Engineering of a 3′-sulpho-Galβ1-4GlcNAc-specific probe by a single amino acid substitution of a fungal galectin

Abstract

Among sulphated glycans, little is known about 3'-sulphation because of the lack of useful probes. In the course of molecular engineering of a fungal galectin from Agrocybe cylindracea, we found that a single substitution of Glu86 with alanine resulted in acquisition of specific binding for the 3'-sulpho-Galβ1-4GlcNAc structure. Extensive glyco-technological analysis revealed that this property was obtained in a 'loss-of-function' manner. Though this mutant (E86A) had low total affinity, it showed substantial binding to a naturally occurring N-glycan, of which the terminal galactose is 3-sulphated. Moreover, E86A specifically bound to HeLa cells, in which galactose-3-O-sulfotransferases (Gal3ST2 or Gal3ST3) were over-expressed.

Studying the Structural Significance of Galectin Design by Playing a Modular Puzzle: Homodimer Generation from Human Tandem-Repeat-Type (Heterodimeric) Galectin-8 by Domain Shuffling

“…Furthermore, such experimental work can also help to answer the question why Gal-8N is toxic, whereas Gal-8 (NN) has no such activity on the tested human colon cancer cells. Considering the versatility of branch-end sialylation/sulfation as a recognition signal, generating new tools for detection and isolation of distinct negatively charged glycans has principal merit [ 77 , 78 , 79 ]. Toward this aim, this variant can serve as platform for further mutational adaption of the human galectin.…”

Lectin Engineering, a Molecular Evolutionary Approach to Expanding the Lectin Utilities

“…In addition, a single amino-acid substitution of Glu at position 86 with Ala (E86A) in ACG conferred a highly specific binding for 3'-sulfo-Galβ1-4GlcNAc (3'S-LacNAc). The derived E86A mutant could behave as a useful probe for the detection of 3'S-LacNAc-containing glycans, which are relatively rare in nature, and thus, only poor information of this glyco-epitope has been available to date [47].…”

“…In contrast, a number of lectins from the galectin, R-type lectin, and β-propeller lectin families have been successfully produced in E. coli , suggesting they are suitable scaffolds for engineering. In our studies, we chose an R-type lectin (EW29Ch) and a fungus-derived galectin (ACG) as templates, and successfully tailored several lectins with practical utility [22,45,47,55]. Recently, more and more lectins have been discovered from microorganisms such as bacteria, algae and fungi, and most of them can be expressed well in E. coli [64,65].…”

“…Site-directed mutagenesis is the simplest and most commonly used method in lectin engineering, particularly in regards to lectins whose crystal structures have been elucidated with their glycan ligands. As described above, we recently conducted site-directed mutagenesis on a fungus galectin (ACG) by replacing each of the amino acid residues involved in the Gal-binding with Ala [45,47]. Among the five resulting mutants, two (N46A and E86A) acquired altered specificity.…”

“…Imamura et al carried out this strategy by targeting Asp86 in ACG, and tailored a mutant E86D with improved specificity for α2-3-sialic acid [43]. However, in that study, they failed to discover the specific binding ability of E86A to 3'S-LacNAc, which was revealed in our later study using glycoconjugate microarray [47], suggesting that adopting a high-throughput method for sugar-binding analysis is quite important for success in lectin engineering.…”