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Glycobiology Advance Access published online on June 13, 2007
Glycobiology, doi:10.1093/glycob/cwm055
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© The Author 2007. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Molecular basis for acceptor substrate specificity of the human ß1,3-glucuronosyltransferases glcat-i and glcat-p involved in glycosaminoglycan and hnk-1 carbohydrate epitope biosynthesis, respectively

Magali Fondeur-Gelinotte1, Virginie Lattard1, Sandrine Gulberti1, Rafael Oriol2, Guillermo Mulliert3, Michael W.H. Coughtrie4, Jacques Magdalou1, Patrick Netter1, Mohamed Ouzzine1 and Sylvie Fournel-Gigleux1,*

1 UMR 7561 CNRS-Université Henri Poincaré Nancy I, Faculté de Médecine, 54505 Vandœuvre-lès-Nancy, France
2 INSERM U602, Université de Paris Sud XI, 94807 Villejuif, France
3 UMR 7036 CNRS-Université Henri Poincaré Nancy I, Faculté des Sciences, 54505 Vandœuvre-lès-Nancy, France,
4 Department of Pathology and Neurosciences, University of Dundee, DD1 9SY Dundee, U.K.

* To whom correspondence should be addressed: UMR CNRS 7561-Université Henri Poincaré Nancy 1, Faculté de Médecine, BP 184, 54505 Vandœuvre-lès-Nancy, France. Tel: 33 383 68 39 72. Fax: 33 383 68 39 59. E-mail: sfg{at}medecine.uhp-nancy.fr

Received on March 8, 2007; revised on May 7, 2007; accepted on May 11, 2007

The human ß1,3-glucuronosyltransferases GlcAT-I and GlcAT-P are key enzymes involved in proteoglycan and HNK-1 carbohydrate epitope synthesis, respectively. Analysis of their acceptor specificity revealed that GlcAT-I was selective towards Galß1,3Gal (referred as Gal2-Gal1), whereas GlcAT-P presented a broader profile. To understand molecular basis of acceptor substrate recognition, we constructed mutants and chimeric enzymes based on multiple sequence alignment and structural information. The drastic effect of mutations of Glu227, Arg247, Asp252 and Glu281 on GlcAT-I activity indicated a key role of the hydrogen bond network formed by these four conserved residues in dictating Gal2 binding. Investigation of GlcAT-I determinants governing Gal1 recognition showed that Trp243 could not be replaced by its counterpart Phe in GlcAT-P. This result combined to molecular modelling provided evidence for the importance of stacking interactions with Trp at position 243 in the selectivity of GlcAT-I towards Galß1,3Gal. Mutation of Gln318 predicted as hydrogen-bonded with 6-hydroxyl of Gal1 had little effect on GlcAT-I activity, reinforcing the role of Trp243 in Gal1 binding. Substitution of Phe245 in GlcAT-P to Ala selectively abolished Galß1,3Gal activity, also highlighting the importance of an aromatic residue at this position in defining the specificity of GlcAT-P. Finally, substituting Phe245, Val320 or Asn321 in GlcAT-P predicted to interact with GlcNAc, to their counterpart in GlcAT-I, moderately affected the activity towards the reference substrate of GlcAT-P, Galß1,4GlcNAc, indicating that its active site tolerates amino acid substitutions, an observation that parallels its promiscuous substrate profile. Taken together, the data clearly define key residues governing ß1,3-glucuronosyltransferases specificity.

Key words: ß1,3-glucuronosyltransferases / acceptor substrate specificity / kinetics / site-directed mutagenesis / glycosaminoglycans


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