Molecular basis for acceptor substrate specificity of the human beta1,3-glucuronosyltransferases GlcAT-I and GlcAT-P involved in glycosaminoglycan and HNK-1 carbohydrate epitope biosynthesis, respectively

Magali Fondeur-Gelinotte, Virginie Lattard, Sandrine Gulberti, Rafael Oriol, Guillermo Mulliert, Michael W. H. Coughtrie, Jacques Magdalou, Patrick Netter, Mohamed Ouzzine, Sylvie Fournel-Gigleux

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    10 Citations (Scopus)

    Abstract

    The human ß1,3-glucuronosyltransferases galactose-ß1,3-glucuronosyltransferase I (GlcAT-I) and galactose-ß1,3-glucuronosyltransferase P (GlcAT-P) are key enzymes involved in proteoglycan and HNK-1 carbohydrate epitope synthesis, respectively. Analysis of their acceptor specificity revealed that GlcAT-I was selective toward Galß1,3Gal (referred to as Gal2-Gal1), whereas GlcAT-P presented a broader profile. To understand the molecular basis of acceptor substrate recognition, we constructed mutants and chimeric enzymes based on multiple sequence alignment and structural information. The drastic effect of mutations of Glu227, Arg247, Asp252, and Glu281 on GlcAT-I activity indicated a key role for the hydrogen bond network formed by these four conserved residues in dictating Gal2 binding. Investigation of GlcAT-I determinants governing Gal1 recognition showed that Trp243 could not be replaced by its counterpart Phe in GlcAT-P. This result combined with molecular modeling provided evidence for the importance of stacking interactions with Trp at position 243 in the selectivity of GlcAT-I toward Galß1,3Gal. Mutation of Gln318 predicted to be hydrogen-bonded to 6-hydroxyl of Gal1 had little effect on GlcAT-I activity, reinforcing the role of Trp243 in Gal1 binding. Substitution of Phe245 in GlcAT-P by Ala selectively abolished Galß1,3Gal activity, also highlighting the importance of an aromatic residue at this position in defining the specificity of GlcAT-P. Finally, substituting Phe245, Val320, or Asn321 in GlcAT-P predicted to interact with N-acetylglucosamine (GlcNAc), by their counterpart in GlcAT-I, moderately affected the activity toward the reference substrate of GlcAT-P, N-acetyllactosamine, indicating that its active site tolerates amino acid substitutions, an observation that parallels its promiscuous substrate profile. Taken together, the data clearly define key residues governing the specificity of ß1,3-glucuronosyltransferases.
    Original languageEnglish
    Pages (from-to)857-867
    Number of pages11
    JournalGlycobiology
    Volume17
    Issue number8
    DOIs
    Publication statusPublished - Aug 2007

    Keywords

    • β1,3-glucuronosyltransferases
    • Acceptor substrate specificity
    • Glycosaminoglycans
    • Kinetics
    • Site-directed mutagenesis

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