Functional Identification of Two Nonredundant Arabidopsis α(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 plantspecific organs such as the cell wall. Our working hypothesis is that AtFUT4 and AtFUT6 genes encode ␣(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 ␣-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 ␣(1,2)fucosidase, and not to ␣(1,3/4)fucosidase, indicated that an ␣(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.

Metabolic click-labeling with a fucose analog reveals pectin delivery, architecture, and dynamics in Arabidopsis cell walls

“…Finally, FucAl incorporation into fut4;fut6 double-mutant seedlings was identical to wild-type (Fig. S3); the products of these two genes have recently been shown to fucosylate AGPs (21). Taken together, the above results indicate that the majority of FucAl is incorporated into pectic RG-I in Arabidopsis rather than xyloglucan, N-linked glycans, RG-II, or AGPs.…”

“…In Arabidopsis cell walls, fucose is present in the hemicellulose xyloglucan (6), the pectins rhamnogalacturonan-I (RG-I) (25) and rhamnogalacturonan-II (RG-II) (26), arabinogalactan proteins (AGPs) (21), and N-linked glycoproteins (22). Each of these cell-wall components has a characteristic extraction pattern (27) and can be analytically digested by a specific set of enzymes (8).…”

“…FucAl was chosen to investigate click chemistry-mediated labeling of plant cell-wall components because several of these glycans are fucosylated at known positions (20)(21)(22). Arabidopsis Col-0 seedlings were incubated in liquid Murashige and Skoog (MS) mineral medium containing 2.5 μM FucAl for 4 h, labeled with Alexa-488 or Alexa-594 azide by the copper-catalyzed reaction, and observed by epifluorescence microscopy.…”

An Arabidopsis Cell Wall Proteoglycan Consists of Pectin and Arabinoxylan Covalently Linked to an Arabinogalactan Protein

“…Pectin and xylan synthesis occurs in the Golgi (Mohnen, 2008;Brown et al, 2011), and AGP synthesis initiates in the endoplasmic reticulum and continues in the Golgi (Oka et al, 2010;Wu et al, 2010). Thus, one possible mechanism for APAP1 biosynthesis is the addition of glycans onto AGP57C by either the consecutive addition of one residue at a time or by block addition, possibly occurring within the Golgi apparatus (Atmodjo et al, 2011(Atmodjo et al, , 2013.…”

Identification and Characterization of in Vitro Galactosyltransferase Activities Involved in Arabinogalactan-Protein Glycosylation in Tobacco and Arabidopsis

“…In other species, additional transferases are possible, as in Arabidopsis (Arabidopsis thaliana), where a-(1,2)fucosyltransferase is involved in AGP glycosylation (van Hengel and Roberts, 2002;Wu et al, 2010).…”