Proteomic analysis of N-glycosylation in mosquito dopachrome conversion enzyme.
A novel dopachrome conversion enzyme (DCE) is present in insects and involved in their melanization pathway. DCE shares no sequence homology with any noninsect species from bacteria to humans. Several DCE sequences have been available, but enzyme structure and catalytic mechanism are unclear. This study concerns DCE PTMs, especially glycosylation. A mosquito DCE was purified and its monosaccharide composition, N-glycosylation site, and oligosaccharide structures were determined. Results showed that N-acetyl D-glucosamine and D-mannose are the major monosaccharides and L-fucose, D-xylose, and D-arabinose are the minor ones in mosquito DCE. Glycosylation site and oligosaccharide structures were elucidated from MS and MS/MS spectra of trypsin-digested DCE glycopeptides. A single N-glycosylation site (Asn285 -Glu-Thr) was identified in DCE and was proven to be fully glycosylated. Man3GlcNAc2, Man3(Fuc)1-2GlcNAc2, and their truncated structures were the dominant oligosaccharides. In addition, high mannose-type structures (Man4-7(Fuc)GlcNAc2) were also identified. Removal of DCE N-oligosaccharides with peptide N-glycosidase (PNGase F) decreased its activity and thermal stability. However, partial DCE deglycosylation with alpha-mannosidase or alpha-fucosidase somewhat stimulated its activity and improved its thermal stability. During mass spectrometric analysis of DCE glycopeptides, their CID patterns were highly intriguing, in that some glycopeptides underwent both C-terminal rearrangement and formation of dimeric structures during CID. Results of this study provide an interesting example in terms of potential complexity of the glycopeptide CID fragmentation pattern.
Li
Junsuo S
JS
Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA. lij@vt.edu
Vavricka
Christopher J
CJ
Christensen
Bruce M
BM
Li
Jianyong
J
D000330:Aedes / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004795:Enzyme Stability; D006031:Glycosylation; D006358:Hot Temperature; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009005:Monosaccharides / Q000032:analysis / Q000737:chemistry*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000494:pharmacology
D009005:Monosaccharides; D020543:Proteome; D014357:Trypsin; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase