title: Expression of human β-N-acetylhexosaminidase B in yeast eases the search for selective inhibitors.
abstract: Human lysosomal β-N-acetylhexosaminidases from the family 20 of glycoside hydrolases are dimeric enzymes catalysing the cleavage of terminal β-N-acetylglucosamine and β-N-acetylgalactosamine residues from a broad spectrum of glycoconjugates. Here, we present a facile, robust, and cost-effective extracellular expression of human β-N-acetylhexosaminidase B in Pichia pastoris KM71H strain. The prepared Hex B was purified in a single step with 33% yield obtaining 10mg of the pure enzyme per 1L of the culture media. The enzyme was used in the inhibition assays with the known mechanism-based inhibitor NAG-thiazoline and a wide variety of its derivatives in the search for specific inhibitors of the human GH20 β-N-acetylhexosaminidases over the human GH84 β-N-acetylglucosaminidase, which was expressed, purified and used in the inhibition experiments as well. Moreover, enzyme-inhibitor complexes were analysed employing computational tools in order to reveal the structural basis of the results of the inhibition assays, showing the importance of water-mediated interactions between the enzyme and respective ligands. The presented method for the heterologous expression of human Hex B is robust, it significantly reduces the costs and equipment demands in comparison to the expression in mammalian cell lines. This will enhance accessibility of this human enzyme to the broad scientific community and may speed up the research of specific inhibitors of this physiologically important glycosidase family.
authors.affiliation: Laboratory of Biotransformation, Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ 14220 Praha 4, Czech Republic; Department of Biochemistry and Microbiology, University of Chemistry and Technology Prague, Technická 5, CZ 16628 Praha 6, Czech Republic. Electronic address: janakrejza@gmail.com.
authors.forename: Jana
authors.initials: J
authors.lastname: Krejzová
authors.affiliation: Department of Structure and Function of Proteins, Institute of Microbiology, Czech Academy of Sciences, Zámek 136, CZ 37333 Nové Hrady, Czech Republic. Electronic address: kulik@nh.cas.cz.
authors.forename: Natallia
authors.initials: N
authors.lastname: Kulik
authors.affiliation: Laboratory of Biotransformation, Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ 14220 Praha 4, Czech Republic. Electronic address: slamova@biomed.cas.cz.
authors.forename: Kristýna
authors.initials: K
authors.lastname: Slámová
authors.affiliation: Laboratory of Biotransformation, Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ 14220 Praha 4, Czech Republic. Electronic address: kren@biomed.cas.cz.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Křen
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D013844:Thiazoles / Q000494:pharmacology; D054821:beta-Hexosaminidase beta Chain / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013844:Thiazoles; C513386:HEXB protein, human; D054821:beta-Hexosaminidase beta Chain; C487818:N-acetylglucosamine thiazoline; D000117:Acetylglucosamine
keywords: Enzyme inhibition; Hex B; O-GlcNAcase; Pichia pastoris; β-N-acetylhexosaminidase