Glycosylation of the murine estrogen receptor-α
Identifieur interne : 000824 ( Istex/Corpus ); précédent : 000823; suivant : 000825Glycosylation of the murine estrogen receptor-α
Auteurs : Xiaogang Cheng ; Gerald W. HartSource :
- Journal of Steroid Biochemistry and Molecular Biology [ 0960-0760 ] ; 2000.
English descriptors
- KwdEn :
- 575-mER-α, mER-α O-GlcNAc site mutant, CHCA, α-cyano-4-hydroxylcinnamic acid, CID-MS, collision induced dissociation of tandem mass spectrometry, Estrogen, Estrogen receptor, Gal, galactose, HPLC, high performance liquid chromatography, LC/ESI–MS, liquid chromatography coupled electrospray ionization mass spectrometry, MALDI-TOF, matrix-assisted laser desorption ionization time of flight, O-GlcNAc, O-GlcNAc, O-linked N-acetylglucosamine, O-glycosylation, PAGE, polyacrylamide gel electrophoresis, PEST domain, Phosphorylation, Post-translational modification, RP, reverse phase, SDS, sodium dodecyl sulfate, TFA, trifluoroacetic acid, WGA, wheat germ agglutinin, galactosyltransferase, Galβ (1-4) galactosyltransferase, mER-α, murine estrogen receptor-α, wt, wild type.
- Teeft :
- Acetonitrile, Additional sites, Aliquot, Amino, Amino acid, Amino acids, Amino terminus, Biochemistry, Biol, Boehringer mannheim, Carboxyl terminus, Casein kinase, Cdna, Cell viability, Chem, Cheng, Coactivator turnover, Cold acetone, Cytoskeletal proteins, Degradation, Domain deletion, Dynamic glycosylation, Ecori site, Edman, Elsevier science, Estrogen, Estrogen receptor, Estrogen receptors, Galactosyltransferase, Glcnac, Glycopeptide, Glycopeptides, Glycosylation, Glycosylation sites, Hart journal, Hplc, Human estrogen receptor, Insect cells, Kinase, Linear gradient, Liquid chromatography, Liquid scintillation, Major site, Major tritium peak, Major tritium peaks, Manual edman degradation, Mass species, Mass spectrometry, Metal resin, Moiety, Monoclonal antibody, Murine estrogen, Mutant, Nuclear proteins, Oxidized peptide, Peptide, Pest domains, Pest regions, Pest sequences, Phase sequencing, Phosphorylation, Previous studies, Proline endopeptidase, Protein kinase, Rapid protein turnover, Receptor, Recombinant virus, Salicylic acid, Same condition, Same hydroxyl moiety, Second dimension, Shallow gradient, Spectrometry, Steroid, Steroid biochem, Steroid biochemistry, Transactivation domain, Transcription, Transcription factors, Tritium, Tritium counts, Tritium peak fraction, Tryptic, Tryptic fragments, Tryptic peptides, Vivo sites, Western blot, Wild type.
Abstract
Abstract: O-linked N-acetylglucosamine (O-GlcNAc) is a highly dynamic and abundant modification found on nuclear and cytoplasmic proteins of nearly all eukaryotes. O-GlcNAc addition is required for life at the single cell level and is analogous to protein phosphorylation in most respects. In a previous study (M.S. Jiang, G.W. Hart, A subpopulation of estrogen receptors are modified by O-linked N-acetylglucosamine. J. Biol. Chem. 270 (1997) 2421–2428), we demonstrated that a subpopulation of the murine estrogen receptor-α (mER-α) is modified by O-GlcNAc at Thr575. Here we mutated mER-α to convert Thr575 and Ser576 to Val and Ala, respectively. Surprisingly, this glycosylation-site mutant is still extensively modified by O-GlcNAc. Analyses of glycopeptides identified two additional sites of modification on mER-α, at Ser10 and Thr50 near the N-terminus. The major glycosylation sites are within or near PEST regions, suggesting that O-GlcNAc may regulate mER-α turnover.
Url:
DOI: 10.1016/S0960-0760(00)00167-9
Links to Exploration step
ISTEX:80DD63B048E65F8E013413F093CC3E50F9B017CALe document en format XML
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Hart</name><affiliation><mods:affiliation>E-mail: gwhart@jhmi.edu</mods:affiliation></affiliation><affiliation><mods:affiliation>Department of Biological Chemistry, School of Medicine, Johns Hopkins University, 725 N Wolfe St., Baltimore, MD 21205-2185, USA</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:80DD63B048E65F8E013413F093CC3E50F9B017CA</idno><date when="2001" year="2001">2001</date><idno type="doi">10.1016/S0960-0760(00)00167-9</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-6LH2X1L0-Q/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000824</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000824</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Glycosylation of the murine estrogen receptor-α</title><author><name sortKey="Cheng, Xiaogang" sort="Cheng, Xiaogang" uniqKey="Cheng X" first="Xiaogang" last="Cheng">Xiaogang Cheng</name><affiliation><mods:affiliation>Department of Biological Chemistry, School of Medicine, Johns Hopkins University, 725 N Wolfe St., Baltimore, MD 21205-2185, USA</mods:affiliation></affiliation></author><author><name sortKey="Hart, Gerald W" sort="Hart, Gerald W" uniqKey="Hart G" first="Gerald W." last="Hart">Gerald W. Hart</name><affiliation><mods:affiliation>E-mail: gwhart@jhmi.edu</mods:affiliation></affiliation><affiliation><mods:affiliation>Department of Biological Chemistry, School of Medicine, Johns Hopkins University, 725 N Wolfe St., Baltimore, MD 21205-2185, USA</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Steroid Biochemistry and Molecular Biology</title><title level="j" type="abbrev">SBMB</title><idno type="ISSN">0960-0760</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000">2000</date><biblScope unit="volume">75</biblScope><biblScope unit="issue">2–3</biblScope><biblScope unit="page" from="147">147</biblScope><biblScope unit="page" to="158">158</biblScope></imprint><idno type="ISSN">0960-0760</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0960-0760</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>575-mER-α, mER-α O-GlcNAc site mutant</term><term>CHCA, α-cyano-4-hydroxylcinnamic acid</term><term>CID-MS, collision induced dissociation of tandem mass spectrometry</term><term>Estrogen</term><term>Estrogen receptor</term><term>Gal, galactose</term><term>HPLC, high performance liquid chromatography</term><term>LC/ESI–MS, liquid chromatography coupled electrospray ionization mass spectrometry</term><term>MALDI-TOF, matrix-assisted laser desorption ionization time of flight</term><term>O-GlcNAc</term><term>O-GlcNAc, O-linked N-acetylglucosamine</term><term>O-glycosylation</term><term>PAGE, polyacrylamide gel electrophoresis</term><term>PEST domain</term><term>Phosphorylation</term><term>Post-translational modification</term><term>RP, reverse phase</term><term>SDS, sodium dodecyl sulfate</term><term>TFA, trifluoroacetic acid</term><term>WGA, wheat germ agglutinin</term><term>galactosyltransferase, Galβ (1-4) galactosyltransferase</term><term>mER-α, murine estrogen receptor-α</term><term>wt, wild type</term></keywords><keywords scheme="Teeft" xml:lang="en"><term>Acetonitrile</term><term>Additional sites</term><term>Aliquot</term><term>Amino</term><term>Amino acid</term><term>Amino acids</term><term>Amino terminus</term><term>Biochemistry</term><term>Biol</term><term>Boehringer mannheim</term><term>Carboxyl terminus</term><term>Casein kinase</term><term>Cdna</term><term>Cell viability</term><term>Chem</term><term>Cheng</term><term>Coactivator turnover</term><term>Cold acetone</term><term>Cytoskeletal proteins</term><term>Degradation</term><term>Domain deletion</term><term>Dynamic glycosylation</term><term>Ecori site</term><term>Edman</term><term>Elsevier science</term><term>Estrogen</term><term>Estrogen receptor</term><term>Estrogen receptors</term><term>Galactosyltransferase</term><term>Glcnac</term><term>Glycopeptide</term><term>Glycopeptides</term><term>Glycosylation</term><term>Glycosylation sites</term><term>Hart journal</term><term>Hplc</term><term>Human estrogen receptor</term><term>Insect cells</term><term>Kinase</term><term>Linear gradient</term><term>Liquid chromatography</term><term>Liquid scintillation</term><term>Major site</term><term>Major tritium peak</term><term>Major tritium peaks</term><term>Manual edman degradation</term><term>Mass species</term><term>Mass spectrometry</term><term>Metal resin</term><term>Moiety</term><term>Monoclonal antibody</term><term>Murine estrogen</term><term>Mutant</term><term>Nuclear proteins</term><term>Oxidized peptide</term><term>Peptide</term><term>Pest domains</term><term>Pest regions</term><term>Pest sequences</term><term>Phase sequencing</term><term>Phosphorylation</term><term>Previous studies</term><term>Proline endopeptidase</term><term>Protein kinase</term><term>Rapid protein turnover</term><term>Receptor</term><term>Recombinant virus</term><term>Salicylic acid</term><term>Same condition</term><term>Same hydroxyl moiety</term><term>Second dimension</term><term>Shallow gradient</term><term>Spectrometry</term><term>Steroid</term><term>Steroid biochem</term><term>Steroid biochemistry</term><term>Transactivation domain</term><term>Transcription</term><term>Transcription factors</term><term>Tritium</term><term>Tritium counts</term><term>Tritium peak fraction</term><term>Tryptic</term><term>Tryptic fragments</term><term>Tryptic peptides</term><term>Vivo sites</term><term>Western blot</term><term>Wild type</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: O-linked N-acetylglucosamine (O-GlcNAc) is a highly dynamic and abundant modification found on nuclear and cytoplasmic proteins of nearly all eukaryotes. O-GlcNAc addition is required for life at the single cell level and is analogous to protein phosphorylation in most respects. In a previous study (M.S. Jiang, G.W. Hart, A subpopulation of estrogen receptors are modified by O-linked N-acetylglucosamine. J. Biol. Chem. 270 (1997) 2421–2428), we demonstrated that a subpopulation of the murine estrogen receptor-α (mER-α) is modified by O-GlcNAc at Thr575. Here we mutated mER-α to convert Thr575 and Ser576 to Val and Ala, respectively. Surprisingly, this glycosylation-site mutant is still extensively modified by O-GlcNAc. Analyses of glycopeptides identified two additional sites of modification on mER-α, at Ser10 and Thr50 near the N-terminus. The major glycosylation sites are within or near PEST regions, suggesting that O-GlcNAc may regulate mER-α turnover.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>estrogen</json:string><json:string>glycopeptide</json:string><json:string>biol</json:string><json:string>tryptic</json:string><json:string>receptor</json:string><json:string>tritium</json:string><json:string>phosphorylation</json:string><json:string>chem</json:string><json:string>peptide</json:string><json:string>biochemistry</json:string><json:string>mutant</json:string><json:string>steroid biochemistry</json:string><json:string>acetonitrile</json:string><json:string>hart journal</json:string><json:string>glycosylation</json:string><json:string>hplc</json:string><json:string>spectrometry</json:string><json:string>edman</json:string><json:string>kinase</json:string><json:string>galactosyltransferase</json:string><json:string>cdna</json:string><json:string>steroid</json:string><json:string>glcnac</json:string><json:string>aliquot</json:string><json:string>glycopeptides</json:string><json:string>moiety</json:string><json:string>manual edman degradation</json:string><json:string>cheng</json:string><json:string>shallow gradient</json:string><json:string>liquid scintillation</json:string><json:string>estrogen receptor</json:string><json:string>tritium counts</json:string><json:string>amino</json:string><json:string>mass spectrometry</json:string><json:string>tryptic peptides</json:string><json:string>amino acids</json:string><json:string>major site</json:string><json:string>estrogen receptors</json:string><json:string>carboxyl terminus</json:string><json:string>metal resin</json:string><json:string>pest sequences</json:string><json:string>degradation</json:string><json:string>proline endopeptidase</json:string><json:string>insect cells</json:string><json:string>western blot</json:string><json:string>phase sequencing</json:string><json:string>previous studies</json:string><json:string>human estrogen receptor</json:string><json:string>additional sites</json:string><json:string>mass species</json:string><json:string>transcription factors</json:string><json:string>domain deletion</json:string><json:string>linear gradient</json:string><json:string>protein kinase</json:string><json:string>transcription</json:string><json:string>rapid protein turnover</json:string><json:string>boehringer mannheim</json:string><json:string>cytoskeletal proteins</json:string><json:string>pest regions</json:string><json:string>wild type</json:string><json:string>ecori site</json:string><json:string>same hydroxyl moiety</json:string><json:string>murine estrogen</json:string><json:string>amino acid</json:string><json:string>liquid chromatography</json:string><json:string>tryptic fragments</json:string><json:string>major tritium peaks</json:string><json:string>cell viability</json:string><json:string>nuclear proteins</json:string><json:string>recombinant virus</json:string><json:string>vivo sites</json:string><json:string>second dimension</json:string><json:string>major tritium peak</json:string><json:string>casein kinase</json:string><json:string>monoclonal antibody</json:string><json:string>oxidized peptide</json:string><json:string>amino terminus</json:string><json:string>salicylic acid</json:string><json:string>cold acetone</json:string><json:string>same condition</json:string><json:string>tritium peak fraction</json:string><json:string>coactivator turnover</json:string><json:string>transactivation domain</json:string><json:string>pest domains</json:string><json:string>elsevier science</json:string><json:string>steroid biochem</json:string><json:string>dynamic glycosylation</json:string><json:string>glycosylation sites</json:string></teeft></keywords><author><json:item><name>Xiaogang Cheng</name><affiliations><json:string>Department of Biological Chemistry, School of Medicine, Johns Hopkins University, 725 N Wolfe St., Baltimore, MD 21205-2185, USA</json:string></affiliations></json:item><json:item><name>Gerald W. Hart</name><affiliations><json:string>E-mail: gwhart@jhmi.edu</json:string><json:string>Department of Biological Chemistry, School of Medicine, Johns Hopkins University, 725 N Wolfe St., Baltimore, MD 21205-2185, USA</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Estrogen receptor</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>O-glycosylation</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>O-GlcNAc</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>PEST domain</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Post-translational modification</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Phosphorylation</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Estrogen</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>CHCA, α-cyano-4-hydroxylcinnamic acid</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>CID-MS, collision induced dissociation of tandem mass spectrometry</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>galactosyltransferase, Galβ (1-4) galactosyltransferase</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Gal, galactose</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>HPLC, high performance liquid chromatography</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>LC/ESI–MS, liquid chromatography coupled electrospray ionization mass spectrometry</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>MALDI-TOF, matrix-assisted laser desorption ionization time of flight</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>mER-α, murine estrogen receptor-α</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>575-mER-α, mER-α O-GlcNAc site mutant</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>O-GlcNAc, O-linked N-acetylglucosamine</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>PAGE, polyacrylamide gel electrophoresis</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>RP, reverse phase</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>SDS, sodium dodecyl sulfate</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>TFA, trifluoroacetic acid</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>WGA, wheat germ agglutinin</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>wt, wild type</value></json:item></subject><arkIstex>ark:/67375/6H6-6LH2X1L0-Q</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: O-linked N-acetylglucosamine (O-GlcNAc) is a highly dynamic and abundant modification found on nuclear and cytoplasmic proteins of nearly all eukaryotes. O-GlcNAc addition is required for life at the single cell level and is analogous to protein phosphorylation in most respects. In a previous study (M.S. Jiang, G.W. Hart, A subpopulation of estrogen receptors are modified by O-linked N-acetylglucosamine. J. Biol. Chem. 270 (1997) 2421–2428), we demonstrated that a subpopulation of the murine estrogen receptor-α (mER-α) is modified by O-GlcNAc at Thr575. Here we mutated mER-α to convert Thr575 and Ser576 to Val and Ala, respectively. Surprisingly, this glycosylation-site mutant is still extensively modified by O-GlcNAc. Analyses of glycopeptides identified two additional sites of modification on mER-α, at Ser10 and Thr50 near the N-terminus. The major glycosylation sites are within or near PEST regions, suggesting that O-GlcNAc may regulate mER-α turnover.</abstract><qualityIndicators><score>8.692</score><pdfWordCount>5742</pdfWordCount><pdfCharCount>35680</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>12</pdfPageCount><pdfPageSize>552 x 768 pts</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>141</abstractWordCount><abstractCharCount>979</abstractCharCount><keywordCount>23</keywordCount></qualityIndicators><title>Glycosylation of the murine estrogen receptor-α</title><pmid><json:string>11226831</json:string></pmid><pii><json:string>S0960-0760(00)00167-9</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Journal of Steroid Biochemistry and Molecular Biology</title><language><json:string>unknown</json:string></language><publicationDate>2000</publicationDate><issn><json:string>0960-0760</json:string></issn><pii><json:string>S0960-0760(00)X0070-2</json:string></pii><volume>75</volume><issue>2–3</issue><pages><first>147</first><last>158</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>2001</json:string><json:string>35S</json:string><json:string>26S</json:string></date><geogName></geogName><orgName><json:string>Seikagaku Corp., Tokyo, Japan</json:string><json:string>Department of Biochemistry and Molecular Genetics</json:string><json:string>Clontech, Palo Alto</json:string><json:string>Elsevier Science Ltd.</json:string><json:string>Abbott Laboratories</json:string></orgName><orgName_funder></orgName_funder><orgName_provider></orgName_provider><persName><json:string>B. X. Cheng</json:string><json:string>J. Biol</json:string><json:string>X. Cheng</json:string><json:string>The</json:string><json:string>M.S. Jiang</json:string><json:string>M.G. Parker</json:string><json:string>G.W. 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The wt–mER-α cDNA was mutated at 575 (Thr→Val) and 576 (Ser→Ala). The mutated mER-α was designated 575-mER-α. Both cDNAs were engineered with a PCR mediated method to incorporate His6 tag at the carboxyl terminal end.</note><note type="content">Fig. 2: Characterization of 575-mER-α by WGA chromatography. The mER-α cDNAs were in vitro translated with rabbit reticulocyte lysate in presence of 35S-Met. The translated proteins were individually loaded onto WGA columns and challenged with 1 M Gal. Glycosylated pools of ERs were eluted with 1 M GlcNAc. Fractions (0.5 ml) were collected and monitored by liquid scintillation counting. (A) 35S-WGA chromatography profiles. Upper panel, 575-mER-α; lower panel, wt–mER-α. (B) Proteins in each major fraction were precipitated and resolved by 8% SDS-PAGE gel. The gels were dried and exposed to X-ray film. Lane L, load; lane FT, flow through; lane W, 1 M Gal challenge; lane E, 1 M GlcNAc elute.</note><note type="content">Fig. 3: Purification of His6 tagged mER-α from Sf9 cells. mER-α expressed in Sf9 cells was purified with metal affinity resin (lane L, FT, and E are load, flow through and elute) and subsequently purified with preparative SDS-PAGE (lane E–P is the prep-cell elute). Aliquots of each fraction were resolved on an analytical 8% SDS-PAGE gel. The gel was either stained with Coomassie Blue or immunoblotted with monoclonal antibody H222 against ER.</note><note type="content">Fig. 4: Galactosyltransferase labeling and tryptic maps of wt–mER-α and 575-mER-α. (A) Purified 575-mER-α was labeled with UDP-[3H]Gal using galactosyltransferase and subsequently desalted over a Sephadex G50 column. The protein was precipitated by cold acetone, resuspended in 0.1 M sodium bicarbonate (pH 7.0), and digested with trypsin at a ratio of 1:50 (w/w). Aliquots of each sample were examined by 8% SDS-PAGE gel. The gel was stained with Coomassie Blue R250, impregnated with 1 M salicylic acid, dried down, and exposed to X-ray film at −70°C overnight. (B) [3H]Gal-labeled 575-mER-α tryptic peptides were applied to a C18 RP column. Peptides were eluted with a 90-min linear gradient of 0–60% acetonitrile (v/v) in 0.1% TFA from 6 to 96 min at a flow rate of 0.1 ml min−1. Fractions (0.2 ml) were collected and 1% of each was counted. Two major tritium peaks were designated as A and B, respectively. (C) Wild type mER-α was purified and labeled under the same condition as the mutant 575-mER-α. [3H]Gal-labeled wt–mER-α tryptic glycopeptides were applied to a C18 RP column. The column was developed under the same condition as panel B.</note><note type="content">Fig. 5: Thr575 is the major O-GlcNAcylation site on wt–mER-α. (A) The major [3H] Gal-labeled wt–mER-α tryptic fragment eluting from a C18 RP column at 55 min (Fig. 4C). This was further purified by a shallow gradient of 15–45% acetonitrile in 0.1% TFA over 90 min at a flow rate of 0.1 ml min−1 on a C18 RP column. The fraction carrying the most tritium counts was subjected to gas phase sequencing. The identity of the glycopeptide is indicated. (B) An aliquot of the tritium peak fraction from panel A was mixed with the matrix CHCA (5 mg ml−1 in 0.3% TFA/50% acetonitrile) and detected by MALDI TOF. Two mass species were detected and analyzed. (C) The major [3H]Gal-labeled mER-α tryptic C-terminal fragment was digested with proline specific endopeptidase at 37°C overnight and purified by a shallow gradient of 0–30% (v/v) acetonitrile in 0.1% TFA over 90 min at a flow rate of 0.1 ml min−1 on a C18 RP column. (D) The fraction of [3H]Gal-labeled glycopeptide from panel C was coupled to Sequelon-AA disk (Millipore) and subjected to manual Edman degradation. The tritium counts released from each cycle were monitored using liquid scintillation counting. The major O-GlcNAcylation site on wt–mER-α was identified at Thr575.</note><note type="content">Fig. 6: Ser10 is the O-GlcNAcylation site on glycopeptide A. (A) [3H] Gal-labeled 575-mER-α tryptic glycopeptide A, eluting from a C18 RP column at 52 min (Fig. 4B), was further purified by a shallow gradient of 15–45% (v/v) acetonitrile in 0.1% TFA over 90 min at a flow rate of 0.1 ml min−1 on a C18 RP column. (B) An aliquot of the tritium peak fraction from panel A was mixed with CHCA as reported in Fig. 5 and detected by MALDI-TOF. Three mass species were detected and their deduced identity is indicated. (C) An aliquot of the fraction A from the second dimension (A) was subjected to LC/ESI-MS/MS analysis. For clarification, predicted b-and y-ions of the oxidized peptide are listed below the spectrum. The methionine at the position four was oxidized resulting in a mass addition of 16 Da. The matched ions are indicated in the spectrum. Some unmatched ions are probably due to either internal cleavage or loss of H2O during the fragmentation process. (D) The fraction of [3H]Gal-labeled glycopeptide A from panel A was coupled to Sequelon-AA disk (Millipore) and subjected to manual Edman degradation. The tritium counts released in each cycle were monitored using liquid scintillation counting. The O-GlcNAc site on glycopeptide A was identified at Ser10.</note><note type="content">Fig. 7: Thr50 is the O-GlcNAcylation site on glycopeptide B. (A) [3H]Gal-labeled 575-mER-α glycopeptide B eluted at 74 min (Fig. 4B) was further digested with endopeptidase Glu-C in presence of 2 M urea and applied to a C18 RP column. The column was developed with a 90-min linear gradient of 0–60% (v/v) acetonitrile in 0.1% TFA at a flow rate of 0.1 ml min−1. The major tritium peak was designated as Glu-C B. (B) An aliquot of the fraction containing Glu-C B was subjected to LC/ESI-MS/MS analysis. The ESI-MS/MS spectrum of the peptide modified by O-GlcNAc is shown. For clarification, predicted b- and y-ions of the peptide are listed below the spectrum. (C) The Glu-C B fraction was coupled to Sequelon-AA disk (Millipore) and subjected to manual Edman degradation. The tritium counts released in each cycle were monitored using liquid scintillation counting. The O-GlcNAc site on glycopeptide B was identified at Thr50.</note><note type="content">Fig. 8: Summary of O-GlcNAcylation sites on mER-α. mER-α's domain structure is indicated. N′ and C′ indicate amino terminal and carboxyl terminal ends. In this study, two O-GlcNAc sites on mER-α were identified near amino terminus at Ser10 and Thr50. The PEST scores regarding the regions underlined near O-GlcNAc sites are also indicated.</note><note type="content">Table 1: Summary of O-GlcNAcylated proteins and in vivo attachment sitesa</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Glycosylation of the murine estrogen receptor-α</title><author xml:id="author-0000"><persName><forename type="first">Xiaogang</forename><surname>Cheng</surname></persName><affiliation>Department of Biological Chemistry, School of Medicine, Johns Hopkins University, 725 N Wolfe St., Baltimore, MD 21205-2185, USA</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Gerald W.</forename><surname>Hart</surname></persName><email>gwhart@jhmi.edu</email><note type="correspondence"><p>Corresponding author. Tel.: +1-410-6145993; fax: +1-410-6148804</p></note><affiliation>Department of Biological Chemistry, School of Medicine, Johns Hopkins University, 725 N Wolfe St., Baltimore, MD 21205-2185, USA</affiliation></author><idno type="istex">80DD63B048E65F8E013413F093CC3E50F9B017CA</idno><idno type="ark">ark:/67375/6H6-6LH2X1L0-Q</idno><idno type="DOI">10.1016/S0960-0760(00)00167-9</idno><idno type="PII">S0960-0760(00)00167-9</idno></analytic><monogr><title level="j">Journal of Steroid Biochemistry and Molecular Biology</title><title level="j" type="abbrev">SBMB</title><idno type="pISSN">0960-0760</idno><idno type="PII">S0960-0760(00)X0070-2</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000"></date><biblScope unit="volume">75</biblScope><biblScope unit="issue">2–3</biblScope><biblScope unit="page" from="147">147</biblScope><biblScope unit="page" to="158">158</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2001</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: O-linked N-acetylglucosamine (O-GlcNAc) is a highly dynamic and abundant modification found on nuclear and cytoplasmic proteins of nearly all eukaryotes. O-GlcNAc addition is required for life at the single cell level and is analogous to protein phosphorylation in most respects. In a previous study (M.S. Jiang, G.W. Hart, A subpopulation of estrogen receptors are modified by O-linked N-acetylglucosamine. J. Biol. Chem. 270 (1997) 2421–2428), we demonstrated that a subpopulation of the murine estrogen receptor-α (mER-α) is modified by O-GlcNAc at Thr575. Here we mutated mER-α to convert Thr575 and Ser576 to Val and Ala, respectively. Surprisingly, this glycosylation-site mutant is still extensively modified by O-GlcNAc. Analyses of glycopeptides identified two additional sites of modification on mER-α, at Ser10 and Thr50 near the N-terminus. The major glycosylation sites are within or near PEST regions, suggesting that O-GlcNAc may regulate mER-α turnover.</p></abstract><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>Estrogen receptor</term></item><item><term>O-glycosylation</term></item><item><term>O-GlcNAc</term></item><item><term>PEST domain</term></item><item><term>Post-translational modification</term></item><item><term>Phosphorylation</term></item><item><term>Estrogen</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Abbreviations</head><item><term>CHCA, α-cyano-4-hydroxylcinnamic acid</term></item><item><term>CID-MS, collision induced dissociation of tandem mass spectrometry</term></item><item><term>galactosyltransferase, Galβ (1-4) galactosyltransferase</term></item><item><term>Gal, galactose</term></item><item><term>HPLC, high performance liquid chromatography</term></item><item><term>LC/ESI–MS, liquid chromatography coupled electrospray ionization mass spectrometry</term></item><item><term>MALDI-TOF, matrix-assisted laser desorption ionization time of flight</term></item><item><term>mER-α, murine estrogen receptor-α</term></item><item><term>575-mER-α, mER-α O-GlcNAc site mutant</term></item><item><term>O-GlcNAc, O-linked N-acetylglucosamine</term></item><item><term>PAGE, polyacrylamide gel electrophoresis</term></item><item><term>RP, reverse phase</term></item><item><term>SDS, sodium dodecyl sulfate</term></item><item><term>TFA, trifluoroacetic acid</term></item><item><term>WGA, wheat germ agglutinin</term></item><item><term>wt, wild type</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2000">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-6LH2X1L0-Q/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="gr5"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="gr6"></istex:entity><istex:entity SYSTEM="gr7" NDATA="IMAGE" name="gr7"></istex:entity><istex:entity SYSTEM="gr8" NDATA="IMAGE" name="gr8"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>SBMB</jid><aid>1482</aid><ce:pii>S0960-0760(00)00167-9</ce:pii><ce:doi>10.1016/S0960-0760(00)00167-9</ce:doi><ce:copyright type="full-transfer" year="2001">Elsevier Science Ltd</ce:copyright></item-info><head><ce:title>Glycosylation of the murine estrogen receptor-α</ce:title><ce:author-group><ce:author><ce:given-name>Xiaogang</ce:given-name><ce:surname>Cheng</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="AFF2"><ce:sup>b</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Gerald W.</ce:given-name><ce:surname>Hart</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="CORR1">*</ce:cross-ref><ce:e-address>gwhart@jhmi.edu</ce:e-address></ce:author><ce:affiliation id="AFF1"><ce:label>a</ce:label><ce:textfn>Department of Biological Chemistry, School of Medicine, Johns Hopkins University, 725 N Wolfe St., Baltimore, MD 21205-2185, USA</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>b</ce:label><ce:textfn>Graduate Program of Department of Biochemistry and Molecular Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294, USA</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Corresponding author. Tel.: +1-410-6145993; fax: +1-410-6148804</ce:text></ce:correspondence></ce:author-group><ce:date-received day="22" month="5" year="2000"></ce:date-received><ce:date-accepted day="15" month="9" year="2000"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para><ce:italic>O</ce:italic>-linked <ce:italic>N</ce:italic>-acetylglucosamine (<ce:italic>O</ce:italic>-GlcNAc) is a highly dynamic and abundant modification found on nuclear and cytoplasmic proteins of nearly all eukaryotes. <ce:italic>O</ce:italic>-GlcNAc addition is required for life at the single cell level and is analogous to protein phosphorylation in most respects. In a previous study (M.S. Jiang, G.W. Hart, A subpopulation of estrogen receptors are modified by <ce:italic>O</ce:italic>-linked <ce:italic>N</ce:italic>-acetylglucosamine. J. Biol. Chem. 270 (1997) 2421–2428), we demonstrated that a subpopulation of the murine estrogen receptor-α (mER-α) is modified by <ce:italic>O</ce:italic>-GlcNAc at Thr<ce:sup>575</ce:sup>. Here we mutated mER-α to convert Thr<ce:sup>575</ce:sup> and Ser<ce:sup>576</ce:sup> to Val and Ala, respectively. Surprisingly, this glycosylation-site mutant is still extensively modified by <ce:italic>O</ce:italic>-GlcNAc. Analyses of glycopeptides identified two additional sites of modification on mER-α, at Ser<ce:sup>10</ce:sup> and Thr<ce:sup>50</ce:sup> near the N-terminus. The major glycosylation sites are within or near PEST regions, suggesting that <ce:italic>O</ce:italic>-GlcNAc may regulate mER-α turnover.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords class="keyword"><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>Estrogen receptor</ce:text></ce:keyword><ce:keyword><ce:text><ce:italic>O</ce:italic>-glycosylation</ce:text></ce:keyword><ce:keyword><ce:text><ce:italic>O</ce:italic>-GlcNAc</ce:text></ce:keyword><ce:keyword><ce:text>PEST domain</ce:text></ce:keyword><ce:keyword><ce:text>Post-translational modification</ce:text></ce:keyword><ce:keyword><ce:text>Phosphorylation</ce:text></ce:keyword><ce:keyword><ce:text>Estrogen</ce:text></ce:keyword></ce:keywords><ce:keywords class="abr"><ce:section-title>Abbreviations</ce:section-title><ce:keyword><ce:text>CHCA, α-cyano-4-hydroxylcinnamic acid</ce:text></ce:keyword><ce:keyword><ce:text>CID-MS, collision induced dissociation of tandem mass spectrometry</ce:text></ce:keyword><ce:keyword><ce:text>galactosyltransferase, Galβ (1-4) galactosyltransferase</ce:text></ce:keyword><ce:keyword><ce:text>Gal, galactose</ce:text></ce:keyword><ce:keyword><ce:text>HPLC, high performance liquid chromatography</ce:text></ce:keyword><ce:keyword><ce:text>LC/ESI–MS, liquid chromatography coupled electrospray ionization mass spectrometry</ce:text></ce:keyword><ce:keyword><ce:text>MALDI-TOF, matrix-assisted laser desorption ionization time of flight</ce:text></ce:keyword><ce:keyword><ce:text>mER-α, murine estrogen receptor-α</ce:text></ce:keyword><ce:keyword><ce:text>575-mER-α, mER-α <ce:italic>O</ce:italic>-GlcNAc site mutant</ce:text></ce:keyword><ce:keyword><ce:text><ce:italic>O</ce:italic>-GlcNAc, <ce:italic>O</ce:italic>-linked <ce:italic>N</ce:italic>-acetylglucosamine</ce:text></ce:keyword><ce:keyword><ce:text>PAGE, polyacrylamide gel electrophoresis</ce:text></ce:keyword><ce:keyword><ce:text>RP, reverse phase</ce:text></ce:keyword><ce:keyword><ce:text>SDS, sodium dodecyl sulfate</ce:text></ce:keyword><ce:keyword><ce:text>TFA, trifluoroacetic acid</ce:text></ce:keyword><ce:keyword><ce:text>WGA, wheat germ agglutinin</ce:text></ce:keyword><ce:keyword><ce:text>wt, wild type</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Glycosylation of the murine estrogen receptor-α</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Glycosylation of the murine estrogen receptor-α</title></titleInfo><name type="personal"><namePart type="given">Xiaogang</namePart><namePart type="family">Cheng</namePart><affiliation>Department of Biological Chemistry, School of Medicine, Johns Hopkins University, 725 N Wolfe St., Baltimore, MD 21205-2185, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Gerald W.</namePart><namePart type="family">Hart</namePart><affiliation>E-mail: gwhart@jhmi.edu</affiliation><affiliation>Department of Biological Chemistry, School of Medicine, Johns Hopkins University, 725 N Wolfe St., Baltimore, MD 21205-2185, USA</affiliation><description>Corresponding author. Tel.: +1-410-6145993; fax: +1-410-6148804</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued><copyrightDate encoding="w3cdtf">2001</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: O-linked N-acetylglucosamine (O-GlcNAc) is a highly dynamic and abundant modification found on nuclear and cytoplasmic proteins of nearly all eukaryotes. O-GlcNAc addition is required for life at the single cell level and is analogous to protein phosphorylation in most respects. In a previous study (M.S. Jiang, G.W. Hart, A subpopulation of estrogen receptors are modified by O-linked N-acetylglucosamine. J. Biol. Chem. 270 (1997) 2421–2428), we demonstrated that a subpopulation of the murine estrogen receptor-α (mER-α) is modified by O-GlcNAc at Thr575. Here we mutated mER-α to convert Thr575 and Ser576 to Val and Ala, respectively. Surprisingly, this glycosylation-site mutant is still extensively modified by O-GlcNAc. Analyses of glycopeptides identified two additional sites of modification on mER-α, at Ser10 and Thr50 near the N-terminus. The major glycosylation sites are within or near PEST regions, suggesting that O-GlcNAc may regulate mER-α turnover.</abstract><note type="content">Fig. 1: Schematic of mER-α constructs used in this study. The wt–mER-α cDNA was mutated at 575 (Thr→Val) and 576 (Ser→Ala). The mutated mER-α was designated 575-mER-α. Both cDNAs were engineered with a PCR mediated method to incorporate His6 tag at the carboxyl terminal end.</note><note type="content">Fig. 2: Characterization of 575-mER-α by WGA chromatography. The mER-α cDNAs were in vitro translated with rabbit reticulocyte lysate in presence of 35S-Met. The translated proteins were individually loaded onto WGA columns and challenged with 1 M Gal. Glycosylated pools of ERs were eluted with 1 M GlcNAc. Fractions (0.5 ml) were collected and monitored by liquid scintillation counting. (A) 35S-WGA chromatography profiles. Upper panel, 575-mER-α; lower panel, wt–mER-α. (B) Proteins in each major fraction were precipitated and resolved by 8% SDS-PAGE gel. The gels were dried and exposed to X-ray film. Lane L, load; lane FT, flow through; lane W, 1 M Gal challenge; lane E, 1 M GlcNAc elute.</note><note type="content">Fig. 3: Purification of His6 tagged mER-α from Sf9 cells. mER-α expressed in Sf9 cells was purified with metal affinity resin (lane L, FT, and E are load, flow through and elute) and subsequently purified with preparative SDS-PAGE (lane E–P is the prep-cell elute). Aliquots of each fraction were resolved on an analytical 8% SDS-PAGE gel. The gel was either stained with Coomassie Blue or immunoblotted with monoclonal antibody H222 against ER.</note><note type="content">Fig. 4: Galactosyltransferase labeling and tryptic maps of wt–mER-α and 575-mER-α. (A) Purified 575-mER-α was labeled with UDP-[3H]Gal using galactosyltransferase and subsequently desalted over a Sephadex G50 column. The protein was precipitated by cold acetone, resuspended in 0.1 M sodium bicarbonate (pH 7.0), and digested with trypsin at a ratio of 1:50 (w/w). Aliquots of each sample were examined by 8% SDS-PAGE gel. The gel was stained with Coomassie Blue R250, impregnated with 1 M salicylic acid, dried down, and exposed to X-ray film at −70°C overnight. (B) [3H]Gal-labeled 575-mER-α tryptic peptides were applied to a C18 RP column. Peptides were eluted with a 90-min linear gradient of 0–60% acetonitrile (v/v) in 0.1% TFA from 6 to 96 min at a flow rate of 0.1 ml min−1. Fractions (0.2 ml) were collected and 1% of each was counted. Two major tritium peaks were designated as A and B, respectively. (C) Wild type mER-α was purified and labeled under the same condition as the mutant 575-mER-α. [3H]Gal-labeled wt–mER-α tryptic glycopeptides were applied to a C18 RP column. The column was developed under the same condition as panel B.</note><note type="content">Fig. 5: Thr575 is the major O-GlcNAcylation site on wt–mER-α. (A) The major [3H] Gal-labeled wt–mER-α tryptic fragment eluting from a C18 RP column at 55 min (Fig. 4C). This was further purified by a shallow gradient of 15–45% acetonitrile in 0.1% TFA over 90 min at a flow rate of 0.1 ml min−1 on a C18 RP column. The fraction carrying the most tritium counts was subjected to gas phase sequencing. The identity of the glycopeptide is indicated. (B) An aliquot of the tritium peak fraction from panel A was mixed with the matrix CHCA (5 mg ml−1 in 0.3% TFA/50% acetonitrile) and detected by MALDI TOF. Two mass species were detected and analyzed. (C) The major [3H]Gal-labeled mER-α tryptic C-terminal fragment was digested with proline specific endopeptidase at 37°C overnight and purified by a shallow gradient of 0–30% (v/v) acetonitrile in 0.1% TFA over 90 min at a flow rate of 0.1 ml min−1 on a C18 RP column. (D) The fraction of [3H]Gal-labeled glycopeptide from panel C was coupled to Sequelon-AA disk (Millipore) and subjected to manual Edman degradation. The tritium counts released from each cycle were monitored using liquid scintillation counting. The major O-GlcNAcylation site on wt–mER-α was identified at Thr575.</note><note type="content">Fig. 6: Ser10 is the O-GlcNAcylation site on glycopeptide A. (A) [3H] Gal-labeled 575-mER-α tryptic glycopeptide A, eluting from a C18 RP column at 52 min (Fig. 4B), was further purified by a shallow gradient of 15–45% (v/v) acetonitrile in 0.1% TFA over 90 min at a flow rate of 0.1 ml min−1 on a C18 RP column. (B) An aliquot of the tritium peak fraction from panel A was mixed with CHCA as reported in Fig. 5 and detected by MALDI-TOF. Three mass species were detected and their deduced identity is indicated. (C) An aliquot of the fraction A from the second dimension (A) was subjected to LC/ESI-MS/MS analysis. For clarification, predicted b-and y-ions of the oxidized peptide are listed below the spectrum. The methionine at the position four was oxidized resulting in a mass addition of 16 Da. The matched ions are indicated in the spectrum. Some unmatched ions are probably due to either internal cleavage or loss of H2O during the fragmentation process. (D) The fraction of [3H]Gal-labeled glycopeptide A from panel A was coupled to Sequelon-AA disk (Millipore) and subjected to manual Edman degradation. The tritium counts released in each cycle were monitored using liquid scintillation counting. The O-GlcNAc site on glycopeptide A was identified at Ser10.</note><note type="content">Fig. 7: Thr50 is the O-GlcNAcylation site on glycopeptide B. (A) [3H]Gal-labeled 575-mER-α glycopeptide B eluted at 74 min (Fig. 4B) was further digested with endopeptidase Glu-C in presence of 2 M urea and applied to a C18 RP column. The column was developed with a 90-min linear gradient of 0–60% (v/v) acetonitrile in 0.1% TFA at a flow rate of 0.1 ml min−1. The major tritium peak was designated as Glu-C B. (B) An aliquot of the fraction containing Glu-C B was subjected to LC/ESI-MS/MS analysis. The ESI-MS/MS spectrum of the peptide modified by O-GlcNAc is shown. For clarification, predicted b- and y-ions of the peptide are listed below the spectrum. (C) The Glu-C B fraction was coupled to Sequelon-AA disk (Millipore) and subjected to manual Edman degradation. The tritium counts released in each cycle were monitored using liquid scintillation counting. The O-GlcNAc site on glycopeptide B was identified at Thr50.</note><note type="content">Fig. 8: Summary of O-GlcNAcylation sites on mER-α. mER-α's domain structure is indicated. N′ and C′ indicate amino terminal and carboxyl terminal ends. In this study, two O-GlcNAc sites on mER-α were identified near amino terminus at Ser10 and Thr50. The PEST scores regarding the regions underlined near O-GlcNAc sites are also indicated.</note><note type="content">Table 1: Summary of O-GlcNAcylated proteins and in vivo attachment sitesa</note><subject lang="en"><genre>Keywords</genre><topic>Estrogen receptor</topic><topic>O-glycosylation</topic><topic>O-GlcNAc</topic><topic>PEST domain</topic><topic>Post-translational modification</topic><topic>Phosphorylation</topic><topic>Estrogen</topic></subject><subject lang="en"><genre>Abbreviations</genre><topic>CHCA, α-cyano-4-hydroxylcinnamic acid</topic><topic>CID-MS, collision induced dissociation of tandem mass spectrometry</topic><topic>galactosyltransferase, Galβ (1-4) galactosyltransferase</topic><topic>Gal, galactose</topic><topic>HPLC, high performance liquid chromatography</topic><topic>LC/ESI–MS, liquid chromatography coupled electrospray ionization mass spectrometry</topic><topic>MALDI-TOF, matrix-assisted laser desorption ionization time of flight</topic><topic>mER-α, murine estrogen receptor-α</topic><topic>575-mER-α, mER-α O-GlcNAc site mutant</topic><topic>O-GlcNAc, O-linked N-acetylglucosamine</topic><topic>PAGE, polyacrylamide gel electrophoresis</topic><topic>RP, reverse phase</topic><topic>SDS, sodium dodecyl sulfate</topic><topic>TFA, trifluoroacetic acid</topic><topic>WGA, wheat germ agglutinin</topic><topic>wt, wild type</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Steroid Biochemistry and Molecular Biology</title></titleInfo><titleInfo type="abbreviated"><title>SBMB</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued></originInfo><identifier type="ISSN">0960-0760</identifier><identifier type="PII">S0960-0760(00)X0070-2</identifier><part><date>2000</date><detail type="volume"><number>75</number><caption>vol.</caption></detail><detail type="issue"><number>2–3</number><caption>no.</caption></detail><extent unit="issue-pages"><start>91</start><end>208</end></extent><extent unit="pages"><start>147</start><end>158</end></extent></part></relatedItem><identifier type="istex">80DD63B048E65F8E013413F093CC3E50F9B017CA</identifier><identifier type="ark">ark:/67375/6H6-6LH2X1L0-Q</identifier><identifier type="DOI">10.1016/S0960-0760(00)00167-9</identifier><identifier type="PII">S0960-0760(00)00167-9</identifier><accessCondition type="use and reproduction" contentType="copyright">©2001 Elsevier Science Ltd</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Elsevier Science Ltd, ©2001</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-6LH2X1L0-Q/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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