title: Molecular diversity of the two sugar-binding sites of the β-trefoil lectin HA33/C (HA1) from Clostridium botulinum type C neurotoxin.
abstract: A critical role in internalizing the Clostridium botulinum neurotoxin into gastrointestinal cells is played by nontoxic components complexed with the toxin. One of the components, a β-trefoil lectin has been known as HA33 or HA1. The HA33 from C. botulinum type A (HA33/A) has been predicted to have a single sugar-binding site, while type C HA33 (HA33/C) has two sites. Here we constructed HA33/C mutants and evaluated the binding capacities of the individual sites through mucin-assay and isothermal titration calorimetry. The mutant W176A (site I knockout) had a K(d) value of 31.5mM for galactose (Gal) and 61.3mM for N-acetylgalactosamine (GalNAc), while the K(d) value for N-acetylneuraminic acid (Neu5Ac) was too high to be determined. In contrast, the double mutant N278A/Q279A (site II knockout) had a K(d) value of 11.8mM for Neu5Ac. We also determined the crystal structures of wild-type and the F179I mutant in complex with GalNAc at site II. The results suggest that site I of HA33/C is quite unique in that it mainly recognizes Neu5Ac, and site II seems less important for the lectin specificity. The architectures and the properties of the sugar-binding sites of HA33/C and HA33/A were shown to be drastically different.
authors.affiliation: Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Nakamura
authors.forename: Takashi
authors.initials: T
authors.lastname: Tonozuka
authors.forename: Sakae
authors.initials: S
authors.lastname: Ito
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Takeda
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Sato
authors.forename: Ichiro
authors.initials: I
authors.lastname: Matsuo
authors.forename: Yukishige
authors.initials: Y
authors.lastname: Ito
authors.forename: Keiji
authors.initials: K
authors.lastname: Oguma
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nishikawa
mesh_terms: D000116:Acetylgalactosamine / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001906:Botulism / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D047968:Clostridium botulinum type C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D005690:Galactose / Q000378:metabolism; D006388:Hemagglutinins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D037102:Lectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009077:Mucins / Q000378:metabolism*; D009154:Mutation; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009498:Neurotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013552:Swine
chemical_list: D001426:Bacterial Proteins; D006388:Hemagglutinins; D037102:Lectins; D009077:Mucins; D009498:Neurotoxins; D019158:N-Acetylneuraminic Acid; D000116:Acetylgalactosamine; D005690:Galactose