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J. Biol. Chem., Vol. 279, Issue 14, 13461-13468, April 2, 2004

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Structure-Function Analysis of the Human Sialyltransferase ST3Gal I

ROLE OF N-GLYCOSYLATION AND A NOVEL CONSERVED SIALYLMOTIF^*

Charlotte Jeanneau, Valérie Chazalet, Claudine Augé, Dikeos Mario Soumpasis¶, Anne Harduin-Lepers||, Philippe Delannoy||, Anne Imberty, and Christelle Breton**

From the Centre de Recherches sur les Macromolécules Végétales (affiliated to Joseph Fourier University), GDR CNRS n° 2590, F-38041 Grenoble, France, the UMR CNRS 8614, Paris-Sud University, F-91405 Orsay, France, the ^¶Center for Biological Sequence Analysis, Technical University of Denmark, DK-2800 Lyngby, Denmark, and the ^||UMR CNRS 8576, USTL, F-59655 Villeneuve d'Ascq, France

All eukaryotic sialyltransferases have in common the presence in their catalytic domain of several conserved peptide regions (sialylmotifs L, S, and VS). Functional analysis of sialylmotifs L and S previously demonstrated their involvement in the binding of donor and acceptor substrates. The region comprised between the sialylmotifs S and VS contains a stretch of four highly conserved residues, with the following consensus sequence (H/y)Y(Y/F/W/h)(E/D/q/g). (Capital letters and lowercase letters indicate a strong or low occurrence of the amino acid, respectively.) The functional importance of these residues and of the conserved residues of motif VS (HX₄E) was assessed using as a template the human ST3Gal I. Mutational analysis showed that residues His²⁹⁹ and Tyr³⁰⁰ of the new motif, and His³¹⁶ of the VS motif, are essential for activity since their substitution by alanine yielded inactive enzymes. Our results suggest that the invariant Tyr residue (Tyr³⁰⁰) plays an important conformational role mainly attributable to the aromatic ring. In contrast, the mutants W301F, E302Q, and E321Q retained significant enzyme activity (25–80% of the wild type). Kinetic analyses and CDP binding assays showed that none of the mutants tested had any significant effect in nucleotide donor binding. Instead the mutant proteins were affected in their binding to the acceptor and/or demonstrated lower catalytic efficiency. Although the human ST3Gal I has four N-glycan attachment sites in its catalytic domain that are potentially glycosylated, none of them was shown to be necessary for enzyme activity. However, N-glycosylation appears to contribute to the proper folding and trafficking of the enzyme.

Received for publication, October 27, 2003 , and in revised form, December 24, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: CERMAV-CNRS, BP 53, F-38041 Grenoble cedex 9, France. Tel.: 33-4-7603-7635; Fax: 33-4-7654-7203; E-mail: Christelle.Breton{at}cermav.cnrs.fr.

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