AtFXG1, an Arabidopsis gene encoding alpha-L-fucosidase active against fucosylated xyloglucan oligosaccharides

Abstract

An alpha-L-fucosidase (EC 3.2.1.51) able to release the t-fucosyl residue from the side chain of xyloglucan oligosaccharides has been detected in the leaves of Arabidopsis plants. Moreover, an alpha-L-fucosidase with similar substrate specificity was purified from cabbage (Brassica oleracea) leaves to render a single band on SDS-PAGE. Two peptide sequences were obtained from this protein band, and they were used to identify an Arabidopsis gene coding for an alpha-fucosidase that we propose to call AtFXG1. In addition, an Arabidopsis gene with homology with known alpha-L-fucosidases has been also found, and we proposed to name it as AtFUC1. Both AtFXG1 and ATFUC1 were heterologously expressed in Pichia pastoris cells and the alpha-L-fucosidase activities secreted to the culture medium. The alpha-L-fucosidase encoded by AtFXG1 was active against the oligosaccharides from xyloglucan XXFG as well as against 2'-fucosyl-lactitol but not against p-nitrophenyl-alpha-L-fucopyranoside. However, the AtFUC1 heterologously expressed was active only against 2'-fucosyl-lactitol. Thus, the former must be related to xyloglucan metabolism.

Figure 1

α-Fucosidase activity extracted from Arabidopsis leaves. The leaves from 21-d-old plants were sorted according to their appearance order. α-Fucosidase activity was measured against [³H]Fuc-labeled XXFG (white bars) and 2′-fucosyl-lactitol (black bars). Mean values with ses are given (n = 3).

Figure 2

Hydrophobic-interaction chromatography. The cabbage protein extract partially purified after ammonium sulfate precipitation and cation-exchange chromatography was chromatographed on a Phenyl-Sepharose HR column. The column was eluted with a gradient between 2 and 0 m of (NH₄)₂SO₄ (upper panel) dissolved in 0.1 m sodium acetate pH 5.5 containing 1 mm dithiotreitol (DTT). Four-milliliter fractions were collected and the α-fucosidase activity (●) was measured against 2′-fucosyl-lactitol. Protein was measured by A₂₈₀ (○).

Figure 3

Preparative IEF. The partially purified extract from cabbage leaves after affinity chromatography was subjected to a preparative IEF using a Mini-Rotofor equipment with an ampholytes pH range between 6.7 and 8.0. One-milliliter fractions were collected and the α-fucosidase activity (●) was measured against 2′-fucosyl-lactitol. Protein was measured by the Coomassie protein assay reagent (○).

Figure 4

Gel permeation chromatography. Fractions 7 to 19 from IEF were pooled and chromatographed on a Sephacryl S-200 HR column. The column was eluted with 0.1 m sodium acetate, pH 5.5, containing 1 mm DTT. Four-milliliter fractions were collected. α-Fucosidase activity (●) was measured against 2′-fucosyl-lactitol, and protein was measured by the Coomassie protein assay reagent.

Figure 5

SDS-PAGE. Selected fractions from the gel permeation chromatography were analyzed by SDS-PAGE on 12% (w/v) polyacrylamide gels. M, Molecular markers.

Figure 6

Schematic representation of the AtFXG1 protein. Amino acid sequence of the two peptides obtained from purified cabbage α-fucosidase are given. Identical amino acids are noted as asterisks. ♦, Putative N-glycosylation sites.

Figure 7

Comparison of a putative gene from Arabidopsis (accession no. AC005851) with known fucosidases from different sources. Bold capital letters mean identical or conservative substitutions, lowercase letters mean non-conserved residues. Fuc_Rat, α-l-Fucosidase from Rattus norvegicus (S10235); Fuc_Hom, α-l-fucosidase from Homo sapiens (NP 000138); Fuc_Can, α-l-fucosidase from Canis familiaris (P48300); and Fuc_Str, α-l-fucosidase from Streptomyces sp. (AAD10477). ♦, Putative N-glycosylation sites.

Figure 8

α-Fucosidase activity measured against 2′-fucosyl-lactitol in the culture medium of P. pastoris cells transformed with Arabidopsis α-fucosidase genes. ○, AtFUC1; ●, AtFXG1.