title: Novel glycosidic linkage in Aedes aegypti chorion peroxidase: N-mannosyl tryptophan.
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 beta-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.
authors.affiliation: Department of Pathobiology, University of Illinois, Urbana, Illinois 61802, USA.
authors.forename: Junsuo S
authors.initials: JS
authors.lastname: Li
authors.forename: Liwang
authors.initials: L
authors.lastname: Cui
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Rock
authors.forename: Jianyong
authors.initials: J
authors.lastname: Li
mesh_terms: D000330:Aedes / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D002021:Buffers; D002853:Chromatography, Liquid; D019343:Citric Acid / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006027:Glycosides / Q000737:chemistry; D006031:Glycosylation; D006868:Hydrolysis; D019476:Insect Proteins; D008358:Mannose / Q000737:chemistry*; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology; D014364:Tryptophan / Q000737:chemistry
chemical_list: D002021:Buffers; D004220:Disulfides; D006027:Glycosides; D019476:Insect Proteins; D010455:Peptides; D010710:Phosphates; D019343:Citric Acid; D014364:Tryptophan; D010544:Peroxidases; C506779:chorion peroxidase, Aedes aegypti; D014357:Trypsin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D008358:Mannose