Biochemical and thermodynamic characterization of mutated beta1,4-galactosyltransferase 7 involved in the progeroid form of the Ehlers-Danlos syndrome.
Fiche publication
Date publication
décembre 2010
Auteurs
Membres identifiés du Cancéropôle Est :
Pr MAGDALOU Jacques, Dr RAHUEL-CLERMONT Sophie
Tous les auteurs :
Rahuel-Clermont S, Daligault F, Piet MH, Gulberti S, Netter P, Branlant G, Magdalou J, Lattard V
Lien Pubmed
Résumé
Three mutations of the B4GALT7 gene [encoding beta1,4-GalT7 (beta1,4-galactosyltransferase 7)], corresponding to A186D, L206P and R270C, have been identified in patients with the progeroid form of the Ehlers-Danlos syndrome and are described as being associated with the reduction or loss of beta1,4-GalT7 activity. However, the molecular basis of the reduction or loss of activity remained to be determined. In the present study, wild-type, A186D, L206P and R270C beta1,4-GalT7 were expressed in CHO618 cells as membrane proteins and in Escherichia coli as soluble proteins fused to MBP (maltose-binding protein). The ability of the expressed proteins to transfer galactose from donor to acceptor substrates was systematically characterized by kinetic analysis. The physicochemical properties of soluble proteins were explored by isothermal titration calorimetry, which is a method of choice when determining the thermodynamic parameters of the binding of substrates. Together, the results showed that: (i) the L206P mutation abolished the activity when L206P beta1,4GalT7 was either inserted in the membrane or expressed as a soluble MBP-full-length fusion protein; (ii) the A186D mutation weakly impaired the binding of the donor substrate; and (iii) the R270C mutation strongly impaired the binding of the acceptor substrate. Moreover, the ex vivo consequences of the mutations were investigated by evaluating the priming efficiency of xylosides on GAG (glycosaminoglycan) chain initiation. The results demonstrate a quantitative effect on GAG biosynthesis, depending on the mutation; GAG biosynthesis was fully inhibited by the L206P mutation and decreased by the R270C mutation, whereas the A186D mutation did not affect GAG biosynthesis severely.
Référence
Biochem J. 2010 Dec 1;432(2):303-11.