Neutral glycosphingolipids in human blood: a precise mass spectrometry analysis with special reference to lipoprotein-associated Shiga toxin receptors

Abstract

This article is available online at http://www.jlr.org Glycosphingolipids (GSLs) are amphipathic molecules that contain a hydrophilic oligosaccharide residue and a hydrophobic ceramide moiety ( 1, 2 ). The ceramide consists of a sphingoid base that is linked with a fatty acid via N-acylation forming the lipid anchor of GSLs. Much effort is currently spent to analyze the biodiversity of sphingolipid structure, metabolism, and function ( 3, 4 ). Neutral GSLs as well as sialic acid carrying gangliosides are most prominently located in the outer leafl et of the plasma membrane of animal cells where they are clustered in lipid rafts ( 5, 6 ), but recent studies have also pointed to important functional roles in the nucleus ( 7 ). Their oligosaccharides protrude from the cell surface rendering GSLs candidates for cell-cell interactions ( 8 ), particularly in the development and pathogenesis of organs ( 9, 10 ). Furthermore, GSLs play important biological roles in the pathophysiology of many infections and serve as receptors for bacteria ( 11 ) and bacterial toxins, including Shiga toxins (Stxs) ( 12, 13 ).Stxs are AB 5 holotoxins, which have been divided into two families, Stx1 and Stx2, each of which consists of the major Stx type and several variants ( 14, 15 ). The pentameric B-subunit of Stx1 binds to globotriaosylceramide (Gb3Cer/CD77) ( 16 )

Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains

“…The specifi cities of polyclonal chicken IgY anti-Gb3Cer and anti-Gb4Cer antibodies have been reported in previous publications (57)(58)(59)(60) …”

Shiga toxin glycosphingolipid receptors of Vero-B4 kidney epithelial cells and their membrane microdomain lipid environment

“…These three Stx-variants, combined with anti-Stx1 and anti-Stx2 antibody, as well as polyclonal chicken anti-GSL antibodies specifi c for Gb3Cer and Gb4Cer, were used in solid-phase binding assays (see TLC overlay assay of GSLs below) for the detection of Stx-receptors in GSL preparations of Vero-B4 cells, according to protocols published previously ( 39,62,(69)(70)(71). The monoclonal mouse IgGantibodies against Stx1 (clone VT109/4-E9) and against Stx2 (clone VT135/6-B9) were purchased from SIFIN GmbH (Berlin, Germany) ( 39,63,69,72 ). Alkaline phosphatase-conjugated goat anti-mouse IgG (code 115-055-003) and rabbit anti-chicken IgY (code 303-055-003) secondary antibodies were from Dianova (Hamburg, Germany).…”

“…For this purpose, the chromatographed unstained high-performance TLC plate was chromatographed in 0.5% (w/v) Plexigum solution in dried hexane until the solvent front had reached the top (after approximately 40 min). TLC immunostainings were done as described in several previous publications ( 62,63,70,72 ). In short, Stx1a-, Stx2a-, or Stx2e-containing supernatants (see Stx1a, Stx2a, Stx2e, anti-Stx, anti-GSL, and secondary antibodies above) were employed using the "microscale method" ( 39,77 ).…”

Association of Shiga toxin glycosphingolipid receptors with membrane microdomains of toxin-sensitive lymphoid and myeloid cells

“…Stx2 combined with anti-Stx2 antibody and polyclonal chicken antibodies against Lc2Cer, Gb3Cer, and Gb4Cer were used in solid-phase binding assays (see "TLC overlay assay" below) for the detection of Lc2Cer and Stx-receptors Gb3Cer and Gb4Cer in the GSL preparations of lymphoid and myeloid cell lines following protocols established previously (40,44,64,(72)(73)(74). The monoclonal mouse IgG-antibody against Stx2 (clone cells/ml in serum-free medium as described previously.…”

“…no. ab29431; Abcam, Cambridge, MA) containing fl otillin proteins VT135/6-B9; 2.75 mg/ml) was from SIFIN GmbH (Berlin, Germany) ( 73,74 …”