title: The high specificities of Phaseolus vulgaris erythro- and leukoagglutinating lectins for bisecting GlcNAc or beta 1-6-linked branch structures, respectively, are attributable to loop B.
abstract: Despite very similar tertiary structures based upon a common framework, legume lectins exhibit an amazing variety of sugar binding specificities. While most of these lectins recognize rather discrete sugar linkages, Phaseolus vulgaris erythroagglutinating and leukoagglutinating lectins (E(4)- and L(4)-PHA) are unique in recognizing larger structures. E(4)- and L(4)-PHA are known to recognize complex type N-glycans containing bisecting GlcNAc or a beta1,6-linked branch, respectively. However, the detailed mechanisms of molecular recognition are poorly understood. In order to dissect the contributions of different portions of each lectin, we carried out region-swapping mutagenesis between E(4)- and L(4)-PHA. We prepared six chimeric lectins by exchanging different combinations of loop B and the central portion of loop C, two of four loops thought to be important for the recognition of monosaccharides (Sharma, V., and Surolia, A. (1997) J. Mol. Biol. 267, 433-445). The chimeric lectins' sugar binding activities were evaluated quantitatively by surface plasmon resonance. These comparisons indicate that the high specificities of E(4)- and L(4)-PHA toward bisecting GlcNAc and beta1,6-linked branch structures are almost solely attributable to loop B. The contribution of the central portion of loop C to the recognition of those structural motifs was found to be negligible. Instead, it modulates affinity toward LacNAc residues present at the nonreducing terminus. Moreover, some of the chimeric lectins prepared in this study showed even higher specificities/affinities than native E(4)- and L(4)-PHA toward complex sugar chains containing either a bisecting GlcNAc residue or a beta1,6-linked branch.
authors.affiliation: Tokyo Research and Development, Amersham Biosciences, 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo, 169-0073 Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Kaneda
authors.forename: Robert F
authors.initials: RF
authors.lastname: Whittier
authors.forename: Hidenori
authors.initials: H
authors.lastname: Yamanaka
authors.forename: Enrique
authors.initials: E
authors.lastname: Carredano
authors.forename: Masanori
authors.initials: M
authors.lastname: Gotoh
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sota
authors.forename: Yukio
authors.initials: Y
authors.lastname: Hasegawa
authors.forename: Yasuro
authors.initials: Y
authors.lastname: Shinohara
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D037102:Lectins / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D027805:Phaseolus / Q000378:metabolism*; D037121:Plant Lectins; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors
chemical_list: D037102:Lectins; D008024:Ligands; D009844:Oligosaccharides; D037121:Plant Lectins; D011993:Recombinant Fusion Proteins; D012313:RNA; D000117:Acetylglucosamine