title: A sensitive gel-based method combining distinct cyclophellitol-based probes for the identification of acid/base residues in human retaining β-glucosidases.
abstract: Retaining β-exoglucosidases operate by a mechanism in which the key amino acids driving the glycosidic bond hydrolysis act as catalytic acid/base and nucleophile. Recently we designed two distinct classes of fluorescent cyclophellitol-type activity-based probes (ABPs) that exploit this mechanism to covalently modify the nucleophile of retaining β-glucosidases. Whereas β-epoxide ABPs require a protonated acid/base for irreversible inhibition of retaining β-glucosidases, β-aziridine ABPs do not. Here we describe a novel sensitive method to identify both catalytic residues of retaining β-glucosidases by the combined use of cyclophellitol β-epoxide- and β-aziridine ABPs. In this approach putative catalytic residues are first substituted to noncarboxylic amino acids such as glycine or glutamine through site-directed mutagenesis. Next, the acid/base and nucleophile can be identified via classical sodium azide-mediated rescue of mutants thereof. Selective labeling with fluorescent β-aziridine but not β-epoxide ABPs identifies the acid/base residue in mutagenized enzyme, as only the β-aziridine ABP can bind in its absence. The Absence of the nucleophile abolishes any ABP labeling. We validated the method by using the retaining β-glucosidase GBA (CAZy glycosylhydrolase family GH30) and then applied it to non-homologous (putative) retaining β-glucosidases categorized in GH1 and GH116: GBA2, GBA3, and LPH. The described method is highly sensitive, requiring only femtomoles (nanograms) of ABP-labeled enzymes.
authors.affiliation: From the Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands and.
authors.forename: Wouter W
authors.initials: WW
authors.lastname: Kallemeijn
authors.affiliation: Bioorganic Synthesis, Leiden Institute of Chemistry, P. O. box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Martin D
authors.initials: MD
authors.lastname: Witte
authors.affiliation: From the Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands and.
authors.forename: Tineke M
authors.initials: TM
authors.lastname: Voorn-Brouwer
authors.affiliation: Bioorganic Synthesis, Leiden Institute of Chemistry, P. O. box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Marthe T C
authors.initials: MT
authors.lastname: Walvoort
authors.affiliation: Bioorganic Synthesis, Leiden Institute of Chemistry, P. O. box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Kah-Yee
authors.initials: KY
authors.lastname: Li
authors.affiliation: Bioorganic Synthesis, Leiden Institute of Chemistry, P. O. box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Jeroen D C
authors.initials: JD
authors.lastname: Codée
authors.affiliation: Bioorganic Synthesis, Leiden Institute of Chemistry, P. O. box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Gijsbert A
authors.initials: GA
authors.lastname: van der Marel
authors.affiliation: From the Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands and.
authors.forename: Rolf G
authors.initials: RG
authors.lastname: Boot
authors.affiliation: Bioorganic Synthesis, Leiden Institute of Chemistry, P. O. box 9502, 2300 RA Leiden, The Netherlands h.s.overkleeft@chem.leidenuniv.nl.
authors.forename: Herman S
authors.initials: HS
authors.lastname: Overkleeft
authors.affiliation: From the Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands and j.m.aerts@amc.uva.nl.
authors.forename: Johannes M F G
authors.initials: JM
authors.lastname: Aerts
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001388:Aziridines / Q000737:chemistry / Q000378:metabolism; D019556:COS Cells; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D003511:Cyclohexanols / Q000737:chemistry / Q000378:metabolism*; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting / Q000379:methods; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D015203:Reproducibility of Results; D019810:Sodium Azide / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D001388:Aziridines; D003511:Cyclohexanols; D004852:Epoxy Compounds; D015335:Molecular Probes; C063285:cyclophellitol; C033132:aziridine; D019810:Sodium Azide; D001617:beta-Glucosidase
keywords: Acid/Base; Activity-based Probe; Gaucher Disease; Glycobiology; Glycoconjugate; Glycolipid; Glycosidase; Labeling; Nucleophile