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Characterization of N-linked oligosaccharides in chorion peroxidase of Aedes aegypti mosquito

Department of Pathobiology, University of Illinois, Urbana, Illinois 61802, USA

(RECEIVED February 16, 2005; FINAL REVISION June 5, 2005; ACCEPTED June 13, 2005)

A peroxidase is present in the chorion of Aedes aegypti eggs and catalyzes chorion protein cross-linking during chorion hardening, which is critical for egg survival in the environment. The unique chorion peroxidase (CPO) is a glycoprotein. This study deals with the N-glycosylation site, structures, and profile of CPO-associated oligosaccharides using mass spectrometric techniques and enzymatic digestion. CPO was isolated from chorion by solubilization and several chromatographic methods. Mono-saccharide composition was analyzed by HPLC with fluorescent detection. Our data revealed that carbohydrate (D-mannose, N-acetyl D-glucosamine, D-arabinose, N-acetyl D-galactosamine, and L-fucose) accounted for 2.24% of the CPO molecular weight. A single N-glycosylation site (Asn³²⁸-Cys- Thr) was identified by tryptic peptide mapping and de novo sequencing of native and PNGase A-deglycosylated CPO using matrix-assisted laser/desorption/ionization time-of-flight mass spectrometry (MALDI/TOF/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The Asn³²⁸ was proven to be a major fully glycosylated site. Potential tryptic glycopeptides and profile were first assessed by MALDI/TOF/MS and then by precursor ion scanning during LC/MS/MS. The structures of N-linked oligosaccharides were elucidated from the MS/MS spectra of glycopeptides and exoglycosidase sequencing of PNGase A-released oligosaccharides. These CPO-associated oligosaccharides had dominant Man₃GlcNAc₂ and Man₃ (Fuc) GlcNAc₂ and high mannose-type structures (Man_4–8GlcNAc₂). The truncated structures, Man₂GlcNAc₂ and Man₂ (Fuc) GlcNAc₂, were also identified. Comparison of CPO activity and Stokes radius between native and deglycosylated CPO suggests that the N-linked oligosaccharides influence the enzyme activity by stabilizing its folded state.

Keywords: Aedes aegypti; chorion peroxidase; glycosylation

Abbreviations: CPO, chorion peroxidase • -CN, -cyano-4-hydoxycinnamic acid • DTT, dithiothreitol • DHB, 2,5-dihydroxylbenzoic acid • LC/ESI/MS/MS, liquid chromatography/electrospray ionization/tandem mass spectrometry • MALDI/TOF/MS, matrix-assisted laser/desorption/ionization time-of-flight mass spectrometry • PMSF, phenyl methyl sulforyl fluoride • PVDF, polyvinylidene difluoride • SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis • TIC, total ion current • TFA, trifluoroacetic acid

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051419105.

Reprint requests to: Jianyong Li, Department of Pathobiology, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61802, USA; e-mail: jli2{at}uiuc.edu; fax: (217) 244-3913.

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				J. S. Li, L. Cui, D. L. Rock, and J. Li Novel Glycosidic Linkage in Aedes aegypti Chorion Peroxidase: N-MANNOSYL TRYPTOPHAN J. Biol. Chem., November 18, 2005; 280(46): 38513 - 38521. [Abstract] [Full Text] [PDF]