N-glycans in Toxicodendron vernicifluum lacquer laccase

The N-glycans in Toxicodendron vernicifluum (Rhus vernicifera) lacquer laccase was elucidated for the first time through a combination of enzymatic digestion and subsequent mass spectrometry measurements using LC-MS/MS and MALDI-TOF MS. Lacquer laccase was isolated from a Japanese lacquer acetone powder from consecutive Sephadex C-50 and DEAE A-50 column chromatography. Trypsin and chymotrypsin digestions of the lacquer laccase resulted in a mixture of peptides and N-glycopeptides, which were treated with peptide-N-glycosidases and then N-alpha-(aminooxyacetyl)tryptophanylarginine methyl ester (aoWR) to give the aoWR-labelled N-glycans. The MS measurements revealed that GlcNAc(4)Hex(5)Fuc(3)Xyl(1) N-glycan was attached at 12 N-glycosylation sites (Asn 5, 14, 180, 194, 233, 274, 284, 347, 364, 381, 398, and 519), GlcNAc(3)Hex(4)Fuc(2)Xyl(1) N-glycan at two sites (Asn 124 and 454), and GlcNAc(3)Hex(6)Fuc(1)Xyl(1) N-glycan at one site (Asn 28). A database search (Mascot search) of the peptides also suggested the presence of N-glycans at the 15 potential N-glycosylation sites (Asn-X-Ser/Thr).