N-glycans in Toxicodendron vernicifluum lacquer laccase

Abstract

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^α-(aminooxyacetyl)tryptophanylarginine methyl ester (aoWR) to give the aoWR-labelled N-glycans. The MS measurements revealed that GlcNAc₄Hex₅Fuc₃Xyl₁ N-glycan was attached at 12 N-glycosylation sites (Asn 5, 14, 180, 194, 233, 274, 284, 347, 364, 381, 398, and 519), GlcNAc₃Hex₄Fuc₂Xyl₁ N-glycan at two sites (Asn 124 and 454), and GlcNAc₃Hex₆Fuc₁Xyl₁ 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).

Keywords: Enzymatic digestion; Lacquer laccase; MS spectrum; Mascot search; N-Glycan.