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Originally published In Press as doi:10.1074/jbc.M002182200 on June 6, 2000

J. Biol. Chem., Vol. 275, Issue 36, 28254-28260, September 8, 2000
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Structure/Function of the Human Galbeta 1,3-glucuronosyltransferase
DIMERIZATION AND FUNCTIONAL ACTIVITY ARE MEDIATED BY TWO CRUCIAL CYSTEINE RESIDUES*

Mohamed OuzzineDagger , Sandrine Gulberti, Patrick Netter, Jacques Magdalou, and Sylvie Fournel-Gigleux

From the UMR CNRS 7561-Université Henri Poincaré Nancy 1, Faculté de Médecine, BP 184, 54505 Vandoeuvre-lès-Nancy, France

Galbeta 1,3-glucuronosyltransferase (GlcAT-I) that catalyzes the transfer of a glucuronic acid residue onto the trisaccharide primer of the glycosaminoglycan-protein linkage region plays an essential role in the early steps of the biosynthesis of glycosaminoglycans. In order to gain insight into the structure/function of the enzyme, the human recombinant GlcAT-I was successfully expressed in the yeast Pichia pastoris, with an apparent molecular mass of 43 kDa. Analysis of the electrophoretic mobility of the membrane-bound protein in nonreducing and reducing conditions, together with cross-linking studies, indicated that the membrane-bound GlcAT-I formed active disulfide-linked dimers. GlcAT-I expressed without the predicted N-terminal cytoplasmic tail or secreted as a polypeptide lacking the cytoplasmic tail and transmembrane domain was similarly organized as dimers, suggesting that the structural determinants for the dimerization state are localized in the luminal domain of the protein. In addition, the role of Cys33 and Cys301 in that process was investigated by site-directed mutagenesis combined with chemical modification of GlcAT-I by N-phenylmaleimide. Replacement of Cys33 with alanine abolished the formation of dimers with a concomitant decrease in the catalytic efficiency mainly due to a decrease in apparent maximal velocity and in affinity for UDP-glucuronic acid. On the other hand, N-phenylmaleimide treatment or alanine substitution of the Cys301 residue inactivated the enzyme. Our study demonstrates that GlcAT-I is organized as a homodimer as a result of disulfide bond formation mediated by Cys33 localized in the stem region, whereas the residue Cys301 localized in a conserved C-terminal domain is strictly required for the functional integrity of the enzyme.

* This work was supported in part by Région Lorraine and by a grant from FR 42 "Protéines."The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: UMR CNRS 7561-Université Henri Poincaré Nancy 1, Faculté de Médecine, BP 184, 54505 Vandoeuvre-lès-Nancy, France. Tel.: 33 3 83 59 27 49; Fax: 33 3 83 59 26 21; E-mail: ouzzine@pharmaco-med.u-nancy.fr.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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