Biochem. J. (2003) 375
(61–73) (Printed in Great Britain)
N-linked glycosylation of native and recombinant cauliflower xyloglucan endotransglycosylase 16A
Hongbin HENRIKSSON*, Stuart E. DENMAN*, Iain D. G. CAMPUZANO†, Pia ADEMARK*, Emma R. MASTER*, Tuula T. TEERI* and Harry BRUMER, III*1
*Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, 106 91 Stockholm, Sweden, and †Waters Corporation, Micromass MS Technologies, Atlas Park, Simonsway, Manchester M22 5PP, U.K.
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 GlcNAc2Man6. 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.
Key words: glycoform, plant N-glycan, protein glycosylation, transglycosylation, xyloglucan endotransglycosylase (XET).
Abbreviations used: BCA, disodium 2,2´-bicinchoninate; DDATM, Data Directed AnalysisTM; Endo H, Streptomyces plicatus endoglycosidase H (EC 3.2.1.96); ESI, electrospray ionization; GH16, glycosyl hydrolase family 16; Gol, glucitol (in reduced oligosaccharides); MaxEntTM, Maximum EntropyTM algorithm; MS/MS, tandem MS; PNGase F, Flavobacterium meningosepticum N-glycosidase F (EC 3.5.1.52); RACE, rapid amplification of cDNA ends; TOF, time-of-flight; XET, xyloglucan endotransglycosylase; XGO, xyloglucan oligosaccharides; XTH, xyloglucan endotransglycosylase/hydrolase.
1To whom correspondence should be addressed (e-mail harry@biotech.kth.se).
The nucleotide sequence of Brassica oleracea var. botrytis XET16A has been submitted to the GenBank®, DDBJ, EMBL and GSDB Nucleotide Sequence Databases under the accession number AY156708.
Received 31 March 2003/2 June 2003; accepted 25 June 2003
Published as BJ Immediate Publication 26 June 2003, DOI 10.1042/BJ20030485
The Biochemical Society, London ©2003
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