Biochemical and thermodynamic characterization of mutated β1,4-galactosyltransferases 7 involved in the progeroid form of the Ehlers-Danlos syndrome
Résumé
Three mutations of the β4GALT7 gene corresponding to A186D, L206P and R270C were identified in patients with the progeroid form of the Ehlers-Danlos syndrome and described to be associated with reduction or loss of activity of the β1,4-galactosyltransferase7. But the molecular bases of the reduction or loss of activity remain to be determined. In the present study, wild-type, A186D, L206P and R270C β1,4-GalT7s were expressed in CHO618 cells as membrane proteins and in Escherichia coli as soluble maltose binding-fusion proteins. Ability of expressed proteins to transfer galactose from donor to acceptor susbtrate was systematically characterized by kinetic analysis. The physicochemical properties of soluble proteins were explored by isothermal titration calorimetry which is a method of choice to have access to the thermodynamic parameters of the binding of substrates. Altogether, the results showed that (i) the L206P mutation abolished the activity when L206P β1,4GalT7 was either inserted in the membrane or expressed as a MBP-full length β1,4GalT7, (ii) the A186D mutation weakly impaired the binding of the donor substrate, (iii) the R270C mutation strongly impaired the binding of the acceptor substrate. Moreover, ex vivo consequences of the mutations were investigated by evaluating the priming efficiency of xylosides on glycosaminoglycan chain initiation. The data demonstrate (iv) a quantitative effect on the glycosaminoglycan biosynthesis depending on the mutations. While the GAG biosynthesis was fully inhibited by the L206P mutation and decreased by the R270C mutation, the A186D mutation did not affect the GAG biosynthesis severely. .
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