Arabidopsis thaliana β1,2-xylosyltransferase: an unusual glycosyltransferase with the potential to act at multiple stages of the plant N-glycosylation pathway

Abstract

XylT (beta1,2-xylosyltransferase) is a unique Golgi-bound glycosyltransferase that is involved in the biosynthesis of glycoprotein-bound N-glycans in plants. To delineate the catalytic domain of XylT, a series of N-terminal deletion mutants was heterologously expressed in insect cells. Whereas the first 54 residues could be deleted without affecting the catalytic activity of the enzyme, removal of an additional five amino acids led to the formation of an inactive protein. Characterization of the N-glycosylation status of recombinant XylT revealed that all three potential N-glycosylation sites of the protein are occupied by N-linked oligosaccharides. However, an unglycosylated version of the enzyme displayed substantial catalytic activity, demonstrating that N-glycosylation is not essential for proper folding of XylT. In contrast with most other glycosyltransferases, XylT is enzymatically active in the absence of added metal ions. This feature is not due to any metal ion directly associated with the enzyme. The precise acceptor substrate specificity of XylT was assessed with several physiologically relevant compounds and the xylosylated reaction products were subsequently tested as substrates of other Golgi-resident glycosyltransferases. These experiments revealed that the substrate specificity of XylT permits the enzyme to act at multiple stages of the plant N-glycosylation pathway.

Reduced Immunogenicity of Arabidopsis hgl1 Mutant N-Glycans Caused by Altered Accessibility of Xylose and core Fucose Epitopes

“…In addition, we obtained evidence that prexylosylation is important for efficient fucosylation, because mutants lacking xylose residues reveal decreased amounts of core fucose residues in their N-glycans. This contrasts with the report of Bencúr et al (43), which showed that fucosyltransferase activity is inhibited by presence of a xylose residue in the acceptor N-glycans. This apparent inconsistency may have been caused by two GlcNac-terminated antennae used as substrate for their in vitro study.…”

A Unique β1,3-Galactosyltransferase Is Indispensable for the Biosynthesis of N-Glycans Containing Lewis a Structures in Arabidopsis thaliana

“…Our data indicate that GALT1 rather than FUT13 is the rate-limiting enzyme. This is supported by the following lines of evidence: (1) GALT1 is expressed in a tissue-specific manner, and its expression levels correlate with the Le a levels of the respective organs; (2) overexpression of GALT1 increases the occurrence of Le a epitopes in tissues with otherwise low Le a contents (e.g., leaves); (3) the catalytic proficiency of GALT1, at least in vitro, is rather low compared with other plant glycosyltransferases (Bencú r et al, 2005;Strasser et al, 2005); (4) while the activities of most other glycosyltransferases involved in N-glycan processing can be readily measured in plant extracts (e.g., Staudacher et al, 1995;Palma et al, 2001;Lé onard et al, 2002;Strasser et al, 2004b), direct detection of endogenous GALT1 activity has not yet been achieved.…”

“…This band was identified as GALT1 itself. Since endogenous and heterologous glycoproteins synthesized by Sf21 cells usually lack N-glycans with terminal galactose residues (e.g., Kubelka et al, 1994;Bencú r et al, 2005), we concluded that GALT1 is capable of autogalactosylation during its production in these cells. In Figure 7B, the autogalactosylation of GALT1 was only detectable upon further incubation with recombinant FUT13 in the presence of the donor substrate GDP-fucose.…”

“…The PCR product was cleaved with PstI and KpnI restriction enzymes and ligated into pVTBacHis1 baculovirus transfer vector, digested with the same enzymes. In this construct, the truncated GALT1 protein is placed downstream of the melittin signal peptide, a 6xHis tag, and an enterokinase cleavage site (Bencú r et al, 2005). Fragments encoding N-terminally truncated versions of Mm-B3GALT1 (amino acids 34 to 326) and FUT13 (amino acids 32 to 401) were generated and subcloned essentially as above using primers MGAL-4F/-5R (Mm-B3GALT1) and primers AthFTC-8F/-11R (FUT13, At1g71990).…”

A proteomic study of the major allergens from yellow jacket venoms

“…Expression in Spodoptera frugiperda Sf21 cells was performed exactly as described previously [21]. Briefly, the recombinant transfer vector (1 mg) was co-transfected with 200 ng of BaculoGold viral DNA (BD Biosciences, Erembodegem, Belgium) into Sf9 cells using Lipofectin (Invitrogen) as recommended by the manufacturer.…”