Molecular cloning and characterization of a plant alpha1,3/4-fucosidase based on sequence tags from almond fucosidase I.
Our work with almond peptide N-glycosidase A made us interested also in the alpha1,3/4-fucosidase which is used as a specific reagent for glycoconjugate analysis. The enzyme was purified to presumed homogeneity by a series of chromatographic steps including dye affinity and fast-performance anion exchange chromatography. The 63 kDa band was analyzed by tandem mass spectrometry which yielded several partial sequences. A homology search retrieved the hypothetical protein Q8GW72 from Arabidopsis thaliana. This protein has recently been described as being specific for alpha1,2-linkages. However, cDNA cloning and expression in Pichia pastoris of the A. thaliana fucosidase showed that it hydrolyzed fucose in 3- and 4-linkage to GlcNAc in Lewis determinants whereas neither 2-linked fucose nor fucose in 3-linkage to the innermost GlcNAc residue were attacked. This first cloning of a plant alpha1,3/4-fucosidase also confirmed the identity of the purified almond enzyme and thus settles the notorious uncertainty about its molecular mass. The alpha1,3/4-fucosidase from Arabidopsis exhibited striking sequence similarity with an enzyme of similar substrate specificity from Streptomyces sp. (Q9Z4I9) and with putative proteins from rice.
Zeleny
Reinhard
R
Department of Chemistry, University of Natural Resources and Applied Biosciences Vienna, Muthgasse, A-1190 Vienna, Austria.
Leonard
Renaud
R
Dorfner
Georg
G
Dalik
Thomas
T
Kolarich
Daniel
D
Altmann
Friedrich
F
D000117:Acetylglucosamine / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D005643:Fucose / Q000378:metabolism; D006868:Hydrolysis; D007983:Lewis Blood Group Antigens / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012275:Oryza / Q000201:enzymology; D010843:Pichia / Q000201:enzymology; D027861:Prunus / Q000201:enzymology*; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity; D005644:alpha-L-Fucosidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D018076:DNA, Complementary; D007983:Lewis Blood Group Antigens; D005643:Fucose; D005644:alpha-L-Fucosidase; D000117:Acetylglucosamine