Interference of the galactose-dependent binding of lectins by novel pentapeptide ligands
Christopher J. ArnuschSabine AndréPaola ValentiniMartin LenschRoland RusswurmHans‐Christian SiebertMarcel J.E. FischerHans‐Joachim GabiusRoland J. Pieters
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Keywords:
Pentapeptide repeat
Galectin
Molecular mimicry
Sequence (biology)
The pregnancy-specific beta 1-globulin (SBG) reacts with 10 out of 11 lectins which have affinity to monosaccharides (glucose, mannose, galactose and fucose) and acetylamino sugars. The affinity to ConA and PHA-P was the most pronounced while this protein did not react with the pea lectin. The SBG reactions are specific for every lectin (even with the identical carbohydrate specificity). Peculiarities of the SBG reactions with lectins made it possible to reveal a few forms of this protein which differ in the carbohydrate composition and conformation of carbohydrate radicals.
Monosaccharide
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Pentapeptide repeat
Galectin
Molecular mimicry
Sequence (biology)
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Galectin
Isothermal Titration Calorimetry
Residue (chemistry)
Binding selectivity
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Monosaccharide
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Sephadex
Galactosamine
Amino sugar
Galactosides
Rhamnose
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Galectin
Glycoconjugate
Molecular Recognition
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Galectins exhibit multiple roles through recognition of diverse structures of β-galactosides. However, this broad specificity often hinders their practical use as probes. In the present study we report a dramatic improvement in the carbohydrate specificity of a multi-specific fungal galectin from the mushroom Agrocybe cylindricea, which binds not only to simple β-galactosides, but also to their derivatives. Site-directed mutagenesis targeting five residues involved in β-galactose binding revealed that replacement of Asn46 with alanine (N46A) increased the binding to GalNAcα1-3Galβ-containing glycans, while eliminating binding to all other β-galactosides, as shown by glycoconjugate microarray analysis. Quantitative analysis by frontal affinity chromatography showed that the mutant N46A had enhanced affinity towards blood group A tetraose (type 2), A hexaose (type 1) and Forssman pentasaccharide with dissociation constants of 5.0 × 10⁻⁶ M, 3.8 × 10⁻⁶ M and 1.0 × 10⁻⁵ M respectively. Surprisingly, all the other mutants generated by saturation mutagenesis of Asn46 exhibited essentially the same specificity as N46A. Moreover, alanine substitution for Pro45, which forms the cis-conformation upon β-galactose binding, exhibited the same specificity as N46A. From a practical viewpoint, the derived N46A mutant proved to be unique as a specific probe to detect GalNAcα1-3Galβ-containing glycans by methods such as flow cytometry, cell staining and lectin microarray.
Galectin
Galactosides
Glycoconjugate
Binding selectivity
Alanine
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The trimeric C-type lectin MGL is involved in activating immune defence and virus uptake, thus becoming a target for inhibitor design. A tetravalent cluster with α-S -GalNAc proved to be effective in the nM range.
Galectin
C-type lectin
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Galactose oxidase
Sialidase
Agglutination (biology)
Affinity electrophoresis
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