scholar
  25. Krieger E, Koraimann G, Vriend G (2002) Increasing the precision of comparative models with YASARA NOVA a self parameterizing force field. Proteins 47:393–402.  https://doi.org/10.1002/prot.10104 CrossRefPubMedGoogle Scholar
  26. Kulik N, Slámová K, Ettrich R, Křen V (2015) Computational study of β-N-acetylhexosaminidase from Talaromyces flavus, a glycosidase with high substrate flexibility. BMC Bioinformatics 16:1–15.  https://doi.org/10.1186/s12859-015-0465-8. CrossRefGoogle Scholar
  27. Laaf D, Bojarová P, Mikulová B, Pelantová H, Křen V, Elling L (2017a) Two-step enzymatic synthesis of β-d-N-acetylgalactosamine-(1-4)-d-N-acetylglucosamine (LacdiNAc) chitooligomers for deciphering galectin binding behavior. Adv Synth Catal 359:2101–2108.  https://doi.org/10.1002/adsc.201700331 CrossRefGoogle Scholar
  28. Laaf D, Bojarová P, Pelantová H, Křen V, Elling L (2017b) Tailored multivalent neo-glycoproteins: synthesis, evaluation, and application of a library of galectin-3-binding glycan ligands. Bioconjug Chem 28:2832–2840.  https://doi.org/10.1021/acs.bioconjchem.7b00520 CrossRefPubMedGoogle Scholar
  29. Macauley MS, Whitworth GE, Debowski AW, Chin D, Vocadlo DJ (2005) O-GlcNAcase uses substrate-assisted catalysis; kinetic analysis and development of highly selective mechanism-inspired inhibitors. J Biol Chem 280:25313–25,322.  https://doi.org/10.1074/jbc.M413819200 CrossRefPubMedGoogle Scholar
  30. Notredame C, Higgins DG, Heringa J (2000) T-coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302:205–217.  https://doi.org/10.1006/jmbi.2000.4042. CrossRefPubMedGoogle Scholar
  31. Plíhal O, Sklenář J, Hofbauerová K, Novák P, Man P, Pompach P, Ryšlavá H, Charvátová-Pišvejcová A, Křen V, Bezouška K (2007) Large propeptides of fungal β-N-acetylhexosaminidases are novel enzyme regulators that must be processed intracellularly to control activity, dimerization, and secretion into the extracellular environment. Biochemistry 46:2719–2734.  https://doi.org/10.1021/bi061828m CrossRefPubMedGoogle Scholar
  32. Raval A, Piana S, Eastwood MP, Dror RO, Shaw DE (2012) Refinement of protein structure homology models via long, all-atom molecular dynamics simulations. Proteins 80:2071–2079.  https://doi.org/10.1002/prot.24098. CrossRefPubMedGoogle Scholar
  33. Robert X, Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42:W320–W324.  https://doi.org/10.1093/nar/gku316 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Ryšlavá H, Kalendová A, Doubnerová V, Skočdopol P, Kumar V, Kukačka Z, Pompach P, Vaněk O, Slámová K, Bojarová P, Kulík N, Ettrich R, Křen V, Bezouška K (2011) Enzymatic characterization and molecular modeling of an evolutionarily interesting fungal β-N-acetylhexosaminidase. FEBS J 278:2469–2484.  https://doi.org/10.1111/j.1742-4658.2011.08173.x CrossRefPubMedGoogle Scholar
  35. Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815.  https://doi.org/10.1006/jmbi.1993.1626 CrossRefPubMedGoogle Scholar
  36. Šimonová A, Kupper CE, Böcker S, Müller A, Hofbauerová K, Pelantová H, Elling L, Křen V, Bojarová P (2014) Chemo-enzymatic synthesis of LacdiNAc dimers of varying length as novel galectin ligands. J Mol Catal B Enzym 101:47–55.  https://doi.org/10.1016/j.molcatb.2013.12.018 CrossRefGoogle Scholar
  37. Škerlová J, Bláha J, Pachl P, Hofbauerová K, Kukačka Z, Man P, Pompach P, Novák P, Otwinowski Z, Brynda J, Vaněk O, Řezáčová P (2018) Crystal structure of native β-N-acetylhexosaminidase isolated from Aspergillus oryzae sheds light onto its substrate specificity, high stability, and regulation by propeptide. FEBS J 285:580–598.  https://doi.org/10.1111/febs.14360 CrossRefPubMedGoogle Scholar
  38. Slámová K, Bojarová P (2017) Engineered N-acetylhexosamine-active enzymes in glycoscience. Biochim Biophys Acta Gen Subj 1861:2070–2087.  https://doi.org/10.1016/j.bbagen.2017.03.019 CrossRefPubMedGoogle Scholar
  39. Slámová K, Bojarová P, Petrásková L, Křen V (2010) β-N-Acetylhexosaminidase: what‘s in a name...? Biotechnol Adv 28:682–693.  https://doi.org/10.1016/j.biotechadv.2010.04.004 CrossRefPubMedGoogle Scholar
  40. Slámová K, Bojarová P, Gerstorferová D, Fliedrová B, Hofmeisterová J, Fiala M, Pompach P, Křen V (2012) Sequencing, cloning and high-yield expression of a fungal β-N-acetylhexosaminidase in Pichia pastoris. Prot Express Purif 82:212–217.  https://doi.org/10.1016/j.pep.2012.01.004 CrossRefGoogle Scholar
  41. Slámová K, Krejzová J, Marhol P, Kalachova L, Kulik N, Pelantová H, Cvačka J, Křen V (2015) Synthesis of derivatized chitooligomers using transglycosidases engineered from the fungal GH20 β-N-acetylhexosaminidase. Adv Synth Catal 357:1941–1950.  https://doi.org/10.1002/adsc.201500075 CrossRefGoogle Scholar
  42. Tews I, Perrakis A, Oppenheim A, Dauter Z, Wilson KS, Vorgias CE (1996) Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease. Nat Struct Biol 3:638–648.  https://doi.org/10.1038/nsb0796-638 CrossRefPubMedGoogle Scholar
  43. Wang GN, André S, Gabius HJ, Murphy PV (2012) Bi- to tetravalent glycoclusters: synthesis, structure-activity profiles as lectin inhibitors and impact of combining both valency and headgroup tailoring on selectivity. Org Biomol Chem 10:6893–6907.  https://doi.org/10.1039/c2ob25870f CrossRefPubMedGoogle Scholar
  44. Weignerová L, Vavrušková P, Pišvejcová A, Thiem J, Křen V (2003) Fungal β-N-acetylhexosaminidases with high β-N-acetylgalactosaminidase activity and their use for synthesis of β-GalNAc-containing oligosaccharides. Carbohydr Res 338:1003–1008.  https://doi.org/10.1016/S0008-6215(03)00044-2 CrossRefPubMedGoogle Scholar
  45. Wiederstein M, Sippl MJ (2007) ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res 35:W407–W410.  https://doi.org/10.1093/nar/gkm290 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Xie J, Hu L, Shi W, Deng X, Cao Z, Shen Q (2008) Synthesis and characterization of hyperbranched polytriazole via an ‘A2 + B3’ approach based on click chemistry. Polym Int 57:965–974.  https://doi.org/10.1002/pi.2433 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Pavla Bojarová
    • 1
    Email author
  • Natallia Kulik
    • 2
  • Kristýna Slámová
    • 1
  • Martin Hubálek
    • 3
  • Michael Kotik
    • 1
  • Josef Cvačka
    • 3
  • Helena Pelantová
    • 1
  • Vladimír Křen
    • 1
  1. 1.Laboratory of Biotransformation, Institute of MicrobiologyCzech Academy of SciencesPrague 4Czech Republic
  2. 2.Laboratory of Structure and Function of Proteins, Institute of MicrobiologyCzech Academy of SciencesNové HradyCzech Republic
  3. 3.Institute of Organic Chemistry and BiochemistryCzech Academy of SciencesPrague 6Czech Republic

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