title: Identification of key functional residues in the active site of human {beta}1,4-galactosyltransferase 7: a major enzyme in the glycosaminoglycan synthesis pathway.
abstract: Glycosaminoglycans (GAGs) play a central role in many pathophysiological events, and exogenous xyloside substrates of β1,4-galactosyltransferase 7 (β4GalT7), a major enzyme of GAG biosynthesis, have interesting biomedical applications. To predict functional peptide regions important for substrate binding and activity of human β4GalT7, we conducted a phylogenetic analysis of the β1,4-galactosyltransferase family and generated a molecular model using the x-ray structure of Drosophila β4GalT7-UDP as template. Two evolutionary conserved motifs, (163)DVD(165) and (221)FWGWGREDDE(230), are central in the organization of the enzyme active site. This model was challenged by systematic engineering of point mutations, combined with in vitro and ex vivo functional assays. Investigation of the kinetic properties of purified recombinant wild-type β4GalT7 and selected mutants identified Trp(224) as a key residue governing both donor and acceptor substrate binding. Our results also suggested the involvement of the canonical carboxylate residue Asp(228) acting as general base in the reaction catalyzed by human β4GalT7. Importantly, ex vivo functional tests demonstrated that regulation of GAG synthesis is highly responsive to modification of these key active site amino acids. Interestingly, engineering mutants at position 224 allowed us to modify the affinity and to modulate the specificity of human β4GalT7 toward UDP-sugars and xyloside acceptors. Furthermore, the W224H mutant was able to sustain decorin GAG chain substitution but not GAG synthesis from exogenously added xyloside. Altogether, this study provides novel insight into human β4GalT7 active site functional domains, allowing manipulation of this enzyme critical for the regulation of GAG synthesis. A better understanding of the mechanism underlying GAG assembly paves the way toward GAG-based therapeutics.
authors.affiliation: Faculté de Médecine, UMR 7561 CNRS-Université de Nancy I, BP 184, 54505 Vandoeuvre-lès-Nancy, France.
authors.forename: Ibtissam
authors.initials: I
authors.lastname: Talhaoui
authors.forename: Catherine
authors.initials: C
authors.lastname: Bui
authors.forename: Rafael
authors.initials: R
authors.lastname: Oriol
authors.forename: Guillermo
authors.initials: G
authors.lastname: Mulliert
authors.forename: Sandrine
authors.initials: S
authors.lastname: Gulberti
authors.forename: Patrick
authors.initials: P
authors.lastname: Netter
authors.forename: Michael W H
authors.initials: MW
authors.lastname: Coughtrie
authors.forename: Mohamed
authors.initials: M
authors.lastname: Ouzzine
authors.forename: Sylvie
authors.initials: S
authors.lastname: Fournel-Gigleux
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D005700:Galactosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000096:biosynthesis*; D006801:Humans; D007448:Invertebrates / Q000737:chemistry / Q000145:classification / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D013379:Substrate Specificity; D014714:Vertebrates / Q000145:classification / Q000235:genetics / Q000378:metabolism
chemical_list: D006025:Glycosaminoglycans; D005700:Galactosyltransferases; C063053:xylosylprotein 4-beta-galactosyltransferase