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.

Production of a human epidermal growth factor fusion protein and its degradation in rat gastrointestinal flushings

1996 ◽  
Vol 16 (1) ◽  
pp. 89-97 ◽  
Author(s):  
C J Xian ◽  
Z Upton ◽  
C Goddard ◽  
C A Shoubridge ◽  
K A McNeil ◽  
...  
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Small Bowel ◽  
Epithelial Cell ◽  
Epidermal Growth Factor ◽  
Fusion Protein ◽  
Egf Receptor ◽  
Human Epidermal Growth Factor ◽  
Epidermal Growth ◽  
Egf Domain

ABSTRACT This study describes the biosynthesis of a human epidermal growth factor fusion protein, Long EGF, that has a 53 amino acid extension peptide derived from the 46 N-terminal amino acids of porcine GH. The approach allowed the production of Long EGF at high efficiency due to the expression of the fusion protein in high yield as inclusion bodies in Escherichia coli. Long EGF had a slightly lower potency compared with native EGF in a range of assays, including binding to anti-EGF antibodies or the EGF receptor, stimulation of Balb/3T3 fibroblast and rat intestinal epithelial cell growth, as well as counteracting the inhibition of mink lung epithelial cell proliferation by transforming growth factor-β1. Degradation of Long EGF and native EGF was compared in gastrointestinal flushings as an indication of whether the EGF domain of the fusion protein would be protected from proteolytic cleavage and be useful as a trophic agent in the gut. Incubation with flushings from the stomach or jejunum of rats caused rapid cleavage of the extension peptide, releasing native EGF. A C-terminal truncation of Arg53 in the stomach and a removal of the C-terminal pentapeptide (49Trp-Trp-Glu-Leu-Arg53) in the small bowel was demonstrated by N-terminal sequencing and mass spectrometry. The degradation patterns were reflected by changes in migration of products on SDS-PAGE and in subsequent binding activities to the EGF receptor and anti-EGF antibodies. The data show that a human EGF fusion protein can be produced efficiently in a bacterial expression system and that it retains biological activity in vitro. Although the extension peptide was rapidly cleaved from Long EGF in both stomach and small bowel producing similar biological activity to native EGF, it could not prevent subsequent degradation of the EGF domain. Other strategies are being investigated to develop an effective oral form of EGF that resists digestion by proteases in the gastrointestinal tract.

scholarly journals Critical cysteine residues for regulation of integrin alphaIIbbeta3 are clustered in the epidermal growth factor domains of the beta3 subunit

2004 ◽  
Vol 378 (3) ◽  
pp. 1079-1082 ◽  
Author(s):  
Tetsuji KAMATA ◽  
Hironobu AMBO ◽  
Wilma PUZON-McLAUGHLIN ◽  
Kenneth Khiem TIEU ◽  
Makoto HANDA ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cysteine Residue ◽  
Cysteine Residues ◽  
Disulphide Bonds ◽  
Binding Function ◽  
Beta3 Subunit ◽  
Epidermal Growth ◽  
Egf Domain

Chemical or enzymic reduction/oxidation of integrin cysteine residues (e.g. by reducing agents and protein disulphide isomerase) may be a mechanism for regulating integrin function. It has also been proposed that unique cysteine residues in the integrin β3 subunit are involved in the regulation of αIIbβ3. In the present study, we studied systematically the role of disulphide bonds in β3 on the ligand-binding function of αIIbβ3 by mutating individual cysteine residues of β3 to serine. We found that the disulphide bonds that are critical for αIIbβ3 regulation are clustered within the EGF (epidermal growth factor) domains. Interestingly, disrupting only a single disulphide bond in the EGF domains was enough to activate αIIbβ3 fully. In contrast, only two (of 13) disulphide bonds tested outside the EGF domains activated αIIbβ3. These results suggest that the disulphide bonds in the EGF domains should be intact to keep αIIbβ3 in an inactive state, and that there is no unique cysteine residue in the EGF domain critical for regulating the receptor. The cysteine residues in the EGF domains are potential targets for chemical or enzymic reduction.

scholarly journals Structure, function, and pathology of protein O-glucosyltransferases

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Muhammad Zubair Mehboob ◽  
Minglin Lang
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cancer Biology ◽  
Transforming Growth Factor ◽  
Consensus Sequence ◽  
Human Diseases ◽  
Systemic Review ◽  
Cell Proliferation Inhibition ◽  
Epidermal Growth ◽  
Aberrant Protein

AbstractProtein O-glucosylation is a crucial form of O-glycosylation, which involves glucose (Glc) addition to a serine residue within a consensus sequence of epidermal growth factor epidermal growth factor (EGF)-like repeats found in several proteins, including Notch. Glc provides stability to EGF-like repeats, is required for S2 cleavage of Notch, and serves to regulate the trafficking of Notch, crumbs2, and Eyes shut proteins to the cell surface. Genetic and biochemical studies have shown a link between aberrant protein O-glucosylation and human diseases. The main players of protein O-glucosylation, protein O-glucosyltransferases (POGLUTs), use uridine diphosphate (UDP)-Glc as a substrate to modify EGF repeats and reside in the endoplasmic reticulum via C-terminal KDEL-like signals. In addition to O-glucosylation activity, POGLUTs can also perform protein O-xylosylation function, i.e., adding xylose (Xyl) from UDP-Xyl; however, both activities rely on residues of EGF repeats, active-site conformations of POGLUTs and sugar substrate concentrations in the ER. Impaired expression of POGLUTs has been associated with initiation and progression of human diseases such as limb-girdle muscular dystrophy, Dowling–Degos disease 4, acute myeloid leukemia, and hepatocytes and pancreatic dysfunction. POGLUTs have been found to alter the expression of cyclin-dependent kinase inhibitors (CDKIs), by affecting Notch or transforming growth factor-β1 signaling, and cause cell proliferation inhibition or induction depending on the particular cell types, which characterizes POGLUT’s cell-dependent dual role. Except for a few downstream elements, the precise mechanisms whereby aberrant protein O-glucosylation causes diseases are largely unknown, leaving behind many questions that need to be addressed. This systemic review comprehensively covers literature to understand the O-glucosyltransferases with a focus on POGLUT1 structure and function, and their role in health and diseases. Moreover, this study also raises unanswered issues for future research in cancer biology, cell communications, muscular diseases, etc.

scholarly journals Factor IXHollywood: substitution of Pro55 by Ala in the first epidermal growth factor-like domain

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1530-1537 ◽  
Author(s):  
SG Spitzer ◽  
MN Kuppuswamy ◽  
R Saini ◽  
CK Kasper ◽  
JJ Birktoft ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Binding Site ◽  
Structural Changes ◽  
Factor Ix ◽  
Single Mutation ◽  
Beta Turn ◽  
Factor Viiia ◽  
Epidermal Growth ◽  
Egf Domain

Factor IX is a multidomain protein essential for hemostasis. We describe a mutation in a patient affecting the first epidermal growth factor (EGF)-like domain of the protein. All exons and the promoter region of the gene were amplified by the polymerase chain reaction method, and sequenced. Only a single mutation (C----G), that predicts the substitution of Pro55 by Ala in the first EGF domain was found in the patient's gene. This mutation leads to new restriction sites for four enzymes. One new site (Nsi) was tested in the amplified exon IV fragment and was shown to provide a rapid and reliable marker for carrier detection and prenatal diagnosis in the affected family. The factor IX protein, termed factor IXHollywood (IXHW), was isolated to homogeneity from the patient's plasma. As compared with normal factor IX (IXN), IXHW contained the same amount of gamma-carboxy glutamic acid but twice the amount of beta-OH aspartic acid. Both IXHW and IXN contained no detectable free -SH groups. Further, IXHW could be readily cleaved to yield a factor IXa-like molecule by factor Xla/Ca2+. However, IXaHW (compared with IXaN) activated factor X approximately twofold slower in the presence of Ca2+ and phospholipid (PL), and 8- to 12-fold slower in the presence of Ca2+, PL, and factor VIIIa. Additionally, IXaHW had only approximately 10% of the activity of IXaN in an aPTT assay. In agreement with the nuclear magnetic resonance- derived structure of EGF, the Chou-Fasman algorithm strongly predicted a beta turn involving residues Asn-Pro55-Cys-Leu in IXN. Replacement of Pro55 by Ala gave a fourfold decrease in the beta turn probability for this peptide, suggesting a change(s) in the secondary structure in the EGF domain of IXHW. Since this domain of IXN is thought to have one high-affinity Ca2+ binding site and may be involved in PL and/or factor VIIIa binding, the localized secondary structural changes in IXHW could lead to distortion of the binding site(s) for the cofactor(s) and, thus, a dysfunctional molecule.

scholarly journals Factor VII Deficiency Caused by a Structural Variant N57D of the First Epidermal Growth Factor Domain

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 142-148
Author(s):  
Blair J.N. Leonard ◽  
Qi Chen ◽  
Morris A. Blajchman ◽  
Frederick A. Ofosu ◽  
Sampath Sridhara ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tissue Factor ◽  
Procoagulant Activity ◽  
Factor Vii ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
293 Cells ◽  
Epidermal Growth ◽  
Egf Domain

We have previously described a kindred with factor VII (FVII) deficiency whose members exhibited reduced procoagulant activity relative to FVII antigen concentration. In this report, the molecular genetic basis of the FVII defect has been determined to be a heterozygous substitution of Asp for Asn at position 57 in the first epidermal growth factor (EGF) domain. Recombinant FVII (N57D) cDNA was created by site-directed mutagenesis and transiently expressed in human 293 cells. The transfected cells synthesized an immunoprecipitable protein with an apparent molecular weight of 50 kD. Quantitation of expression by FVII enzyme-linked immunosorbent assay indicated that mutant protein yields were consistently low, typically 10% to 30% of wild-type FVII. FVII (N57D) protein did not accumulate intracellularly, and Northern blot analysis indicated equivalent FVII mRNA levels in 293 cells expressing either wild-type FVII or FVII (N57D). Secreted FVII (N57D) protein did not bind tissue factor, exhibited no procoagulant activity, and failed to bind a conformation-dependent monoclonal antibody specific for the first EGF domain of FVII. Molecular modeling of the first EGF domain of FVII predicted that the N57D amino acid substitution would disrupt tertiary bonding structure. We conclude that the N57D mutation affects folding of the first EGF domain of FVII resulting in decreased cellular secretion of a mutant FVII molecule, which is unable to bind tissue factor and is therefore biologically inactive.

scholarly journals Studies on the mechanisms of autophagy: formation of the autophagic vacuole.

1990 ◽  
Vol 110 (6) ◽  
pp. 1923-1933 ◽  
Author(s):  
W A Dunn
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Membrane Proteins ◽  
Epidermal Growth Factor ◽  
Golgi Apparatus ◽  
Integral Membrane Proteins ◽  
Secretory Proteins ◽  
Rat Serum ◽  
Autophagic Vacuoles ◽  
Epidermal Growth

Autophagic vacuoles form within 15 min of perfusing a liver with amino acid-depleted medium. These vacuoles are bound by a "smooth" double membrane and do not contain acid phosphatase activity. In an attempt to identify the membrane source of these vacuoles, I have used morphological techniques combined with immunological probes to localize specific membrane antigens to the limiting membranes of newly formed or nascent autophagic vacuoles. Antibodies to three integral membrane proteins of the plasma membrane (CE9, HA4, and epidermal growth factor receptor) and one of the Golgi apparatus (sialyltransferase) did not label these vacuoles. Internalized epidermal growth factor and its membrane receptor were not found in nascent autophagic vacuoles but were present in lysosome-like degradative autophagic vacuoles. All these results suggested that autophagic vacuoles were not formed from plasma membrane, Golgi apparatus, or endosome constituents. Antisera prepared against integral membrane proteins (14, 25, and 40 kD) of the RER was found to label the inner and outer limiting membranes of almost all nascent autophagic vacuoles. In addition, ribophorin II was identified at the limiting membranes of many nascent autophagic vacuoles. Finally, secretory proteins, rat serum albumin and alpha 2u-globulin, were localized to the lumen of the RER and to the intramembrane space between the inner and outer membranes of some of these vacuoles. The results were consistent with the formation of autophagic vacuoles from ribosome-free regions of the RER.

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