The fuc1 gene product (20 kDa FUC1) of Pisum sativum has no alpha-L-fucosidase activity.
An alpha-L-fucosidase purified from pea (Pisum sativum L. cv Alaska) epicotyl was previously described as a cell wall enzyme of 20 kDa that hydrolyses terminal alpha-L-fucosidic linkages from oligosaccharide fragments of xyloglucan. cDNA and genomic copies were further isolated and sequenced. The predicted product of the cDNA and the genomic clone (fuc1), was a 20 kDa protein containing a signal peptide and five cysteines. This was the first alpha-L-fucosidase gene to be cloned in plants but its fucosidase activity has not been demonstrated. Here, our biochemical and immuno analyses suggest that fuc1 does not encode an alpha-L-fucosidase. Pea fuc1 expressed in Escherichia coli, insect cells and Arabidopsis thaliana produced recombinant proteins without alpha-L-fucosidase activity. Pea plants had endogenous alpha-L-fucosidase activity, but the enzyme was not recognised by an antibody produced against recombinant FUC1 protein expressed in E. coli. In contrast, the antibody immunoprecipitated a 20 kDa protein which was inactive. By chromatographic analysis of pea protein extracts, we separated alpha-L-fucosidase-active fractions from the 20 kDa protein fractions. We conclude that the alpha-L-fucosidase activity is not attributable to the 20 kDa FUC1 protein. A new function for fuc1 gene product, now named PIP20 (for protease inhibitor from pea) is proposed.
Tarragó
Teresa
T
Department de Genètica Molecular, Institut de Biologia Molecular de Barcelona, (CSIC), 18-26 Jordi Girona, 08034 Barcelona, Spain.
Martínez
Immaculada
I
Torrent
Margarita
M
Codina
Anna
A
Giralt
Ernest
E
Puigdomènech
Pere
P
Ludevid
Dolores
D
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D017360:Arabidopsis / Q000235:genetics; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015151:Immunoblotting; D018532:Pisum sativum / Q000201:enzymology / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018411:Spodoptera; D005644:alpha-L-Fucosidase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D010940:Plant Proteins; D011994:Recombinant Proteins; D005644:alpha-L-Fucosidase