Characterization of N-linked oligosaccharides in chorion peroxidase of Aedes aegypti mosquito.
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(4-8)GlcNAc2). 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.
Li
Junsuo S
JS
Department of Pathobiology, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61802, USA.
Li
Jianyong
J
D000330:Aedes / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002823:Chorion / Q000201:enzymology*; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000032:analysis; D009844:Oligosaccharides / Q000737:chemistry*; D009195:Peroxidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry
D009005:Monosaccharides; D009844:Oligosaccharides; D011134:Polysaccharides; D009195:Peroxidase