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

Abstract

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 (Asn 328 ‐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 Asn 328 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 Man 3 GlcNAc 2 and Man 3 (Fuc) GlcNAc 2 and high mannose‐type structures (Man 4–8 GlcNAc 2 ). The truncated structures, Man 2 GlcNAc 2 and Man 2 (Fuc) GlcNAc 2 , 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.