Analysis of microdomain-association of Shiga toxin glycosphingolipid receptors in primary human endothelial cells
Final Report Abstract
Shiga toxin (Stx)-mediated injury to microvascular endothelial cells in the kidney and the brain underlies the pathogenesis of the hemolytic-uremic syndrome (HUS) caused by enterohemorrhagic Escherichia coli (EHEC). Stxs are AB5 toxins and the B-pentamers of the two major Stx subtypes, Stx1a and Stx2a, preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and to less extent to globotetraosylceramide (Gb4Cer) exposed on endothelial cells. Several studies performed with nonendothelial cells at the beginning of the project suggested the association of Stx receptors with lipid rafts as a functional requirement for internalization and retrograde transport of Stx to intracellular targets. In this project we performed comprehensive structural analyses of the different Gb3Cer and Gb4Cer receptor lipoforms and their association with lipid rafts of primary human microvascular endothelial cells of the kidney (primary human renal glomerular endothelial cells, pHRGECs) and the brain (primary human brain microvascular endothelial cells, pHBMECs). Furthermore, we explored the association of Stx receptor GSLs with the lipid raft marker protein flotillin-2 and the canonical raft markers cholesterol and sphingomyelin (SM) in microdomain preparations using detergent-resistent membranes (DRMs). We furthermore evaluated the effect of inhibition of the GSL biosynthesis using the ceramide analogon D-PDMP on the lipid composition of microdomains. The core project was accompanied by several minor projects, which related to the main goals and benefited from newly developed technologies throughout the project period. In extension of the primary issue we included, for instance, porcine primary endothelial cells and kidney epithelial cells in the Stx binding studies and identified, for example, erythrocyte precursor cells as newly detected targets of Stxs. A technological highlight is the partial contribution to novel mass spectrometry imaging based on postionization MALDI technology for efficient detection of membrane lipids in tissue slices and cell cultures.
Publications
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(2008) Glycosphingolipids in vascular endothelial cells: relationship of heterogeneity in Gb3Cer/CD77 receptor expression with differential Shiga toxin 1 cytotoxicity. Glycoconj. J. 25, 291-304
Schweppe, C.H., Bielaszewska, M., Pohlentz, G., Friedrich, A.W., Büntemeyer, H., Schmidt, M.A., Kim, K.S., Peter-Katalinić, J., Karch, H. and Müthing, J.
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(2009). Shiga toxins, glycosphingolipid diversity, and endothelial cell injury. Thromb. Haemost. 101, 252-264
Müthing, J., Schweppe, C.H., Karch, H. and Friedrich, A.W.
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(2010) Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy. J. Biomed. Opt. 15, 036009
Kemper, B., Bauwens, A., Vollmer, A., Ketelhut, S., Langehanenberg, P., Müthing, J., Karch, H. and von Bally, G.
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(2010) Neutral glycosphingolipids in human blood: a precise mass spectrometry analysis with special reference to lipoprotein-associated Shiga toxin receptors. J. Lipid Res. 51, 2282-2294
Schweppe, C.H., Hoffmann, P., Nofer, J.R., Pohlentz, G., Mormann, M., Karch, H., Friedrich. A.W. and Müthing, J.
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(2010) On the structural diversity of Shiga toxin glycosphingolipid receptors in lymphoid and myeloid cells determined by nanoelectrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 24, 2295-2304
Hoffmann, P., Hülsewig, M., Duvar, S., Ziehr, H., Mormann, M., Peter-Katalinić, J., Friedrich, A.W., Karch, H. and Müthing, J.
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(2011) Differential cytotoxic actions of Shiga toxin 1 and Shiga toxin 2 on microvascular and macrovascular endothelial cells. Thromb. Haemost. 105, 515-528
Bauwens, A., Bielaszewska, M., Kemper, B., Langehanenberg, P., von Bally, G., Reichelt, R., Mulac, D., Humpf, H.U., Friedrich, A.W., Kim, K.S., Karch, H. and Müthing, J.
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(2011) Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains. J. Lipid Res. 52, 618-634
Betz, J., Bielaszewska, M., Thies, A., Humpf, H.U., Dreisewerd, K., Karch, H., Kim, K.S., Friedrich, A.W. and Müthing, J.
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(2011) Struktur-Funktions-Analysen von Shiga Toxin-Rezeptoren mikro- und makrovaskulärer Endothelzellen. PhD thesis, University of Münster
Betz, J.
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(2012) Promiscuous Shiga toxin 2e and its intimate relationship to Forssman. Glycobiology 22, 849-862
Müthing, J., Meisen, I., Zhang, W., Bielaszewska, M., Mormann, M., Bauerfeind, R., Schmidt, M.A., Friedrich, A.W. and Karch, H.
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(2012) Uncommon membrane distribution of Shiga toxin glycosphingolipid receptors in toxin-sensitive human glomerular microvascular endothelial cells. Biol. Chem. 393, 133-147
Betz, J., Bauwens, A., Kunsmann, L., Bielaszewska, M., Mormann, M., Humpf, H.U., Karch, H., Friedrich, A.W. and Müthing, J.
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(2013) Facing glycosphingolipid-Shiga toxin interaction: dire straits for endothelial cells of the human vasculature. Cell. Mol. Life Sci. 70, 425-457
Bauwens, A., Betz, J., Meisen, I., Kemper, B., Karch, H. and Müthing, J.
