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Glycobiology Advance Access published online on May 24, 2004
Glycobiology, doi:10.1093/glycob/cwh097
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Received March 4, 2004
Revised May 15, 2004
Accepted May 16, 2004

ORIGINAL ARTICLES

Structural characterization by 13C-NMR spectroscopy of products synthesized in vitro by polysaccharide synthases using 13C-enriched glycosyl donors: application to a UDP-glucose:(1->3)-{beta}-D-glucan synthase from blackberry (Rubus fruticosus)

Jon K. Fairweather 1, Joséphine Lai Kee Him 1, Laurent Heux 1, Hugues Driguez 1, Vincent Bulone 1*

1 Centre de Recherches sur les Macromolécules Végétales (CERMAV-UPR CNRS 5301), affiliated with the Joseph Fourier University of Grenoble, B.P. 53, 38041 Grenoble cedex 9, France

* To whom correspondence should be addressed. E-mail: Vincent.Bulone{at}univ-lyon1.fr.


  Abstract

A simple and sensitive method for the characterization of products synthesized in vitro by polysaccharide synthases is described. It relies on the use of 13C-enriched nucleotide sugars as substrates and on the analysis of the newly synthesized polysaccharides by 13C nuclear magnetic resonance (NMR) spectroscopy. The method was validated with a (1->3)-{beta}-D-glucan synthase from blackberry, but it may be applied to the study of any glycosyltransferase. The chemical synthesis of UDP-D-[U-13C]glucose was achieved in a classical procedure, with an overall yield of 50%. A uniformly labeled (1->3)-{beta}-D-glucan was synthesized from this substrate, using detergent extracts of blackberry cell membranes as a source of synthase. One hundred µg of product was sufficient for liquid and solid-state 13C-NMR spectroscopy analyses. The method is at least 100 times more sensitive than in the case of non-enriched polysaccharides. It allows the unequivocal identification and direct structural characterization of the products synthesized in vitro, as opposed to conventional methods that rely on the use of radioactive substrates and enzymatic hydrolysis of the polysaccharides with specific glycoside hydrolases. The method proves that the glycan analyzed was synthesized de novo since the final product is enriched in 13C. Information on the three-dimensional organization of the polymer may also be obtained by solid-state NMR spectroscopy.

Key words: 13C-NMR spectroscopy, in vitro synthesis of (1->3)-{beta}-D-glucan, polysaccharide synthases, structural analysis of polysaccharides, UDP-[U-13C]glucose


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