19 results on '"Zachara N"'
Search Results
2. Dynamic interplay between O-GlcNAc and phosphorylation: roles in signaling, stress responses, and transcription: SL09-055
- Author
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Hart, G. W., Zachara, N., Wells, L., Vosseller, K., Iyer, S., Kamemura, K., Sakabe, K., Cheung, W., Slawson, C., Whelan, S., and Lakshmanan, T.
- Published
- 2003
3. SNAI1 Regulates the Hexosamine Biosynthesis Pathway to Promote Kras Mutant Lung Tumorigenesis
- Author
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Taparra, K.T., Wang, H., Malek, R., Nugent, K., Groves, J., Yildirir, G., Simons, B., Felsher, D., Zachara, N., and Tran, P.T.
- Published
- 2016
- Full Text
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4. Nucleocytoplasmic Glycosylation: O-Linked β-N-Acetylglucosamine.
- Author
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Zachara, N. E., Cheung, W. D., and Hart, G. W.
- Published
- 2004
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5. Aberrant N-glycosylation is a therapeutic target in carriers of a common and highly pleiotropic mutation in the manganese transporter ZIP8.
- Author
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Tomar V, Kang J, Lin R, Brant SR, Lazarev M, Tressler C, Glunde K, Zachara N, and Melia J
- Abstract
The treatment of defective glycosylation in clinical practice has been limited to patients with rare and severe phenotypes associated with congenital disorders of glycosylation (CDG). Carried by approximately 5% of the human population, the discovery of the highly pleiotropic, missense mutation in a manganese transporter ZIP8 has exposed under-appreciated roles for Mn homeostasis and aberrant Mn-dependent glycosyltransferases activity leading to defective N-glycosylation in complex human diseases. Here, we test the hypothesis that aberrant N-glycosylation contributes to disease pathogenesis of ZIP8 A391T-associated Crohn's disease. Analysis of N-glycan branching in intestinal biopsies demonstrates perturbation in active Crohn's disease and a genotype-dependent effect characterized by increased truncated N-glycans. A mouse model of ZIP8 391-Thr recapitulates the intestinal glycophenotype of patients carrying mutations in ZIP8. Borrowing from therapeutic strategies employed in the treatment of patients with CDGs, oral monosaccharide therapy with N-acetylglucosamine ameliorates the epithelial N-glycan defect, bile acid dyshomeostasis, intestinal permeability, and susceptibility to chemical-induced colitis in a mouse model of ZIP8 391-Thr. Together, these data support ZIP8 391-Thr alters N-glycosylation to contribute to disease pathogenesis, challenging the clinical paradigm that CDGs are limited to patients with rare diseases. Critically, the defect in glycosylation can be targeted with monosaccharide supplementation, providing an opportunity for genotype-driven, personalized medicine.
- Published
- 2024
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6. Cataloging natural sialic acids and other nonulosonic acids (NulOs), and their representation using the Symbol Nomenclature for Glycans.
- Author
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Lewis AL, Toukach P, Bolton E, Chen X, Frank M, Lütteke T, Knirel Y, Schoenhofen I, Varki A, Vinogradov E, Woods RJ, Zachara N, Zhang J, Kamerling JP, and Neelamegham S
- Subjects
- Animals, Polysaccharides chemistry, Monosaccharides, Cataloging, Sialic Acids chemistry, N-Acetylneuraminic Acid
- Abstract
Nonulosonic acids or non-2-ulosonic acids (NulOs) are an ancient family of 2-ketoaldonic acids (α-ketoaldonic acids) with a 9-carbon backbone. In nature, these monosaccharides occur either in a 3-deoxy form (referred to as "sialic acids") or in a 3,9-dideoxy "sialic-acid-like" form. The former sialic acids are most common in the deuterostome lineage, including vertebrates, and mimicked by some of their pathogens. The latter sialic-acid-like molecules are found in bacteria and archaea. NulOs are often prominently positioned at the outermost tips of cell surface glycans, and have many key roles in evolution, biology and disease. The diversity of stereochemistry and structural modifications among the NulOs contributes to more than 90 sialic acid forms and 50 sialic-acid-like variants described thus far in nature. This paper reports the curation of these diverse naturally occurring NulOs at the NCBI sialic acid page (https://www.ncbi.nlm.nih.gov/glycans/sialic.html) as part of the NCBI-Glycans initiative. This includes external links to relevant Carbohydrate Structure Databases. As the amino and hydroxyl groups of these monosaccharides are extensively derivatized by various substituents in nature, the Symbol Nomenclature For Glycans (SNFG) rules have been expanded to represent this natural diversity. These developments help illustrate the natural diversity of sialic acids and related NulOs, and enable their systematic representation in publications and online resources., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2023
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7. Transient receptor potential canonical type 6 (TRPC6) O-GlcNAcylation at Threonine-221 plays potent role in channel regulation.
- Author
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Mishra S, Ma J, McKoy D, Sasaki M, Farinelli F, Page RC, Ranek MJ, Zachara N, and Kass DA
- Abstract
Transient receptor potential canonical type 6 (TRPC6) is a non-voltage-gated channel that principally conducts calcium. Elevated channel activation contributes to fibrosis, hypertrophy, and proteinuria, often coupled to stimulation of nuclear factor of activated T-cells (NFAT). TRPC6 is post-translationally regulated, but a role for O-linked β-N-acetyl glucosamine (O-GlcNAcylation) as elevated by diabetes, is unknown. Here we show TRPC6 is constitutively O-GlcNAcylated at Ser14, Thr70, and Thr221 in the N-terminus ankryn-4 (AR4) and linker (LH1) domains. Mutagenesis to alanine reveals T221 as a critical controller of resting TRPC6 conductance, and associated NFAT activity and pro-hypertrophic signaling. T→A mutations at sites homologous in closely related TRPC3 and TRPC7 also increases their activity. Molecular modeling predicts interactions between Thr221- O -GlcNAc and Ser199, Glu200, and Glu246, and combined alanine substitutions of the latter similarly elevates resting NFAT activity. Thus, O-GlcNAcylated T221 and interactions with coordinating residues is required for normal TRPC6 channel conductance and NFAT activation., Competing Interests: D.A.K. receives grant support from Boehringer Ingelheim pursuing the use of a selective TRPC6 antagonist for treatment of Duchenne Muscular Dystrophy., (© 2023 The Authors.)
- Published
- 2023
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8. Serum amyloid A1 (SAA1) protein in human colostrum.
- Author
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Sack GH Jr, Zachara N, Rosenblum N, Talbot CC Jr, Kreimer S, Cole R, and McDonald TL
- Abstract
Proteins of the serum amyloid A (SAA) family have been remarkably conserved in evolution. Their biologic function(s) are not fully defined but they are likely to be a part of primordial host defense. We have detected a ∼ 12-kDa protein reacting with antibodies against serum amyloid A (SAA) in human colostrum by western blotting. Mass spectrometry identified the reactive species as SAA1, previously identified as a prominent member of the acute-phase response in serum. Our finding SAA1 in human colostrum contrasts with bovine, caprine and ovine colostrum where a species corresponding to putative SAA3 is uniformly present. SAA1 protein in human colostrum presumably contributes to neonatal protection.
- Published
- 2018
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9. Post-translational O-GlcNAcylation is essential for nuclear pore integrity and maintenance of the pore selectivity filter.
- Author
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Zhu Y, Liu TW, Madden Z, Yuzwa SA, Murray K, Cecioni S, Zachara N, and Vocadlo DJ
- Subjects
- Animals, Cell Line, Glycosylation, Mice, Nuclear Pore Complex Proteins metabolism, Protein Processing, Post-Translational genetics, Protein Processing, Post-Translational physiology, Protein Stability, Ubiquitination genetics, Ubiquitination physiology, Nuclear Pore metabolism
- Abstract
O-glycosylation of the nuclear pore complex (NPC) by O-linked N-acetylglucosamine (O-GlcNAc) is conserved within metazoans. Many nucleoporins (Nups) comprising the NPC are constitutively O-GlcNAcylated, but the functional role of this modification remains enigmatic. We show that loss of O-GlcNAc, induced by either inhibition of O-GlcNAc transferase (OGT) or deletion of the gene encoding OGT, leads to decreased cellular levels of a number of natively O-GlcNAcylated Nups. Loss of O-GlcNAc enables increased ubiquitination of these Nups and their increased proteasomal degradation. The decreased half-life of these deglycosylated Nups manifests in their gradual loss from the NPC and a downstream malfunction of the nuclear pore selective permeability barrier in both dividing and post-mitotic cells. These findings define a critical role of O-GlcNAc modification of the NPC in maintaining its composition and the function of the selectivity filter. The results implicate NPC glycosylation as a regulator of NPC function and reveal the role of conserved glycosylation of the NPC among metazoans., (© The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.)
- Published
- 2016
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10. O-linked N-acetylglucosamine (O-GlcNAc) protein modification is increased in the cartilage of patients with knee osteoarthritis.
- Author
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Tardio L, Andrés-Bergós J, Zachara NE, Larrañaga-Vera A, Rodriguez-Villar C, Herrero-Beaumont G, and Largo R
- Subjects
- Acylation, Adult, Cartilage, Articular pathology, Case-Control Studies, Cell Differentiation physiology, Cells, Cultured, Chondrocytes drug effects, Chondrocytes metabolism, Female, Humans, Inflammation Mediators pharmacology, Interleukin-1 pharmacology, Isoenzymes biosynthesis, Male, Middle Aged, N-Acetylglucosaminyltransferases biosynthesis, Osteoarthritis, Knee pathology, Protein Modification, Translational drug effects, beta-N-Acetylhexosaminidases biosynthesis, Acetylglucosamine metabolism, Cartilage, Articular metabolism, Osteoarthritis, Knee metabolism, Protein Modification, Translational physiology
- Abstract
Objective: There is increasing evidence that the addition of O-linked N-acetylglucosamine (O-GlcNAc) to proteins plays an important role in cell signaling pathways. In chondrocytes, accumulation of O-GlcNAc-modified proteins induces hypertrophic differentiation. Osteoarthritis (OA) is characterized by cartilage degradation, and hypertrophic-like changes in hyaline chondrocytes. However, the mechanisms responsible for these changes have not been described. Our aim was to study whether O-GlcNAcylation and the enzymes responsible for this modification are dysregulated in the cartilage of patients with knee OA and whether interleukin-1 could induce these modifications in cultured human OA chondrocytes (HOC)., Design: Human cartilage was obtained from patients with knee OA and from age and sex-matched healthy donors. HOC were cultured and stimulated with the catabolic cytokine IL-1α. Global protein O-GlcNAcylation and the synthesis of the key enzymes responsible for this modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), were assessed by western blot., Results: OA was associated with a 4-fold increase in the global O-GlcNAcylation in the cartilage. OA cartilage showed a re-distribution of the OGT and OGA isoforms, with a net increase in the presence of both enzymes, in comparison to healthy cartilage. In HOC, IL-1α stimulation rapidly increased O-GlcNAcylation and OGT and OGA synthesis., Conclusions: Our results indicate that a proinflammatory milieu could favor the accumulation of O-GlcNAcylated proteins in OA cartilage, together with the dysregulation of the enzymes responsible for this modification. The increase in O-GlcNAcylation could be responsible, at least partially, for the re-expression of hypertrophic differentiation markers that have been observed in OA., (Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2014
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11. Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis.
- Author
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Tarrant MK, Rho HS, Xie Z, Jiang YL, Gross C, Culhane JC, Yan G, Qian J, Ichikawa Y, Matsuoka T, Zachara N, Etzkorn FA, Hart GW, Jeong JS, Blackshaw S, Zhu H, and Cole PA
- Subjects
- Animals, Casein Kinase II biosynthesis, Casein Kinase II chemistry, Cell Line, Humans, NIMA-Interacting Peptidylprolyl Isomerase, Peptidylprolyl Isomerase chemistry, Peptidylprolyl Isomerase metabolism, Phosphorylation, Rats, Serine metabolism, Acetylglucosamine metabolism, Casein Kinase II metabolism
- Abstract
Protein serine-threonine kinase casein kinase II (CK2) is involved in a myriad of cellular processes including cell growth and proliferation through its phosphorylation of hundreds of substrates, yet how CK2 function is regulated is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-linked β-N-acetyl-glucosamine (O-GlcNAc) on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344). We use protein semisynthesis to show that phosphorylation of Thr344 increases the cellular stability of CK2α by strengthening its interaction with Pin1, whereas glycosylation of Ser347 seems to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degradation. By performing kinase assays with site-specifically phospho- and glyco-modified CK2α in combination with CK2β and Pin1 binding partners on human protein microarrays, we show that the kinase substrate selectivity of CK2 is modulated by these specific post-translational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biological outputs.
- Published
- 2012
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12. The coactivator of transcription CREB-binding protein interacts preferentially with the glycosylated form of Stat5.
- Author
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Gewinner C, Hart G, Zachara N, Cole R, Beisenherz-Huss C, and Groner B
- Subjects
- Acetylglucosamine pharmacology, Active Transport, Cell Nucleus, Alanine chemistry, Animals, CREB-Binding Protein, Cell Differentiation, Cell Division, Cell Line, Cell Nucleus metabolism, Cytoplasm metabolism, DNA-Binding Proteins metabolism, Dimerization, Epithelial Cells cytology, Galactosyltransferases metabolism, Glycosylation, Growth Substances metabolism, Humans, Insecta, Interferons metabolism, Lectins metabolism, Ligands, Luciferases metabolism, Mass Spectrometry, Microscopy, Fluorescence, Mutation, Nuclear Proteins chemistry, Phosphorylation, Prolactin metabolism, Promoter Regions, Genetic, Protein Binding, Protein Structure, Tertiary, Recombinant Proteins chemistry, STAT5 Transcription Factor, Serine chemistry, Threonine chemistry, Trans-Activators chemistry, Transcription, Genetic, Transfection, Tumor Suppressor Proteins, Tyrosine chemistry, DNA-Binding Proteins physiology, Milk Proteins, Nuclear Proteins metabolism, Trans-Activators metabolism, Trans-Activators physiology
- Abstract
The signal transducer and activator of transcription (Stat) gene family comprises seven members with similarities in their domain structure and a common mode of activation. Members of this gene family mediate interferon induction of gene transcription and the response to a large number of growth factors and hormones. Extracellular ligand binding to transmembrane receptors causes the intracellular activation of associated tyrosine kinases, phosphorylation of Stat molecules, dimerization, and translocation to the nucleus. Prolactin-induced phosphorylation of Stat5 is a key event in the development and differentiation of mammary epithelial cells. In addition to the crucial phosphorylation at tyrosine 694, we have identified an O-linked N-acetylglucosamine (O-GlcNAc) as another secondary modification essential for the transcriptional induction by Stat5. This modification was only found on nuclear Stat5 after cytokine activation. Similar observations were made with Stat1, Stat3, and Stat6. Glycosylation of Stat5, however, does not seem to be a prerequisite for nuclear translocation. Mass spectrometric analysis revealed a glycosylated peptide in the N-terminal region of Stat5. Replacement of threonine 92 by an alanine residue (Stat5a-T92A) strongly reduced the prolactin induction of Stat5a glycosylation and abolished transactivation of a target gene promoter. Only the glycosylated form of Stat5 was able to bind the coactivator of transcription CBP, an essential interaction for Stat5-mediated gene transcription.
- Published
- 2004
- Full Text
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13. Detection and analysis of proteins modified by O-linked N-acetylglucosamine.
- Author
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Zachara NE, Cole RN, Hart GW, and Gao Y
- Subjects
- Chromatography, Affinity, Immunoblotting, Lectins chemistry, Protein Processing, Post-Translational, Proteins isolation & purification, Acetylglucosamine chemistry, Proteins analysis, Proteins chemistry
- Abstract
First, a protocol for increasing the stoichiometry of O-GlcNAc on proteins is given. This is followed by simple techniques for the detection/screening of O-GlcNAc-modified proteins either by immunoblotting or lectin affinity chromatography. Separate protocols verify that the glycan is O-linked GlcNAc. These methods are followed by protocols for more comprehensive analysis of O-GlcNAc modified proteins, including labeling of O-GlcNAc residues with [3H]Gal, and subsequent product analysis. The final two protocols assay for O-GlcNAc transferase and O-GlcNAcase activity, respectively.
- Published
- 2001
- Full Text
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14. Identification of glycosylation sites in mucin peptides by edman degradation.
- Author
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Zachara NE and Gooley AA
- Subjects
- Animals, Binding Sites, Glycosylation, Humans, Indicators and Reagents, Peptides, Mucins analysis
- Published
- 2000
- Full Text
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15. Structural heterogeneity in the core oligosaccharide of the S-layer glycoprotein from Aneurinibacillus thermoaerophilus DSM 10155.
- Author
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Wugeditsch T, Zachara NE, Puchberger M, Kosma P, Gooley AA, and Messner P
- Subjects
- Bacterial Proteins isolation & purification, Carbohydrate Conformation, Carbohydrate Sequence, Chromatography, Gel, Chromatography, High Pressure Liquid, Glycopeptides chemistry, Glycopeptides isolation & purification, Membrane Glycoproteins isolation & purification, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Oligosaccharides isolation & purification, Pronase, Spectrometry, Mass, Secondary Ion, Bacillus chemistry, Bacterial Proteins chemistry, Membrane Glycoproteins chemistry, Oligosaccharides chemistry
- Abstract
The surface layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 has a total carbohydrate content of 15% (by mass), consisting of O-linked oligosaccharide chains. After proteolytic digestion of the S-layer glycoprotein byPronase E and subsequent purification of the digestion products by gel permeation chromatography, chromatofocusing and high-performance liquid chromatography two glycopeptide pools A and B with identical glycans and the repeating unit structure -->4)-alpha-l-Rha p -(1-->3)-beta-d- glycero -d- manno -Hep p -(1--> (Kosma et al., 1995b, Glycobiology, 5, 791-796) were obtained. Combined evidence from modified Edman-degradation in combination with liquid chromatography electrospray mass-spectrometry and nuclear magnetic resonance spectroscopy revealed that both glycopeptides contain equal amounts of the complete core structure alpha-l-Rha p -(1-->3)-alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and the truncated forms alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and beta-d-Gal p NAc-(1-->O)-Thr/Ser. All glycopeptides possessed the novel linkage types beta-d-Gal p NAc-(1-->O)-Thr/Ser. The different cores were substituted with varying numbers of disaccharide repeating units. By 300 MHz proton nuclear magnetic resonance spectroscopy the complete carbohydrate core structure of the fluorescently labeled glyco-peptide B was determined after Smith-degradation of its glycan chain. The NMR data confirmed and complemented the results of the mass spectroscopy experiments. Based on the S-layer glycopeptide structure, a pathway for its biosynthesis is suggested.
- Published
- 1999
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16. High density O-glycosylation on tandem repeat peptide from secretory MUC1 of T47D breast cancer cells.
- Author
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Müller S, Alving K, Peter-Katalinic J, Zachara N, Gooley AA, and Hanisch FG
- Subjects
- Amino Acid Sequence, Blotting, Western, Carbohydrate Sequence, Carboxypeptidases metabolism, Cathepsin A, Chromatography, High Pressure Liquid, Cysteine Endopeptidases metabolism, Female, Glycosylation, Humans, Mass Spectrometry, Molecular Sequence Data, Tumor Cells, Cultured, Breast Neoplasms chemistry, Mucin-1 chemistry
- Abstract
The site-specific O-glycosylation of MUC1 tandem repeat peptides from secretory mucin of T47D breast cancer cells was analyzed. After affinity isolation on immobilized BC3 antibody, MUC1 was partially deglycosylated by enzymatic treatment with alpha-sialidase/beta-galactosidase and fragmented by proteolytic cleavage with the Arg-C-specific endopeptidase clostripain. The PAP20 glycopeptides were isolated by reversed phase high pressure liquid chromatography and subjected to the structural analyses by quadrupole time-of-flight electrospray ionization mass spectrometry and to the sequencing by Edman degradation. All five positions of the repeat peptide were revealed as O-glycosylation targets in the tumor cell, including the Thr within the DTR motif. The degree of substitution was estimated to average 4.8 glycans per repeat, which compares to 2.6 glycosylated sites per repeat for the mucin from milk (Müller, S., Goletz, S., Packer, N., Gooley, A. A., Lawson, A. M., and Hanisch, F.-G. (1997) J. Biol. Chem. 272, 24780-24793). In addition to a modification by glycosylation, the immunodominant DTR motif on T47D-MUC1 is altered by amino acid replacements (PAPGSTAPAAHGVTSAPESR), which were revealed in about 50% of PAP20 peptides. The high incidence of these replacements and their detection also in other cancer cell lines imply that the conserved tandem repeat domain of MUC1 is polymorphic with respect to the peptide sequence.
- Published
- 1999
- Full Text
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17. Dynamic epigenetic regulation of initial O-glycosylation by UDP-N-Acetylgalactosamine:Peptide N-acetylgalactosaminyltransferases. site-specific glycosylation of MUC1 repeat peptide influences the substrate qualities at adjacent or distant Ser/Thr positions.
- Author
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Hanisch FG, Müller S, Hassan H, Clausen H, Zachara N, Gooley AA, Paulsen H, Alving K, and Peter-Katalinic J
- Subjects
- Amino Acid Sequence, Animals, Carbohydrate Sequence, Chromatography, High Pressure Liquid, Gene Expression Regulation, Glycosylation, Humans, Milk, Human enzymology, Molecular Sequence Data, Protein Conformation, Recombinant Proteins metabolism, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Structure-Activity Relationship, Substrate Specificity, Polypeptide N-acetylgalactosaminyltransferase, Acetylgalactosamine metabolism, Mucin-1 metabolism, N-Acetylgalactosaminyltransferases metabolism, Peptide Fragments metabolism, Serine metabolism, Threonine metabolism
- Abstract
In search of possible epigenetic regulatory mechanisms ruling the initiation of O-glycosylation by polypeptide:N-acetylgalactosaminyltransferases, we studied the influences of mono- and disaccharide substituents of glycopeptide substrates on the site-specific in vitro addition of N-acetylgalactosamine (GalNAc) residues by recombinant GalNAc-Ts (rGalNAc-T1, -T2, and -T3). The substrates were 20-mers (HGV20) or 21-mers (AHG21) of the MUC1 tandem repeat peptide carrying GalNAcalpha or Galbeta1-3GalNAcalpha at different positions. The enzymatic products were analyzed by MALDI mass spectrometry and Edman degradation for the number and sites of incorporated GalNAc. Disaccharide placed on the first position of the diad Ser-16-Thr-17 prevents glycosylation of the second, whereas disaccharide on the second position of Ser-16-Thr-17 and Thr-5-Ser-6 does not prevent GalNAc addition to the first. Multiple disaccharide substituents suppress any further glycosylation at the remaining sites. Glycosylation of Ser-16 is negatively affected by glycosylation at position -6 (Thr-10) or -10 (Ser-6) and is inhibited by disaccharide at position -11 (Thr-5), suggesting the occurrence of glycosylation-induced effects on distant acceptor sites. Kinetic studies revealed the accelerated addition of GalNAc to Ser-16 adjacent to GalNAc-substituted Thr-17, demonstrating positive regulatory effects induced by glycosylation on the monosaccharide level. These antagonistic effects of mono- and disaccharides could underlie a postulated regulatory mechanism.
- Published
- 1999
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18. Modified glycosylation of cellobiohydrolase I from a high cellulase-producing mutant strain of Trichoderma reesei.
- Author
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Harrison MJ, Nouwens AS, Jardine DR, Zachara NE, Gooley AA, Nevalainen H, and Packer NH
- Subjects
- Acetylglucosamine analysis, Amino Acid Sequence, Asparagine chemistry, Cellulose 1,4-beta-Cellobiosidase, Glycopeptides chemistry, Glycosylation, Mass Spectrometry, Membrane Glycoproteins chemistry, Molecular Sequence Data, Peptide Fragments chemistry, Sulfuric Acid Esters chemistry, Cellulase chemistry, Glycoproteins chemistry, Protein Processing, Post-Translational, Trichoderma enzymology
- Abstract
Cellobiohydrolase I is an industrially important exocellulase secreted in high yields by the filamentous fungus Trichoderma reesei. The nature and effect of glycosylation of CBHI and other cellulolytic enzymes is largely unknown, although many other structural and mechanistic aspects of cellulolytic enzymes are well characterised. Using a combination of liquid chromatography, electrospray mass spectrometry, solid-phase Edman degradation, and monosaccharide analysis we have identified every site of glycosylation of CBHI from a high cellulase-producing mutant strain of T. reesei, ALKO2877, and characterised each site in terms of its modifying carbohydrate and site-specific heterogeneity. The catalytic core domain comprises three N-linked glycans which each consist of a single N-acetylglucosamine residue. Within the glycopeptide linker domain, all eight threonines are variably glycosylated with between at least one, and up to three, mannose residues per site. All serines in this domain are at least partially glycosylated with a single mannose residue. This linker region has also been shown to be sulfated by a combination of ion chromatography and collision-induced dissociation electrospray mass spectrometry. The sulfate is probably mannose-linked. The biological significance of N-linked single N-acetylglucosamine in the catalytic core, and mannose sulfation in the linker region, is not known.
- Published
- 1998
- Full Text
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19. Recombinant prespore-specific antigen from Dictyostelium discoideum is a beta-sheet glycoprotein with a spacer peptide modified by O-linked N-acetylglucosamine.
- Author
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Zachara NE, Packer NH, Temple MD, Slade MB, Jardine DR, Karuso P, Moss CJ, Mabbutt BC, Curmi PM, Williams KL, and Gooley AA
- Subjects
- Acetylglucosamine analysis, Amino Acid Sequence, Animals, Antigens, Surface genetics, Carbohydrate Conformation, Chromatography, Gas, Chromatography, High Pressure Liquid, Fungal Proteins genetics, Glycopeptides chemistry, Glycosylation, Magnetic Resonance Spectroscopy, Mass Spectrometry, Membrane Glycoproteins genetics, Molecular Sequence Data, Recombinant Proteins chemistry, Sequence Homology, Amino Acid, Trypsin metabolism, Antigens, Protozoan, Antigens, Surface chemistry, Dictyostelium chemistry, Fungal Proteins chemistry, Membrane Glycoproteins chemistry, Protein Structure, Secondary, Protozoan Proteins
- Abstract
Prespore-specific antigen (PsA) is a putative cell-adhesion molecule of the cellular slime mould Dictyostelium discoideum, which has a similar molecular architecture to several mammalian cell-surface proteins. It has an N-terminal globular domain presented to the extracellular environment on an O-glycosylated stem (glycopeptide) that is attached to the cell membrane through a glycosyl-PtdIns anchor. The sequence of PsA suggests that PsA may belong to a new family of cell-surface molecules and here we present information on the structure of the N-terminal globular domain and determine the reducing-terminal linkage of the O-glycosylation. To obtain a sufficient amount of pure protein, a secreted recombinant form of PsA (rPsA), was expressed in D. discoideum and characterised. 1H-NMR spectra of rPsA contained features consistent with a high degree of beta-sheet in the N-terminal globular domain, a feature commonly observed in cell-adhesion proteins. Solid-phase Edman degradation of the glycopeptide of rPsA indicated that 14 of the 15 threonines and serines in the spacer region were glycosylated. The chemical structures of the O-glycosylations were determined to be single N-acetylglucosamine residues.
- Published
- 1996
- Full Text
- View/download PDF
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