scholarly journals N-Glycans on EGF domain-specific O-GlcNAc transferase (EOGT) facilitate EOGT maturation and peripheral endoplasmic reticulum localization

2020 ◽  
Vol 295 (25) ◽  
pp. 8560-8574 ◽  
Author(s):  
Sayad Md. Didarul Alam ◽  
Yohei Tsukamoto ◽  
Mitsutaka Ogawa ◽  
Yuya Senoo ◽  
Kazutaka Ikeda ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Species ◽  
Cell Periphery ◽  
Consensus Sequences ◽  
Domain Specific ◽  
Glycosylation Sites ◽  
Epidermal Growth ◽  
Egf Domain ◽  
Glcnac Transferase ◽  
And Function

Epidermal growth factor (EGF) domain-specific O-GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER)-resident protein that modifies EGF repeats of Notch receptors and thereby regulates Delta-like ligand-mediated Notch signaling. Several EOGT mutations that may affect putative N-glycosylation consensus sites are recorded in the cancer database, but the presence and function of N-glycans in EOGT have not yet been characterized. Here, we identified N-glycosylation sites in mouse EOGT and elucidated their molecular functions. Three predicted N-glycosylation consensus sequences on EOGT are highly conserved among mammalian species. Within these sites, we found that Asn-263 and Asn-354, but not Asn-493, are modified with N-glycans. Lectin blotting, endoglycosidase H digestion, and MS analysis revealed that both residues are modified with oligomannose N-glycans. Loss of an individual N-glycan on EOGT did not affect its endoplasmic reticulum (ER) localization, enzyme activity, and ability to O-GlcNAcylate Notch1 in HEK293T cells. However, simultaneous substitution of both N-glycosylation sites affected both EOGT maturation and expression levels without an apparent change in enzymatic activity, suggesting that N-glycosylation at a single site is sufficient for EOGT maturation and expression. Accordingly, a decrease in O-GlcNAc stoichiometry was observed in Notch1 co-expressed with an N263Q/N354Q variant compared with WT EOGT. Moreover, the N263Q/N354Q variant exhibited altered subcellular distribution within the ER in HEK293T cells, indicating that N-glycosylation of EOGT is required for its ER localization at the cell periphery. These results suggest critical roles of N-glycans in sustaining O-GlcNAc transferase function both by maintaining EOGT levels and by ensuring its proper subcellular localization in the ER.

scholarly journals EOGT and O-GlcNAc on secreted and membrane proteins

2017 ◽  
Vol 45 (2) ◽  
pp. 401-408 ◽  
Author(s):  
Shweta Varshney ◽  
Pamela Stanley
Keyword(s):  
Growth Factor ◽  
Membrane Proteins ◽  
Epidermal Growth Factor ◽  
Consensus Sequence ◽  
Transmembrane Proteins ◽  
Domain Specific ◽  
Epidermal Growth ◽  
Egf Domain ◽  
Glcnac Transferase ◽  
Cellular Compartments

Here, we describe a recently discovered O-GlcNAc transferase termed EOGT for EGF domain-specific O-GlcNAc transferase. EOGT transfers GlcNAc (N-acetylglucosamine) to Ser or Thr in secreted and membrane proteins that contain one or more epidermal growth factor-like repeats with a specific consensus sequence. Thus, EOGT is distinct from OGT, the O-GlcNAc transferase, that transfers GlcNAc to Ser/Thr in proteins of the cytoplasm or nucleus. EOGT and OGT are in separate cellular compartments and have mostly distinct substrates, although both can act on cytoplasmic (OGT) and lumenal (EOGT) domains of transmembrane proteins. The present review will describe known substrates of EOGT and biological roles for EOGT in Drosophila and humans. Mutations in EOGT that give rise to Adams–Oliver Syndrome in humans will also be discussed.

scholarly journals Structural Divergence in O-GlcNAc Glycans Displayed on Epidermal Growth Factor-like Repeats of Mammalian Notch1

Molecules ◽  
2018 ◽  
Vol 23 (7) ◽  
pp. 1745 ◽  
Author(s):  
Mitsutaka Ogawa ◽  
Yuya Senoo ◽  
Kazutaka Ikeda ◽  
Hideyuki Takeuchi ◽  
Tetsuya Okajima
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cultured Cells ◽  
Domain Specific ◽  
Notch Receptors ◽  
Terminal Galactose ◽  
Structural Divergence ◽  
Epidermal Growth ◽  
Glcnac Transferase ◽  
And Function

Extracellular O-GlcNAc is a novel class of modification catalyzed by epidermal growth factor-like (EGF)-domain specific O-GlcNAc transferase (EOGT). In mammals, EOGT is required for ligand-mediated Notch signaling for vascular development. Previous studies have revealed that O-GlcNAc in mammalian cultured cells is subject to subsequent glycosylation, which may impose additional layers of regulation. This study aimed to analyze the O-GlcNAc glycans of Drosophila EGF20 as model substrates and mouse Notch1 EGF repeats by mass-spectrometry. The analysis of Drosophila EGF20 expressed in HEK293T cells revealed that the majority of the proteins are modified with an elongated form of O-GlcNAc glycan comprising terminal galactose or sialic acid residues. In contrast, recombinant Notch1 EGF repeats isolated from HEK293T cells revealed structural divergence of O-GlcNAc glycans among the different EGF domains. Although the majority of Notch1 EGF2 and EGF20 domains contained the extended forms of the glycan, the O-GlcNAc in many other domains mostly existed as a monosaccharide irrespective of the exogenous EOGT expression. Our results raised a hypothesis that an array of O-GlcNAc monosaccharides may impact the structure and function of Notch receptors.

scholarly journals Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Heather J Tarbet ◽  
Lee Dolat ◽  
Timothy J Smith ◽  
Brett M Condon ◽  
E Timothy O'Brien ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transferase Activity ◽  
Protein Protein Interactions ◽  
Site Specific ◽  
Form And Function ◽  
Glycosylation Sites ◽  
Wide Range ◽  
Dynamics And Function ◽  
Glcnac Transferase ◽  
And Function

Intermediate filaments (IF) are a major component of the metazoan cytoskeleton and are essential for normal cell morphology, motility, and signal transduction. Dysregulation of IFs causes a wide range of human diseases, including skin disorders, cardiomyopathies, lipodystrophy, and neuropathy. Despite this pathophysiological significance, how cells regulate IF structure, dynamics, and function remains poorly understood. Here, we show that site-specific modification of the prototypical IF protein vimentin with O-linked β-N-acetylglucosamine (O-GlcNAc) mediates its homotypic protein-protein interactions and is required in human cells for IF morphology and cell migration. In addition, we show that the intracellular pathogen Chlamydia trachomatis, which remodels the host IF cytoskeleton during infection, requires specific vimentin glycosylation sites and O-GlcNAc transferase activity to maintain its replicative niche. Our results provide new insight into the biochemical and cell biological functions of vimentin O-GlcNAcylation, and may have broad implications for our understanding of the regulation of IF proteins in general.

scholarly journals Human α-galactosidase A: glycosylation site 3 is essential for enzyme solubility

1998 ◽  
Vol 332 (3) ◽  
pp. 789-797 ◽  
Author(s):  
Yiannis A. IOANNOU ◽  
Ken M. ZEIDNER ◽  
Marie E. GRACE ◽  
Robert J. DESNICK
Keyword(s):  
Endoplasmic Reticulum ◽  
Site Occupancy ◽  
Glycosylation Site ◽  
Enzymic Activity ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Consensus Sequences ◽  
Hybrid Type ◽  
Type Site ◽  
And Function

Human α-galactosidase A (EC 3.2.1.22; α-Gal A) is the homodimeric glycoprotein that hydrolyses the terminal α-galactosyl moieties from glycolipids and glycoproteins. The type, site occupancy and function of the N-linked oligosaccharide chains on this lysosomal hydrolase were determined. Endoglycosidase treatment of the purified recombinant enzyme and mutagenesis studies indicated that three (Asn-139, Asn-192 and Asn-215) of the four potential N-glycosylation consensus sequences were occupied by complex, high-mannose and hybrid-type oligosaccharides respectively. When expressed in COS-1 cells, glycoforms with glycosylation site 1 or 2 obliterated had more than 70% of wild-type activity, and both glycoforms were secreted. In contrast, the glycoform with only site 3 eliminated had decreased activity (less than 40%); little, if any, was secreted. Expressed mutant glycoforms in which site 3 and site 1 or 2 were obliterated had little, if any, intracellular or secreted enzymic activity, and immunofluorescence microscopy revealed that the expressed mutant glycoforms were retained in the endoplasmic reticulum, presumably where they were degraded. Thus glycosylation at site 3 was crucial to the formation of soluble, active enzyme, as well as transport to the lysosome. Absence of the site 3 hybrid-type oligosaccharide exposed an adjacent, normally protected, hydrophobic region, resulting in aggregation of the enzyme polypeptide in the endoplasmic reticulum. In support of this concept, endoglycosidase H-treated enzyme or mannose-terminated enzyme expressed in Autographa californica cells also aggregated when concentrated, emphasizing that site 3 occupancy by a hybrid-type oligosaccharide was required for enzyme solubility.

scholarly journals Autosomal recessive Adams–Oliver syndrome caused by homozygous mutation in EOGT, encoding an EGF domain-specific O-GlcNAc transferase

2013 ◽  
Vol 22 (3) ◽  
pp. 374-378 ◽  
Author(s):  
Idan Cohen ◽  
Eldad Silberstein ◽  
Yonatan Perez ◽  
Daniella Landau ◽  
Khalil Elbedour ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Homozygous Mutation ◽  
Domain Specific ◽  
Egf Domain ◽  
Glcnac Transferase

A Mucin-Specific Protease Enables Molecular and Functional Analysis of Human Cancer-Associated Mucins

2018 ◽  
Author(s):  
Stacy A. Malaker ◽  
Kayvon Pedram ◽  
Michael J. Ferracane ◽  
Elliot C. Woods ◽  
Jessica Kramer ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
High Resolution Mass Spectrometry ◽  
Human Cancer ◽  
Glycosylation Sites ◽  
Bacterial Protease ◽  
Specific Protease ◽  
To Come ◽  
And Function

<div> <div> <div> <p>Mucins are a class of highly O-glycosylated proteins that are ubiquitously expressed on cellular surfaces and are important for human health, especially in the context of carcinomas. However, the molecular mechanisms by which aberrant mucin structures lead to tumor progression and immune evasion have been slow to come to light, in part because methods for selective mucin degradation are lacking. Here we employ high resolution mass spectrometry, polymer synthesis, and computational peptide docking to demonstrate that a bacterial protease, called StcE, cleaves mucin domains by recognizing a discrete peptide-, glycan-, and secondary structure- based motif. We exploited StcE’s unique properties to map glycosylation sites and structures of purified and recombinant human mucins by mass spectrometry. As well, we found that StcE will digest cancer-associated mucins from cultured cells and from ovarian cancer patient-derived ascites fluid. Finally, using StcE we discovered that Siglec-7, a glyco-immune checkpoint receptor, specifically binds sialomucins as biological ligands, whereas the related Siglec-9 receptor does not. Mucin-specific proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of glycoprotein structure and function and for deorphanizing mucin-binding receptors. </p> </div> </div> </div>

scholarly journals Maturation of the Na,K-ATPase in the Endoplasmic Reticulum in Health and Disease

2021 ◽  
Author(s):  
Vitalii Kryvenko ◽  
Olga Vagin ◽  
Laura A. Dada ◽  
Jacob I. Sznajder ◽  
István Vadász
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Signaling ◽  
Loss Of Function ◽  
Α Subunit ◽  
Rate Limiting Step ◽  
Atpase Subunits ◽  
A Cell ◽  
Health And Disease ◽  
And Function ◽  
Rate Limiting

Abstract The Na,K-ATPase establishes the electrochemical gradient of cells by driving an active exchange of Na+ and K+ ions while consuming ATP. The minimal functional transporter consists of a catalytic α-subunit and a β-subunit with chaperon activity. The Na,K-ATPase also functions as a cell adhesion molecule and participates in various intracellular signaling pathways. The maturation and trafficking of the Na,K-ATPase include co- and post-translational processing of the enzyme in the endoplasmic reticulum (ER) and the Golgi apparatus and subsequent delivery to the plasma membrane (PM). The ER folding of the enzyme is considered as the rate-limiting step in the membrane delivery of the protein. It has been demonstrated that only assembled Na,K-ATPase α:β-complexes may exit the organelle, whereas unassembled, misfolded or unfolded subunits are retained in the ER and are subsequently degraded. Loss of function of the Na,K-ATPase has been associated with lung, heart, kidney and neurological disorders. Recently, it has been shown that ER dysfunction, in particular, alterations in the homeostasis of the organelle, as well as impaired ER-resident chaperone activity may impede folding of Na,K-ATPase subunits, thus decreasing the abundance and function of the enzyme at the PM. Here, we summarize our current understanding on maturation and subsequent processing of the Na,K-ATPase in the ER under physiological and pathophysiological conditions. Graphic Abstract

Sex Steroids and Human Behavior: Implications for Developmental Psychopathology

CNS Spectrums ◽  
2001 ◽  
Vol 6 (1) ◽  
pp. 75-88 ◽  
Author(s):  
Gerianne M. Alexander ◽  
Bradley S. Peterson
Keyword(s):  
Sex Differences ◽  
Human Behavior ◽  
Sex Steroids ◽  
Developmental Psychopathology ◽  
Mammalian Species ◽  
Brain Structures ◽  
Postnatal Life ◽  
Brain Structure And Function ◽  
Atypical Development ◽  
And Function

AbstractIn a variety of mammalian species, prenatal androgens organize brain structures and functions that are later activated by steroid hormones in postnatal life. In humans, studies of individuals with typical and atypical development suggest that sex differences in reproductive and nonreproductive behavior derive in part from similar prenatal and postnatal steroid effects on brain development. This paper provides a summary of research investigating hormonal influences on human behavior and describes how sex differences in the prevalences and natural histories of developmental psychopathologies may be consistent with these steroid effects. An association between patterns of sexual differentiation and specific forms of psychopathology suggests novel avenues for assessing the effects of sex steroids on brain structure and function, which may in turn improve our understanding of typical and atypical development in women and men.

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