Search PubMed⌕ Search

Biomedical subjects

G W Hart

Publications and source records attributed to G W Hart.

At least 37 records · Page 2Linked to original sources
PubMed1999

Regulation of a cytosolic and nuclear O-GlcNAc transferase. Role of the tetratricopeptide repeats.

The O-GlcNAc transferase (OGT) is a unique nuclear and cytosolic glycosyltransferase that contains multiple tetratricopeptide repeats. We have begun to characterize the mechanisms regulating OGT using a combination of deletion analysis and kinetic studies. Here we show that the p110 subunit of the enzyme forms both homo- and heterotrimers that appear to have different binding affinities for UDP-GlcNAc. The multimerization domain of OGT lies within the tetratricopeptide repeat domain and is not necessary for activity. Kinetic analyses of the full-length trimer and the truncated monomer forms of OGT suggest that both forms function through a random bi-bi kinetic mechanism. Both the monomer and trimer have similar specific activities and similar K(m) values for peptide substrates. However, they differ in their binding affinities for UDP-GlcNAc, indicating that subunit interactions affect enzyme activity. The findings that recombinant OGT has three distinct K(m) values for UDP-GlcNAc and that UDP-GlcNAc concentrations modulates the affinity of OGT for peptides suggest that OGT is exquisitely regulated by the levels of UDP-GlcNAc within the nucleus and cytoplasm.

Animals↗
PubMed1999

Determination of beta1,4-galactosyltransferase enzymatic activity by capillary electrophoresis and laser-induced fluorescence detection.

We have developed a nonradioactive method to assay UDP-Gal:beta-d-GlcNAcbeta1,4-galactosyltransferase (beta4GalT-I) enzymatic activity. Capillary electrophoresis combined with laser-induced fluorescence detection (CE-LIF) was employed to provide a baseline separation of FITC-conjugated O-GlcNAc-containing substrate peptides and galactose-capped product peptides, while at the same time allowing a level of detection in the low attomole range (10(-18)). The addition of 2 mM hexamethylene diamine to the borate-based capillary electrophoretic buffer modulated the electroosmotic flow, resulting in optimum separation of the glycopeptide product from reactant. beta4GalT-I activity was dependent upon the addition of both manganese and UDP-galactose. Using this assay, we show that two beta4GalT-I constructs, predicted to localize to different intracellular compartments, are enzymatically active when expressed in vitro using a rabbit reticulocyte transcription-translation system. The high sensitivity of product detection by CE-LIF in combination with in vitro transcription-translation is applicable to the facile determination of the enzymatic activity of other newly cloned glycosyltransferases.

Amino Acid Sequence↗
PubMed1999

Glycosylation sites flank phosphorylation sites on synapsin I: O-linked N-acetylglucosamine residues are localized within domains mediating synapsin I interactions.

Synapsin I is concentrated in nerve terminals, where it appears to anchor synaptic vesicles to the cytoskeleton and thereby ensures a steady supply of fusion-competent synaptic vesicles. Although phosphorylation-dependent binding of synapsin I to cytoskeletal elements and synaptic vesicles is well characterized, little is known about synapsin I's O-linked N-acetylglucosamine (O-GlcNAc) modifications. Here, we identified seven in vivo O-GlcNAcylation sites on synapsin I by analysis of HPLC-purified digests of rat brain synapsin I. The seven O-GlcNAcylation sites (Ser55, Thr56, Thr87, Ser516, Thr524, Thr562, and Ser576) in synapsin I are clustered around its five phosphorylation sites in domains B and D. The proximity of phosphorylation sites to O-GlcNAcylation sites in the regulatory domains of synapsin I suggests that O-GlcNAcylation may modulate phosphorylation and indirectly affect synapsin I interactions. With use of synthetic peptides, however, the presence of an O-GlcNAc at sites Thr562 and Ser576 resulted in only a 66% increase in the Km of calcium/calmodulin-dependent protein kinase II phosphorylation of site Ser566 with no effect on its Vmax. We conclude that O-GlcNAcylation likely plays a more direct role in synapsin I interactions than simply modulating the protein's phosphorylation.

Acetylglucosamine↗
PubMed1999

Localization of the O-linked N-acetylglucosamine transferase in rat pancreas.

O-linked N-acetylglucosamine transferase (OGT) catalyzes the attachment ofN-acetylglucosamine (GlcNAc) monosaccharides to the hydroxyl group of serine or threonine residues of intracellular proteins and may play an important role in the hexosamine pathway. Glucose-induced insulin resistance is mediated by increased activity of the hexosamine pathway. In the present study, we examined the localization of OGT mRNA and OGT protein in the rat pancreas. The sites of OGT mRNA expression were determined by in situ hybridization histochemistry with a digoxigenin (DIG)-labeled antisense cRNA probe. Intense hybridization signals were present in the exocrine acinar cells, while weaker ones were detected in the islets of Langerhans. This distribution was confirmed using additional antisense cRNA or oligo-cDNA probes complementary to different regions of OGT mRNA. In addition, immunofluorescence staining with antibody raised against OGT stained both the exocrine acinar cells and endocrine islet cells. In the acinar cell nucleus, the zymogen granule region and contour of the cell were intensely stained. In the islets of Langerhans, especially in the alpha-cells, intense staining with anti-OGT antibody was observed. These staining patterns were almost identical to those seen when staining for the O-linked GlcNAc (O-GlcNAc) modification. Immuno-electron microscopy showed that OGT is localized to the euchromatin of the nucleus and around the secretory granules of exocrine acinar cells and endocrine islet cells. These results suggest that OGT is involved in the regulation of transcription and of granular secretion. Thus, one or more O-GlcNAcylated proteins may be important components of the glucose-sensing mechanism in the pancreas.

Animals↗
PubMed1998

Nuclear and cytoplasmic glycosylation.

O-GlcNAcylation is a form of cytoplasmic and nuclear glycosylation that is found on many diverse proteins of the cell including RNA polymerase II and its associated transcription factors, cytoskeletal proteins, nucleoporins, viral proteins, heat shock proteins, tumor suppressors, and oncogenes. It involves the attachment of a single, unmodified N-acetylglucosaminyl residue O-glycosidically linked to the hydroxyl groups of serine and threonine moieties of proteins. It is a highly abundant and dynamic form of posttranslational modification that appears to modulate function in a manner similar to phosphorylation. All O-GlcNAc-containing proteins are phosphoproteins that are involved in the formation of multimeric complexes, suggesting that O-GlcNAc may play a role in mediating protein-protein interactions. O-GlcNAc sites resemble phosphorylation sites and in many cases the two modifications are mutually exclusive; therefore, O-GlcNAcylation may act as an antagonist of phosphorylation and help to mediate many essential functions of the cell.

Acetylglucosamine↗
PubMed1998

Recycling cell surface glycoproteins undergo limited oligosaccharide reprocessing in LEC1 mutant Chinese hamster ovary cells.

The ability of particular cell surface glycoproteins to recycle and become exposed to individual Golgi enzymes has been demonstrated. This study was designed to determine whether endocytic trafficking includes significant reentry into the overall oligosaccharide processing pathway. The Lec1 mutant of Chinese hamster ovary (CHO) cells lack N -acetylglucosaminyltransferase I (GlcNAc-TI) activity resulting in surface expression of incompletely processed Man5GlcNAc2 N -linked oligosaccharides. An oligosaccharide tracer was created by exoglycosylation of cell surface glycoproteins with purified porcine GlcNAc-TI and UDP-[3H]GlcNAc. Upon reculturing, all cell surface glycoproteins that acquired [3H]GlcNAc were acted upon by intracellular mannosidase II, the next enzyme in the Golgi processing pathway of complex N -linked oligosaccharides (t1/2= 3-4 h). That all radiolabeled cell surface glycoproteins were included in this endocytic pathway indicates a common intracellular compartment into which endocytosed cell surface glycoproteins return. Significantly, no evidence was found for continued oligosaccharide processing consistent with transit through the latter cisternae of the Golgi apparatus. These data indicate that, although recycling plasma membrane glycoproteins can be reexposed to individual Golgi-derived enzymes, significant reentry into the overall contiguous processing pathway is not evident.

Acetylglucosamine↗
PubMed1997

Dynamic glycosylation of nuclear and cytosolic proteins. Cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeats.

O-Linked N-acetylglucosamine (O-GlcNAc) glycosylation is a dynamic modification of eukaryotic nuclear and cytosolic proteins analogous to protein phosphorylation. We have cloned and characterized a novel gene for an O-GlcNAc transferase (OGT) that shares no sequence homology or structural similarities with other glycosyltransferases. The OGT gene is highly conserved (up to 80% identity) in all eukaryotes examined. Unlike previously described glycosyltransferases, OGT is localized to the cytosol and nucleus. The OGT protein contains multiple tandem repeats of the tetratricopeptide repeat motif. The presence of tetratricopeptide repeats, which can mediate protein-protein interactions, suggests that OGT may be regulated by protein interactions that are independent of the enzyme's catalytic site. The OGT is also modified by tyrosine phosphorylation, indicating that tyrosine kinase signal transduction cascades may play a role in modulating OGT activity.

Amino Acid Sequence↗
PubMed1997

A subpopulation of estrogen receptors are modified by O-linked N-acetylglucosamine.

Estrogen receptors (ER) are ligand-inducible transcription factors regulated by Ser(Thr)-O-phosphorylation. Many transcription factors and eukaryotic RNA polymerase II itself are also dynamically modified by Ser(Thr)-O-linked N-acetylglucosamine moieties (O-GlcNAc). Here we report that subpopulations of murine, bovine, and human estrogen receptors are modified by O-GlcNAc. O-GlcNAc moieties were detected on insect cell-expressed, mouse ER (mER) by probing with bovine milk galactosyltransferase, followed by structural analysis. Wheat germ agglutinin-Sepharose affinity chromatography also readily detected terminal GlcNAc residues on subpopulations of ER purified from calf uterus, from human breast cancer cells (MCF-7), or from mER produced by in vitro translation. These data suggest that greater than 10% of these populations of estrogen receptors bear O-GlcNAc. Site mapping of insect cell expressed mER localized one major site of O-GlcNAc addition to Thr-575, within a PEST region of the carboxyl-terminal F domain. Based upon their relative resistance to both hexosaminidase and to in vitro galactosylation, O-GlcNAc moieties appear to be largely buried on native mER. This dynamic saccharide modification, like phosphorylation, may play a role in modulating the dimerization, stability, or transactivation functions of estrogen receptors.

Acetylglucosamine↗
PubMed1997

Dynamic O-linked glycosylation of nuclear and cytoskeletal proteins.

Modification of Ser and Thr residues by attachment of O-linked N-acetylglucos-amine [Ser(Thr)-O-GlcNAcylation] to eukaryotic nuclear and cytosolic proteins is as dynamic and possibly as abundant as Ser(Thr) phosphorylation. Known O-GlcNAcylated proteins include cytoskeletal proteins and their regulatory proteins; viral proteins; nuclear-pore, heat-shock, tumor-suppressor, and nuclearoncogene proteins; RNA polymerase II catalytic subunit; and a multitude of transcription factors. Although functionally diverse, all of these proteins are also phosphoproteins. Most O-GlcNAcylated proteins form highly regulated multimeric associations that are dependent upon their posttranslational modifications. Evidence is mounting that O-GlcNAcylation is an important regulatory modification that may have a reciprocal relationship with O-phosphorylation and may modulate many biological processes in eukaryotes.

Animals↗
PubMed1996

The microtubule-associated protein tau is extensively modified with O-linked N-acetylglucosamine.

Tau is a family of phosphoproteins that are important in modulating microtubule stability in neurons. In Alzheimer's disease tau is abnormally hyperphosphorylated, no longer binds microtubules, and self-assembles to form paired helical filaments that likely contribute to neuron death. Here we demonstrate that normal bovine tau is multiply modified by Ser(Thr)-O-linked N-acetylglucosamine, a dynamic and abundant post-translational modification that is often reciprocal to Ser(Thr)-phosphorylation. O-GlcNAcylation of tau was demonstrated by blotting with succinylated wheat germ agglutinin and by probing with bovine milk beta(1,4)galactosyltransferase. Structural analyses confirm the linkage and the saccharide structure. Tau splicing variants are multiply O-GlcNAcylated at similar sites, with an average stoichiometry of greater than 4 mol of O-linked N-acetylglucosamine/mol of tau. However, the number of sites occupied appears to be greater than 12, suggesting substoichiometric occupancy at any given site. A similar relationship between average stoichiometry and site-occupancy has also been described for the phosphorylation of tau. Site-specific or stoichiometric changes in O-GlcNAcylation may not only modulate tau function but may also play a role in the formation of paired helical filaments.

Acetylglucosamine↗
PubMed1996

Cytoplasmic O-GlcNAc modification of the head domain and the KSP repeat motif of the neurofilament protein neurofilament-H.

Neurofilaments, the major intermediate filaments in large myelinated neurons, are essential for specifying proper axonal caliber. Mammalian neurofilaments are obligate heteropolymers assembled from three polypeptides, neurofilament (NF)-H, NF-M, and NF-L, each of which undergoes phosphorylation at multiple sites. NF-M and NF-L are known to be modified by O-linked N-acetylglucosamine (O-GlcNAc) (Dong, D. L.-Y., Xu, Z.-S., Chevrier, M. R., Cotter, R. J., Cleveland, D. W., and Hart, G. W. (1993) J. Biol. Chem. 268, 16679-16687). Here we further report that NF-H is extensively modified by O-GlcNAc at Thr53, Ser54, and Ser56 in the head domain and, somewhat surprisingly, at multiple sites within the Lys-Ser-Pro repeat motif in the tail domain, a region in assembled neurofilaments known to be nearly stoichiometrically phosphorylated on each of the approximately 50 KSP repeats. Beyond the earlier identified sites on NF-M and NF-L, O-GlcNAc sites on Thr19 and Ser34 of NF-M and Ser34 and Ser48 of NF-L are also determined here, all of which are localized in head domain sequences critical for filament assembly. The proximity of O-GlcNAc and phosphorylation sites in both head and tail domains of each subunit indicates that these modifications may influence one another and play a role in filament assembly and network formation.

Acetylglucosamine↗
PubMed1996

Dynamic O-GlcNAcylation of the small heat shock protein alpha B-crystallin.

alphaB-Crystallin, originally described as a structural lens protein, is now known to be a member of the small heat shock protein family and is expressed in a number of nonlens tissues. This highly conserved 20 kDa protein aggregates with homologous proteins, including alphaA-crystallin and the small heat shock protein HSP28, to form large heteromeric complexes. Recently, Roquemore et al. (1992) have established that both phosphorylated and unphosphorylated forms of lens alphaB-crystallin are modified with O-linked N-acetylglucosamine, a dynamic posttranslational modification abundant on nuclear and cytoplasmic proteins. In this paper, we have identified the major site of O-GlcNAcylation on lens alphaB as Thr 170. We have further shown that this modification is not restricted to lens alphaB-crystallin but occurs on alphaB isolated from rat heart tissue and human astroglioma cells. Two-dimensional electrophoresis of rat heart alphaB-crystallin revealed two O-GlcNAcylated forms with mobilities corresponding to the unphosphorylated form (alphaB2) and an unidentified, slightly more acidic form. Phosphorylated alphaB-crystallin (alphaB1) was not detected in the rat heart preparation. The major O-GlcNAcylation site on alphaB-crystallins from rat heart also appears to be at Thr 170. Metabolic pulse-chase labeling studies of U373-MG astroglioma cells indicated that turnover of the carbohydrate on alphaB-crystallin is not static but proceeds many-fold more rapidly than turnover of the protein backbone itself, consistent with a regulatory role for O-GlcNAc on this small heat shock protein.

Acetylglucosamine↗
PubMed1996

Selective detection and site-analysis of O-GlcNAc-modified glycopeptides by beta-elimination and tandem electrospray mass spectrometry.

Over the past decade, a number of nuclear and cytoplasmic proteins have been identified that are modified by single N-acetylglucosamine residues attached to the hydroxyl side chain of serines or threonines (O-GlcNAc). O-GlcNAc is a dynamic modification and therefore may act in a regulatory capacity analogous to phosphorylation. To undertake site-directed mutagenesis studies of O-GlcNAc's function, it is necessary to identify the sites of glycosylation on various proteins. The current method of site mapping, which involves galactosyltransferase labeling, generation of glycopeptides by proteolysis, purification by several rounds of HPLC, and gas-phase and manual Edman sequencing, is very tedious and requires about 10 pmol of pure, labeled glycopeptide. In this report, synthetic glycopeptides were generated and used to demonstrate that O-GlcNAc-modified peptides can be rapidly identified in complex mixtures by HPLC-coupled electrospray mass spectrometry due to the partial loss of the O-linked glycan (204 amu) at a modest orifice potential. Furthermore, the exact site of glycosylation was directly identified in the low picomole range by collision-induced dissociation (CID) of the glycopeptide after removal of the O-GlcNAc by alkaline beta-elimination. The conversion of glycosylserine to 2-aminopropenoic acid (2-ap) by beta-elimination both decreased the mass of the glycopeptide by 222 amu and resulted in a CID fragment ion representing the loss of 69 amu (2-ap) instead of 87 amu (Ser) at the position of the glycosylserine. Finally, we tested this method on an identical synthetic, alpha-linked O-GalNAc-modified peptide. Like O-GlcNAc, the O-GalNAc moiety was selectively removed at a modest orifice potential; however, the beta-elimination conditions that efficiently removed the O-GlcNAc only liberated about 20% of the O-GalNAc. We conclude that the selectivity and the sensitivity of this method will make it a powerful tool for determining the sites of O-GlcNAc modification on proteins of low abundance such as transcription factors and oncogenes.

Acetylglucosamine↗
PubMed1995

c-Myc is glycosylated at threonine 58, a known phosphorylation site and a mutational hot spot in lymphomas.

c-Myc is a helix-loop leucine zipper phosphoprotein that heterodimerizes with Max and regulates gene transcription in cell proliferation, cell differentiation, and programmed cell death. Previously, we demonstrated that c-Myc is modified by O-linked N-acetylglucosamine (O-GlcNAc) within or nearby the N-terminal transcriptional activation domain (Chou, T.-Y., Dang, C.V., and Hart, G.W. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4417-4421). In this paper, we identified the O-GlcNAc attachment site(s) on c-Myc. c-Myc purified from sf9 insect cells was trypsinized, and its GlcNAc moieties were enzymically labeled with [3H]galactose. The [3H]galactose-labeled glycopeptides were isolated by reverse phase high performance liquid chromatography and then subjected to gas-phase sequencing, manual Edman degradation, and laser desorption/ionization mass spectrometry. These analyses show that threonine 58, an in vivo phosphorylation site in the transactivation domain, is the major O-GlcNAc glycosylation site of c-Myc. Mutation of threonine 58, frequently found in retroviral v-Myc proteins and in human Burkitt and AIDS-related lymphomas, is associated with enhanced transforming activity and tumorigenicity. The reciprocal glycosylation and phosphorylation at this biologically significant amino acid residue may play an important role in the regulation of the functions of c-Myc.

Amino Acid Sequence↗