70-kDa-heat shock protein presents an adjustable lectinic activity towards O-linked N-acetylglucosamine

Numerous works demonstrated that the dynamic O-GlcNAc glycosylation could protect against the proteasomal degradation by modifying the target proteins and the proteasome itself. Considering that Hsp70 is a crucial component in the quality control of protein conformation in the proteasomal pathway, we investigated the possibility that Hsp70 physically interacts with O-GlcNAc proteins through a lectinic activity. First, we demonstrate that in HepG2 cells, Hsp70 can specifically bind to O-GlcNAc residues but also is itself modified by O-GlcNAc. Second, when cells were deprived of glucose (nutrient stress), Hsp70 lectinic activity markedly increased whereas its glycosylation dramatically decreased. On the other hand, a 42 degrees C thermic stress did not affect any of these features. Lastly, the nature of O-GlcNAc modified proteins co-immunoprecipitating with Hsp70 was similar for cells submitted to the thermic and to nutrient stress. These results strongly suggest that O-GlcNAc influences protein stability through specific interaction with 70-kDa-heat shock protein members.     

Does O-GlcNAc play a role in neurodegenerative diseases?

There are several lines of evidence that the modification of proteins by cytosolic- and nuclear-specific O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is closely related to neuropathologies, particularly Alzheimer's disease. Several neuronal proteins have been identified as being modified with O-GlcNAc; these proteins could form part of the inclusion bodies found, for example, in the most frequently observed neurologic disorder (i.e., Alzheimer's disease; Tau protein and beta-amyloid peptide are the well known aggregated proteins). O-GlcNAc proteins are also implicated in synaptosomal transport (e.g., synapsins and clathrin-assembly proteins). Inclusion bodies are partly characterized by a deficiency in the ubiquitin-proteasome system, avoiding the degradation of aggregated proteins. From this perspective, it appears interesting that substrate proteins could be protected against proteasomal degradation by being covalently modified with single N-acetylglucosamine on serine or threonine, and that the proteasome itself is modified and regulated by O-GlcNAc (in this case the turnover of neuronal proteins correlates with extracellular glucose). Interestingly, glucose uptake and metabolism are impaired in neuronal disorders, and this phenomenon is linked to increased phosphorylation. In view of the existence of the dynamic interplay between O-GlcNAc and phosphorylation, it is tempting to draw a parallel between the use of glucose, O-GlcNAc glycosylation and phosphorylation. Lastly, the two enzymes responsible for O-GlcNAc dynamism (i.e., O-GlcNAc transferase and glucosaminidase) are both enriched in the brain and genes that encode the two enzymes are located in two regions that are found to be frequently mutated in neurologic disorders. The data presented in this review strongly suggest that O-GlcNAc could play an active role in neurodegenerative diseases.     

Evidence of a balance between phosphorylation and O-GlcNAc glycosylation of Tau proteins--a role in nuclear localization

Both phosphorylation and O-GlcNAc glycosylation posttranslationally modify microtubule-associated Tau proteins. Whereas the hyperphosphorylation of these proteins that occurs in Alzheimer's disease is well characterized, little is known about the O-GlcNAc glycosylation. The present study demonstrates that a balance exists between phosphorylation and O-GlcNAc glycosylation of Tau proteins, and furthermore that a dysfunction of this balance correlates with reduced nuclear localization.The affinity of Tau proteins for WGA lectin, together with evidence from [3H]-galactose transfer and analysis of beta-eliminated products, demonstrated the presence of O-GlcNAc residues on both cytosolic and nuclear Tau proteins. In addition, our data indicated the existence of a balance between phosphorylation and O-GlcNAc glycosylation events. Indeed, as demonstrated by 2D-electrophoresis and Western blotting, O-GlcNAc residues were mainly located on the less phosphorylated Tau 441 variants, whereas the more phosphorylated forms were devoid of O-GlcNAc residues. Furthermore, the Tau protein hyperphosphorylation induced by cellular okadaic acid treatment was correlated with reduced incorporation of O-GlcNAc residues into Tau proteins and with diminished Tau transfer into the nucleus. Hence, this paper establishes a direct relationship between O-GlcNAc glycosylation, phosphorylation and cellular localization of Tau proteins.     

O-GlcNAc-specific antibody CTD110.6 cross-reacts with N-GlcNAc2-modified proteins induced under glucose deprivation

Modification of serine and threonine residues in proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is a feature of many cellular responses to the nutritional state and to stress. O-GlcNAc modification is reversibly regulated by O-linked β-N-acetylglucosamine transferase (OGT) and β-D-N-acetylglucosaminase (O-GlcNAcase). O-GlcNAc modification of proteins is dependent on the concentration of uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), which is a substrate of OGT and is synthesized via the hexosamine biosynthetic pathway. Immunoblot analysis using the O-GlcNAc-specific antibody CTD110.6 has indicated that glucose deprivation increases protein O-GlcNAcylation in some cancer cells. The mechanism of this paradoxical phenomenon has remained unclear. Here we show that the increased glycosylation induced by glucose deprivation and detected by CTD110.6 antibodies is actually modification by N-GlcNAc(2), rather than by O-GlcNAc. We found that this induced glycosylation was not regulated by OGT and O-GlcNAcase, unlike typical O-GlcNAcylation, and it was inhibited by treatment with tunicamycin, an N-glycosylation inhibitor. Proteomics analysis showed that proteins modified by this induced glycosylation were N-GlcNAc(2)-modified glycoproteins. Furthermore, CTD110.6 antibodies reacted with N-GlcNAc(2)-modified glycoproteins produced by a yeast strain with a ts-mutant of ALG1 that could not add a mannose residue to dolichol-PP-GlcNAc(2). Our results demonstrated that N-GlcNAc(2)-modified glycoproteins were induced under glucose deprivation and that they cross-reacted with the O-GlcNAc-specific antibody CTD110.6. We therefore propose that the glycosylation status of proteins previously classified as O-GlcNAc-modified proteins according to their reactivity with CTD110.6 antibodies must be re-examined. We also suggest that the repression of mature N-linked glycoproteins due to increased levels of N-GlcNAc(2)-modified proteins is a newly recognized pathway for effective use of sugar under stress and deprivation conditions. Further research is needed to clarify the physiological and pathological roles of N-GlcNAc(2)-modified proteins.     

Topology of O-linked N-acetylglucosamine in murine lymphocytes

A unique form of protein glycosylation in which N-acetylglucosamine monosaccharides are O-glycosidically linked to serine or threonine residues (O-GlcNAc) was initially reported in studies that used purified bovine milk galactosyltransferase to exogenously probe living populations of murine lymphocytes. However, in this same study, detergent latency experiments surprisingly indicated that unlike other known forms of protein glycosylation, O-GlcNAc-modified proteins occur predominantly intracellularly. Since we now know that as little as 5% lysis could have accounted for the putative cell surface O-GlcNAc seen in these earlier studies, and also in the light of recent data on the subcellular localization of the O-GlcNAc glycosyltransferase(s), we decided to critically reexamine the topology and polypeptide distribution of O-GlcNAc in primary cultures of murine lymphocytes that were prepared using improved cell selection techniques that do not involve complement-mediated lysis for cell enrichment. We have also examined two well-characterized T cell hybridoma lines. Under these highly stringent conditions of cell viability, we were unable to detect O-GlcNAc bearing proteins on the cell surfaces of any of these cell types. Also, O-GlcNAc was found on a similar subset of proteins in all of the various lymphocyte cell types. These data suggest that O-GLcNAc is highly restricted to the cytoplasmic/nucleoplasmic compartment of the cell and is found on a similar subset of nuclear and cytoplasmic proteins in functionally different types of lymphocytes.     

Use of galactosyltransferase to assess the biological function of O-linked N-acetyl-d-glucosamine: a potential role for O-GlcNAc during cell division

Many cytosolic and nuclear proteins are modified by monomeric O-linked N-acetyl-d-glucosamine (O-GlcNAc). The biological functions of this form of glycosylation are unclear but evidence suggests that it heightens regulation of protein function. To assess the biological function of O-GlcNAc addition, we examined the biological effects of galactosyltransferase (GalT) microinjected into the cytoplasm of Xenopus ovarian oocytes. GalT, which catalyzes beta1-4-galactose addition to O-GlcNAc, should inhibit deglycosylation and lectin-like interactions requiring unmodified O-GlcNAc residues. Although GalT injection into diplotene-arrested oocytes has no detectable effects on cell viability, it is toxic to oocytes entering meiosis. Cell-cycle-specific toxicity is recapitulated in vitro as GalT inhibits formation of nuclei and microtubule asters from cell-free extracts of ovulated frog eggs. These observations suggest that regulation of O-GlcNAc is important for cell cycle progression and may be important in diseases in which O-GlcNAc metabolism is abnormal. The methods described here outline a viable experimental scheme for ascribing a biological function to this form of glycosylation.     

Modulation of O-GlcNAc glycosylation during Xenopus oocyte maturation

O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is a post-translational modification, which is believed antagonises phosphorylation. We have studied the O-GlcNAc level during Xenopus oocyte meiotic resumption, taking advantage of the high synchrony of this model which is dependent upon a burst of phosphorylation. Stimulation of immature stage VI oocytes using progesterone was followed by a 4.51 +/- 0.32 fold increase in the GlcNAc content, concomitantly to an increase in phosphorylation, notably on two cytoplasmic proteins of 66 and 97 kDa. The increase of O-GlcNAc for the 97 kDa protein, which we identified as beta-catenin was partly related to its accumulation during maturation, as was demonstrated by the use of the protein synthesis inhibitor--cycloheximide. Microinjection of free GlcNAc, which inhibits O-glycosylated proteins-lectins interactions, delayed the progesterone-induced maturation without affecting the O-GlcNAc content. Our results suggest that O-GlcNAc glycosylation could regulate protein-protein interactions required for the cell cycle kinetic.     

O-GlcNAc level variations are associated with the development of skeletal muscle atrophy.

O-linked N-acetylglucosaminylation (O-GlcNAc) is a regulatory posttranslational modification of nucleocytoplasmic proteins, which consists of the attachment of N-acetylglucosamine to serine or threonine residues of a protein. This glycosylation is a ubiquitous posttranslational modification, which probably plays important roles in many aspects of protein function. Our laboratory has previously reported that, in skeletal muscle, proteins of the glycolytic pathway and energetic metabolism and contractile proteins were O-GlcNAc modified (Cieniewski-Bernard C, Bastide B, Lefebvre T, Lemoine J, Mounier Y, and Michalski JC. Mol Cell Proteomics 3: 577-585, 2004). O-GlcNAc has been recently demonstrated to play a role in modulating cellular function in response to nutrition and also in stress conditions. Therefore, we have investigated here the implication of the glycosylation/deglycosylation process in the development of atrophy in rat skeletal muscle after hindlimb unloading. The high O-GlcNAc level found in control soleus [compared with control extensor digitorum longus (EDL)] becomes lower in atrophied soleus. On the opposite side, the low rate of O-GlcNAc in control EDL reaches higher levels in EDL, not atrophied after hindlimb unloading. These variations in O-GlcNAc level are correlated with a variation of the O-GlcNAc process enzyme activities and could be associated with a differential expression of heat shock proteins. Our results suggest that O-GlcNAc variations could control the muscle protein homeostasis and be implicated in the regulation of muscular atrophy.     

O-linked N-acetylglucosaminylation is involved in the Ca2+ activation properties of rat skeletal muscle

O-Linked N-acetylglucosaminylation termed O-GlcNAc is a dynamic cytosolic and nuclear glycosylation that is dependent both on glucose flow through the hexosamine biosynthesis pathway and on phosphorylation because of the existence of a balance between phosphorylation and O-GlcNAc. This glycosylation is a ubiquitous post-translational modification, which probably plays an important role in many aspects of protein functions. We have previously reported that, in skeletal muscle, proteins of the glycolytic pathway, energetic metabolism, and contractile proteins were O-GlcNAc-modified and that O-Glc-NAc variations could control the muscle protein homeostasis and be implicated in the regulation of muscular atrophy. In this paper, we report O-N-acetylglucosaminylation of a number of key contractile proteins (i.e. myosin heavy and light chains and actin), which suggests that this glycosylation could be involved in skeletal muscle contraction. Moreover, our results showed that incubation of skeletal muscle skinned fibers in N-acetyl-d-glucosamine, in a concentration solution known to inhibit O-GlcNAc-dependent interactions, induced a decrease in calcium sensitivity and affinity of muscular fibers, whereas the cooperativity of the thin filament proteins was not modified. Thus, our results suggest that O-GlcNAc is involved in contractile protein interactions and could thereby modulate muscle contraction.     

Use of Galactosyltransferase to Assess the Biological Function of O-linked N-Acetyl- -Glucosamine: A Potential Role for O-GlcNAc during Cell Division

Many cytosolic and nuclear proteins are modified by monomeric O-linked N-acetyl- -glucosamine (O-GlcNAc). The biological functions of this form of glycosylation are unclear but evidence suggests that it heightens regulation of protein function. To assess the biological function of O-GlcNAc addition, we examined the biological effects of galactosyltransferase (GalT) microinjected into the cytoplasm of Xenopus ovarian oocytes. GalT, which catalyzes β1-4-galactose addition to O-GlcNAc, should inhibit deglycosylation and lectin-like interactions requiring unmodified O-GlcNAc residues. Although GalT injection into diplotene-arrested oocytes has no detectable effects on cell viability, it is toxic to oocytes entering meiosis. Cell-cycle-specific toxicity is recapitulated in vitro as GalT inhibits formation of nuclei and microtubule asters from cell-free extracts of ovulated frog eggs. These observations suggest that regulation of O-GlcNAc is important for cell cycle progression and may be important in diseases in which O-GlcNAc metabolism is abnormal. The methods described here outline a viable experimental scheme for ascribing a biological function to this form of glycosylation.     

O-linked N-acetylglucosamine is present on the extracellular domain of notch receptors

Rare types of glycosylation often occur in a domain-specific manner and are involved in specific biological processes. In particular, O-fucose glycans are reported to regulate the functions of EGF domain-containing proteins such as Notch receptors. In the course of mass spectrometric analysis of O-glycans displayed on Drosophila Notch receptors expressed in S2 cells, we found an unusual O-linked N-acetylhexosamine (HexNAc) modification which occurs at a site distinct from those of O-fucose and O-glucose glycosylations. Modification site mapping by mass spectrometry and amino acid substitution studies revealed that O-HexNAc modification occurs on a serine or threonine located between the fifth and sixth cysteines within the EGF domain. This modification occurs simultaneously along with other closely positioned O-glycosylations. This modification was determined to be O-beta-GlcNAc by galactosyltransferase labeling and beta-N-acetyl-hexosaminidase digestion experiments and by immunoblotting with a specific antibody. O-GlcNAc modification occurs at multiple sites on Notch epidermal growth factor repeats. O-GlcNAc modification was also found on the extracellular domain of Delta, a ligand for Notch receptors. Although the O-GlcNAc modification is known to regulate a wide range of cellular processes, the list of known modified proteins has previously been limited to intracellular proteins in animals. Thus, the finding of O-GlcNAc modification in extracellular environments predicts a distinct glycosylation process that might be associated with a novel regulatory mechanism for Notch receptor activity.     

Glycosylation of the murine estrogen receptor-alpha

O-linked N-acetylglucosamine (O-GlcNAc) is a highly dynamic and abundant modification found on nuclear and cytoplasmic proteins of nearly all eukaryotes. O-GlcNAc addition is required for life at the single cell level and is analogous to protein phosphorylation in most respects. In a previous study (M.S. Jiang, G.W. Hart, A subpopulation of estrogen receptors are modified by O-linked N-acetylglucosamine. J. Biol. Chem. 270 (1997) 2421-2428), we demonstrated that a subpopulation of the murine estrogen receptor-alpha (mER-alpha) is modified by O-GlcNAc at Thr(575). Here we mutated mER-alpha to convert Thr(575) and Ser(576) to Val and Ala, respectively. Surprisingly, this glycosylation-site mutant is still extensively modified by O-GlcNAc. Analyses of glycopeptides identified two additional sites of modification on mER-alpha, at Ser(10) and Thr(50) near the N-terminus. The major glycosylation sites are within or near PEST regions, suggesting that O-GlcNAc may regulate mER-alpha turnover.     

Defining the regulated secreted proteome of rodent adipocytes upon the induction of insulin resistance

Insulin resistance defines the metabolic syndrome and precedes, as well is the hallmark of, type II diabetes. Adipocytes, besides being a major site for energy storage, are endocrine in nature and secrete a variety of proteins, adipocytokines (adipokines), that can modulate insulin sensitivity, inflammation, obesity, hypertension, food intake (anorexigenic and orexigenic), and general energy homeostasis. Recent data demonstrates that increased intracellular glycosylation of proteins via O-GlcNAc can induce insulin resistance and that a rodent model with genetically elevated O-GlcNAc levels in muscle and fat displays hyperleptinemia. The link between O-GlcNAc levels, insulin resistance, and adipocytokine secretion is further explored here. First, with the use of immortalized and primary rodent adipocytes, the secreted proteome of differentiated adipocytes is more fully elucidated by the identification of 97 and 203 secreted proteins, respectively. Mapping of more than 80 N-linked glycosylation sites on adipocytokines from the cell lines further defines this proteome. Importantly, adipocytokines that are modulated when cells are shifted from insulin responsive to insulin resistant conditions are determined. By the use of two protocols for inducing insulin resistance, classical hyperglycemia with chronic insulin exposure and pharmacological elevation of O-GlcNAc levels, several proteins are identified that are regulated in a similar fashion under both conditions including HCNP, Quiescin Q6, Angiotensin, lipoprotein lipase, matrix metalloproteinase 2, and slit homologue 3. Detection of these potential prognostic/diagnostic biomarkers for metabolic syndrome, type II diabetes, and the resulting complications of both diseases further establishes the central role of the O-GlcNAc modification of intracellular proteins in the pathophysiology of these conditions.     

Dynamic O-glycosylation of nuclear and cytosolic proteins: further characterization of the nucleocytoplasmic beta-N-acetylglucosaminidase, O-GlcNAcase.

beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and dynamic post-translational modification implicated in protein regulation that appears to be functionally more similar to phosphorylation than to classical glycosylation. There are nucleocytoplasmic enzymes for the attachment and removal of O-GlcNAc. Here, we further characterize the recently cloned beta-N-acetylglucosaminidase, O-GlcNAcase. Both recombinant and purified endogenous O-GlcNAcase rapidly release free GlcNAc from O-GlcNAc-modified peptide substrates. The recombinant enzyme functions as a monomer and has kinetic parameters (K(m) = 1.1 mm for paranitrophenyl-GlcNAc, k(cat) = 1 s(-1)) that are similar to those of lysosomal hexosaminidases. The endogenous O-GlcNAcase appears to be in a complex with other proteins and is predominantly localized to the cytosol. Overexpression of the enzyme in living cells results in decreased O-GlcNAc modification of nucleocytoplasmic proteins. Finally, we show that the enzyme is a substrate for caspase-3 but, surprisingly, the cleavage has no effect on in vitro O-GlcNAcase activity. These studies support the identification of this protein as an O-GlcNAcase and identify important interactions and modifications that may regulate the enzyme and O-GlcNAc cycling.     

O-GlcNAc glycosylation: a signal for the nuclear transport of cytosolic proteins?

Year 2004 marks the 20th anniversary of the discovery of O-linked N-acetylglucosamine (O-GlcNAc) by Gerald W. Hart. Despite interest for O-GlcNAc, the functions played by this single monosaccharide remain poorly understood, though numerous roles have been suggested, among which is the involvement of O-GlcNAc in the nuclear transport of cytosolic proteins. This idea was first sustained by studies on bovine serum albumin that showed that the protein could be actively carried to the nucleus when it was modified with sugars. In this paper, we will review data on this puzzling problem. We will first describe the well-established nuclear localisation signal (NLS)-dependent nuclear transport by presenting the different factors involved, and then, we will examine where and how O-GlcNAc could be involved in nuclear transport. Whereas it has been suggested that O-GlcNAc could interfere at two levels in the nuclear transport both by modifying proteins to be translocated to the nucleus and by modifying the nucleoporins of the nuclear pore complex, according to us, this second idea seems unlikely. Part of this study will also be dedicated to a relatively new concept in the nuclear transport: the role of the 70-kDa heat shock proteins (HSP70). The action of the chaperone in nuclear translocation was put forward 10 years ago, but new findings suggest that this mechanism could be linked to O-GlcNAc glycosylation.