Novel glycosidic linkage in Aedes aegypti chorion peroxidase: N-mannosyl tryptophan

Junsuo S. Li, Liwang Cui, Daniel L. Rock, Jianyong Li

Research output: Contribution to journalArticle

17 Scopus citations

Abstract

Aedes aegypti chorion peroxidase (CPO) plays a crucial role in chorion hardening by catalyzing chorion protein cross-linking through dityrosine formation. The enzyme is extremely resistant to denaturing conditions, which seem intimately related to its post-translational modifications, including disulfide bond formation and glycosylation. In this report, we have provided data that describe a new type of glycosylation in CPO, where a mannose is linked to the N-1 atom of the indole ring of Trp residue. Through liquid chromatography/electrospray ionization/tandem mass spectrometry and de novo sequencing of CPO tryptic peptides, we determined that three of the seven available Trp residues in mature CPO are partially (40-50%) or completely mannosylated. This conclusion is based on the following properties of the electrospray ionization/tandem mass spectrometry spectra and the enzymatic reaction of these peptides: 1) the presence of a 162-Da substituent in each Trp residue; 2) the presence of abundant fragments of m/z 163 ([Hex + H]) and [M + H - 162] (typical for N-glycosides); 3) the absence of a loss of 120 Da (this loss is typical for aromatic C-glycosides); and 4) the cleavage of the glycosidic linkage by PNGase A or F (typical for N-glycans). These results establish that a C-N bond is formed between the anomeric carbon of a mannose residue and the N-1 atom of the indole ring of Trp. This is the first report that provides definitive evidence for N-mannosylation of Trp residues in a protein. In addition, our data demonstrate that PNGase can hydrolyze Trp N-linked mannose in peptides, which is unusual because no typical β-amide bond is present in the Trp-mannosyl moiety. Results of this study should stimulate research toward a comprehensive understanding of physiology and biochemistry of Trp N-mannosylation in proteins and the overall biochemical mechanisms of PNGase-catalyzed reactions.

Original languageEnglish (US)
Pages (from-to)38513-38521
Number of pages9
JournalJournal of Biological Chemistry
Volume280
Issue number46
DOIs
StatePublished - Dec 1 2005

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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