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Glycan-dependent signaling: O-linked N-acetylglucosamine

LCBB, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA

¹Correspondence: Chief, LCBB, NIDDK, National Institutes of Health, Bldg. 8, Rm. 402, Bethesda, MD 20892, USA. E-mail: jah{at}helix.nih.gov

	ABSTRACT

TOP ABSTRACT O-LINKED N-ACETYLGLUCOSAMINE: A... METHODS OF ANALYSIS HOW WIDESPREAD IS O-GlcNAc... CELLULAR PROTEINS MODIFIED BY... THE O-LINKED GlcNAc TRANSFERASE:... O-GlcNAcase (HEXOSAMINIDASE C) THE HEXOSAMINE BIOSYNTHETIC... EVIDENCE FOR AN O-GlcNAc... GLYCAN-DEPENDENT SIGNALING AND... SUMMARY AND FUTURE PROSPECTS REFERENCES

 
The addition of O-linked N-acetylglucosamine (O-GlcNAc) to target proteins may serve as a signaling modification analogous to protein phosphorylation. Like phosphorylation, O-GlcNAc is a dynamic modification occurring in the nucleus and cytoplasm. Various analytical methods have been developed to detect O-GlcNAc and distinguish it from glycosylation in the endomembrane system. Many target molecules have been identified; these targets are typically components of supramolecular complexes such as transcription factors, nuclear pore proteins, or cytoskeletal components. The enzymes responsible for O-GlcNAc addition and removal are highly conserved molecules having molecular features consistent with a signaling role. The O-GlcNAc transferase and O-GlcNAcase are likely to act in consort with kinases and phosphatases generating various isoforms of physiological substrates. These isoforms may differ in such properties as protein–protein interactions, protein stability, and enzymatic activity. Since O-GlcNAc plays a critical role in the regulation of signaling pathways of higher plants, the glycan modification is likely to perform similar signaling functions in mammalian cells. Glucose and amino acid metabolism generates hexosamine precursors that may be key regulators of a nutrient sensing pathway involving O-GlcNAc signaling. Altered O-linked GlcNAc metabolism may also occur in human diseases including neurodegenerative disorders, diabetes mellitus and cancer.—Hanover, J. A. Glycan-dependent signaling: O-linked N-acetylglucosamine.

Key Words: O-GlcNAc • hexosaminidase C • diabetes mellitus • OGT

	O-LINKED N-ACETYLGLUCOSAMINE: A BRIEF HISTORY

TOP ABSTRACT O-LINKED N-ACETYLGLUCOSAMINE: A... METHODS OF ANALYSIS HOW WIDESPREAD IS O-GlcNAc... CELLULAR PROTEINS MODIFIED BY... THE O-LINKED GlcNAc TRANSFERASE:... O-GlcNAcase (HEXOSAMINIDASE C) THE HEXOSAMINE BIOSYNTHETIC... EVIDENCE FOR AN O-GlcNAc... GLYCAN-DEPENDENT SIGNALING AND... SUMMARY AND FUTURE PROSPECTS REFERENCES

 
Although numerous early findings pointed to the existence of short chain saccharides terminating in N-acetylglucosamine (O-GlcNAc) was first formally described in 1984 (1) . This early report focused on O-GlcNAc as a cell surface modification, but it was soon recognized that the bulk of the O-GlcNAc residues were intracellular (2 3 4 5 6) . One particularly striking finding was that glycoproteins bearing O-GlcNAc were present in high concentration at the vertebrate nuclear pore complex (4 5 6 7) . This was followed in close order by the observation that components of the transcription machinery were also modified by O-GlcNAc (8 , 9) . As the number of proteins shown to contain O-GlcNAc continued to grow, interest turned to the enzymes mediating glycan addition and removal. The O-linked GlcNAc transferase was characterized and partially purified from rat liver and rabbit reticulocyte lysate (10 , 11) . Characterization of the hexosaminidase proved more difficult, and a report describing the activity appeared several years later (12) . More recently a protein previously thought to be a hyaluronidase has been tentatively identified as O-GlcNAcase (13) . Analysis of the role of O-GlcNAc in cellular physiology closely paralleled the advances in enzymology and substrate characterization. The modification was shown to play a role in nuclear transport, although a lectin-like interaction was probably not involved (14 , 15) . O-GlcNAc addition and removal were shown to be highly dynamic and changed dramatically during cell activation (16) . In addition, transcription and translation factors were shown to be modified in their activity by O-GlcNAc modification and, in some cases, their activities were modified (8 , 17 18 19 20 21 22 23 24 25) . The O-linked GlcNAc transferase was molecularly cloned and shown to be a member of the tetratricopeptide repeat (TPR) family of proteins and similar to the Spindly gene in Arabidopsis (26 , 27) . More recently, a detailed analysis of the O-GlcNAc transferase revealed more of its structure (28 , 29) . Certain kinases, including GSK-3 and casein kinase, were shown to be substrates for the enzyme (29) . The gene encoding the transferase has also been analyzed in mice and humans and appears to be essential (30 ; J. A. Hanover unpublished results). Thus, for O-GlcNAc, it has been an exciting 2 decades from discovery to gene. The reagents are now available for a more detailed analysis of the functioning of this unique modification.

	METHODS OF ANALYSIS

TOP ABSTRACT O-LINKED N-ACETYLGLUCOSAMINE: A... METHODS OF ANALYSIS HOW WIDESPREAD IS O-GlcNAc... CELLULAR PROTEINS MODIFIED BY... THE O-LINKED GlcNAc TRANSFERASE:... O-GlcNAcase (HEXOSAMINIDASE C) THE HEXOSAMINE BIOSYNTHETIC... EVIDENCE FOR AN O-GlcNAc... GLYCAN-DEPENDENT SIGNALING AND... SUMMARY AND FUTURE PROSPECTS REFERENCES

 
Galactosyltransferase
Because O-GlcNAc is a dynamic modification, its detection on substrate proteins has sometimes proved difficult. The original means of detection was the use of the catalytic subunit of the enzyme lactose synthetase, galactosyltransferase, as a topological probe (1 , 31) . This enzyme transfers galactose to terminal GlcNAc residues, thus creating a ß 1–4 linkage. By using radiochemically labeled UDP-galactose as substrate, acceptor molecules could readily be identified. This technique has its share of limitations, notably, the cryptic nature of some O-GlcNAc sites. It must also be used in combination with other techniques to confirm the identity of O-GlcNAc since the enzyme will modify any terminal GlcNAc, even those present on membrane and secretory N-linked and O-linked oligosaccharides.

Alkaline ß-elimination
Since O-GlcNAc is in an O-glycosidic linkage to Ser and Thr residues on proteins, it is susceptible to removal by reduction under alkaline conditions (1 , 4) . Although not specific for O-GlcNAc, this method can be useful in confirming the identity of the modification after labeling with galactosyltransferase or in tissue extracts. Some destruction of the GlcNAcitol does occur during this reaction.

Antibodies to O-GlcNAc containing proteins
Antibodies generated to GlcNAc-modified proteins have been particularly helpful in identifying this class of proteins (4 5 6 7) . In addition, antibodies to streptococcal antigens have been found to cross-react with O-GlcNAc (32) . Antibodies more specific for O-GlcNAc have recently been prepared in several laboratories; these are likely to help in understanding the cellular regulation of O-GlcNAc addition. Such antibodies may be used much the way Ser and Thr phosphorylation specific antibodies have been used on immunoblots and in combination with immunofluorescence microscopy.

Mass spectrometry
Mass spectrometry has recently been applied to the detection and characterization of O-GlcNAc (17 , 24 , 33 34 35 36 37) . By examining intact proteins using MALDI, it is possible to look at the time course and extent of glycosylation of an O-linked GlcNAc glycoprotein. We have recently used this approach to examine the glycosylation of recombinant nuclear pore proteins (see http://www.cipergen.com/F1 case1.html). This method is both more specific and more quantitative than some of the other analytical approaches and has been described in greater depth elsewhere (36) .

Other analytical approaches
Other approaches such as gel electrophoresis and fluorescent techniques have been used to detect O-GlcNAc. Perhaps the best characterized is the FACE (fluorophore-assisted carbohydrate electrophoresis) procedure in which gel electrophoresis is used to separate a mixture of fluorescently labeled monosaccharides (see http://.www.glyko.com/products/face.html). This approach has recently been applied to O-linked GlcNAc. The difficulty with this procedure is determining what fraction of the total GlcNAc detected is derived from the O-linked GlcNAc linkage. This may be facilitated when recombinant forms of specific O-GlcNAcases become readily available.

	HOW WIDESPREAD IS O-GlcNAc ADDITION?

TOP ABSTRACT O-LINKED N-ACETYLGLUCOSAMINE: A... METHODS OF ANALYSIS HOW WIDESPREAD IS O-GlcNAc... CELLULAR PROTEINS MODIFIED BY... THE O-LINKED GlcNAc TRANSFERASE:... O-GlcNAcase (HEXOSAMINIDASE C) THE HEXOSAMINE BIOSYNTHETIC... EVIDENCE FOR AN O-GlcNAc... GLYCAN-DEPENDENT SIGNALING AND... SUMMARY AND FUTURE PROSPECTS REFERENCES

 
Although most of the characterized proteins modified by O-GlcNAc are from vertebrate sources, it is clear that the modification is much more widespread. Growing knowledge of genome sequences including Arabidopsis, other higher plants, Drosophila, Caenorhabditis, various parasitic protozoa, yeast, and bacteria have provided some clues. The enzymes of O-GlcNAc metabolism (OGT and O-GlcNAcase) have been seen in these organisms as expressed sequence tags, and in some cases the genes have been identified. More problematic has been the demonstration of the carbohydrate linkage itself in these diverse organisms. In plants, there is some evidence that nuclear pore proteins are modified by O-GlcNAc (38 , 39) , but the modification is capped with addition saccharide moieties. Enzymes with features suggesting they mediate O-GlcNAc addition are present in Arabidopsis (40) . In protozoa, O-GlcNAc addition has been well documented (41 , 42 , 44) . There is no clear-cut evidence for O-GlcNAc transferase activity in budding yeast and detection of the linkage has proved difficult. In bacteria, the problem has been less well studied. Some evidence suggests that the enzymes may be present in bacteria, perhaps being derived by lateral transfer from higher organisms (45) . As the role of O-GlcNAc addition comes under closer scrutiny and the number of completely sequenced genomes increases, we are likely to learn more about its phylogenetic distribution.

	CELLULAR PROTEINS MODIFIED BY O-LINKED GLcNAc TRANSFERASE: CLUES TO FUNCTION?

TOP ABSTRACT O-LINKED N-ACETYLGLUCOSAMINE: A... METHODS OF ANALYSIS HOW WIDESPREAD IS O-GlcNAc... CELLULAR PROTEINS MODIFIED BY... THE O-LINKED GlcNAc TRANSFERASE:... O-GlcNAcase (HEXOSAMINIDASE C) THE HEXOSAMINE BIOSYNTHETIC... EVIDENCE FOR AN O-GlcNAc... GLYCAN-DEPENDENT SIGNALING AND... SUMMARY AND FUTURE PROSPECTS REFERENCES

 
As shown in Fig. 1 , many proteins are modified by O-GlcNAc. An analysis of the properties of all of these proteins has suggested some shared features. In general, these proteins are both phosphorylated and glycosylated. They also are present in macromolecular complexes whose assembly may be regulated. A brief description of some of these macromolecular complexes follows.

View larger version (41K): [in this window] [in a new window]   Figure 1. Substrates for O-linked GlcNAc transferase (OGT) and similarity to protein phosphorylation. O-linked GlcNAc has been found to modify components of supramolecular structures such as the nuclear pore, transcription complex, and certain cytoskeletal proteins. The figures on the left depict the supramolecular structures that are targets for O-GlcNAc addition: a reconstruction of the nuclear pore complex, puffs on polytene chromosomes, and atomic force micrographs of a transcription complex and the cytoskeleton, respectively. The figure below illustrates the dynamic relationship between protein phosphorylation and O-linked GlcNAc addition. OGT has a substrate specificity similar to known kinases such as GSK-3 and Casein kinase.

Nuclear pore proteins (the nucleoporins)
One of the best studied classes of substrates are the nuclear pore proteins (4 5 6 7 , 11 , 15 , 27 , 29 , 46 47 48 49 50 51 52 53 54 55 56 57) . At least six major vertebrate nuclear pore proteins are modified by O-GlcNAc. Yet it is still not clear how the modification is involved in the many functions of the nuclear pore complex. Antibodies or lectins that interact with glycosylated nucleoporins interfere with nuclear transport at an energy-dependent step (15 , 46 , 58 59 60 61) . Some evidence suggests that O-GlcNAc may represent an alternative nuclear transport signal (62) . Nuclear transport and nuclear pore assembly can occur when O-GlcNAc is capped with a galactose residue (14 , 54) . Yet cytoplasmic expression of a galactosyltransferase appears to be toxic to cells (63) . Nuclear pore proteins are both glycosylated and phosphorylated (51 , 54 , 64 65 66 67) . Phosphorylation of the nucleoporins is regulated during the cell cycle whereas glycosylation appears stable throughout the cell cycle (54) . Although the precise role of the O-GlcNAc modification in nuclear import/export is poorly understood, the finding that so many nucleoporins are modified in this manner remains provocative.

Transcription machinery (Pol II and transcription factors)
RNA polymerase II and many of its associated transcription factors are modified by O-GlcNAc (8 , 17 18 19 , 22 23 24 25 , 33 , 68 69 70 71 72 73) . Much work has focused on the CTD domain of RNA polymerase, where phosphorylation and glycosylation appear to be reciprocal or mutually exclusive (74) . The precise role of the modification in the functioning of the polymerase has remained elusive, however. Evidence suggests that O-GlcNAc may modulate the turnover and protein–protein interactions of transcription factors such as SP1 and the estrogen receptor (8 , 22 , 23 , 25 , 68 , 69 , 72 , 75) . Like the work on the nuclear transport machinery, efforts to understand the role of glycosylation in the normal function or regulation of the transcription machinery have proved difficult. This is due to the tremendous complexity of that machinery itself, the absence of good in vitro systems, and the finding that so many components are modified by O-GlcNAc.

O-GlcNAc-modified proteins and cellular architecture
Many cytosolic proteins have been identified as substrates for O-GlcNAc addition, as Fig. 1 demonstrates. Some common themes emerge upon examination of these cytoskeletal substrates. Many of the known regulatory proteins that interact with actin and tubulin contain O-GlcNAc (66 , 76 77 78) . Proteins thought to bridge the cytoskeleton to the plasma membrane such as band 4.1, vinculin, talin, and the synapsins are also modified (2 , 79 80 81 82) . Some of the cytokeratins appear to be dynamically modified by O-GlcNAc and phosphate, with some indications of changes during the cell cycle (83 84 85) . The presence of distinct isoforms of these various proteins is consistent with the notion that O-linked GlcNAc addition may a final step in an incompletely defined signal transduction pathway.

	THE O-LINKED GlcNAc TRANSFERASE: A CONSERVED TPR PROTEIN

TOP ABSTRACT O-LINKED N-ACETYLGLUCOSAMINE: A... METHODS OF ANALYSIS HOW WIDESPREAD IS O-GlcNAc... CELLULAR PROTEINS MODIFIED BY... THE O-LINKED GlcNAc TRANSFERASE:... O-GlcNAcase (HEXOSAMINIDASE C) THE HEXOSAMINE BIOSYNTHETIC... EVIDENCE FOR AN O-GlcNAc... GLYCAN-DEPENDENT SIGNALING AND... SUMMARY AND FUTURE PROSPECTS REFERENCES

 
Efforts to understand the dynamic addition of GlcNAc to a large number of substrates has led to the purification and cloning of the enzyme(s) mediating this glycan addition.

Purification, characterization, and molecular identification of O-GlcNAc transferase

ß-N-acetylglucosaminyltransferase (OGT) activity has been detected in a wide range of tissues including rat liver, rabbit blood, and an enriched reticulocyte fraction (10 , 11 , 55) . The strategies used to identify the activity relied heavily on the identification of protein substrates such as the nuclear pore proteins, which provided peptide substrates or could be produced in recombinant form (10 , 11 , 49 , 55 , 67) . Subsequent purification of the enzyme from these sources revealed a catalytic component of 100 kDa, and microsequencing of the protein led rapidly to the molecular cloning of the transferase from various sources (26 , 27) .

O-linked GlcNAc transferase: molecular genetics
Transcripts encoding OGT
O-linked GlcNAc transferase cDNAs and the genes encoding those transcripts have been identified in humans, rats, and Caenorhabditis elegans (26 , 27) . In humans, four predominant transcripts encoding OGT ranging from 10 to 3 kb transcripts were found in most tissues. Transcripts were particularly enriched in the pancreas (27) . Even more striking was the high concentration of OGT transcripts in the ß cells of the islets of Langerhans within the pancreas (72 , 86) (Fig. 2 ). These observations were particularly intriguing in light of our hypothesis that OGT plays a role in glucose sensing. By regulating the secretion of insulin, the pancreatic ß cell is the key regulator of glucose homeostasis. Other tissues such as skeletal muscle, brain, fat, and cardiac muscle also had relatively high mRNA levels. Smaller amounts of the transcript are present in the kidney (26 , 27) . The current databases suggest that many alternatively spliced forms of OGT exist in certain tissues.

View larger version (77K): [in this window] [in a new window]   Figure 2. In situ localization of OGT transcripts in the pancreas to the ß cells of the islet of Langerhans. Double-label immunofluorescent detection of mRNA transcripts within the mouse pancreas with anti DIG antibody (red) and either insulin (at left) or glucagon (at right, green) FITC labeled antibody). Colocalization of the labels in the ß cells suggests enrichment of the OGT transcripts in this cell type. Note the lack of colocalization in the cells marked by anti-glucagon antibody.

The OGT gene
The gene encoding OGT is present as a single copy on the same region of the mouse and human X chromosome (Fig. 3 ; see also ref 30 ). Positional cloning efforts have yet to positively identify any human diseases that precisely map to this region, although the OGT locus is a candidate gene for X-linked Parkinson dystonia (see ‘Neurodegenerative diseases: a role for O-linked GlcNAc?’ below). On ablation in embryos, the gene has been shown to be essential for stem cell viability (J. A. Hanover, unpublished results) and embryonic development (30) . Conditional knockout experiments involving ablation of the gene in the various target tissues are under way. Much remains to be learned about the processing and expression of the OGT gene.

View larger version (26K): [in this window] [in a new window]   Figure 3. Location of the OGT gene on mouse and human chromosome X. The gene encoding OGT spans greater than 50 kb of mouse and human chromosome X. This localization has been determined by fluorescence in situ hybridization (left for mouse) and by radiation hybrid analysis and direct sequencing for the human gene encoding. The gene is essential for both embryonic stem cell viability and early embryonic development.

Domain structure and post-translational modifications
The coding sequence of OGT suggested a tripartite structure consisting of a repetitive domain, a linker and targeting domain, and a catalytic domain (26 , 27) (Fig. 4 ). This overall structure has been largely confirmed as a result of site-directed mutagenesis and expression in Escherichia coli and baculovirus (28 , 29) . The structures shown in Fig. 4 are proteins (whose X-ray structure has recently been solved) that share the conserved domains of OGT (see below).

View larger version (22K): [in this window] [in a new window]   Figure 4. Domain structure of O-linked GlcNAc transferase. OGT has an amino-terminal repeat domain containing tetratricopeptide repeats (TPR), a central linker and targeting domain, and a carboxyl-terminal catalytic domain. A molecular model of the structure of the 9–12 TPR repeats was derived from the crystal structure of the PP1 protein (87) . The structure shown below at right is from the coordinates of N-acetylglucosaminyltransferase I, which represents a new protein superfamily. OGT is likely to adopt a similar structure based on sequence similarity with transferases such as N-Acetylglucosaminyltransferase I.

Tetratricopeptide repeats
The TPR repeats are present in 9–12 copies in the known O-linked GlcNAc transferases (26 , 27 , 29 , 45) (Fig. 4) . The TPR repeat is found in many proteins where it is thought to mediate protein–protein interactions. The TPR repeats of OGT may play a role in substrate recognition and trimerization of the enzyme (28 , 29 , 45) . In general, these repeats act in groups of three, thus forming a groove that will accommodate peptides (45) . The structure of a TPR repeat protein was solved recently, leading to the proposal that repeats may form a superhelix with a diameter of roughly 50 Å (87) . Other studies suggest that certain TPR proteins may serve to bridge other proteins such as those of the heat shock proteins in the multichaperone machine (88) . The precise role of the TPR domain of OGT is currently under active investigation.

Linker/targeting domain
Following the TPR repeats in the central region of C. elegans, OGT is a sequence that appears to be a canonic nuclear targeting signal (Fig. 4) . This motif is interrupted in human and rat OGT, and precise mutagenesis studies have not yet been performed to demonstrate whether this domain is important for nuclear targeting of OGT. Overexpression of the enzyme in C. elegans confirmed that it enters the nucleus when expressed in the nematode (27) . Clearly, some of the enzyme is present in the nucleus and many substrates are nuclear. It is also clear that a pool of OGT exists in the cytoplasm and on the surface of certain cytoplasmic organelles. The regulation of the intracellular trafficking of the enzyme is under intense investigation.

The catalytic domain
We have suggested that the catalytic domain of OGT shares some features with known glycosyltransferases and certain lectins (45) . This similarity is not striking at the primary sequence level, but some motifs present in OGT are also present in these other enzymes (Fig. 4) . The regions of similarity are present in two subdomains designated catalytic domains 1 and 2. Recently, the crystal structure of an O-linked GlcNAc has been solved (89) , and the critical residues interacting with sugar nucleotide appear to be conserved between these enzymes and OGT (such as the ‘... DXD... ’ motif). Mutational analysis suggests that any substantial deletion in this region of the molecule dramatically inhibits the enzymatic activity of OGT (28 , 29 , 45) (Fig. 4) .

Post-translational modifications
Although it is the product of a unique single gene, OGT appears to be subject to proteolysis and post-translational modifications. A 78 kDa form of the enzyme has been detected (10) , which may represent that proteolytic product. In addition, OGT is modified post-translationally in some intriguing ways (26 27 28 29) . These include autoglycosylation with O-GlcNAc and tyrosine phosphorylation. The effects of these modifications on enzyme activity or substrate recognition have not been examined in detail.

	O-GlcNAcase (HEXOSAMINIDASE C)

TOP ABSTRACT O-LINKED N-ACETYLGLUCOSAMINE: A... METHODS OF ANALYSIS HOW WIDESPREAD IS O-GlcNAc... CELLULAR PROTEINS MODIFIED BY... THE O-LINKED GlcNAc TRANSFERASE:... O-GlcNAcase (HEXOSAMINIDASE C) THE HEXOSAMINE BIOSYNTHETIC... EVIDENCE FOR AN O-GlcNAc... GLYCAN-DEPENDENT SIGNALING AND... SUMMARY AND FUTURE PROSPECTS REFERENCES

 
O GlcNAcase: purification and identification
Some studies have been directed at purifying and identifying the enzyme (or enzymes) normally responsible for O-GlcNAc removal (12 , 13 , 86) . This has proved difficult since hexosaminidases exist in the lysosomes of cells that are released upon subcellular fractionation. One candidate enzyme that removed O-GlcNAc was purified from the cytosolic fraction of rat spleen. This preparation was shown to consist of a 51 and 54 kDa subunit (12) . More recently, candidates for the O-GlcNAcase have been examined in a number of tissues, including the brain (13 , 86 , 90) . This latter activity has been attributed to a previously identified protein originally thought to be a hyaluronidase associated with meningioma (13 , 91) . This information suggests that O-GlcNAcase is a 103 kDa protein with an acidic isoelectric point (13 , 91) . Analysis of the protein structure shows no obvious protein motifs. The only similarity occurring with a probability greater than chance is the BglG anti-terminator motif from E. coli. This RNA binding protein prevents transcription termination of the bgl operon that is regulated by ß-glucosides. The significance of this similarity is not yet known. The O-GlcNAcase also shows limited homology (near its amino terminus) to Clostridium perfringens Mu toxin and to a lacto-N-biosidase precursor from Streptomyces. This sequence similarity may reflect the position of the catalytically important residues in the molecule. Another aspect of the regulation of the putative O-GlcNAcase is the existence of at least two isoforms in the brain that are produced by alternative splicing. Both variants have the same amino terminus. This observation, coupled with the sequence similarities mentioned above, allows us to tentatively assign a region corresponding to 63–283 in the amino terminus as the catalytic domain of O-GlcNAcase (Fig. 5 ).

View larger version (30K): [in this window] [in a new window]   Figure 5. Domain structure and isoforms of GlcNAcase. GlcNAcase was recently identified as a previously described ‘hyaluronidase’ (13 , 91) . Little is known about its domain structure since it contains no recognizable common protein motifs. Two isoforms exist in the brain differing only in the length of their carboxyl-terminal extensions. The putative catalytic domain is shown in blue near the amino terminus; this region has limited homology with other glycosidases as mentioned in the text. No homology with other known hexosaminidases (such as lysosomal enzymes hexosaminidase A or B) is evident from examination the primary sequence.

A previous analysis of the tissue distribution of this transcript suggests that it is evolutionarily highly conserved and enriched in the brain, skeletal muscle, and pancreas (91) . This is similar to the known distribution of OGT transcripts (26 , 27) . The gene is present on human chromosome 10 (91) .

Inhibitors of O-GlcNAcase
The best-characterized inhibitor of the O-GlcNAcase is O-(2-acetamido-2-deoxy D-glucopyranosylidene)amino-N-phenylcarbamate, also called PUGNAc (65) . This is a nontoxic compound that leads to the accumulation of O-GlcNAc-modified proteins in treated cells. This increase can be as much as twofold in certain cell types. Another, less potent inhibitor of the O-GlcNAcase is streptozotocin. This O-GlcNAc analog is an irreversible inhibitor of the enzyme and can lead to a dramatic accumulation of O-GlcNAc in certain target tissues such as the pancreas (72 , 86 , 90 , 92 , 93) . Streptozotocin has long been thought to act by inducing fragmentation of DNA in the ß cells of the islet. Some evidence points to the inhibition of O-GlcNAcase as another potential mechanism for selective ß cell destruction in animals treated with streptozotocin (72 , 86 , 92 , 93) . However, this hypothesis has recently been challenged (90) . Future work on these inhibitors may provide additional insight into to the role of O-GlcNAcase in O-GlcNAc metabolism.

	THE HEXOSAMINE BIOSYNTHETIC PATHWAY: GLUCOSE TO UDP-HEXOSAMINES

TOP ABSTRACT O-LINKED N-ACETYLGLUCOSAMINE: A... METHODS OF ANALYSIS HOW WIDESPREAD IS O-GlcNAc... CELLULAR PROTEINS MODIFIED BY... THE O-LINKED GlcNAc TRANSFERASE:... O-GlcNAcase (HEXOSAMINIDASE C) THE HEXOSAMINE BIOSYNTHETIC... EVIDENCE FOR AN O-GlcNAc... GLYCAN-DEPENDENT SIGNALING AND... SUMMARY AND FUTURE PROSPECTS REFERENCES

 
The pathway through which UDP-GlcNAc and UDP-GalNAc are synthesized from glucose is often termed the hexosamine biosynthetic pathway. Only a small percentage of the glucose entering cells is routed to this pathway, yet flux through the pathway is thought to be regulated largely by the levels of glucose and the rate-limiting enzyme GFAT (glutamine:fructose-6-phosphate amidotransferase). An intriguing aspect of hexosamine biosynthesis is its apparent link to cellular glucose sensing pathways, particularly that of insulin (94 95 96 97) . Overexpression of GFAT has been found to recapitulate many of the physiological features of insulin resistance in vertebrates such as glucose transporter translocation and transcriptional effects (98 99 100 101 102 103 104) . Figure 6 illustrates the suspected link between the hexosamine biosynthetic pathway and the glycosylation of substrates by OGT. In this model, glucose entering cells is routed via the hexosamine biosynthetic pathway into the production of UDP-GlcNAc. This elevated UDP-GlcNAc has two effects on the pathway: 1) UDP-GlcNAc feedback inhibits GFAT, thus blunting synthesis, and 2) it accelerates the glycosylation of proteins by OGT. The range of concentrations over which OGT acts matches the cytosolic concentrations of sugar nucleotides (in the micromolar range) in this model. One problem with this type of regulation is that the true cytoplasmic concentration of UDP-GlcNAc has not been determined. The bulk of the sugar nucleotide is transported into the endomembrane system, where it is involved in membrane and secretory glycoprotein biosynthesis (86 , 105) . Another complication is the inhibition of GFAT by excess nucleotide that would severely limit the range over which UDP-GlcNAc concentrations might be expected to accumulate. Despite these complications, a large body of evidence argues that this pathway plays some role in signal transduction (20 , 86 , 92 , 93 , 98 , 100 101 102 103 104 105 106 107 108 109) . The extent to which these biological phenomena can be explained by addition of O-GlcNAc to target substrates remains to be determined.

View larger version (13K): [in this window] [in a new window]   Figure 6. The hexosamine biosynthetic pathway and O-GlcNAc-dependent signaling. One intriguing hypothesis is that the pathway by which glucose is converted to UDP-hexosamine is part of a signaling cascade. A fraction of the glucose entering cells is converted to UDP-GlcNAc through a series of interconversions involving glutamine and catalyzed in part by the enzyme GFAT (glutamine:fructose-6-phosphate amidotransferase). The levels of UDP-GlcNAc would then reflect the flux through the pathway and could serve as a glucose sensor or integrator. UDP-GlcNAc levels would fluctuate in response to glucose and glutamine levels, leading to differential activity of OGT within a physiologically range dictated by the transferase’s Km with respect to the sugar nucleotide (right panel). Increased OGT activity would lead to enhanced O-GlcNAc levels on physiologically relevant substrates. The physiological manifestations of elevation of UDP-GlcNAc levels are summarized in the text and include the phenomenon known as ‘insulin resistance’.

	EVIDENCE FOR AN O-GlcNAc-DEPENDENT SIGNALING PATHWAY

TOP ABSTRACT O-LINKED N-ACETYLGLUCOSAMINE: A... METHODS OF ANALYSIS HOW WIDESPREAD IS O-GlcNAc... CELLULAR PROTEINS MODIFIED BY... THE O-LINKED GlcNAc TRANSFERASE:... O-GlcNAcase (HEXOSAMINIDASE C) THE HEXOSAMINE BIOSYNTHETIC... EVIDENCE FOR AN O-GlcNAc... GLYCAN-DEPENDENT SIGNALING AND... SUMMARY AND FUTURE PROSPECTS REFERENCES

 
In addition to the anecdotal evidence implicating hexosamine biosynthesis in insulin resistance, other features of the modification suggest a role in signal transduction. First, there is the obvious analogy with phosphorylation. Like phosphorylation, O-GlcNAc addition is a dynamic modification (16 , 19 , 67 , 83 , 110 , 111) . Most of the proteins bearing O-GlcNAc are also phosphorylated such that distinct isoforms bearing different constellations of modifications have been documented extensively (17 18 19 , 21 , 23 , 54 , 67 68 69 , 76 , 79 , 112 113 114 115 116 117 118) . The substrate specificity of OGT with respect to peptide recognition is similar to certain kinases such as GSK-3. Taken together, these observations suggest that O-GlcNAc addition may be an integral component of certain signaling cascades, much like phosphorylation. This hypothesis is further strengthened by certain experiments that appear to link the glycosylation of certain transcription factors with an alteration in transcription (8 , 17 18 19 20 , 25 , 68 , 70 , 71 , 119) . At least one form of bacterial toxin has striking similarity to OGT and may represent an O-linked GlcNAc transferase (120 , 121) . The Clostridium novyi toxin, like many bacterial toxins, interferes with a normal cellular mechanism leading to selective cell toxicity in this modification of the Rho proteins (120 , 121) .

The most direct evidence that O-GlcNAc addition plays a role in signaling comes from studies with the higher plant Arabidopsis. One of the key regulators of plant growth and development is gibberellin, a hormone that promotes cell elongation and modulates germination and flowering. A mutation termed Spindly (Spy) is a recessive mutation leading to a constitutive activation of the gibberellin signaling pathway (40 , 122) . Thus, the genetic evidence suggests that SPY encodes a negative regulator of GA signal transduction acting early in the pathway (123) . The Spindly protein was shown to have a great deal of similarity to OGT (26 , 27) . Although it has not yet been demonstrated to mediate O-Linked GlcNAc transfer, the genetic and sequence data implicating OGT in this signal transduction cascade are compelling.

	GLYCAN-DEPENDENT SIGNALING AND HUMAN DISEASES

TOP ABSTRACT O-LINKED N-ACETYLGLUCOSAMINE: A... METHODS OF ANALYSIS HOW WIDESPREAD IS O-GlcNAc... CELLULAR PROTEINS MODIFIED BY... THE O-LINKED GlcNAc TRANSFERASE:... O-GlcNAcase (HEXOSAMINIDASE C) THE HEXOSAMINE BIOSYNTHETIC... EVIDENCE FOR AN O-GlcNAc... GLYCAN-DEPENDENT SIGNALING AND... SUMMARY AND FUTURE PROSPECTS REFERENCES

 
Neurodegenerative diseases: a role for O-linked GlcNAc?
The enzymes of O-GlcNAc metabolism are enriched in the brain and O-GlcNAc is abundant in that tissue (26 , 27 , 91) . Many proteins important for neuronal function are modified by O-GlcNAc including neurofilaments, clathrin assembly proteins, and the ß-amyloid precursor protein (76 , 78 , 79 , 110 , 115 , 116 , 118 , 124 , 125) . In Alzheimer brains, Tau is hyperphosphorylated and AP-3 is underglycosylated (76 , 115 , 116 , 124) . Since glucose metabolism is essential for neural function, this tissue may be particularly susceptible to lowered glycosylation by the O-GlcNAc pathway. This could lead to the altered post-translational modifications observed in Alzheimer’s and could be part of the pathological manifestation of the disease. The distribution of OGT transcripts in the brain is certainly not uniform (S. M. Sato, J. A. Hanover, and Z. Lai, unpublished results), making certain neurons more susceptible to such damage. Another intriguing finding is the observation that X-linked Parkinson dystonia maps to the location of the X chromosome corresponding roughly to OGT (J. A. Hanover, and S. Yu, unpublished results; ref 30 ). This disorder, also termed Lubing, has been extensively studied recently and is one of the hereditary movement disorders. It will be of interest to determine whether the pathology correlates with altered O-GlcNAc metabolism (126 127 128 129) .

Diabetes mellitus and O-linked GlcNAc
As described above, several lines of indirect evidence points to a role for O-linked GlcNAc in mammalian insulin resistance. There is also evidence that O-linked GlcNAc may be part of the glucose sensing mechanism in the ß cells of the pancreas. Transcripts encoding OGT are enriched in the ß cells (86 , 92) . In addition, glucosamine can blunt insulin secretion under some circumstances (130) . Streptozotocin, a ß cell toxin, has recently been shown to inhibit O-GlcNAcase; the resulting elevation of O-GlcNAc levels has been suggested to be part of the mechanism of selective ß cell destruction (86 , 92) . The hyperglycemia resulting from NIDDM could also lead to a chronic elevation of GlcNAc levels and result in ß cell loss. The progressive loss of insulin secreting cells is well documented in NIDDM. Additional studies are required to determine the extent to which O-GlcNAc metabolism is involved in ß cell function and in insulin action in peripheral tissues.

Cancer, tumor suppression, and O-linked GlcNAc
The striking changes in cell morphology, gene expression, and energy utilization observed in transformed cells may be associated with changes in O-GlcNAc metabolism. Proteins known to be oncogenes, such as c-myc and the SV40 T antigen, are O-GlcNAc modified (17 , 18 , 131) . The c-myc transcription factor is involved in transcriptional regulation of some glucose-responsive genes. In the case of the myc oncogene, the glycosylation and phosphorylation occur at residue Thr 58 in the transactivation domain, a mutational hotspot for Burkitt’s lymphoma. The problem in interpreting much of the mutational analysis performed on oncogenes such as myc is that any given site can bear both phosphate and GlcNAc. Thus, the mutational analysis must be interpreted with caution. Like the oncogenes, tumor suppressor genes may also be modified by O-GlcNAc. Evidence suggests that p53, the most extensively studied tumor suppressor, bears O-GlcNAc (132) . The role of this modification in the many cellular functions of p53 such as tumor suppression, apoptosis, and gene regulation is presently unknown.

Tumor cells are known to have dramatically altered glucose metabolism. Transformed cells switch from oxidative to glycolytic metabolism, where energy requirements of the rapidly dividing cells are provided by glycolysis-derived ATP. This phenomena, termed the ‘Crabtree effect’, has puzzled researchers for years, and many explanations have been offered for the metabolic transition. Given the central role of glucose in regulating O-GlcNAc metabolism in cells, future research no doubt will focus on the significance of O-GlcNAc modification in the Crabtree effect.

	SUMMARY AND FUTURE PROSPECTS

TOP ABSTRACT O-LINKED N-ACETYLGLUCOSAMINE: A... METHODS OF ANALYSIS HOW WIDESPREAD IS O-GlcNAc... CELLULAR PROTEINS MODIFIED BY... THE O-LINKED GlcNAc TRANSFERASE:... O-GlcNAcase (HEXOSAMINIDASE C) THE HEXOSAMINE BIOSYNTHETIC... EVIDENCE FOR AN O-GlcNAc... GLYCAN-DEPENDENT SIGNALING AND... SUMMARY AND FUTURE PROSPECTS REFERENCES

 
A model for O-linked GlcNAc-dependent cell signaling
The molecular characterization of the enzymes involved O-GlcNAc metabolism, and the growing list of known substrates have led to a simple model for how O-GlcNAc may function to modulate various cellular functions (Fig. 7 ). The O-GlcNAc transferase and O-GlcNAcase are likely to act in consort with kinases to generate various isoforms of physiological substrates. The TPR repeats of OGT, in particular, may be involved in substrate recognition and perhaps bridging of substrates in larger assemblies. The catalytic domain of OGT is directly responsive to the levels of UDP-GlcNAc and perhaps is regulated by autoglycosylation or phosphorylation of the transferase. The subcellular localization of OGT may also be modulated; this relocalization may play a key role in OGT function. The O-GlcNAcase may be subject to additional forms of regulation including post-translational modifications or changes in subcellular localization. The result of these complex interactions would be modulation of such parameters as protein–protein interactions, protein stability, and perhaps enzymatic activity of the substrates themselves. Thus, the system would provide a means for integrating information derived from the levels of key components of the nutrient sensing hexosamine pathway such as glucose and glutamine. This model suggests that O-GlcNAc, like phosphorylation, performs a variety of functions depending on the substrate recognized and modified.

View larger version (48K): [in this window] [in a new window]   Figure 7. Summary of the central role of O-GlcNAc metabolism in cell signaling. This model for how O-GlcNAc may modulate various cellular functions reflects many of the current findings. The levels of UDP-GlcNAc appear to be responsive to nutrient concentrations within cells that include glucose, essential amino acids (glutamine in particular), and fatty acids. Each has been shown to elevate UDP-GlcNAc levels. Other forms of regulation may result form the localization or modifications of the enzymes of O-GlcNAc metabolism, as shown. The enzymes OGT and GlcNAcase oppose each other but are independently regulated. There is clearly a dynamic interplay between the action of cellular kinases, phosphatases, and the enzymes of O-GlcNAc metabolism. Certain kinases are known to be substrates for OGT; OGT may be modified by specific kinases. This dynamic interplay creates tremendous potential for intracellular regulation. Among those cellular functions modulated by O-GlcNAc metabolism are nuclear transport, transcription, translation, cell signaling, apoptosis, and cell shape. Each function is described in greater detail in the text. Current evidence suggests that defects in O-GlcNAc metabolism are associated with human diseases such as neurodegeneration, diabetes mellitus, and cancer.

Future prospects
It is now clear that O-GlcNAc is involved in key cellular events such as transcription, translation, nuclear transport, and cell signaling. Dysregulation of O-GlcNAc metabolism is likely to be involved in certain human diseases. Given these possibilities, analytical approaches to accelerate research in this area will be increasingly important. This brief review was written in the hope that young investigators may find some aspect of glycan-dependent signaling to be of interest and begin to explore some of these possibilities.

	REFERENCES

TOP ABSTRACT O-LINKED N-ACETYLGLUCOSAMINE: A... METHODS OF ANALYSIS HOW WIDESPREAD IS O-GlcNAc... CELLULAR PROTEINS MODIFIED BY... THE O-LINKED GlcNAc TRANSFERASE:... O-GlcNAcase (HEXOSAMINIDASE C) THE HEXOSAMINE BIOSYNTHETIC... EVIDENCE FOR AN O-GlcNAc... GLYCAN-DEPENDENT SIGNALING AND... SUMMARY AND FUTURE PROSPECTS REFERENCES

 

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