Kinetic Analyses of Bindings of Shiga-like Toxin to Clustered and Dispersed Gb3 Glyco-Arrays on a Quartz-Crystal Microbalance

Abstract

One-, two-, four-, and eight-branched globotriaosyl saccharides (Gb(3): Gal-alpha1,4-Gal-beta1,4-Glc), whose reducing ends were biotinylated, were prepared (1Gb(3)-bio, 2Gb(3)-bio, 4Gb(3)-bio, and 8Gb(3)-bio, respectively). They are dispersively immobilized as a glyco-array in the matrix of biotinylated maltotriose (Glc(3)-bio) on a streptavidin-covered 27 MHz quartz-crystal microbalance (QCM). The binding kinetics of the verotoxin B subunit (VTB) to various branched Gb(3)-bio ligands in the Glc(3)-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. VTB can recognize the Gb(3) unit but not the Glc(3) unit, where VTB is a pentamer having five binding sites for one Gb(3) unit per each B subunit (having a total of 15 binding sites for Gb(3)). By changing the Gb(3) multivalency, the Gb(3) packing density, and the Gb(3) cluster size in the Glc(3) matrix, association constants (K(a)), maximum amounts bound (Delta m(max)), and binding and dissociation rate constants (k(on) and k(off)) were obtained. When 15 sites of VTB were recognized by 16 Gb(3) units, K(a) was 100 times larger than that when 15 sites of VTB were recognized by only 2 Gb(3) units, with a 6-fold-larger k(on) and a 25-fold-smaller k(off). When the Gb(3) multivalency was changed by covering with two 1Gb(3)-bio, 2Gb(3)-bio, 4Gb(3)-bio, or 8Gb(3)-bio ligands on two pockets of one streptavidin, the K(a) values increased with increasing branch number from one to eight. When the Gb(3) cluster size was changed from eight 1Gb(3)-bio units to one 8Gb(3)-bio unit in the matrix, the K(a) values increased but the Delta m(max) values decreased with increasing cluster size from eight 1Gb(3)-bio units to one 8Gb(3)-bio unit. This is the first example of systematically obtaining all kinetic parameters of sugar-binding proteins to sugars on a glyco-array by changing the sugar multivalency, the sugar packing density, and the sugar cluster size in the matrix.