N-linked glycosylation of native and recombinant cauliflower xyloglucan endotransglycosylase 16A

H Henriksson, SE Denman, IDG Campuzano, P Ademark, ER Master, TT Teeri, H Brumer*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

Abstract

The gene encoding a XET (xyloglucan endotransglycosylase) from cauliflower (Brassica oleracea var. botrytis) florets has been cloned and sequenced. Sequence analysis indicated a high degree of similarity to other XET enzymes belonging to glycosyl hydrolase family 16 (GH16). In addition to the conserved GH16 catalytic sequence motif EIDFE, there exists one potential N-linked glycosylation site. which is also highly conserved in XET enzymes from this family. Purification of the corresponding protein from extracts of cauliflower florets allowed the fractionation of a single, pure glycoform. which was analysed by MS techniques. Accurate protein mass determination following the enzymic deglycosylation of this glycoform indicated the presence of a high-mannose-type glycan of the general structure GlcNAc(2)Man(6). LC/MS and MS/MS (tandem MS) analysis provided supporting evidence for this structure and confirmed that the glycosylation site (underlined) was situated close to the predicted catalytic residues in the conserved sequence YLSSTNNEHDEIDFEFLGNRTGQPVILQTNVFTGGK. Heterologous expression in Pichia pastoris produced a range of protein glycoforms, which were, on average, more highly mannosylated than the purified native enzyme. This difference in glycosylation did not influence the apparent enzymic activity of the enzyme significantly. However, the removal of high-mannose glycosylation in recombinant cauliflower XET by endoglycosidase H, quantified by electrospray-ionization MS, caused a 40 % decrease in the transglycosylation activity of the enzyme. No hydrolytic activity was detected in native or heterologously expressed BobXET16A, even when almost completely deglycosylated.

Original languageEnglish
Pages (from-to)61-73
Number of pages13
JournalBIOCHEMICAL JOURNAL
Volume375
DOIs
Publication statusPublished - 1 Oct 2003
MoE publication typeA1 Journal article-refereed

Keywords

  • glycoform
  • plant N-glycan
  • protein glycosylation
  • transglycosylation
  • xyloglucan endotransglycosylase (XET)
  • PLANT-CELL WALLS
  • AZUKI-BEAN EPICOTYLS
  • ENDOXYLOGLUCAN TRANSFERASE
  • MOLECULAR CHARACTERIZATION
  • OLIGOSACCHARIDE STRUCTURES
  • ENDO-TRANSGLYCOSYLASE
  • ENZYME INTERMEDIATE
  • COLORIMETRIC ASSAY
  • TRANSGENIC PLANTS
  • NASTURTIUM SEEDS

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