Histidine-Tagged Shiga Toxin B Subunit Binding Assay: Simple and Specific Determination of Gb3 Content in Mammalian Cells

Abstract

The recombinant 1B-His, in which 6 histidine residues were added at the carboxy termini of the B subunits, was prepared as described previously.15) The BL21DE cells expressing 1B-His were cultured in 300 ml of LB broth (Difco laboratories, Detroit, MI, U.S.A.) supplemented with 50 mg/ml kanamycin (Nacalai Tesque, Inc., Kyoto, Japan) at 37°C for 2 h. The cells were subsequently treated with 1.0 mmol/l isopropyl b-D(Ϫ)thiogalactopyranoside (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 37°C for 4 h. Cells were pooled by centrifugation at 6000 rpm for 15 min at 4°C. The 1B-His was extracted from cell pellets with the Bugbuster protein extraction reagent (Novagen, Madison, WI, U.S.A.) and purified by using the His-bind purification kit (Novagen) according to manufacturer's recommendations. Purified 1B-His fractions were applied to an NAP10 column (Amersham Biosciences, Uppsala, Sweden) equilibrated with phosphate-buffered saline (PBS). The purified 1B-His was revealed as a single band on SDS-PAGE (data not shown) and its aliquots were stored at Ϫ20°C.Materials HisProbe-HRP was purchased from Pierce Biotechnology, 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 m mg/ml) to the standard Gb3 adsorbed on a multi-well H type plate was observed within 30 min at 37°C; and its binding could be visualized by the following applications of HisProbe-HRP (8 m mg/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.

Determination of globotriaosylceramide analogs in the organs of a mouse model of Fabry disease

“…Approximately 5 g of each organ from 20-week-old Gla tm Tg(CAG-A4GALT) mice was collected and transferred to Oita University for Gb3 extraction. The organs were homogenized in H2O, glycosphingolipids were isolated and purified by solvent extraction and mild alkaline treatment, and Gb3 was purified by Iatrobeads column chromatography, as previously described (23).…”

“…Metabolomic studies focusing on the Gb3 structure (18,19) also identified Gb3 isoforms (Gb3 containing various fatty acids without sphingosine modifications) and Gb3 analogs (Gb3 with various sphingosine modifications) in the plasma and urine of patients with Fabry disease. These variations in sphingosine and fatty acid moieties contribute to the structural heterogeneity of Gb3. Several assays for Gb3 determination have been previously described, such as highperformance liquid chromatography (HPLC) of benzoyl-derivatives (20), thin layer chromatography (TLC)-orcinol staining (21), and Shiga toxin B subunit binding assays (22,23); however, recently, Gb3 analysis was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) because of its high specificity and sensitivity (13). Gb3 concentrations in the plasma of patients with Fabry disease are often assayed by measuring the ten most abundant Gb3 isoforms (13,24); however, Manwaring et al (19) described the presence of >24 Gb3 isoforms/analogs in the plasma of these patients.…”

Increased globotriaosylceramide levels in a transgenic mouse expressing human 1,4-galactosyltransferase and a mouse model for treating Fabry disease

“…The Stx1B binding assay was performed as described previously (23), with some modifications. After glycosphingolipids were separated by TLC as described above, a TLC plate was sunk in a 0.4% polyisobutylmethacrylate (GlycoTech, Rockville, MD, USA) solution (2.5% polyisobutylmethacrylate in chloroform was diluted to 0.4% with hexane) and then blocked with 1% bovine serum albumin in PBS (BSA-PBS).…”

Globotriaosylceramide (Gb3) content in HeLa cells is correlated to Shiga toxin-induced cytotoxicity and Gb3 synthase expression

“…Glycosphingolipids were visualized by spraying orcinol-sulfuric acid reagent for 5-10 min at 100 o C as previously described (32).…”

“…The content of Gb3 in the HLCC lines was determined via 1B-His binding assay, as described in our previous study [32]. In order to determine Gb4 content, TLC analysis was conducted quantitatively with TLC plates using a solvent system of chloroform-methanol-water (60:35:8, v/v/v).…”