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

Abstract

Membrane microdomains, also named lipid rafts ( 1 ), are enriched in glycosphingolipids (GSLs) and cholesterol ( 2 ), and assembled with membrane-integrated proteins, such as caveolins or fl otillins, with affi nity for particular lipid species ( 3 ). Caveolins are cholesterol-binding proteins ( 4 ) and occur in fl ask-shaped invaginations named caveolae ( 5 ). Flotillin proteins, also called reggie proteins, are found in distinct membrane microdomains ( 6 ). Caveolae are particularly abundant in endothelial cells and are involved in many cellular processes, including cholesterol homeostasis, endocytosis, and transcytosis of macromolecules ( 7-9 ). While numerous proteomic studies have been conducted to identify lipid raft-or caveolae-associated proteins of various cell types, including endothelial cells ( 10-12 ), the GSL composition of lipid rafts or caveolae, particularly of endothelial cells, has attracted less attention, despite their outstanding role in membrane microdomain formation

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

“…Incubation was stopped by removal of the diluted supernatants. Remaining adherent cells were fi xed with formalin, stained with crystal violet, and densitometrically quantifi ed (61)(62)(63). Results represent the mean ± SD of quadruplicate determinations and are portrayed as percentage values of untreated control cells.…”

“…Cultures were routinely passaged every 2 to 3 days using 0.25% trypsin-EDTA (Invitrogen, Karlsruhe, Germany; catalog number 25200) before cells became confl uent. Appropriate cell production for the isolation of preparative amounts of GSLs from total cells (see Purifi cation of neutral GSLs from Vero-B4 cells below) and for the preparation of sucrose density gradient fractions (see Preparation of sucrose density gradient fractions: DRMs and nonDRM fractions below) was performed in 175 cm 2 tissue culture fl asks (Greiner Bio-One, Frickenhausen, Germany), as previously described for endothelial cells (61)(62)(63).…”

“…Indirect determination of cell viability was performed using the crystal violet assay, as previously described (61)(62)(63). Briefl y, Vero-B4 cells were grown until subconfl uence in tissue culture fl asks (Greiner Bio-One), trypsinized and seeded in 100 l volumes in 96-well tissue culture plates (Corning Inc., Corning, NY) (initial cell seeding density of 4 × 10 3 cells/well).…”

“…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 ).…”

“…Plasma membrane organization in microdomains seems to be a functional requirement for binding and effi cient internalization of pathogens and toxins, such as Vibrio cholerae (30/9/25/18/6, each by volume) and stained with molybdenum blue Dittmer-Lester reagent ( 73,74 ). Cholesterol was stained upon TLC separation in chloroform/acetone (96/4, v/v) with manganese(II)chloride ( 62,75 ). Neutral GSLs were separated in chloroform/methanol/water (120/70/17, each by volume) and stained with orcinol ( 76 ).…”