Three mutations of the B4GALT7 gene [encoding β1,4-GalT7 (β1,4-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 β1,4-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 β1,4-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 β1,4GalT7 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.
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Research Article|
November 12 2010
Biochemical and thermodynamic characterization of mutated β1,4-galactosyltransferase 7 involved in the progeroid form of the Ehlers–Danlos syndrome
Sophie Rahuel-Clermont;
Sophie Rahuel-Clermont
*ARN-RNP-structure fonction-maturation, Enzymologie Moléculaire et Structurale, Unité Mixte de Recherche 7214, CNRS-Nancy Université, Faculté des Sciences et Techniques, BP 239 Vandoeuvre-Les-Nancy, France
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Franck Daligault;
Franck Daligault
†Physiopathologie, Pharmacologie et Ingénierie Articulaires, Unité Mixte de Recherche 7561, CNRS-Nancy Université, Faculté de Médecine, BP 184 Vandoeuvre-Les-Nancy, France
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Marie-Helene Piet;
Marie-Helene Piet
†Physiopathologie, Pharmacologie et Ingénierie Articulaires, Unité Mixte de Recherche 7561, CNRS-Nancy Université, Faculté de Médecine, BP 184 Vandoeuvre-Les-Nancy, France
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Sandrine Gulberti;
Sandrine Gulberti
†Physiopathologie, Pharmacologie et Ingénierie Articulaires, Unité Mixte de Recherche 7561, CNRS-Nancy Université, Faculté de Médecine, BP 184 Vandoeuvre-Les-Nancy, France
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Patrick Netter;
Patrick Netter
†Physiopathologie, Pharmacologie et Ingénierie Articulaires, Unité Mixte de Recherche 7561, CNRS-Nancy Université, Faculté de Médecine, BP 184 Vandoeuvre-Les-Nancy, France
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Guy Branlant;
Guy Branlant
*ARN-RNP-structure fonction-maturation, Enzymologie Moléculaire et Structurale, Unité Mixte de Recherche 7214, CNRS-Nancy Université, Faculté des Sciences et Techniques, BP 239 Vandoeuvre-Les-Nancy, France
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Jacques Magdalou;
Jacques Magdalou
†Physiopathologie, Pharmacologie et Ingénierie Articulaires, Unité Mixte de Recherche 7561, CNRS-Nancy Université, Faculté de Médecine, BP 184 Vandoeuvre-Les-Nancy, France
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Virginie Lattard
Virginie Lattard
1
†Physiopathologie, Pharmacologie et Ingénierie Articulaires, Unité Mixte de Recherche 7561, CNRS-Nancy Université, Faculté de Médecine, BP 184 Vandoeuvre-Les-Nancy, France
1To whom correspondence should be addressed (email v.lattard@vetagro-sup.fr).
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Biochem J (2010) 432 (2): 303–311.
Article history
Received:
June 22 2010
Revision Received:
August 27 2010
Accepted:
September 01 2010
Accepted Manuscript online:
September 01 2010
Citation
Sophie Rahuel-Clermont, Franck Daligault, Marie-Helene Piet, Sandrine Gulberti, Patrick Netter, Guy Branlant, Jacques Magdalou, Virginie Lattard; Biochemical and thermodynamic characterization of mutated β1,4-galactosyltransferase 7 involved in the progeroid form of the Ehlers–Danlos syndrome. Biochem J 1 December 2010; 432 (2): 303–311. doi: https://doi.org/10.1042/BJ20100921
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