Arabidopsis thaliana beta1,2-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.

Figure 1. Characterization of N-terminally truncated variants of A. thaliana XylT produced in insect cells

(A) Heterologous expression of soluble forms of A. thaliana XylT in insect cells. Equivalent amounts of culture supernatants (sn) and protein extracts from Sf21 insect cells (c) infected with recombinant baculoviruses encoding the indicated N-terminally truncated versions of A. thaliana XylT lacking between 39 (Δ39) and 79 (Δ79) residues were separated under reducing conditions by SDS/PAGE, transferred on to a nitrocellulose membrane and probed with mouse anti-enterokinase recognition site antibodies. Migration positions of selected prestained molecular-mass standards are indicated. The detected sizes of the recombinant polypeptides were in close agreement with their theoretical molecular masses (not accounting for any N-glycans). No specific signals were obtained with lysates and conditioned media of uninfected Sf21 cells. (B) Analysis of A. thaliana XylT after endoglycosidase treatment. Protein extracts from Sf21 insect cells infected with recombinant baculoviruses encoding N-terminally truncated versions of A. thaliana XylT lacking 54 (Δ54) and 59 (Δ59) residues were incubated overnight in the presence (+) and absence (−) of PNGase F or endo H. Proteins separated by SDS/PAGE were transferred on to a nitrocellulose membrane and probed with anti-enterokinase recognition site antibodies as above.

Figure 2. Limited proteolysis of purified recombinant A. thaliana XylT by a co-purifying proteinase

(A) Generation of two-chain A. thaliana XylT on prolonged storage. Purified recombinant A. thaliana XylT was stored in the absence (−) or presence (+) of proteinase inhibitors (E-64, leupeptin and PMSF) for 3 months at 4 °C. Samples were analysed by SDS/PAGE under reducing conditions and silver staining. The migration positions of selected molecular-mass standards are indicated. (B) The light chain of two-chain XylT is derived from the N-terminal part of the native enzyme. Purified recombinant A. thaliana XylT stored in the absence of proteinase inhibitors for 3 months was incubated overnight in the presence (+) or absence (−) of PNGase F. Samples were then analysed by SDS/PAGE under reducing conditions and silver staining or subjected to Western-blot analysis with anti-enterokinase recognition site antibodies. *, the migration position of PNGase F.

Figure 3. All three potential N-glycosylation sites of A. thaliana XylT are modified with N-linked oligosaccharides

The two-chain form of purified recombinant A. thaliana XylT was incubated overnight in the absence (A, C) or presence (B) of PNGase F before SDS/PAGE analysis. Bands corresponding to the XylT heavy chain were excised and subjected to trypsin treatment (A, B) or trypsin/endoproteinase Glu-C (C) treatment. Peptides thus generated were analysed by MALDI–TOF-MS. (A, B) The peptide N³⁰¹FTKPVCFR³⁰⁹ is modified at Asn³⁰¹ with N-linked oligosaccharides (A). The GP is partially modified at its cysteine residue with acrylamide instead of being carbamidomethylated, resulting in double peaks. The corresponding unglycosylated peptide was not detected in the sample. The N-glycan structures found were MM [Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc], MMX {Xylβ1-2[Manα1-6(Manα1-3)]Manβ1-4GlcNAcβ1-4GlcNAc} and MMF⁶ [Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc]. These GP peaks are absent from the deglycosylated sample (B). (C) The peptide A⁴⁵⁹SVIIGAHGAGLTHIVSATPN⁴⁷⁹TTIFE⁴⁸⁴ is modified at Asn⁴⁷⁹ with N-linked oligosaccharides. Two main N-glycan structures could be detected (MM and MMX). The corresponding unglycosylated peptide was not detected in the sample.

Figure 4. Synthesis of unglycosylated A. thaliana XylT in insect cells

Sf21 insect cells infected with recombinant baculoviruses encoding Δ39 XylT were cultured in the absence (–) or presence (+) of tunicamycin. Cell extracts were then incubated overnight in the presence (+) or absence (–) of PNGase F before SDS/PAGE and immunoblot analysis with anti-enterokinase recognition site antibodies. The migration positions of selected prestained molecular-mass standards are indicated.

Figure 5. Acceptor substrate specificities of XylT and GnT II

(A) Purified recombinant A. thaliana XylT was incubated with the indicated acceptor substrates and UDP-[¹⁴C]xylose. The samples were then analysed by TLC before orcinol/H₂SO₄ staining (lanes 1–5) and autoradiography (lanes 6 and 7). The migration direction is indicated by an arrow. (B) Purified recombinant human GnT II was incubated with the indicated acceptor substrates and UDP-[¹⁴C]GlcNAc. The samples were then analysed by TLC before orcinol/H₂SO₄ staining (lanes 1–5) and autoradiography (lanes 6 and 7).

Scheme 1. XylT may act at multiple stages of the plant N-glycosylation pathway

The standard pathway is shown in black and novel steps are displayed in grey. Reactions still awaiting experimental verification are highlighted with a question mark. Man II, α-mannosidase II.