Elsevier

Carbohydrate Research

Volume 344, Issue 12, 17 August 2009, Pages 1487-1493
Carbohydrate Research

Synthesis of Lewis X epitopes on plant N-glycans

Dedicated to Professor Hans Kamerling on the occasion of his 65th birthday
https://doi.org/10.1016/j.carres.2009.05.003Get rights and content

Abstract

Glycoproteins from tobacco line xFxG1, in which expression of a hybrid β-(1→4)-galactosyltransferase (GalT) and a hybrid α-(1→3)-fucosyltransferase IXa (FUT9a) is combined, contained an abundance of hybrid N-glycans with Lewis X (LeX) epitopes. A comparison with N-glycan profiles from plants expressing only the hybrid β-(1→4)-galactosyltransferase suggested that the fucosylation of the LacNAc residues in line xFxG1 protected galactosylated N-glycans from endogenous plant β-galactosidase activity.

Introduction

N-Glycosylation in plants and animals is similar up to the Golgi complex, where this process starts to diverge between and within both types of organisms depending on tissue type and developmental stage. The differences are mainly due to differences in the repertoire of glycosyltransferases encoded by these organisms and to some extent to differential compartmentalization.1, 2 A whole range of N-acetylglucosaminyltransferases and galactosyltransferases such as β-(1→4)-galactosyltransferase (GalT) do not occur in plants, although GalT can be expressed ectopically in tobacco using genetic modification as was shown in our laboratory.3, 4 Conversely, β-(1→2)-xylosyltransferase (XylT) appears to be ubiquitous in plants and seems to be absent from most animals except snails5 and trematodes.6

There is at least one modification at the N-glycan antennae that is shared by plants and animals, that is, the Lea epitope that is the result of the consecutive linkage of β-(1→3)-galactose and α-(1→4)-fucose to the terminal GlcNAc.7 A plant homologue of the fucosyltransferase responsible for this addition has been cloned in our laboratory.8 In our efforts to enable production of ‘humanized’ antibodies in plants, the human β-(1→4)-galactosyltransferase 1 (GalT)3, and later a hybrid GalT comprising the N-terminal domain of the Arabidopsis XylT and the catalytic domain of GalT, were introduced in tobacco4 and shown to induce synthesis of N-glycans with LacNAc structures that are typical of animal N-glycans. In tobacco plants expressing the hybrid GalT, we found a remarkable accumulation of the hybrid N-glycan GlcNAcMan5GlcNAc2, which was very likely to be due to de-galactosylation of the main galactosylated N-linked oligosaccharide GalGlcNAcMan5GlcNAc2. Under normal circumstances GlcNAcMan5GlcNAc2 is a transient intermediate in the synthesis of complex N-glycans and does not accumulate to a detectable level.

Plant β-galactosidases that might account for this de-galactosylation of β-(1→4)-galactosylated N-glycans, such as jack bean β-galactosidase, have been described decades ago.9 In general, α-(1→3)-fucosylation of a Galβ-(1→4)-GlcNAc (LacNAc) structure renders the galactose residue insensitive to a range of β-galactosidases10, a fact that has turned out to be very useful for sequential glycosidase sequencing of oligosaccharides.11 Hence, it was deemed useful to try to modify the ectopic LacNAc structures in plants expressing the hybrid GalT by expression of a suitable α-(1→3)-fucosyltransferase from an animal source and thus create an LeX epitope.

The mouse FUT9 gene was the first of its kind to be cloned12 and was subsequently shown to be highly homologous to its human counterpart.13 It proved to be a distant member of a family of at least six mammalian α-(1→3)-fucosyltransferases, five of which were capable of fucosylating the nonreducing ends of glycans with type 2 chains yielding LeX epitopes.14

In this paper we describe the results of MALDI-TOFMS analysis of N-glycans from tobacco plants transformed with a construct comprising two genes, the hybrid GalT used in earlier experiments and a hybrid FUT9. Both genes featured the N-terminal domain of Arabidopsis XylT. The stem region and catalytic domain of the hybrid FUT9 were derived from a pufferfish Tetraodon nigroviridis FUT9 homologue displaying 57% homology at the amino acid level with the corresponding segment of the mouse and human FUT9. One transgenic tobacco line was identified that synthesizes N-glycans with LeX epitopes. Five other lines were found that lack galactosylation of N-glycans, but contain typical wild-type plant N-glycans with one or two additional fucose residues.

Section snippets

Construction of plant transformation vectors

The gene fragment encoding the catalytic domain comprising residues 38–359 of a T. nigroviridis FUT9a homologue was obtained by PCR using genomic DNA and primers based on accession AJ783833. It was fused N-terminally with the sequence encoding the first 53 aa of the Arabidopsis thaliana XylT gene. Sequencing of a number of independent clones containing the truncated FUT9 gene revealed two non-silent mutations in all of them that lead to aa substitutions at positions 65 (I to V) and 176 (A to

Discussion

The enzymatic characteristics of the pufferfish FUT9 homologue employed in this study had not been verified experimentally, but the segment we used was 57% homologous at the amino acid level to both the mouse and human FUT9 proteins. As the human enzyme was shown to preferentially fucosylate terminal LacNAc structures,14 unlike the other human FUT genes capable of fucosylating lactosamine, we considered it potentially useful for synthesizing LeX on plant N-glycans. The choice of

Construction of plant transformation vectors

A DNA fragment comprising the stem region and catalytic domain of T. nigroviridis FUT9a (accession no. AJ783833; Martinez-Duncker, I., Oriol, R. and Mollicone, R., unpublished) was amplified from genomic DNA using the following primers: FUTup, TGACCATGGCGTCTCACATGACTGAATTCTCCTCCGGACCAGTGGAGACAGGACTGA; FUTdw, GTGACGGATCCAATCAACCCCAGTACCACTTGTTAAG. The Nco I/Bam HI digested fragment was cloned into a likewise digested pMTL2336 derivative lacking the Eco 31I site, giving clone pMTLtrFUT9. A cDNA

Acknowledgements

We thank Dr. A. Roesner and Professor T. Burmester (Johannes Gutenberg-University, Mainz, Germany) for providing us with Tetraodon nigroviridis genomic DNA and Dr. C.H. Hokke (University of Leiden, The Netherlands) and Dr. A. Streit (King’s College. London, United Kingdom) for providing monoclonal antibodies 128-4F9-A, 291-4D10-A, 291-2G3-A and anti-L5, respectively.

References (40)

  • I.B.H. Wilson

    Curr. Opin. Struct. Biol.

    (2002)
  • V. Gomord et al.

    Curr. Opin. Plant Biol.

    (2004)
  • H. Bakker et al.

    FEBS Lett.

    (2001)
  • R. Zeleny et al.

    Anal. Biochem.

    (1997)
  • A. Kobata

    Anal. Biochem.

    (1979)
  • T. Kudo et al.

    J. Biol. Chem.

    (1998)
  • M. Kaneko et al.

    FEBS Lett.

    (1999)
  • S. Nishihara et al.

    FEBS Lett.

    (1999)
  • C. Brito et al.

    Biochimie

    (2008)
  • E.V. Chandrasekaran et al.

    Carbohydr. Res.

    (2003)
  • R. Zeleny et al.

    Phytochemistry

    (2006)
  • Y.O. Ahn et al.

    Phytochemistry

    (2007)
  • P.J. Tacken et al.

    Blood

    (2005)
  • S.-C. Hsu et al.

    J. Aller. Clin. Immunol.

    (2007)
  • S.P. Chambers et al.

    Gene

    (1988)
  • H. Bakker et al.

    Proc. Nat. Acad. Sci. U.S.A.

    (2001)
  • H. Bakker et al.

    Proc. Nat. Acad. Sci. U.S.A.

    (2006)
  • J.A. van Kuik et al.

    J. Biol. Chem.

    (1985)
  • K.-H. Khoo et al.

    Glycobiology

    (1997)
  • A.-C. Fitchette-Lainé et al.

    Plant J.

    (1997)
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