title: Directed evolution of lectins with sugar-binding specificity for 6-sulfo-galactose.
abstract: 6-sulfo-galactose (6S-Gal) is a prevalent motif observed in highly sulfated keratan sulfate, which is closely associated with the glioblastoma malignancy while acting as a critical determinant for endogenous lectins. However, facile detection of this unique glycoepitope is greatly hampered because of a lack of appropriate probes. We have previously reported tailoring an α2-6-linked sialic acid-binding lectin from a ricin-B chain-like galactose-binding protein, EW29Ch, by a reinforced ribosome display system following an error-prone PCR. In this study, we challenged the creation of novel lectins to recognize 6S-Gal-terminated glycans by incorporating a high-throughput screening system with a glycoconjugate microarray. After two rounds of selection procedures, 20 mutants were obtained and 12 were then successfully expressed in Escherichia coli, 8 of which showed a significant affinity for 6'-Sulfo-LN (6-O-sulfo-Galβ1-4GlcNAc), which the parental EW29Ch lacked. Analysis of two representative mutants by frontal affinity chromatography revealed a substantial affinity (K(d) ∼3 μm) for a 6S-Gal-terminated glycan. On the basis of the observation that all eight mutants have a common mutation at Glu-20 to Lys, site-directed mutagenesis experiments were performed focusing on this aspect. The results clearly indicated that the E20K mutation is necessary and sufficient to acquire the specificity for 6S-Gal. We also confirmed a difference in binding between E20K and EW29Ch to CHO cells, in which enzymes to catalyze the synthesis of 6S-Gal were overexpressed. The results clearly demonstrate that these mutants have potential to distinguish between cells containing different amounts of 6S-Gal-terminated glycans. This new technology will be used to provide novel tools essential for sulfoglycomics.
authors.affiliation: Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Dan
authors.initials: D
authors.lastname: Hu
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tateno
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kuno
authors.forename: Rikio
authors.initials: R
authors.lastname: Yabe
authors.forename: Jun
authors.initials: J
authors.lastname: Hirabayashi
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000737:chemistry* / Q000378:metabolism; D037161:Galectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054794:Glycomics / Q000379:methods; D007632:Keratan Sulfate / Q000737:chemistry* / Q000378:metabolism; D020125:Mutation, Missense; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C542531:EW29 lectin, Lumbricus terrestris; D037161:Galectins; D011994:Recombinant Proteins; D007632:Keratan Sulfate; D005690:Galactose