TY - JOUR A1 - Fink, Julian A1 - Schumacher, Fabian A1 - Schlegel, Jan A1 - Stenzel, Philipp A1 - Wigger, Dominik A1 - Sauer, Markus A1 - Kleuser, Burkhard A1 - Seibel, Jürgen T1 - Azidosphinganine enables metabolic labeling and detection of sphingolipid de novo synthesis JF - Organic & biomolecular chemistry : an international journal of synthetic, physical and biomolecular organic chemistry N2 - Here were report the combination of biocompatible click chemistry of omega-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that omega-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, omega-azidosphinganine represents a useful biofunctional tool for metabolic investigations both by in vivo fluorescence imaging of the sphingolipid subcellular localization in the ER and by in vitro high-resolution mass spectrometry analysis. This should reveal novel insights of the molecular mechanisms sphingolipids and their processing enzymes have e.g. in infection. Y1 - 2021 U6 - https://doi.org/10.1039/d0ob02592e SN - 1477-0520 SN - 1477-0539 VL - 19 IS - 10 SP - 2203 EP - 2212 PB - Royal Society of Chemistry CY - Cambridge ER - TY - GEN A1 - Lang, Judith A1 - Bohn, Patrick A1 - Bhat, Hilal A1 - Jastrow, Holger A1 - Walkenfort, Bernd A1 - Cansiz, Feyza A1 - Fink, Julian A1 - Bauer, Michael A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Lang, Karl S. T1 - Acid ceramidase of macrophages traps herpes simplex virus in multivesicular bodies and protects from severe disease T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1(-/-) mice results in replication of HSV-1 and Asah1(-/-) mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1400 KW - immunology KW - infection KW - membrane fusion KW - phagocytosis KW - sphingolipids Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-515661 SN - 1866-8372 IS - 1 ER - TY - JOUR A1 - Lang, Judith A1 - Bohn, Patrick A1 - Bhat, Hilal A1 - Jastrow, Holger A1 - Walkenfort, Bernd A1 - Cansiz, Feyza A1 - Fink, Julian A1 - Bauer, Michael A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Lang, Karl S. T1 - Acid ceramidase of macrophages traps herpes simplex virus in multivesicular bodies and protects from severe disease JF - Nature Communications N2 - Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1(-/-) mice results in replication of HSV-1 and Asah1(-/-) mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol. KW - immunology KW - infection KW - membrane fusion KW - phagocytosis KW - sphingolipids Y1 - 2020 U6 - https://doi.org/10.1038/s41467-020-15072-8 SN - 2041-1723 VL - 11 IS - 1 SP - 1 EP - 15 PB - Nature Publishing Group UK CY - London ER - TY - JOUR A1 - Solger, Franziska A1 - Kunz, Tobias C. A1 - Fink, Julian A1 - Paprotka, Kerstin A1 - Pfister, Pauline A1 - Hagen, Franziska A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Seibel, Jürgen A1 - Rudel, Thomas T1 - A role of sphingosine in the intracellular survival of Neisseria gonorrhoeae JF - Frontiers in Cellular and Infection Microbiology N2 - Obligate human pathogenic Neisseria gonorrhoeae are the second most frequent bacterial cause of sexually transmitted diseases. These bacteria invade different mucosal tissues and occasionally disseminate into the bloodstream. Invasion into epithelial cells requires the activation of host cell receptors by the formation of ceramide-rich platforms. Here, we investigated the role of sphingosine in the invasion and intracellular survival of gonococci. Sphingosine exhibited an anti-gonococcal activity in vitro. We used specific sphingosine analogs and click chemistry to visualize sphingosine in infected cells. Sphingosine localized to the membrane of intracellular gonococci. Inhibitor studies and the application of a sphingosine derivative indicated that increased sphingosine levels reduced the intracellular survival of gonococci. We demonstrate here, that sphingosine can target intracellular bacteria and may therefore exert a direct bactericidal effect inside cells. KW - Neisseria gonorrhoeae KW - sphingosine KW - sphingolipids KW - sphingosine kinases KW - invasion KW - survival KW - click chemistry Y1 - 2020 U6 - https://doi.org/10.3389/fcimb.2020.00215 SN - 2235-2988 VL - 10 PB - Frontiers Media CY - Lausanne ER -