Functional identification of two nonredundant Arabidopsis alpha(1,2)fucosyltransferases specific to arabinogalactan proteins

Abstract

Virtually nothing is known about the mechanisms and enzymes responsible for the glycosylation of arabinogalactan proteins (AGPs). The glycosyltransferase 37 family contains plant-specific enzymes, which suggests involvement in plant-specific organs such as the cell wall. Our working hypothesis is that AtFUT4 and AtFUT6 genes encode alpha(1,2)fucosyltransferases (FUTs) for AGPs. Multiple lines of evidence support this hypothesis. First, overexpression of the two genes in tobacco BY2 cells, known to contain nonfucosylated AGPs, resulted in a staining of transgenic cells with eel lectin, which specifically binds to terminal alpha-linked fucose. Second, monosaccharide analysis by high pH anion exchange chromatography and electrospray ionization mass spectrometry indicated the presence of fucose in AGPs from transgenic cell lines but not in AGPs from wild type cells. Third, detergent extracts from microsomal membranes prepared from transgenic lines were able to fucosylate, in vitro, purified AGPs from BY2 wild type cells. Susceptibility of [(14)C]fucosylated AGPs to alpha(1,2)fucosidase, and not to alpha(1,3/4)fucosidase, indicated that an alpha(1,2) linkage is formed. Furthermore, dearabinosylated AGPs were not substrate acceptors for these enzymes, indicating that arabinosyl residues represent the fucosylation sites on these molecules. Testing of several polysaccharides, oligosaccharides, and glycoproteins as potential substrate acceptors in the fucosyl transfer reactions indicated that the two enzymes are specific for AGPs but are not functionally redundant because they differentially fucosylate certain AGPs. AtFUT4 and AtFUT6 are the first enzymes to be characterized for AGP glycosylation and further our understanding of cell wall biosynthesis.

FIGURE 1.

Detection of eel (A. anguilla) lectin epitope in tobacco BY2 cells. Eel lectin (anti-H agglutinin) linked to a fluorescent dye (Texas Red) was used to stain (5 h) tobacco BY2 cells from 1-, 6-, and 9-day-old cultures of WT or transgenic cells expressing the AtFUT4.1 or AtFUT6 genes. The cells were observed under fluorescent light (FL) and white light (WL) as indicated. Scale bars, 20 μm.

FIGURE 2.

Expression of His-tagged versions of AtFUT4.1 and AtFUT6 proteins in tobacco BY2 cells. A, dry weight of the cells was determined after 1, 3, 6, 9, 11, and 14 days of culture to monitor growth stages of the cells. The growth was steady until 9 days of culture and then stabilized, indicating a separation of cell division stage (3–9 days) from cell elongation stage (9–14 days). B, 7- and 14-day-old cultures of BY2 WT cells or transgenic BY2 cells were screened by Western blotting using anti-His₆ tag antibodies (Clontech). The cells were harvested and directly ground in liquid nitrogen before extraction with SDS-PAGE loading buffer, and then the proteins were separated on 10% gels. The protein bands detected had an estimated size of ∼60 kDa in agreement with the predicted size of AtFUT4.1 (∼61 kDa) and AtFUT6 (∼59 kDa) proteins. Marker sizes (kDa) are indicated on the left. MW, molecular mass; d, days.

FIGURE 3.

Determination of the presence of Fuc in purified AGPs by HPAEC and ESI-MS. Purified AGPs from tobacco BY2 cells expressing the AtFUT4.1 or AtFUT6 genes or from WT cells were subjected to acid hydrolysis (2 m trifluoroacetic acid), and the resulting monosaccharides were fractionated by HPAEC on a CarboPac PA20 column (A). Fractions eluting between 2 and 6 min were collected, and the peak corresponding to Fuc is indicated by an asterisk in A. HPAEC fractions were then analyzed by ESI-MS (B). The ion at m/z 165 ([M+H]⁺) confirmed the presence of Fuc in the purified AGPs from transgenic BY2 cells and not in AGPs from the BY2 WT cells. Purified l-Fuc was used as standard.

FIGURE 4.

Fucosyltransferase activity in detergent extracts from tobacco BY2 cells expressing the AtFUT4.1 or AtFUT6 gene and from BY2 WT cells. Fucosyltransferase activity was monitored by measuring [¹⁴C]Fuc transfer onto AGPs from GDP-[¹⁴C]Fuc as described under “Experimental Procedures.” Enzyme sources were prepared from microsomal membranes solubilized with 0.5% digitonin from WT cells and transgenic cells. A–C show the fucosyltransferase activity resulting from using AGP substrate acceptors purified from wild type (AGPs-BY2WT), from cells expressing the AtFUT6 gene (AGPs-BY2:F6), and from cells expressing the AtFUT4.1 gene (AGPs-BY2:F4), respectively. All of the AGPs were used at 0, 50, and 100 μg/reaction. Each value is an average based on at least three experiment sets. The error bars represent the S.E.

FIGURE 5.

A simplified representation of an AGP glycomodule (A), and the effect of arabinose (Ara) removal on the ability of these glycomodules to act as acceptors in FUT assay (B). A, the simplified structure of arabinogalactan polymers in BY2 AGPs was adapted from Ref. . The Araf residues susceptible to mild acid treatment are indicated by asterisks. B, fucosyltransferase assays were performed using as acceptors mild untreated and acid-treated AGPs-BY2WT (100 μg) in the presence of GDP-[¹⁴C]Fuc and detergent extracts from BY2 WT cells or transgenic BY2 cell lines expressing the AtFUT4.1 or AtFUT6 genes. The reactions containing no protein extracts were used as negative controls. Each value is an average based on at least three experimental sets. The error bars represent the S.E.

FIGURE 6.

Bio-gel P2 fractionation of the [¹⁴C]Fuc-labeled AGPs-BY2WT after treatment with α(1,2)fucosidase II or α(1,3/4)fucosidase from almond meal as described under “Experimental Procedures.” The [¹⁴C]Fuc-labeled AGPs-BY2WT were formed by detergent extracts from microsomal membranes of BY2 cells expressing AtFUT4.1 or AtFUT6 genes. Bio-gel P2 columns were eluted with degassed water, and the elution volumes of dextran (V_o) (average molecular weight 500,000) and sugars with degree of polymerization (DP) of 1, 2, 4, 6, and 7–9 are indicated with arrows at the top.

FIGURE 7.

Subcellular localization of AtFUT6-GFP in N. tabacum leaves. AtFUT6-GFP and ST-YFP (a Golgi marker) fusion proteins were transiently co-expressed in tobacco plant leaves. Fluorescence spots are seen following the expression of AtFUT6-GFP (a) and ST-YFP (b). Fluorescence spots observed for both AtFUT6-GFP and ST-YFP are co-localized as shown in the overlay (c), suggesting a Golgi localization of the AtFUT6 protein. The scale bars are 8 μm.