Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Oct 1;375(Pt 1):61–73. doi: 10.1042/BJ20030485

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

Hongbin Henriksson 1, Stuart E Denman 1, Iain D G Campuzano 1, Pia Ademark 1, Emma R Master 1, Tuula T Teeri 1, Harry Brumer 3rd 1
PMCID: PMC1223658  PMID: 12826015

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 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.

Full Text

The Full Text of this article is available as a PDF (240.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arrowsmith D. A., de Silva J. Characterisation of two tomato fruit-expressed cDNAs encoding xyloglucan endo-transglycosylase. Plant Mol Biol. 1995 Jun;28(3):391–403. doi: 10.1007/BF00020389. [DOI] [PubMed] [Google Scholar]
  2. Baran R., Sulová Z., Stratilová E., Farkas V. Ping-pong character of nasturtium-seed xyloglucan endotransglycosylase (XET) reaction. Gen Physiol Biophys. 2000 Dec;19(4):427–440. [PubMed] [Google Scholar]
  3. Bardor M, Faye Lc, Lerouge P. Analysis of the N-glycosylation of recombinant glycoproteins produced in transgenic plants. Trends Plant Sci. 1999 Sep;4(9):376–380. doi: 10.1016/s1360-1385(99)01461-2. [DOI] [PubMed] [Google Scholar]
  4. Bourquin Veronica, Nishikubo Nobuyuki, Abe Hisashi, Brumer Harry, Denman Stuart, Eklund Marlin, Christiernin Maria, Teeri Tunla T., Sundberg Björn, Mellerowicz Ewa J. Xyloglucan endotransglycosylases have a function during the formation of secondary cell walls of vascular tissues. Plant Cell. 2002 Dec;14(12):3073–3088. doi: 10.1105/tpc.007773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bransome E. D., Jr, Grower M. F. Liquid scintillation counting of (3H) and (14C) on solid supports: a warning. Anal Biochem. 1970 Dec;38(2):401–408. doi: 10.1016/0003-2697(70)90464-1. [DOI] [PubMed] [Google Scholar]
  6. Brummell D. A., Harpster M. H. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Mol Biol. 2001 Sep;47(1-2):311–340. [PubMed] [Google Scholar]
  7. Campbell P., Braam J. Co- and/or post-translational modifications are critical for TCH4 XET activity. Plant J. 1998 Aug;15(4):553–561. doi: 10.1046/j.1365-313x.1998.00239.x. [DOI] [PubMed] [Google Scholar]
  8. Campbell P., Braam J. In vitro activities of four xyloglucan endotransglycosylases from Arabidopsis. Plant J. 1999 May;18(4):371–382. doi: 10.1046/j.1365-313x.1999.00459.x. [DOI] [PubMed] [Google Scholar]
  9. Campbell P., Braam J. Xyloglucan endotransglycosylases: diversity of genes, enzymes and potential wall-modifying functions. Trends Plant Sci. 1999 Sep;4(9):361–366. doi: 10.1016/s1360-1385(99)01468-5. [DOI] [PubMed] [Google Scholar]
  10. Catalá C., Rose J. K., York W. S., Albersheim P., Darvill A. G., Bennett A. B. Characterization of a tomato xyloglucan endotransglycosylase gene that is down-regulated by auxin in etiolated hypocotyls. Plant Physiol. 2001 Nov;127(3):1180–1192. doi: 10.1104/pp.010481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Edwards M., Dea I. C., Bulpin P. V., Reid J. S. Purification and properties of a novel xyloglucan-specific endo-(1----4)-beta-D-glucanase from germinated nasturtium seeds (Tropaeolum majus L.). J Biol Chem. 1986 Jul 15;261(20):9489–9494. [PubMed] [Google Scholar]
  12. Fry S. C., Smith R. C., Renwick K. F., Martin D. J., Hodge S. K., Matthews K. J. Xyloglucan endotransglycosylase, a new wall-loosening enzyme activity from plants. Biochem J. 1992 Mar 15;282(Pt 3):821–828. doi: 10.1042/bj2820821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gavel Y., von Heijne G. Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering. Protein Eng. 1990 Apr;3(5):433–442. doi: 10.1093/protein/3.5.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gemmill T. R., Trimble R. B. Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim Biophys Acta. 1999 Jan 6;1426(2):227–237. doi: 10.1016/s0304-4165(98)00126-3. [DOI] [PubMed] [Google Scholar]
  15. Henrissat B., Davies G. Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol. 1997 Oct;7(5):637–644. doi: 10.1016/s0959-440x(97)80072-3. [DOI] [PubMed] [Google Scholar]
  16. Henrissat B., Teeri T. T., Warren R. A. A scheme for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants. FEBS Lett. 1998 Mar 27;425(2):352–354. doi: 10.1016/s0014-5793(98)00265-8. [DOI] [PubMed] [Google Scholar]
  17. Johansson Patrik, Denman Stuart, Brumer Harry, Kallas Asa M., Henriksson Hongbin, Bergfors Terese, Teeri Tuula T., Jones T. Alwyn. Crystallization and preliminary X-ray analysis of a xyloglucan endotransglycosylase from Populus tremula x tremuloides. Acta Crystallogr D Biol Crystallogr. 2003 Feb 21;59(Pt 3):535–537. doi: 10.1107/s090744490202348x. [DOI] [PubMed] [Google Scholar]
  18. Jonsson A. P., Carlquist M., Husman B., Ljunggren J., Jörnvall H., Bergman T., Griffiths W. J. Structural analysis of the thyroid hormone receptor ligand binding domain: studies using a quadrupole time-of-flight tandem mass spectrometer. Rapid Commun Mass Spectrom. 1999;13(18):1782–1791. doi: 10.1002/(SICI)1097-0231(19990930)13:18<1782::AID-RCM714>3.0.CO;2-W. [DOI] [PubMed] [Google Scholar]
  19. Kohen A., Jonsson T., Klinman J. P. Effects of protein glycosylation on catalysis: changes in hydrogen tunneling and enthalpy of activation in the glucose oxidase reaction. Biochemistry. 1997 Mar 4;36(9):2603–2611. doi: 10.1021/bi962492r. [DOI] [PubMed] [Google Scholar]
  20. Lloyd R. C., Davis B. G., Jones J. B. Site-selective glycosylation of subtilisin Bacillus lentus causes dramatic increases in esterase activity. Bioorg Med Chem. 2000 Jul;8(7):1537–1544. doi: 10.1016/s0968-0896(00)00084-5. [DOI] [PubMed] [Google Scholar]
  21. McDougall G. J., Fry S. C. Xyloglucan oligosaccharides promote growth and activate cellulase: evidence for a role of cellulase in cell expansion. Plant Physiol. 1990 Jul;93(3):1042–1048. doi: 10.1104/pp.93.3.1042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Medford J. I., Elmer J. S., Klee H. J. Molecular cloning and characterization of genes expressed in shoot apical meristems. Plant Cell. 1991 Apr;3(4):359–370. doi: 10.1105/tpc.3.4.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Montesino R., García R., Quintero O., Cremata J. A. Variation in N-linked oligosaccharide structures on heterologous proteins secreted by the methylotrophic yeast Pichia pastoris. Protein Expr Purif. 1998 Nov;14(2):197–207. doi: 10.1006/prep.1998.0933. [DOI] [PubMed] [Google Scholar]
  24. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  25. Nishitani K., Tominaga R. Endo-xyloglucan transferase, a novel class of glycosyltransferase that catalyzes transfer of a segment of xyloglucan molecule to another xyloglucan molecule. J Biol Chem. 1992 Oct 15;267(29):21058–21064. [PubMed] [Google Scholar]
  26. Okazawa K., Sato Y., Nakagawa T., Asada K., Kato I., Tomita E., Nishitani K. Molecular cloning and cDNA sequencing of endoxyloglucan transferase, a novel class of glycosyltransferase that mediates molecular grafting between matrix polysaccharides in plant cell walls. J Biol Chem. 1993 Dec 5;268(34):25364–25368. [PubMed] [Google Scholar]
  27. Pauly M., Albersheim P., Darvill A., York W. S. Molecular domains of the cellulose/xyloglucan network in the cell walls of higher plants. Plant J. 1999 Dec;20(6):629–639. doi: 10.1046/j.1365-313x.1999.00630.x. [DOI] [PubMed] [Google Scholar]
  28. Pauly M., Qin Q., Greene H., Albersheim P., Darvill A., York W. S. Changes in the structure of xyloglucan during cell elongation. Planta. 2001 Apr;212(5-6):842–850. doi: 10.1007/s004250000448. [DOI] [PubMed] [Google Scholar]
  29. Perkins D. N., Pappin D. J., Creasy D. M., Cottrell J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis. 1999 Dec;20(18):3551–3567. doi: 10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  30. Rose J. K., Brummell D. A., Bennett A. B. Two divergent xyloglucan endotransglycosylases exhibit mutually exclusive patterns of expression in nasturtium. Plant Physiol. 1996 Feb;110(2):493–499. doi: 10.1104/pp.110.2.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rose JK, Bennett AB. Cooperative disassembly of the cellulose-xyloglucan network of plant cell walls: parallels between cell expansion and fruit ripening. Trends Plant Sci. 1999 May;4(5):176–183. doi: 10.1016/s1360-1385(99)01405-3. [DOI] [PubMed] [Google Scholar]
  32. Rose Jocelyn K. C., Braam Janet, Fry Stephen C., Nishitani Kazuhiko. The XTH family of enzymes involved in xyloglucan endotransglucosylation and endohydrolysis: current perspectives and a new unifying nomenclature. Plant Cell Physiol. 2002 Dec;43(12):1421–1435. doi: 10.1093/pcp/pcf171. [DOI] [PubMed] [Google Scholar]
  33. Schröder R., Atkinson R. G., Langenkämper G., Redgwell R. J. Biochemical and molecular characterisation of xyloglucan endotransglycosylase from ripe kiwifruit. Planta. 1998 Feb;204(2):242–251. doi: 10.1007/s004250050253. [DOI] [PubMed] [Google Scholar]
  34. Steele N. M., Fry S. C. Differences in catalytic properties between native isoenzymes of xyloglucan endotransglycosylase (XET). Phytochemistry. 2000 Aug;54(7):667–680. doi: 10.1016/s0031-9422(00)00203-x. [DOI] [PubMed] [Google Scholar]
  35. Steele N. M., Fry S. C. Purification of xyloglucan endotransglycosylases (XETs): a generally applicable and simple method based on reversible formation of an enzyme-substrate complex. Biochem J. 1999 May 15;340(Pt 1):207–211. [PMC free article] [PubMed] [Google Scholar]
  36. Steele N. M., Sulová Z., Campbell P., Braam J., Farkas V., Fry S. C. Ten isoenzymes of xyloglucan endotransglycosylase from plant cell walls select and cleave the donor substrate stochastically. Biochem J. 2001 May 1;355(Pt 3):671–679. doi: 10.1042/bj3550671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sulová Z., Farkas V. Kinetic evidence of the existence of a stable enzyme-glycosyl intermediary complex in the reaction catalyzed by endotransglycosylase. Gen Physiol Biophys. 1998 Jun;17(2):133–142. [PubMed] [Google Scholar]
  38. Sulová Z., Farkas V. Purification of xyloglucan endotransglycosylase based on affinity sorption of the active glycosyl-enzyme intermediate complex to cellulose. Protein Expr Purif. 1999 Jul;16(2):231–235. doi: 10.1006/prep.1999.1043. [DOI] [PubMed] [Google Scholar]
  39. Sulová Z., Lednická M., Farkas V. A colorimetric assay for xyloglucan-endotransglycosylase from germinating seeds. Anal Biochem. 1995 Jul 20;229(1):80–85. doi: 10.1006/abio.1995.1381. [DOI] [PubMed] [Google Scholar]
  40. Sulová Z., Takácová M., Steele N. M., Fry S. C., Farkas V. Xyloglucan endotransglycosylase: evidence for the existence of a relatively stable glycosyl-enzyme intermediate. Biochem J. 1998 Mar 15;330(Pt 3):1475–1480. doi: 10.1042/bj3301475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tabuchi A., Mori H., Kamisaka S., Hoson T. A new type of endo-xyloglucan transferase devoted to xyloglucan hydrolysis in the cell wall of azuki bean epicotyls. Plant Cell Physiol. 2001 Feb;42(2):154–161. doi: 10.1093/pcp/pce016. [DOI] [PubMed] [Google Scholar]
  42. Thompson J. E., Fry S. C. Restructuring of wall-bound xyloglucan by transglycosylation in living plant cells. Plant J. 2001 Apr;26(1):23–34. doi: 10.1046/j.1365-313x.2001.01005.x. [DOI] [PubMed] [Google Scholar]
  43. Thompson J. E., Smith R. C., Fry S. C. Xyloglucan undergoes interpolymeric transglycosylation during binding to the plant cell wall in vivo: evidence from 13C/3H dual labelling and isopycnic centrifugation in caesium trifluoroacetate. Biochem J. 1997 Nov 1;327(Pt 3):699–708. doi: 10.1042/bj3270699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Vierhuis E., York W. S., Kolli V. S., Vincken J., Schols H. A., Van Alebeek G. W., Voragen A. G. Structural analyses of two arabinose containing oligosaccharides derived from olive fruit xyloglucan: XXSG and XLSG. Carbohydr Res. 2001 Jun 4;332(3):285–297. doi: 10.1016/s0008-6215(01)00096-9. [DOI] [PubMed] [Google Scholar]
  45. Vincken J. P., Beldman G., Voragen A. G. Substrate specificity of endoglucanases: what determines xyloglucanase activity? Carbohydr Res. 1997 Mar 13;298(4):299–310. doi: 10.1016/s0008-6215(96)00325-4. [DOI] [PubMed] [Google Scholar]
  46. Vincken J. P., York W. S., Beldman G., Voragen A. G. Two general branching patterns of xyloglucan, XXXG and XXGG. Plant Physiol. 1997 May;114(1):9–13. doi: 10.1104/pp.114.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vincken J. P., de Keizer A., Beldman G., Voragen A. G. Fractionation of xyloglucan fragments and their interaction with cellulose. Plant Physiol. 1995 Aug;108(4):1579–1585. doi: 10.1104/pp.108.4.1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Vissenberg K., Fry S. C., Verbelen J. P. Root hair initiation is coupled to a highly localized increase of xyloglucan endotransglycosylase action in Arabidopsis roots. Plant Physiol. 2001 Nov;127(3):1125–1135. [PMC free article] [PubMed] [Google Scholar]
  49. Vissenberg K., Martinez-Vilchez I. M., Verbelen J. P., Miller J. G., Fry S. C. In vivo colocalization of xyloglucan endotransglycosylase activity and its donor substrate in the elongation zone of Arabidopsis roots. Plant Cell. 2000 Jul;12(7):1229–1237. doi: 10.1105/tpc.12.7.1229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Whitney SE, Gothard MG, Mitchell JT, Gidley MJ. Roles of cellulose and xyloglucan in determining the mechanical properties of primary plant cell walls. Plant Physiol. 1999 Oct;121(2):657–664. doi: 10.1104/pp.121.2.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yokoyama R., Nishitani K. A comprehensive expression analysis of all members of a gene family encoding cell-wall enzymes allowed us to predict cis-regulatory regions involved in cell-wall construction in specific organs of Arabidopsis. Plant Cell Physiol. 2001 Oct;42(10):1025–1033. doi: 10.1093/pcp/pce154. [DOI] [PubMed] [Google Scholar]
  52. de Silva J., Jarman C. D., Arrowsmith D. A., Stronach M. S., Chengappa S., Sidebottom C., Reid J. S. Molecular characterization of a xyloglucan-specific endo-(1-->4)-beta-D-glucanase (xyloglucan endo-transglycosylase) from nasturtium seeds. Plant J. 1993 May;3(5):701–711. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES