Structure/function of the human Gal beta 1,3-glucuronosyltransferase - Dimerization and functional activity are mediated by two crucial cysteine residues

Gal beta 1,3-glucuronosyltransferase (GlcAT-I) that catalyzes the transfer of a glucuronic acid residue onto the trisaccharide primer of the glycosami noglycan-protein Linkage region plays an essential role in the early steps of the biosynthesis of glycosaminoglycans. In order to gain insight into th e structure/function of the enzyme, the human recombinant GlcAT-I was succe ssfully expressed in the yeast Pichia pastoris, with an apparent molecular mass of 43 kDa, Analysis of the electrophoretic mobility of the membrane-bo und protein in nonreducing and reducing conditions, together with cross-lin king studies, indicated that the membrane-bound GlcAT-I formed active disul fide-linked dimers, GlcAT-I expressed without the predicted N-terminal cyto plasmic 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 lu minal domain of the protein. In addition, the role of Cys(33) and Cys(301) in that process was investigated by site-directed mutagenesis combined with chemical modification of GlcAT-I by N-phenylmaleimide. Replacement of Cys( 33) with alanine abolished the formation of dimers with a concomitant decre ase in the catalytic efficiency mainly due to a decrease in apparent maxima l velocity and in affinity for UDP-glucuronic acid. On the other hand, N-ph enylmaleimide treatment or alanine substitution of the Cys(301) residue ina ctivated the enzyme, Our study demonstrates that GlcAT-I is organized as a homodimer as a result of disulfide bond formation mediated by Cys(33) local ized in the stem region, whereas the residue Cys(301) localized in a conser ved C-terminal domain is strictly required for the functional integrity of the enzyme.