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

Abstract

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.

Cross-linking of glycosphingolipids at the plasma membrane: consequences for intracellular signaling and traffic

“…One might speculate that if cPLA 2 a indeed is translocated at least partly to the Golgi apparatus in response to the toxin-induced Ca 2? influx, cPLA 2 a could be involved in toxin transport to and through the Golgi, as we have previously found that inhibition of cPLA 2 reduces Stx transport to the Golgi [12]. The fractionation data also show that AnxA1 is translocated to membranes upon StxB-S2 treatment, and the immunofluorescence data suggest that these membranes are at least partly late endosomal/lysosomal membranes as an increase in colocalization with LAMP1 was seen.…”

“…We previously showed that a modified Stx B-subunit containing two sulfation sites (StxB-S2), induced dissociation of the cPLA 2 a-AnxA1 complex [12]. To investigate whether the intact Stx, containing both Aand B-chains, would induce a similar dissociation of this complex, HeLa cells were incubated with equal molar concentrations of StxB-S2 or SLT1, an enzymatically inactive Stx mutant [20].…”

“…Stx is internalized by endocytosis after binding to the oligosaccharide part of Gb3 and is transported retrogradely through the Golgi apparatus and the endoplasmic reticulum to its final destination, the cytosol, where it inhibits protein synthesis (for recent reviews on Shiga toxin, see [10,11]). We have previously shown that Stx is able to induce dissociation of the cytosolic complex of annexin A1 (AnxA1) and the phospholipase A2, cPLA 2 a [12]. The function of annexins is linked to their ability to bind membrane phospholipids in a dynamic and reversible fashion through their Ca 2?…”

“…signaling and membrane functions [13]. Studies from our laboratory have shown that AnxA1 plays a role in the retrograde transport of Stx, where depletion of AnxA1 results in increased transport of Stx to the Golgi, an effect that is dependent on cPLA 2 a activity [12]. Phospholipase A2 (PLA 2 ) is a class of enzymes that removes a fatty acid from phospholipids at the sn-2 position, converting cone-or cylindrical-shaped phospholipids to inverted cone-shaped lysophospholipids [14].…”

Plasma membrane reorganization: A glycolipid gateway for microbes

“…One of the main evidence of clathrin-independent endocytosis of Shiga toxins is the formation of long surface-connected membrane tubules [33]. Toxin internalization is independent of raft-associated flotillins or scaffolding proteins like annexins, as inhibition of these two proteins in two independent experiments did not affect toxin entry [50,51]. The toxininduced activation of receptor tyrosine kinases such as Yes, Src, Syk and Lyn in different cell lines mediates the formation of clathrin coated pits to increase Shiga toxin uptake [44,52,53].…”