title: Alteration of the carbohydrate-binding specificity of a C-type lectin CEL-I mutant with an EPN carbohydrate-binding motif.
abstract: CEL-I is a Gal/GalNAc-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-recognition domains (CRDs) with the carbohydrate-recognition motif QPD (Gln-Pro- Asp), which is generally known to exist in galactose-specific C-type CRDs. In the present study, a mutant CEL-I with EPN (Glu-Pro-Asn) motif, which is thought to be responsible for the carbohydrate-recognition of mannose-specific Ctype CRDs, was produced in Escherichia coli, and its effects on the carbohydrate-binding specificity were examined using polyamidoamine dendrimer (PD) conjugated with carbohydrates. Although wild-type CEL-I effectively formed complexes with N-acetylgalactosamine (GalNAc)-PD but not with mannose-PD, the mutant CEL-I showed relatively weak but definite affinity for mannose-PD. These results indicated that the QPD and EPN motifs play a significant role in the carbohydrate-recognition mechanism of CEL-I, especially in the discrimination of galactose and mannose. Additional mutations in the recombinant CEL-I binding site may further increase its specificity for mannose, and should provide insights into designing novel carbohydrate-recognition proteins.
authors.affiliation: Biomolecular Chemistry Laboratory, Graduate School of Engineering, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. thata@nagasaki-u.ac.jp
authors.forename: Tomomitsu
authors.initials: T
authors.lastname: Hatakeyama
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Ishimine
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Baba
authors.forename: Masanari
authors.initials: M
authors.lastname: Kimura
authors.forename: Hideaki
authors.initials: H
authors.lastname: Unno
authors.forename: Shuichiro
authors.initials: S
authors.lastname: Goda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D048016:Cucumaria; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000737:chemistry / Q000378:metabolism*; D037181:Lectins, C-Type / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008358:Mannose / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D037181:Lectins, C-Type; D011994:Recombinant Proteins; D008358:Mannose; D005690:Galactose