title: Discoidin I from Dictyostelium discoideum and Interactions with oligosaccharides: specificity, affinity, crystal structures, and comparison with discoidin II.
abstract: Discoidin I (DiscI) and discoidin II (DiscII) are N-acetylgalactosamine (GalNAc)-binding proteins from Dictyostelium discoideum. They consist of two domains: an N-terminal discoidin domain and a C-terminal H-type lectin domain. They were cloned and expressed in high yield in recombinant form in Escherichia coli. Although both lectins bind galactose (Gal) and GalNAc, glycan array experiments performed on the recombinant proteins displayed strong differences in their specificity for oligosaccharides. DiscI and DiscII bind preferentially to Gal/GalNAcbeta1-3Gal/GalNAc-containing and Gal/GalNAcbeta1-4GlcNAcbeta1-6Gal/GalNAc-containing glycans, respectively. The affinity of the interaction of DiscI with monosaccharides and disaccharides was evaluated using isothermal titration calorimetry experiments. The three-dimensional structures of native DiscI and its complexes with GalNAc, GalNAcbeta1-3Gal, and Galbeta1-3GalNAc were solved by X-ray crystallography. DiscI forms trimers with involvement of calcium at the monomer interface. The N-terminal discoidin domain presents a structural similarity to F-type lectins such as the eel agglutinin, where an amphiphilic binding pocket suggests possible carbohydrate-binding activity. In the C-terminal H-type lectin domain, the GalNAc residue establishes specific hydrogen bonds that explain the observed affinity (K(d)=3x10(-4) M). The different specificities of DiscI and DiscII for oligosaccharides were rationalized from the different structures obtained by either X-ray crystallography or molecular modeling.
authors.affiliation: CERMAV-CNRS, 601 rue de la Chimie, BP53, F-38041 Grenoble Cedex 09, France.
authors.forename: Sophie V
authors.initials: SV
authors.lastname: Mathieu
authors.forename: Karoline Saboia
authors.initials: KS
authors.lastname: Aragão
authors.forename: Anne
authors.initials: A
authors.lastname: Imberty
authors.forename: Annabelle
authors.initials: A
authors.lastname: Varrot
mesh_terms: D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004023:Dictyostelium / Q000737:chemistry / Q000378:metabolism*; D000070821:Discoidins; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D037102:Lectins / Q000737:chemistry* / Q000378:metabolism*; D009005:Monosaccharides / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000070821:Discoidins; D037102:Lectins; D009005:Monosaccharides; D009844:Oligosaccharides; D015800:Protozoan Proteins; D011994:Recombinant Proteins