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1.
eNeuro ; 10(10)2023 10.
Article in English | MEDLINE | ID: mdl-37816595

ABSTRACT

Lysosomes are acidic organelles that traffic throughout neurons delivering catabolic enzymes to distal regions of the cell and maintaining degradative demands. Loss of function mutations in the gene GBA encoding the lysosomal enzyme glucocerebrosidase (GCase) cause the lysosomal storage disorder Gaucher's disease (GD) and are the most common genetic risk factor for synucleinopathies like Parkinson's disease (PD) and dementia with Lewy bodies (DLB). GCase degrades the membrane lipid glucosylceramide (GlcCer) and mutations in GBA, or inhibiting its activity, results in the accumulation of GlcCer and disturbs the composition of the lysosomal membrane. The lysosomal membrane serves as the platform to which intracellular trafficking complexes are recruited and activated. Here, we investigated whether lysosomal trafficking in axons was altered by inhibition of GCase with the pharmacological agent Conduritol B Epoxide (CBE). Using live cell imaging in human male induced pluripotent human stem cell (iPSC)-derived forebrain neurons, we demonstrated that lysosomal transport was similar in both control and CBE-treated neurons. Furthermore, we tested whether lysosomal rupture, a process implicated in various neurodegenerative disorders, was affected by inhibition of GCase. Using L-leucyl-L-leucine methyl ester (LLoME) to induce lysosomal membrane damage and immunocytochemical staining for markers of lysosomal rupture, we found no difference in susceptibility to rupture between control and CBE-treated neurons. These results suggest the loss of GCase activity does not contribute to neurodegenerative disease by disrupting either lysosomal transport or rupture.


Subject(s)
Induced Pluripotent Stem Cells , Neurodegenerative Diseases , Male , Humans , Glucosylceramidase/genetics , Glucosylceramidase/metabolism , Neurodegenerative Diseases/metabolism , Axonal Transport , Neurons/metabolism , Prosencephalon/metabolism , Lysosomes/metabolism , alpha-Synuclein/metabolism
2.
J Dent Res ; 102(4): 440-449, 2023 04.
Article in English | MEDLINE | ID: mdl-36749069

ABSTRACT

Osteoclasts play a key role in the regulation of bone mass and are highly active metabolically. Here we show that a metabolic reprogramming toward the hexosamine biosynthetic pathway (HBP) is required not only for osteoclast differentiation but also to determine the bone resorption mode during physiological and pathological bone remodeling. We found that pharmacological inhibition of O-GlcNAc transferase (OGT) significantly reduced protein O-GlcNAcylation and osteoclast differentiation. Accordingly, genetic deletion of OGT also inhibited osteoclast formation and downregulated critical markers related to osteoclasts differentiation and function (NFATc1, αvintegrin, cathepsin K). Indeed, cells treated with OSMI-1, an OGT inhibitor, also reduced nuclear translocation of NFATc1. Furthermore, the addition of exogenous N-acetylglucosamine (GlcNAc) strongly increased osteoclast formation and demineralization ability. Strikingly, our data show for the first time that O-GlcNAcylation facilitates an aggressive trench resorption mode in human cells. The incubation of osteoclasts with exogenous GlcNAc increases the percentage of erosion by trench while having no effect on pit resorption mode. Through time-lapse recording, we documented that osteoclasts making trenches moving across the bone surface are sensitive to GlcNAcylation. Finally, osteoclast-specific Ogt-deficient mice show increased bone density and reduced inflammation-induced bone loss during apical periodontitis model. We show that osteoclast-specific Ogt-deficient mice are less susceptible to develop bacterial-induced periapical lesion. Consistent with this, Ogt-deleted mice showed a decreased number of tartrate-resistant acid phosphatase-positive cells lining the apical periodontitis site. In summary, here we describe a hitherto undiscovered role of the HBP/O-GlcNAcylation axis tuning resorption mode and dictating bone resorption outcome.


Subject(s)
Bone Resorption , Periapical Periodontitis , Mice , Humans , Animals , Hexosamines/metabolism , Biosynthetic Pathways , Bone Resorption/metabolism , Osteoclasts/metabolism , Transcription Factors/metabolism
3.
Org Biomol Chem ; 20(3): 619-629, 2022 01 19.
Article in English | MEDLINE | ID: mdl-34940771

ABSTRACT

We report the synthesis of seven-membered iminosugars derived from a 3S-acetamido-4R,5R,6S-trihydroxyazepane scaffold and their evaluation as inhibitors of functionally related exo-N-acetylhexosaminidases including human O-GlcNAcase (OGA), human lysosomal ß-hexosaminidase (HexAB), and Escherichia coli NagZ. Capitalizing on the flexibility of azepanes and the active site tolerances of hexosaminidases, we explore the effects of epimerization of stereocenters at C-3, C-5 and C-6 and C-alkylation at the C-2 or C-7 positions. Accordingly, epimerization at C-6 (L-ido) and at C-5 (D-galacto) led to selective HexAB inhibitors whereas introduction of a propyl group at C-7 on the C-3 epimer furnished a potent NagZ inhibitor.


Subject(s)
Acetylglucosaminidase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Imino Sugars/pharmacology , beta-N-Acetylhexosaminidases/antagonists & inhibitors , Acetylglucosaminidase/metabolism , Alkylation , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Escherichia coli/enzymology , Humans , Imino Sugars/chemical synthesis , Imino Sugars/chemistry , Molecular Conformation , beta-N-Acetylhexosaminidases/metabolism
4.
Org Biomol Chem ; 15(21): 4609-4619, 2017 May 31.
Article in English | MEDLINE | ID: mdl-28513749

ABSTRACT

The synthesis of a series of d-gluco-like configured 4,5,6-trihydroxyazepanes bearing a triazole, a sulfonamide or a fluorinated acetamide moiety at C-3 is described. These synthetic derivatives have been tested for their ability to selectively inhibit the muropeptide recycling glucosaminidase NagZ and to thereby increase sensitivity of Pseudomonas aeruginosa to ß-lactams, a pathway with substantial therapeutic potential. While introduction of triazole and sulfamide groups failed to lead to glucosaminidase inhibitors, the NHCOCF3 analog proved to be a selective inhibitor of NagZ over other glucosaminidases including human O-GlcNAcase and lysosomal hexosaminidases HexA and B.


Subject(s)
Anti-Bacterial Agents/pharmacology , Azepines/chemistry , Azepines/pharmacology , Glycoside Hydrolases/antagonists & inhibitors , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , beta-Lactams/pharmacology , Azepines/chemical synthesis , Azepines/metabolism , Ceftazidime/pharmacology , Drug Synergism , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/metabolism , Hydroxylation , Molecular Docking Simulation , Protein Conformation
5.
Chem Sci ; 7(6): 3742-3750, 2016 Jun 01.
Article in English | MEDLINE | ID: mdl-29997861

ABSTRACT

The modification of nucleocytoplasmic proteins with O-linked N-acetylglucosamine (O-GlcNAc) plays diverse roles in multicellular organisms. Inhibitors of O-GlcNAc hydrolase (OGA), the enzyme that removes O-GlcNAc from proteins, lead to increased O-GlcNAc levels in cells and are seeing widespread adoption in the field as a research tool used in cells and in vivo. Here we synthesize and study a series of tight binding carbohydrate-based inhibitors of human OGA (hOGA). The most potent of these 2'-aminothiazolines binds with a sub-nanomolar Ki value to hOGA (510 ± 50 pM) and the most selective has greater than 1 800 000-fold selectivity for hOGA over mechanistically related human lysosomal ß-hexosaminidase. Structural data of inhibitors in complex with an hOGA homologue reveals the basis for variation in binding among these compounds. Using linear free energy analyses, we show binding of these 2'-aminothiazoline inhibitors depends on the pKa of the aminothiazoline ring system, revealing the protonation state of the inhibitor is a key driver of binding. Using series of inhibitors and synthetic substrates, we show that 2'-aminothiazoline inhibitors are transition state analogues of hOGA that bind to the enzyme up to 1-million fold more tightly than the substrate. These collective data support an oxazoline, rather than a protonated oxazolinium ion, intermediate being formed along the reaction pathway. Inhibitors from this series will prove generally useful tools for the study of O-GlcNAc. The new insights gained here, into the catalytic mechanism of hOGA and the fundamental drivers of potency and selectivity of OGA inhibitors, should enable tuning of hOGA inhibitors with desirable properties.

6.
Nature ; 412(6849): 835-8, 2001 Aug 23.
Article in English | MEDLINE | ID: mdl-11518970

ABSTRACT

Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray diffraction techniques. A catalytic mechanism, featuring a long-lived oxocarbenium-ion intermediate, was proposed on the basis of model-building studies. The 'Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of beta-glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining beta-glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated. Here we show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. We also show the three-dimensional structure of this intermediate as determined by X-ray diffraction. We formulate a general catalytic mechanism for all retaining beta-glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate.


Subject(s)
Muramidase/metabolism , Animals , Catalysis , Chickens , Crystallography, X-Ray , Models, Molecular , Muramidase/chemistry , Muramidase/genetics , Mutation , Protein Conformation , Spectrometry, Mass, Electrospray Ionization
7.
J Biol Chem ; 276(45): 42131-7, 2001 Nov 09.
Article in English | MEDLINE | ID: mdl-11522797

ABSTRACT

Azasugar inhibitors of the isofagomine class are potent competitive inhibitors of configuration-retaining beta-glycosidases. This potency results from the formation of a strong electrostatic interaction between a protonated endocyclic nitrogen at the "anomeric" center of the inhibitor and the catalytic nucleophile of the enzyme. Although the majority of retaining beta-glycosidases use a mechanism involving a carboxylate residue as a nucleophile, Streptomyces plicatus beta-N-acetylhexos-aminidase (SpHEX) and related family 20 glycosidases lack such a catalytic residue and use instead the carbonyl oxygen of the 2-acetamido group of the substrate as a nucleophile to "attack" the anomeric center. Thus, a strong electrostatic interaction between the inhibitor and enzyme is not expected to occur; nonetheless, the 1-N-azasugar (2R,3R,4S,5R)-2-acetamido-3,4-dihydroxy-5-hydroxymethyl-piperidinium hydrochloride (GalNAc-isofagomine.HCl), which was synthesized and assayed for its ability to inhibit SpHEX, was found to be a potent competitive inhibitor of the enzyme (K(i) = 2.7 microm). A crystallographic complex of GalNAc-isofagomine bound to SpHEX was solved and refined to 1.75 A and revealed that the lack of a strong electrostatic interaction between the "anomeric" center of GalNAc-isofagomine and SpHEX is compensated for by a novel 2.8-A hydrogen bond formed between the equatorial proton of the endocyclic nitrogen of the azasugar ring and the carboxylate of the general acid-base residue Glu-314 of SpHEX. This interaction appears to contribute to the unexpected potency of GalNAc-isofagomine toward SpHEX.


Subject(s)
Enzyme Inhibitors/pharmacology , Piperidines/pharmacology , Streptomyces/enzymology , beta-N-Acetylhexosaminidases/antagonists & inhibitors , Hydrogen Bonding , Imino Pyranoses , Piperidines/chemistry , beta-N-Acetylhexosaminidases/chemistry
8.
Biochemistry ; 40(7): 2201-9, 2001 Feb 20.
Article in English | MEDLINE | ID: mdl-11329289

ABSTRACT

Human beta-hexosaminidase A (alpha beta) and B (beta beta) are composed of subunits (alpha and beta) that are 60% identical and have been grouped with other evolutionarily related glycosidases into "Family 20". The three-dimensional structure of only one Family 20 member has been elucidated, a bacterial chitobiase. This enzyme shares primary structure homology with both the human subunits only in its active-site region, and even in this restricted area, the level of identity is only 26%. Thus, the validity of the molecular model for the active site of the human enzyme based on chitobiase must be determined experimentally. In this report, we analyze highly purified mutant forms of human hexosaminidase B that have had conservative substitutions made at Glu and Asp residues predicted by the chitobiase model to be part of its active site. Mutation of beta Glu(355) to Gln reduces k(cat) 5000-fold with only a small effect on K(m), while also shifting the pH optimum. These effects are consistent with assignment of this residue as the acid/base catalytic residue. Similarly, mutation of beta Asp(354) to Asn reduced k(cat) 2000-fold while leaving K(m) essentially unaltered, consistent with assignment of this residue as the residue that interacts with the substrate acetamide group to promote its attack on the anomeric center. These data in conjunction with the mutagenesis studies of Asp(241) and Glu(491) indicate that the molecular model is substantially accurate in its identification of catalytically important residues.


Subject(s)
Aspartic Acid/metabolism , Glutamic Acid/metabolism , beta-N-Acetylhexosaminidases/metabolism , Acetylglucosaminidase/genetics , Acetylglucosaminidase/metabolism , Amino Acid Substitution/genetics , Animals , Aspartic Acid/genetics , Binding Sites/genetics , CHO Cells , Catalysis , Cricetinae , Glutamic Acid/genetics , Hexosaminidase B , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics , Membrane Proteins/metabolism , Mutagenesis, Site-Directed , Protein Conformation , Reproducibility of Results , Sequence Alignment , Sequence Homology, Amino Acid , Tetraspanins , Transfection , beta-N-Acetylhexosaminidases/genetics
9.
J Biol Chem ; 276(13): 10330-7, 2001 Mar 30.
Article in English | MEDLINE | ID: mdl-11124970

ABSTRACT

beta-Hexosaminidase, a family 20 glycosyl hydrolase, catalyzes the removal of beta-1,4-linked N-acetylhexosamine residues from oligosaccharides and their conjugates. Heritable deficiency of this enzyme results in various forms of GalNAc-beta(1,4)-[N-acetylneuraminic acid (2,3)]-Gal-beta(1,4)-Glc-ceramide gangliosidosis, including Tay-Sachs disease. We have determined the x-ray crystal structure of a beta-hexosaminidase from Streptomyces plicatus to 2.2 A resolution (Protein Data Bank code ). beta-Hexosaminidases are believed to use a substrate-assisted catalytic mechanism that generates a cyclic oxazolinium ion intermediate. We have solved and refined a complex between the cyclic intermediate analogue N-acetylglucosamine-thiazoline and beta-hexosaminidase from S. plicatus to 2.1 A resolution (Protein Data Bank code ). Difference Fourier analysis revealed the pyranose ring of N-acetylglucosamine-thiazoline bound in the enzyme active site with a conformation close to that of a (4)C(1) chair. A tryptophan-lined hydrophobic pocket envelopes the thiazoline ring, protecting it from solvolysis at the iminium ion carbon. Within this pocket, Tyr(393) and Asp(313) appear important for positioning the 2-acetamido group of the substrate for nucleophilic attack at the anomeric center and for dispersing the positive charge distributed into the oxazolinium ring upon cyclization. This complex provides decisive structural evidence for substrate-assisted catalysis and the formation of a covalent, cyclic intermediate in family 20 beta-hexosaminidases.


Subject(s)
beta-N-Acetylhexosaminidases/chemistry , Acetylglucosamine/chemistry , Aspartic Acid/chemistry , Catalysis , Crystallography, X-Ray , Electrons , Escherichia coli/metabolism , Gangliosidoses/genetics , Models, Chemical , Models, Molecular , Protein Binding , Protein Conformation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Spectroscopy, Fourier Transform Infrared , Streptomyces/chemistry , Streptomyces/enzymology , Thiazoles/chemistry , Tryptophan/chemistry
11.
Biochemistry ; 39(1): 117-26, 2000 Jan 11.
Article in English | MEDLINE | ID: mdl-10625486

ABSTRACT

The novel mechanism-based reagent 2-acetamido-2-deoxy-5-fluoro-alpha-L-idopyranosykl fluoride has been synthesized, and the kinetic parameters K(M) = 0.23 mM and K(CAT)= 0.55 min(-1) for its hydrolysis by vibrio furnisi beta-N-acetylglucosaminidase (ExoII) HAVE been determined. Investigation of mixtures of enzyme with this slow substrate by electrospray mass spectrometry revealed a high steady-state population of the 2-acetamido-2-deoxy-5-fluoro-beta-L-idopyranosyl-enzyme, indicating that the hydrolytic mechanism of ExoII involves the formation and rate-determining hydrolysis of a glycosyl-enzyme intermediate. Analysis of a peptic digest of the glycosyl-enzyme by HPLC/ESMS/MS in the netural-loss mode permitted identification of a peptide bearing the 5-fluoro-sugar moiety. Tandem MS sequencing of the labeled peptide, in conjuction with multiple sequence alignmentsS of family 3 members, allowed the identification of ASP242 as the catalytic nucleophile within the sequence IVFSDDLSM.


Subject(s)
Acetylglucosaminidase/chemistry , Aspartic Acid/chemistry , Monosaccharides/chemistry , Vibrio/enzymology , Acetylglucosaminidase/metabolism , Aspartic Acid/metabolism , Binding Sites , Catalysis , Hydrolysis , Indicators and Reagents , Kinetics , Peptides/isolation & purification , Peptides/metabolism , Sequence Alignment , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Structure-Activity Relationship
12.
Biochem J ; 335 ( Pt 2): 449-55, 1998 Oct 15.
Article in English | MEDLINE | ID: mdl-9761746

ABSTRACT

Thermoanaerobacterium saccharolyticum beta-xylosidase is a member of family 39 of the glycosyl hydrolases. This grouping comprises both retaining beta-d-xylosidases and alpha-l-iduronidases. T. saccharolyticum beta-xylosidase catalyses the hydrolysis of short xylo-oligosaccharides into free xylose via a covalent xylosyl-enzyme intermediate. Incubation of T. saccharolyticum beta-xylosidase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-d-xyloside resulted in time-dependent inactivation of the enzyme (inactivation rate constant ki=0.089 min-1, dissociation constant for the inactivator Ki=65 microM) through the accumulation of a covalent 2-deoxy-2-fluoro-alpha-d-xylosyl-enzyme, as observed by electrospray MS. Removal of excess inactivator and regeneration of the free enzyme through transglycosylation with either xylobiose or thiobenzyl xyloside demonstrated that the covalent intermediate was kinetically competent. Peptic digestion of the 2-deoxy-2-fluoro-alpha-d-xylosyl-enzyme intermediate and subsequent analysis by electrospray ionization triple-quadrupole MS in the neutral-loss mode indicated the presence of a 2-deoxy-2-fluoro-alpha-d-xylosyl peptide. Sequence determination of the labelled peptide by tandem MS in the daughter-ion scan mode permitted the identification of Glu-277 (bold and underlined) as the catalytic nucleophile within the sequence IILNSHFPNLPFHITEY.


Subject(s)
Bacteria, Anaerobic/enzymology , Mass Spectrometry/methods , Xylosidases/chemistry , Xylosidases/metabolism , Amino Acid Sequence , Binding Sites , Catalytic Domain , Conserved Sequence , Enzyme Activation , Glutamic Acid , Molecular Sequence Data , Peptide Fragments/chemistry , Sequence Alignment , Sequence Analysis
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