N-linked glycosylation of native and recombinant cauliflower xyloglucan endotransglycosylase 16A
2003 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 375, 61-73 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2003. Vol. 375, 61-73 p.
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
IdentifiersURN: urn:nbn:se:kth:diva-22883DOI: 10.1042/bj20030485ISI: 000185876800007OAI: oai:DiVA.org:kth-22883DiVA: diva2:341581
QC 201005252010-08-102010-08-10Bibliographically approved