Characterization of the Glycosylation Sites in Cyclooxygenase-2 Using Mass Spectrometry

Nemeth, Jennifer F.; Hochgesang, G. Phillip; Marnett, Lawrence J.; Caprioli, Richard M.

doi:10.1021/bi015133r

Cited by 18 publications

(26 citation statements)

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“…The carbohydrate moieties at each site are believed to be high-mannose chains [24,25]. The variability of glycosylation at Asn 580 leads to the production of two distinct glycoforms of 72 and 74 kDa.…”

Section: Introductionmentioning

confidence: 99%

Glycosylation regulates turnover of cyclooxygenase‐2

Sevigny

Alas

et al. 2006

FEBS Letters

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Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in the prostanoid biosynthesis pathway, converting arachidonic acid into prostaglandin H 2 . COX-2 exists as 72 and 74 kDa glycoforms, the latter resulting from an additional oligosaccharide chain at residue Asn 580 . In this study, Asn 580 was mutated to determine the biological significance of this variable glycosylation. COS-1 cells transfected with the mutant gene were unable to express the 74 kDa glycoform and were found to accumulate more COX-2 protein and have five times greater COX-2 activity than cells expressing both glycoforms. Thus, COX-2 turnover appears to depend upon glycosylation of the 72 kDa glycoform. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

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Section: Introductionmentioning

confidence: 99%

Glycosylation regulates turnover of cyclooxygenase‐2

Sevigny

Alas

et al. 2006

FEBS Letters

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“…Glycosylation site analysis is usually performed following proteolytic cleavage of the glycoprotein, such that each glycosylation site is located within a separate peptide. However, this technique is often hampered by poor ionization of glycoproteins or glycopeptides compared with their unmodified forms, thereby limiting the sensitivity of the assay [5,6]. A full characterization of glycoprotein component in complex protein mixtures by ESI and MALDI/MS is a challenging task.…”

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confidence: 99%

Immobilization of aminophenylboronic acid on magnetic beads for the direct determination of glycoproteins by matrix assisted laser desorption ionization mass spectrometry

Lee¹,

Kim²,

et al. 2005

J. Am. Soc. Mass Spectrom.

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Aminophenylboronic acid (APBA) has been immobilized on magnetic beads for the direct determination of glycoprotein by matrix assisted laser desorption/ionizaton time of flight mass spectrometry (MALDI-TOF-MS). An APBA layer was formed on the surface of carboxylic acid terminated magnetic beads by coupling with carbodiimide and subsequently reacted with an N-hydroxysuccinimide moiety. The immobilized APBA was identified by MALDI-TOF-MS without a matrix. Glycoproteins, such as HbA1c, fibrinogen, or RNase B were separated and desalted using APBA magnetic beads by simply washing the magnetic beads and then separating them by external magnet. Proteins can be identified by direct determination of proteins on beads on MALDI plate and confirmed again by peptide mass finger printing after digestion of proteins on magnetic beads by trypsin. Fluorescence image with a FITC tagging protein using confocal laser microscopy showed the difference of immobilization efficiency between glycoproteins and nonglycoproteins. The methods developed within this work allow the simple treatment and enrichment of glycoproteins as well as direct determination of proteins on beads by

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“…Glycosylation of COX-2 is required for normal physiological processes (13). COX-2 has four sites for glycosylation with a subunit molecular mass of 72 kDa (14). Inhibition of glycosylation leads to a reduced molecular mass and activity of COX-2 (15).…”

Section: Discussionmentioning

confidence: 99%

Protein phosphorylation on tyrosine restores expression and glycosylation of cyclooxygenase-2 by 2-deoxy-D-glucose-caused endoplasmic reticulum stress in rabbit articular chondrocyte

Yu¹,

Kim²

2012

BMB Reports

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2-deoxy-D-glucose(2DG)-caused endoplasmic reticulum (ER)stress inhibits protein phosphorylation at tyrosine residues. However, the accurate regulatory mechanisms, which determine the inflammatory response of chondrocytes to ER stress via protein tyrosine phosphorylation, have not been systematically evaluated. Thus, in this study, we examined whether protein phosphorylation at tyrosine residues can modulate the expression and glycosylation of COX-2, which is reduced by 2DG-induced ER stress. We observed that protein tyrosine phosphatase (PTP) inhibitors, sodium orthovanadate (SOV), and phenylarsine oxide (PAO) significantly decreased expression of ER stress inducible proteins, glucose-regulated protein 94 (GRP94), and CCAAT/ enhancer-binding-protein-related gene (GADD153), which was induced by 2DG. In addition, we demonstrated that SOV and PAO noticeably restored the expression and glycosylation of COX-2 after treatment with 2DG. These results suggest that protein phosphorylation of tyrosine residues plays an important role in the regulation of expression and glycosylation during 2DG-induced ER stress in rabbit articular chondrocytes. [BMB reports 2012; 45(5): 317-322]

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Characterization of the Glycosylation Sites in Cyclooxygenase-2 Using Mass Spectrometry

Cited by 18 publications

References 0 publications

Glycosylation regulates turnover of cyclooxygenase‐2

Glycosylation regulates turnover of cyclooxygenase‐2

Immobilization of aminophenylboronic acid on magnetic beads for the direct determination of glycoproteins by matrix assisted laser desorption ionization mass spectrometry

Protein phosphorylation on tyrosine restores expression and glycosylation of cyclooxygenase-2 by 2-deoxy-D-glucose-caused endoplasmic reticulum stress in rabbit articular chondrocyte

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