Histidine-tagged shiga toxin B subunit binding assay: simple and specific determination of gb3 content in mammalian cells.
A two-step binding assay for globotriaosylceramide (Gb3) content was developed by histidine-tagging strategy, which is a well-established method for the purification of recombinant proteins. The complete binding of the recombinant His-tagged Shiga toxin 1B subunit (1B-His) (1 microg/ml) to the standard Gb3 adsorbed on a multi-well H type plate was observed within 30 min at 37 degrees C; and its binding could be visualized by the following applications of HisProbe-HRP (8 microg/ml) and tetramethylbenzidine (TMB) peroxidase substrate. The 1B-His binding assay was linear over the range of 1 to 100 ng of Gb3 per well. The binding of 1B-His was specific to Gb3 separated from HeLa cells, and no major cross-reactivity of other glycolipids in Folch's lower fractions extracted from HeLa cells was detected. The glycolipids in Folch's lower fractions from HeLa cells, human fibroblasts and mouse heart were suitable for this assay, but the further purification was needed for glycolipids from human plasma, thus sample preparation is critical factor for the reliable determination of Gb3 content. The 1B-His binding to Gb3 was inhibited by the addition of galactose, but not mannose. This 1B-His binding assay will be useful not only for the determination of Gb3 content, but also for screening for the compounds which inhibit the toxin-binding to Gb3. The strategy of our present method may be applicable for other binding assay, such as Cholera toxin B-subunit for ganglioside GM1.
Shin
In-Sun
IS
Department of Agricultural and Life Sciences, Obihiro University of Agriculture and Veterinary Medicine, Japan.
Nishikawa
Kiyotaka
K
Maruyama
Hiroki
H
Ishii
Satoshi
S
D000818:Animals; D001560:Benzidines / Q000378:metabolism; D002477:Cells / Q000737:chemistry*; D005347:Fibroblasts / Q000737:chemistry; D006028:Glycosphingolipids / Q000032:analysis / Q000378:metabolism; D006367:HeLa Cells / Q000737:chemistry; D006321:Heart / Q000502:physiology; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D051379:Mice; D009195:Peroxidase / Q000378:metabolism; D022621:Shiga Toxin / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors; D014281:Trihexosylceramides / Q000032:analysis / Q000378:metabolism*
D001560:Benzidines; D006028:Glycosphingolipids; D014281:Trihexosylceramides; C021758:3,3',5,5'-tetramethylbenzidine; D006639:Histidine; C018549:globotriaosylceramide; D022621:Shiga Toxin; D009195:Peroxidase