Annexin A1 and A2: Roles in Retrograde Trafficking of Shiga Toxin

PLOS ONE, Dec 2019

Annexins constitute a family of calcium and membrane binding proteins. As annexin A1 and A2 have previously been linked to various membrane trafficking events, we initiated this study to investigate the role of these annexins in the uptake and intracellular transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin. Once endocytosed, both toxins are retrogradely transported from endosomes to the Golgi apparatus and the endoplasmic reticulum before being targeted to the cytosol where they inhibit protein synthesis. This study was performed to obtain new information both about toxin transport and the function of annexin A1 and annexin A2. Our data show that depletion of annexin A1 or A2 alters the retrograde transport of Stx but not ricin, without affecting toxin binding or internalization. Knockdown of annexin A1 increases Golgi transport of Stx, whereas knockdown of annexin A2 slightly decreases the same transport step. Interestingly, annexin A1 was found in proximity to cytoplasmic phospholipase A2 (cPLA2), and the basal as well as the increased Golgi transport of Stx upon annexin A1 knockdown is dependent on cPLA2 activity. In conclusion, annexin A1 and A2 have different roles in Stx transport to the trans-Golgi network. The most prominent role is played by annexin A1 which normally works as a negative regulator of retrograde transport from the endosomes to the Golgi network, most likely by complex formation and inhibition of cPLA2.

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Annexin A1 and A2: Roles in Retrograde Trafficking of Shiga Toxin

et al. (2012) Annexin A1 and A2: Roles in Retrograde Trafficking of Shiga Toxin. PLoS ONE 7(7): e40429. doi:10.1371/journal.pone.0040429 Annexin A1 and A2: Roles in Retrograde Trafficking of Shiga Toxin Lionel Tcatchoff 0 Sofia Andersson 0 Audrun Utskarpen 0 Tove Irene Klokk 0 Sigrid S. Ska nland 0 Sascha Pust 0 Volker Gerke 0 Kirsten Sandvig 0 Steve H. Caplan, University of Nebraska Medical Center, United States of America 0 1 Department of Biochemistry, Centre for Cancer Biomedicine, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital , Montebello, Oslo , Norway , 2 Department of Molecular Biosciences, University of Oslo , Oslo , Norway , 3 Institute for Medical Chemistry, Center for Molecular Biology of Inflammation, University of Mu nster , M u nster , Germany Annexins constitute a family of calcium and membrane binding proteins. As annexin A1 and A2 have previously been linked to various membrane trafficking events, we initiated this study to investigate the role of these annexins in the uptake and intracellular transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin. Once endocytosed, both toxins are retrogradely transported from endosomes to the Golgi apparatus and the endoplasmic reticulum before being targeted to the cytosol where they inhibit protein synthesis. This study was performed to obtain new information both about toxin transport and the function of annexin A1 and annexin A2. Our data show that depletion of annexin A1 or A2 alters the retrograde transport of Stx but not ricin, without affecting toxin binding or internalization. Knockdown of annexin A1 increases Golgi transport of Stx, whereas knockdown of annexin A2 slightly decreases the same transport step. Interestingly, annexin A1 was found in proximity to cytoplasmic phospholipase A2 (cPLA2), and the basal as well as the increased Golgi transport of Stx upon annexin A1 knockdown is dependent on cPLA2 activity. In conclusion, annexin A1 and A2 have different roles in Stx transport to the trans-Golgi network. The most prominent role is played by annexin A1 which normally works as a negative regulator of retrograde transport from the endosomes to the Golgi network, most likely by complex formation and inhibition of cPLA2. - Funding: This work was supported by grants from the Norwegian Cancer Society, the Norwegian Research Council for Science and the Humanities, the Norwegian Research Council Functional Genomics (NFR-FUGE), and the German Research Council to VG (DFG, SFB 629). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Numerous toxins take advantage of the mammalian cellular machinery to enter and reach their target within the cell. The bacterial Shiga toxin (Stx) and the plant toxin ricin are examples of such toxins, and do therefore represent valuable tools to study endocytic and intracellular transport mechanisms (for recent reviews, see [1,2]). These toxins are built up of an A and a B moiety, where the latter facilitates the binding to cellular receptors, the glycosphingolipid globotriaosylceramide (Gb3) for Stx and terminal galactose residues of glycolipids and glycoproteins for ricin. After entering the cell by endocytosis, the toxins are sorted in the early endosomes and are either recycled, destined for lysosomal degradation or transported retrogradely to the Golgi apparatus and the endoplasmic reticulum (ER). The enzymatically active part of the A subunit is then translocated from the ER to the cytosol where it exerts its cytotoxic effect by inactivating ribosomal function leading to an inhibition of protein synthesis [2,3]. During the past years, a variety of studies have resulted in the identification of proteins that are necessary for the highly regulated transport events used by toxins. These include clathrin and its binding partners [47], sorting nexins [5,811] as well as different lipids [12,13]. Moreover, binding of Stx has been shown to trigger activation of various signalling molecules, including protein kinase Cd, Syk and p38 [1416]. Annexins, a family of structurally related Ca2+-binding proteins [17], are of interest in the search for candidate proteins that take part in the uptake and transport of Stx and ricin. Annexins comprise a conserved C-terminal core domain interacting with membrane phospholipids and a variable N-terminal domain, which is responsible for the specific functions of individual annexins. The N-terminal part of annexin A1 contains three possible phosphorylation sites while annexin A2 contains phosphorylation sites which are presumable targets for PKCa and Src kinases. Both N-terminal domains also harbour interaction motifs for proteins of the S100 family, S100A11 and S100A10 (also named p11) for annexin A1 and A2 respectively (...truncated)


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Lionel Tcatchoff, Sofia Andersson, Audrun Utskarpen, Tove Irene Klokk, Sigrid S. Skånland, Sascha Pust, Volker Gerke, Kirsten Sandvig. Annexin A1 and A2: Roles in Retrograde Trafficking of Shiga Toxin, PLOS ONE, 2012, Volume 7, Issue 7, DOI: 10.1371/journal.pone.0040429