Xyloglucan fucosyltransferase, an enzyme involved in plant cell wall biosynthesis

Science

Volumn 284, Issue 5422, 1999, Pages 1976-1979

  Perrin, Robyn M ^a   DeRocher, Amy E ^a   Bar Peled, Maor ^a,d   Zeng, Weiqing ^a   Norambuena, Lorena ^c   Orellana, Ariel ^c   Raikhel, Natasha V ^a,b   Keegstra, Kenneth ^a,b  

^a MICHIGAN STATE UNIVERSITY   (United States)

^b Michigan State University   (United States)

^c UNIVERSITY OF CHILE   (Chile)

^d MONSANTO COMPANY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CELLULOSE; FUCOSYLTRANSFERASE; GLUCAN; HEMICELLULOSE; MEMBRANE PROTEIN; PECTIN; UNCLASSIFIED DRUG; XYLENE; XYLOGLUCAN FUCOSYLTRANSFERASE;

AMINO ACID SEQUENCE; ARABIDOPSIS; ARTICLE; CELL WALL; DISEASE RESISTANCE; ENZYME ACTIVITY; ENZYME ANALYSIS; NONHUMAN; PLANT; POLYACRYLAMIDE GEL ELECTROPHORESIS; PRIORITY JOURNAL; PROTEIN EXPRESSION; SEQUENCE ANALYSIS; SIGNAL TRANSDUCTION;

AMINO ACID SEQUENCE; ANIMALS; ARABIDOPSIS; CARBOHYDRATE CONFORMATION; CELL WALL; CLONING, MOLECULAR; COS CELLS; DNA, COMPLEMENTARY; EXPRESSED SEQUENCE TAGS; FUCOSYLTRANSFERASES; GENES, PLANT; GLUCANS; MOLECULAR SEQUENCE DATA; PEAS; POLYSACCHARIDES; XYLANS;

ARABIDOPSIS; MAMMALIA; PISUM SATIVUM;

EID: 0033580981     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5422.1976     Document Type: Article

References (40)

1. N. Carpita, in Plant Metabolism, D. Dennis, D. Turpin, D. Lefebvre, D. Layzell, Eds. (Longman, Singapore, ed. 2, 1997), chap. 9.
2. D. J. Cosgrove, Annu. Rev. Cell Dev. Biol. 13, 171 (1997); Plant Physiol. 102, 1 (1993).
3. D. J. Cosgrove, Annu. Rev. Cell Dev. Biol. 13, 171 (1997); Plant Physiol. 102, 1 (1993).
4. T. Arioli et al., Science 279, 717 (1998); J. R. Pear, Y. Kawagoe, W. E. Schreckengost, D. P. Delmer, Proc. Natl. Acad. Sci. U.S.A. 93, 12637 (1996).
5. T. Arioli et al., Science 279, 717 (1998); J. R. Pear, Y. Kawagoe, W. E. Schreckengost, D. P. Delmer, Proc. Natl. Acad. Sci. U.S.A. 93, 12637 (1996).
6. T. Hayashi, Annu. Rev. Plant Phys. Plant Mol. Biol. 40, 139 (1989).
7. E. Zablackis, J. Huang, B. Muller, A. G. Darvill, P. Albersheim, Plant Physiol. 107, 1129 (1995).
8. S. Levy, G. Maclachlan, L. A. Staehelin, Plant J. 11, 373 (1997); S. Levy, W. S. York, R. Struike-Prill, B. Meyer, L. A. Staehelin, ibid. 1, 195 (1991).
9. S. Levy, G. Maclachlan, L. A. Staehelin, Plant J. 11, 373 (1997); S. Levy, W. S. York, R. Struike-Prill, B. Meyer, L. A. Staehelin, ibid. 1, 195 (1991).
10. S. E. C. Whitney, J. E. Brigham, A. H. Darke, J. S. Grant Reid, M. J. Gidley, ibid. 8, 491 (1995).
11. M. M. Purugganan, J. Braam, S. C. Fry, Plant Physiol. 115, 181 (1997).
12. R. A. Creelman and J. E. Mullet, Plant Cell 9, 1211 (1997); S. C. Fry, S. Aldington, P. R. Hetherington, J. Aitken, Plant Physiol. 103, 1 (1993); G. J. McDougall and S. C. Fry, J. Exp. Bot. 40, 233 (1989); W. S. York, A. G. Darvill, P. Albersheim, Plant Physiol. 75, 295 (1984).
13. R. A. Creelman and J. E. Mullet, Plant Cell 9, 1211 (1997); S. C. Fry, S. Aldington, P. R. Hetherington, J. Aitken, Plant Physiol. 103, 1 (1993); G. J. McDougall and S. C. Fry, J. Exp. Bot. 40, 233 (1989); W. S. York, A. G. Darvill, P. Albersheim, Plant Physiol. 75, 295 (1984).
14. R. A. Creelman and J. E. Mullet, Plant Cell 9, 1211 (1997); S. C. Fry, S. Aldington, P. R. Hetherington, J. Aitken, Plant Physiol. 103, 1 (1993); G. J. McDougall and S. C. Fry, J. Exp. Bot. 40, 233 (1989); W. S. York, A. G. Darvill, P. Albersheim, Plant Physiol. 75, 295 (1984).
15. R. A. Creelman and J. E. Mullet, Plant Cell 9, 1211 (1997); S. C. Fry, S. Aldington, P. R. Hetherington, J. Aitken, Plant Physiol. 103, 1 (1993); G. J. McDougall and S. C. Fry, J. Exp. Bot. 40, 233 (1989); W. S. York, A. G. Darvill, P. Albersheim, Plant Physiol. 75, 295 (1984).
16. K. J. Colley, Glycobiology 7, 1 (1997); J. Puhlmann et al., Plant Physiol. 104, 699 (1994); A. Driouich, L. Faye, L. A. Staehelin, Trends Biochem. Sci. 18, 210 (1993); P. J. Moore, K. M. M. Swords, M. A. Lynch, L. A. Staehelin, J. Cell Biol. 112, 589 (1991).
17. K. J. Colley, Glycobiology 7, 1 (1997); J. Puhlmann et al., Plant Physiol. 104, 699 (1994); A. Driouich, L. Faye, L. A. Staehelin, Trends Biochem. Sci. 18, 210 (1993); P. J. Moore, K. M. M. Swords, M. A. Lynch, L. A. Staehelin, J. Cell Biol. 112, 589 (1991).
18. K. J. Colley, Glycobiology 7, 1 (1997); J. Puhlmann et al., Plant Physiol. 104, 699 (1994); A. Driouich, L. Faye, L. A. Staehelin, Trends Biochem. Sci. 18, 210 (1993); P. J. Moore, K. M. M. Swords, M. A. Lynch, L. A. Staehelin, J. Cell Biol. 112, 589 (1991).
19. K. J. Colley, Glycobiology 7, 1 (1997); J. Puhlmann et al., Plant Physiol. 104, 699 (1994); A. Driouich, L. Faye, L. A. Staehelin, Trends Biochem. Sci. 18, 210 (1993); P. J. Moore, K. M. M. Swords, M. A. Lynch, L. A. Staehelin, J. Cell Biol. 112, 589 (1991).
20. A. E. DeRocher, N. V. Raikhel, K. Keegstra, data not shown.
21. R. Kteene and E. G. Berger, Biochem. Biophys. Acta 1154, 283 (1993).
22. Y. Fujiki, A. L. Hubbard, S. Fowler, P. B. Lazarow, J. Cell Biol. 93, 97 (1982).
23. V. Farkas and G. Maclachlan, Arch. Biochem. Biophys. 264, 48 (1988); A. Camirand, D. Brummell, G. Maclachlan, Plant Physiol. 84, 753 (1987).
24. V. Farkas and G. Maclachlan, Arch. Biochem. Biophys. 264, 48 (1988); A. Camirand, D. Brummell, G. Maclachlan, Plant Physiol. 84, 753 (1987).
25. 3H incorporation was measured by scintillation counting. The amount of fucose incorporated into the product was used to calculate activity in nanokats.
26. 3 to strip away peripheral membrane proteins (13). The suspension was centrifuged at 100,000g for 1 hour, and the resulting pellets were washed and resuspended in buffer [50 mM Pipes-KOH (pH 62), 20% glycerol, 1 mM EDTA, 1 mM DTT, 0.1 M PMSF, and 1 μg each of aprotinin, leupeptin, and pepstatin per millimeter]. The suspension was homogenized, Triton X-100 was added to a final volume of 0.8%, and the sample was stirred for 1 to 2 hours to solubilize membrane proteins before centrifugation a final time at 100,000j for 1 hour. Supernatant was collected and saved. When Arabidopsis celt suspension culture was used as a tissue source, the procedure was identical except that the cells were lysed with a French pressure cell at 4000 psi. Pea carbonate-washed supernatants were pooled and separated on a GDP agarose affinity chromatography column, and GDP-binding proteins were eluted by means of excess free GDP. Protein levels were monitored by absorbance at 280 nm. The protein samples were desalted on a Sephadex G-25 column, concentrated, and further separated on a Phenomenex SEC 4000 size exclusion column. Some samples were further purified with a Poros QE or Resource Q anion exchange column and were subsequently separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
27. Tamarind seed XG was fucosylated by purified pea FTase equal to 33 pKat total activity [assay conditions were as follows: tamarind XG (1 mg/ml), 1.5 mM GDP-fucose, and 50 mM Pipes-KOH (pH 6.2)]. The XG product was precipitated with ethanol, resuspended in water, reprecipitated, and sent to the Complex Carbohydrate Research Center (Athens, GA) for linkage analysis. An equal amount of tamarind XG was also submitted for linkage analysis.
28. Proteins in size exclusion column eluate fractions containing peak amounts of FTase activity were concentrated with a Millipore 4-ml 10-kD concentrator and separated by electrophoresis. After brief staining with Coomassie brilliant blue R250 and destaining, the separated proteins were excised, rinsed in 50% acetonitrile, stored at -80°C, and sent to Harvard Microchemistry (Cambridge, MA) for tryptic peptide sequencing. The following six peptide sequences were obtained: VFGFLGR, YLLHPTNNVWGLVVR, AVLITSLSSGYFEK, YYDAYLAK, LLGCLLADGFDEK, and ESILPDVNR (29). Using these peptides as a query, the Blastp program identifed an Arabidopsis EST, 191A6T7, which encoded four out of six peptides.
29. 2-terminus that is similar to a fifth pea peptide (18). The nucleotide sequence of AtFT1 has been assigned the GenBank accession number AF154111.
30. AtFT1 is encoded within BAC T18E12, derived from chromosome II (nucleotides 41209 through 41503 and 41780 through 43252), which has been fully sequenced by the Arabidopsis Genome Initiative. A second open resting frame has been predicted within T18E12 (nucleotides 43562 through 43748 and 43813 through 45215), which shows, 63% similarity to AtFT1 at the deduced amino acid level.
31. R. M. Perrin, N. V. Raikhel, K. Keegstra, C. Wilkerson, unpublished results.
32. 2 and washing one time with 25 mM HepesKOH (pH 7.0) and 8 M urea. The pellet was resuspended in 6 M guanidine-HCl, and protein was precipitated from the supernatant with 10% trichloro-acetic acid. The protein was emulsified with Titermax adjuvent (CytRx Corporation, Norcross, GA) and injected into a rabbit. For protein immunoblotting, 40 μl of carbonate-washed solubilized protein from Arabidopsis and 50 ng of purified antigen were separated by SDS-PAGE and electrobiotted. Antibodies to AtFT1 (dilution, 1:5000) were used for protein immunoblotting. Horseradish peroxidase-conjugated goat antibodies raised against rabbit antibodies were used as secondary antibodies. Signals were detected by the enhanced chemiluminescence method (Pierce, Rockford, IL). Membranes were stained with Coomassie blue to detect protein.
33. For immunoprecipitations, solid NaCl was added to carbonate-washed solubilized Arabidopsis protein to a final concentration of 200 mM. The Arabidopsis protein was precleared by incubation with 1:10 volume of a 50% slurry of protein A-Sepharose beads (Pharmacia) in buffer A [25 mM Pipes-KOH (pH 7.5), 50 mM NaCl, and 2 mM EDTA (pH 8.0)]. The resulting supernatants were incubated with 50 μl of immune or preimmune antiserum to AtFT1 for 1 hour. A 1:5 volume of protein A-Sepharose slurry was added to precipitate the antigen-antibody complexes, and the samples were incubated for an additional 3 hours with rocking at 4°C. Samples were then centrifuged and washed five times in buffer A containing 1% Triton X-100 and two times in buffer A without detergent. The pellets were resuspended in buffer A to a final volume of 120 μl and assayed for AtFTase activity as described above.
34. 2, 20 mM Hepes (pH 7.0),and 0.05% Triton X-100 at 25°C for 90 min. The reaction was halted by the addition of ethanol to a final concentration of 70%. Samples were incubated at 4°C and filtered through 1.5-μm glass fiber filters. The filters were washed with 70% ethanol containing 1 mM EDTA. The filters were then dried and radioactivity was determined by liquid scintillation. A biological control using pea Golgi vesicles was carried out in parallel.
35. R. M. Perrin, K. Keegstra, N. V. Raikhel, data not shown.
36. Three motifs were found to be conserved among several α-1,2-fucosyltransferases, despite low overall homology. One ([IV]G[IV][HQ][VI]R..[DN]) has been described previously (27) (square brackets indicate that either of the indicated amino acids was found at the indicated position; dots indicate that three or more different amino acids were found at the indicated position). In addition, a second motif (D[EK] [MQ][FI)F[CR][EQ].DQ) and a third region (G[LF]G [ND][RC)[IL].[TS][LI)A[SA].[FW][LR][YF]A.[LQ]T[DG] R..[LA].[VI][DE]) were conserved (29).
37. C. Breton, R. Oriol, A. Inberty, Glycobiology 8, 87 (1998).
38. J. E. Varner and L.-S. Lin, Cell 56, 231 (1989).
39. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, IIe; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S. Ser, T, Thr V, Val; W, Trp; and Y, Tyr.
40. The authors acknowledge funding from the Department of Energy (grant DE-FG02-91ER20021), C. Wilkerson for assistance with computer analysis, and members of the Keegstra and Raikhel laboratories for helpful discussions.