AtFXG1, an Arabidopsis Gene Encoding α-l-Fucosidase Active against Fucosylated Xyloglucan Oligosaccharides

Abstract

An ␣-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 ␣-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 ␣-fucosidase that we propose to call AtFXG1. In addition, an Arabidopsis gene with homology with known ␣-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 ␣-l-fucosidase activities secreted to the culture medium. The ␣-l-fucosidase encoded by AtFXG1 was active against the oligosaccharides from xyloglucan XXFG as well as against 2Ј-fucosyl-lactitol but not against p-nitrophenyl-␣-lfucopyranoside. However, the AtFUC1 heterologously expressed was active only against 2Ј-fucosyl-lactitol. Thus, the former must be related to xyloglucan metabolism.Plant cell walls are built by two independent networks, cellulose microfibers cross-linked by xyloglucan chains and cross-linked pectins, both networks contributing to their mechanical and functional properties (Roberts, 2001). The xyloglucan-cellulose complex has been considered as the network responsible for controlling the rate of cell expansion, xyloglucan being the load-bearing component in the primary cell walls because of its proposed cross-linking of the cellulose microfibers (Fry, 1989). It consists of a linearattached to the OH-6 of ␤-glucosyl residues. In addition to its structural role, xyloglucan may act as a source of signaling molecules. Oligosaccharides derived from xyloglucan have been found to be formed in vivo (Fry, 1986), and they have been shown to regulate auxin-induced Fry, 1988, 1990) and acid pH-induced (Lorences et al., 1990) growth, the t-Fuc being necessary for the regulatory effect of the XXFG, a nonasaccharide derived from xyloglucan (Fry et al., 1993). The Fuc-deficient mur mutants of Arabidopsis (Reiter et al., 1993) showed dwarf growth habit and fragile cell walls.Enzymes that modify xyloglucan oligosaccharides have been detected in plant cell walls (Fry, 1995). An ␣-fucosidase that removes the ␣-l-fucosyl residue from XXFG has been purified from pea (Pisum sativum) epicotyls (Farkas et al., 1991; Augur et al., 1993) and has been cloned (Augur et al., 1995). Furthermore, cDNAs encoding ␣-l-fucosidase have been also isolated from human (Fukushima et al., 1985;Occhiodoro et al., 1989) and rat (Fisher and Aronson, 1989) livers. However, no ␣-l-fucosidase has yet been identified in Arabidopsis. Although some sequences homologous to pea ␣-l-fucosidase have been found in leguminous plants, there is no clear candidate for this activity in the whole genome of Arabidopsis.Thus, our aim in the present paper has been to look for an Arabidopsis gene(s) encoding for ␣-fucosidase activity. Two differ...

AXY8 Encodes an α-Fucosidase, Underscoring the Importance of Apoplastic Metabolism on the Fine Structure of Arabidopsis Cell Wall Polysaccharides

“…The AXY8-GFP fusion was present in the apoplast and was retained in the apoplast when the plasma membrane, as indicated by the marker FM 4-64, receded under plasmolysis-inducing conditions ( Figure 3J; see Supplemental Figure 5 online). These experiments confirmed earlier reports that AXY8 represents a soluble protein (Lé onard et al, 2008) and that plant fucosidase activities can be found in apoplastic extracts of plants (de la Torre et al, 2002;Franková and Fry, 2011).…”

“…Two Arabidopsis genes have been proposed to encode XyG: fucosidases: FXG1, as it exhibits a-fucosidase activity against XyG oligosaccharides when heterologously expressed (de la Torre et al, 2002), and Fuc95A that when expressed heterologously as previously shown is able to release Fuc from XyG oligosaccharides and polymers (Lé onard et al, 2008). However, their role in planta has not been established yet.…”

“…Based on previous biochemical studies (de la Torre et al, 2002;Franková and Fry, 2011) and confirmed by GFP experiments presented here, AXY8 is localized in the apoplast ( Figure 3). Based on OLIMP of microsomal fractions, it acts mainly but not exclusively on XyG after secretion into the apoplast (Figure 4; see Supplemental Table 1 online).…”

Quantitative Trait Loci Analysis of Primary Cell Wall Composition in Arabidopsis

“…Any change in the side-chain structures of xyloglucan may result primarily from a net change in biosynthesis, production of the substrate (GDP-Fuc) by fucosyltransferases, or catabolism by fucosidases. A number of enzymes affecting xyloglucan side chains have been identified: MUR1/GMD1, GMD2, and GER are involved in the synthesis of GDP-Fuc (Bonin and Reiter, 2000); MUR2 is a xyloglucan-specific fucosyltransferase (Perrin et al, 1999); MUR3 is a galactosyl transferase, catalyzing the addition of Gal to the third position of XXXG but not to the equivalent position of XLXG (Madson et al, 2003); and FXG1 is a fucosidase that removes fucosyl moieties from XyGOs (De La Torre et al, 2002). Neither MUR2 nor any of the other nine members of the fucosyltransferase gene family map to any of the three QTL.…”

Structure and Substrate Specificity of a Eukaryotic Fucosidase from Fusarium graminearum

“…Crystal structural studies have shown that GH29 enzymes have a conserved catalytic active site formed at one end of a (␤/␣) 8 triosephosphate isomerase (TIM) barrel domain, whereas GH95 enzymes have their active site formed by an (␣/␣) 6 helical barrel domain (22,23,(27)(28)(29). Besides GH29 and GH95 enzymes, A. thaliana also expresses a novel lipase-like fucosidase named AtFXG1, which has not been classified in CAZy (33).…”