Hemolytic uremic syndrome (HUS) is characterized by verotoxin-mediated damage to endothelial cells that results in hemolytic anemia, throbocytopenia, and multisystemic complications including renal failure. In almost all cases, verotoxins (shiga-like toxins) released by enterohemorrhagic E. coli bind to glycolipids on the endothelial cells, are then routed to the endoplasmic reticulum, and thereby inactivate the 28S ribosomes and halt protein synthesis. Although glycolipid receptors are required for VT susceptibility, the degree of susceptibility does not correlate directly with the quantity of receptor. The clinical correlative of these observations is that children and adults differ in their susceptibility to HUS despite expressing similar quantities of VT-receptor and glycolipids in renal cells. The principal investigator therefore hypothesizes that differences in docking to verotoxin receptors or distinct pathways controlling intracellular trafficking of VT and its glycolipid receptors underlie these differences. In preliminary studies VT-susceptible Vero cells were transfected with the cDNA encoding Forssman synthetase (FS). FS-transfected cells were highly resistant to VT, yet still demonstrated toxin binding. Ligand blotting demonstrated the presence of two glycolipid receptors (R1 and R2) whereas only R2, a novel receptor not previously identified, was present in FS-transfected cells.
In Specific Aim #1, the principal investigator will identify glycolipid receptor R2, purify this receptor from VT-resistant cell extracts, confirm its identity by mass spectroscopy, and add purified R2 glycolipid exogenously to other cell types with a priori resistance to verotoxin. Tandem experiments with receptor R1 will serve as a control.
In Specific Aim #2, the principal investigator will analyze internalization and intracellular trafficking of verotoxin and cholera toxin receptor glycolipids in FS-transfected cells and wild-type cells. Immunogold electron microscopy will be used to track the intracellular fate of labeled verotoxin in FS-transfected and WT-cells. Intracellular trafficking of cholera toxin, which binds an altogether different glycolipid (GM1) will be examined in this same system. An inducible promoter will be used to titrate expression of FS to understand whether a minimal amount of the enzyme is required for VT-resistance.
In Specific Aim #3, the principal investigator will use his newly developed transgenic mouse model for overexpression of FS to characterize mRNA expression of this enzyme and alterations in glycolipid expression in various tissues. A murine model for HUS, which fails to mimic precisely the effects of VT because mice express a spectrum of glycolipids different from that in humans, will be adapted using FS transgenic mice and littermate controls to determine whether altered glycolipid expression results in VT resistance in vivo.
| Yu, M; Haslam, R H; Haslam, D B (2000) HEDJ, an Hsp40 co-chaperone localized to the endoplasmic reticulum of human cells. J Biol Chem 275:24984-92 |
| Xu, H; Storch, T; Yu, M et al. (1999) Characterization of the human Forssman synthetase gene. An evolving association between glycolipid synthesis and host-microbial interactions. J Biol Chem 274:29390-8 |
