title: Mannose-recognition mutant of the galactose/N-acetylgalactosamine-specific C-type lectin CEL-I engineered by site-directed mutagenesis.
abstract: BACKGROUND
  CEL-I is a galactose/N-acetylgalactosamine-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. Its carbohydrate-binding site contains a QPD (Gln-Pro-Asp) motif, which is generally recognized as the galactose specificity-determining motif in the C-type lectins. In our previous study, replacement of the QPD motif by an EPN (Glu-Pro-Asn) motif led to a weak binding affinity for mannose. Therefore, we examined the effects of an additional mutation in the carbohydrate-binding site on the specificity of the lectin.
  
  
  METHODS
  Trp105 of EPN-CEL-I was replaced by a histidine residue using site-directed mutagenesis, and the binding affinity of the resulting mutant, EPNH-CEL-I, was examined by sugar-polyamidoamine dendrimer assay, isothermal titration calorimetry, and glycoconjugate microarray analysis. Tertiary structure of the EPNH-CEL-I/mannose complex was determined by X-ray crystallographic analysis.
  
  
  RESULTS
  Sugar-polyamidoamine dendrimer assay and glycoconjugate microarray analysis revealed a drastic change in the specificity of EPNH-CEL-I from galactose/N-acetylgalactosamine to mannose. The association constant of EPNH-CEL-I for mannose was determined to be 3.17×10(3) M(-1) at 25°C. Mannose specificity of EPNH-CEL-I was achieved by stabilization of the binding of mannose in a correct orientation, in which the EPN motif can form proper hydrogen bonds with 3- and 4-hydroxy groups of the bound mannose.
  
  
  CONCLUSIONS
  Specificity of CEL-I can be engineered by mutating a limited number of amino acid residues in addition to the QPD/EPN motifs.
  
  
  CONCLUSIONS
  Versatility of the C-type carbohydrate-recognition domain structure in the recognition of various carbohydrate chains could become a promising platform to develop novel molecular recognition proteins.
authors: Laboratory of Biomolecular Chemistry, Graduate School of Engineering, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
authors: Hiromi
authors: H
authors: Moriuchi
authors: Laboratory of Biomolecular Chemistry, Graduate School of Engineering, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
authors: Hideaki
authors: H
authors: Unno
authors: Laboratory of Biomolecular Chemistry, Graduate School of Engineering, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
authors: Shuichiro
authors: S
authors: Goda
authors: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8568, Japan.
authors: Hiroaki
authors: H
authors: Tateno
authors: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8568, Japan.
authors: Jun
authors: J
authors: Hirabayashi
authors: Laboratory of Biomolecular Chemistry, Graduate School of Engineering, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. Electronic address: thata@nagasaki-u.ac.jp.
authors: Tomomitsu
authors: T
authors: Hatakeyama
mesh_terms: D000116:Acetylgalactosamine / Q000737:chemistry / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D002151:Calorimetry / Q000379:methods; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D048016:Cucumaria / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000737:chemistry / Q000378:metabolism*; D037181:Lectins, C-Type / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008358:Mannose / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary
chemical_list: D037181:Lectins, C-Type; D000116:Acetylgalactosamine; D008358:Mannose; D005690:Galactose
keywords: C-type lectin; Carbohydrate; Site-directed mutagenesis; X-ray crystallography