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Expression analysis of a type S2 EUL-related lectin from rice in Pichia pastoris

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Abstract

Rice (Oryza sativa) expresses different putative carbohydrate-binding proteins belonging to the class of lectins containing an Euonymus lectin (EUL)-related domain, one of them being OrysaEULS2. The OrysaEULS2 sequence consists of a 56 amino acid N-terminal domain followed by the EUL sequence. In this paper the original sequence of the EUL domain of OrysaEULS2 and some mutant forms have been expressed in Pichia pastoris. Subsequently, the recombinant proteins were purified and their carbohydrate binding properties determined. Analysis of the original protein on the glycan array revealed interaction with mannose containing structures and to a lesser extent with glycans containing lactosamine related structures. It was shown that mutation of tryptophan residue 134 into leucine resulted in an almost complete loss of carbohydrate binding activity of OrysaEULS2. Our results show that the EUL domain in OrysaEULS2 interacts with glycan structures, and hence can be considered as a lectin. However, the binding of the protein with the array is much weaker than that of other EUL-related lectins. Furthermore, our results indicate that gene divergence within the family of EUL-related lectins lead to changes in carbohydrate binding specificity.

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Acknowledgments

This work was funded primarily by the Fund for Scientific Research – Flanders (FWO grants G.0022.08 and KAN 1.5.069.09.N.), the Research Council of Ghent University (projects BOF2005 ⁄ GOA ⁄ 008 and BOF2007 ⁄ GOA ⁄ 0017). Bassam Al Atalah is recipient of a doctoral grant from the Special Research Council of Ghent University. The authors want to thank the Consortium for Functional Glycomics funded by the NIGMS GM62116 for the glycan array analysis. The authors also want to thank the Rice Genome Resource Center, National Institute of Agrobiological Sciences, Japan for providing the cDNA clone encoding OrysaEULS2.

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Authors and Affiliations

  1. Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure links 653, 9000, Ghent, Belgium

    Bassam Al Atalah & Els J. M. Van Damme

  2. Signaux et Messages Cellulaires chez les Végétaux, UMR CNRS-UPS 5546, Pole de Biotechnologie végétale, BP 17, 24 Chemin de Borde Rouge, Castanet-Tolosan, 31326, France

    Pierre Rougé

  3. Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, 30322, USA

    David F. Smith & Yi Lasanajak

  4. Laboratory of Molecular Immunology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000, Leuven, Belgium

    Paul Proost

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  1. Bassam Al Atalah
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  2. Pierre Rougé
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  3. David F. Smith
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Correspondence to Els J. M. Van Damme.

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Al Atalah, B., Rougé, P., Smith, D.F. et al. Expression analysis of a type S2 EUL-related lectin from rice in Pichia pastoris . Glycoconj J 29, 467–479 (2012). https://doi.org/10.1007/s10719-012-9405-2

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