A Selective Inhibitor Gal‐PUGNAc of Human Lysosomal β‐Hexosaminidases Modulates Levels of the Ganglioside GM2 in Neuroblastoma Cells

Stubbs, Keith A.; Macauley, Matthew; Vocadlo, David J.

doi:10.1002/ange.200804583

Cited by 18 publications

(17 citation statements)

References 27 publications

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“…The analysis of CTD110.6 immunoreactivity pattern against the proteins of these skinned fibers clearly demonstrated that the modification in the calcium activation parameters was associated with an increase of O-GlcNAcylation on predominant proteins expressed in soleus skinned fibers. Recent data demonstrated that PUGNAc could have an effect by inhibiting lysosomal hexosaminidase [36], [37]. Since no complex glycans were detected on proteins extracted from skinned biopsies, we concluded that the only effect of PUGNAc on skinned fibers (and included in T/pCa experiments) was the increase of O-GlcNAc level.…”

Section: Discussionmentioning

confidence: 74%

Increasing O-GlcNAcylation Level on Organ Culture of Soleus Modulates the Calcium Activation Parameters of Muscle Fibers

et al. 2012

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O-N-acetylglucosaminylation is a reversible post-translational modification which presents a dynamic and highly regulated interplay with phosphorylation. New insights suggest that O-GlcNAcylation might be involved in striated muscle physiology, in particular in contractile properties such as the calcium activation parameters. By the inhibition of O-GlcNAcase, we investigated the effect of the increase of soleus O-GlcNAcylation level on the contractile properties by establishing T/pCa relationships. We increased the O-GlcNAcylation level on soleus biopsies performing an organ culture of soleus treated or not with PUGNAc or Thiamet-G, two O-GlcNAcase inhibitors. The enhancement of O-GlcNAcylation pattern was associated with an increase of calcium affinity on slow soleus skinned fibers. Analysis of the glycoproteins pattern showed that this effect is solely due to O-GlcNAcylation of proteins extracted from skinned biopsies. We also characterized the O-GlcNAcylated contractile proteins using a proteomic approach, and identified among others troponin T and I as being O-GlcNAc modified. We quantified the variation of O-GlcNAc level on all these identified proteins, and showed that several regulatory contractile proteins, predominantly fast isoforms, presented a drastic increase in their O-GlcNAc level. Since the only slow isoform of contractile protein presenting an increase of O-GlcNAc level was MLC2, the effect of enhanced O-GlcNAcylation pattern on calcium activation parameters could involve the O-GlcNAcylation of sMLC2, without excluding that an unidentified O-GlcNAc proteins, such as TnC, could be potentially involved in this mechanism. All these data strongly linked O-GlcNAcylation to the modulation of contractile activity of skeletal muscle.

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Section: Discussionmentioning

confidence: 74%

Increasing O-GlcNAcylation Level on Organ Culture of Soleus Modulates the Calcium Activation Parameters of Muscle Fibers

et al. 2012

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“…In addition to results from in vitro studies, researchers found that increased levels of O-GlcNAcylation lead to insulin resistance in vivo [2,20]. PUGNAc is a potent inhibitor of OGA; however, it has some off-target effects [21,22]. Several papers show that other OGA inhibitors, such as NButGT, do not affect insulin signalling [23,24].…”

Section: Discussionmentioning

confidence: 99%

Obesity resistance and increased energy expenditure by white adipose tissue browning in Oga +/- mice

et al. 2015

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Aims/hypothesis O-GlcNAcylation plays a role as a metabolic sensor regulating cellular signalling, transcription and metabolism. Transcription factors and signalling pathways related to metabolism are modulated by N-acetyl-glucosamine (OGlcNAc) modification. Aberrant regulation of OGlcNAcylation is closely linked to insulin resistance, type 2 diabetes and obesity. Current evidence shows that increased O-GlcNAcylation negatively regulates insulin signalling, which is associated with insulin resistance and type 2 diabetes. Here, we aimed to evaluate the effects of Oga (also known as Mgea5) haploinsufficiency, which causes hyper-OGlcNAcylation, on metabolism. Methods We examined whether Oga +/-mice developed insulin resistance. Metabolic variables were determined including body weight, glucose and insulin tolerance, metabolic rate and thermogenesis. Results Oga deficiency does not affect insulin signalling even at hyper-O-GlcNAc levels. Oga +/-mice are lean with reduced fat mass and improved glucose tolerance. Furthermore, Oga +/-mice resist high-fat diet-induced obesity with ameliorated hepatic steatosis and improved glucose metabolism. Oga haploinsufficiency potentiates energy expenditure through the enhancement of brown adipocyte differentiation from the stromal vascular fraction of subcutaneous white adipose tissue (WAT). Conclusions/interpretation Our observations suggest that O-GlcNAcase (OGA) is essential for energy metabolism via regulation of the thermogenic WAT program.

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“…Together, these mechanistic and structural studies have guided the development of potent and specific inhibitors against β-hexosaminidases, 14 , 16 , 18 − 20 which have acted as powerful tools for probing the cellular function of the enzymes and the roles they play in disease. 21 − 24 …”

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confidence: 99%

Mechanism of Human Nucleocytoplasmic Hexosaminidase D

et al. 2016

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Mammalian β-hexosaminidases have been shown to play essential roles in cellular physiology and health. These enzymes are responsible for the cleavage of the monosaccharides N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) from cellular substrates. One of these β-hexosaminidases, hexosaminidase D (HexD), encoded by the HEXDC gene, has received little attention. No mechanistic studies have focused on the role of this unusual nucleocytoplasmically localized β-hexosaminidase, and its cellular function remains unknown. Using a series of kinetic and mechanistic investigations into HexD, we define the precise catalytic mechanism of this enzyme and establish the identities of key enzymic residues. The preparation of synthetic aryl N-acetylgalactosaminide substrates for HexD in combination with measurements of kinetic parameters for wild-type and mutant enzymes, linear free energy analyses of the enzyme-catalyzed hydrolysis of these substrates, evaluation of the reaction by nuclear magnetic resonance, and inhibition studies collectively reveal the detailed mechanism of action employed by HexD. HexD is a retaining glycosidase that operates using a substrate-assisted catalytic mechanism, has a preference for galactosaminide over glucosaminide substrates, and shows a pH optimum in its second-order rate constant at pH 6.5–7.0. The catalytically important residues are Asp148 and Glu149, with Glu149 serving as the general acid/base residue and Asp148 as the polarizing residue. HexD is inhibited by Gal-NAG-thiazoline (Ki = 420 nM). The fundamental insights gained from this study will aid in the development of potent and selective probes for HexD, which will serve as useful tools to improve our understanding of the physiological role played by this unusual enzyme.

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A Selective Inhibitor Gal‐PUGNAc of Human Lysosomal β‐Hexosaminidases Modulates Levels of the Ganglioside GM2 in Neuroblastoma Cells

Cited by 18 publications

References 27 publications

Increasing O-GlcNAcylation Level on Organ Culture of Soleus Modulates the Calcium Activation Parameters of Muscle Fibers

Increasing O-GlcNAcylation Level on Organ Culture of Soleus Modulates the Calcium Activation Parameters of Muscle Fibers

Obesity resistance and increased energy expenditure by white adipose tissue browning in Oga +/- mice

Mechanism of Human Nucleocytoplasmic Hexosaminidase D

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