||Enzymatic Digestion and Mass Spectroscopies of N-Linked Glycans in Lacquer Stellacyanin and Laccase from Rhus vernicifera
Lacquer sap is water-in-oil emulsion consisted of urushiols, polysaccharides,glycoproteins, laccase, and stellacyanin that have been used as natural paint in Asiancountries. Laccase and stellacyanin are copper?containing glycoproteins, which areheavily glycosylated proteins containing 3 and 15 possible N-glycosylation sites(Asn-x-Thr/Ser), respectively. Structural studies on their carbohydrate structureshave not been accomplished yet.In present study, we isolated laccase and stellacyanin from lacquer acetonepowder by continuous ion exchange chromatography using Sephadex CM-50 andDEAE A-50. Protein glycosylarion was characterized using enzymatic digestion,chemical labeling techniques of released N-linked glycan and subsequent massspectrometric analysis MALDI TOF MS an LC/MS/MS, respectively. We found thatthree N-glycosylation sites (Asn28, 60 and 102) in lacquer stellacyanin allglycosylated with same complex-type N-linked glycan, constituted withGlcNAc4Man3Gal2Fuc3Xyl1.Also lacquer laccase glycosylated with complex- and also hybride-type N-linkedglycans that are common plant-specific structural units of ?-xylose, core ?-fucose andLewis epitope (GlcNAc-Gal-Fuc) were observed by MALDI TOF MS. LC/MS/MSanalysis of glycopeptides revealed that 13 N-glycosylation sites in laccase wereglycosylated frequently with complex type N-glycan, GlcNAc4Man3Gal2Fuc3Xyl1,and among them Asn5, 233 and 381 were also found to be glycosylated withhybride?type N-glycan, mainly GlcNAc2-3Man4-5Fuc1Xyl1. Glycopeptides which carrypossible N-glycosylation sites at Asn 364 and 519 were not confirmed by LC/MS/MSanalysis.