title: The Y54(L)W mutation of anti-leukotriene C4 single-chain antibody increases affinity to leukotriene E4.
abstract: The X-ray crystal structure of an anti-leukotriene (LT) C4monoclonal antibody (mAbLTC) in complex with LTC4was determined, however, crystallographic studies alone are not enough to fully understand the structures of the antigen-binding site. To elucidate the individual contribution of Tyr-54 and Asn-58 in the light chain of mAbLTC, both of which formed a hydrogen bond with glutamic acid of LTC4, we examined whether substitution of the residues affects the antigen binding affinity and specificity using an anti-LTC4single chain variable fragment (scFvLTC). Among the Tyr-54(L) mutants, Y54(L)W showed a dramatic increase in the affinity to LTE4which was comparable to that to LTD4Essentially the same results were obtained using the Y54(L)W mutant expressed in Escherichia coli and Pichia pastoris. The structural modeling suggested the formation of a novel hydrogen bond between the substituted tryptophan in the antibody and the cysteine residue in LTE4The affinity of Y54(L)R, Y54(L)E and Y54(L)L to LTC4was markedly reduced, whereas other tested Tyr-54(L) mutants as well as Asn-58(L) mutants did not show significant change in LT binding. The results may provide an insight into the molecular basis of specific LT recognition by the antibody.
authors.affiliation: Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kawakami
authors.affiliation: Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197, Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Kinoshita
authors.affiliation: Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197, Japan.
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Mori
authors.affiliation: Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Okochi
authors.affiliation: Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197, Japan.
authors.forename: Shiori
authors.initials: S
authors.lastname: Hirano
authors.affiliation: Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197, Japan.
authors.forename: Ichika
authors.initials: I
authors.lastname: Shimoda
authors.affiliation: Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197, Japan.
authors.forename: Keita
authors.initials: K
authors.lastname: Kanzaki
authors.affiliation: Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197, Japan.
authors.forename: Toshiko
authors.initials: T
authors.lastname: Suzuki-Yamamoto
authors.affiliation: Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197, Japan.
authors.forename: Masumi
authors.initials: M
authors.lastname: Kimoto
authors.affiliation: Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1, Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Sugahara
authors.affiliation: Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1, Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Hori
authors.affiliation: Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, 5-10-1 Fuchinobe, Chuo-ku, Sagamihara-shi, Kanagawa 252-5258, Japan.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Saino
authors.affiliation: Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1, Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.|Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, 5-10-1 Fuchinobe, Chuo-ku, Sagamihara-shi, Kanagawa 252-5258, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Miyano
authors.affiliation: Department of Food and Nutrition, Kyoto Women's University, 35 Kitahiyoshi-cho, Imakumano, Higashiyama-ku, Kyoto, 605-8501, Japan.
authors.forename: Shozo
authors.initials: S
authors.lastname: Yamamoto
authors.affiliation: Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197, Japan ytaka@fhw.oka-pu.ac.jp.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Takahashi
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000915:Antibody Affinity / Q000235:genetics*; D017999:Leukotriene E4 / Q000737:chemistry*; D051379:Mice; D020125:Mutation, Missense; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics
chemical_list: D057127:Single-Chain Antibodies; D017999:Leukotriene E4
keywords: Leukotriene; Monoclonal antibody; Recombinant protein expression; Single-chain variable fragment; Site-directed mutagenesis