Membrane glycoprotein receptor and hole-forming properties of a cytolytic protein toxin

Glycosylphosphatidylinositol-anchored proteins may be concentrated in membrane microdomains (lipid rafts) that are also enriched in cholesterol and sphingolipids. The glycosyl anchor of these proteins is a specific, high affinity receptor for the channel-forming protein aerolysin. We wished to determine if the presence of rafts promotes the activity of aerolysin. Treatment of T lymphocytes with methyl-␤-cyclodextrin, which destroys lipid rafts by sequestering cholesterol, had no measurable effect on the sensitivity of the cells to aerolysin; nor did similar treatment of erythrocytes decrease the rate at which they were lysed by the toxin. We also studied the rate of aerolysin-induced channel formation in liposomes containing glycosylphosphatidylinositolanchored placental alkaline phosphatase, which we show is a receptor for aerolysin. In liposomes containing sphingolipids as well as glycerophospholipids and cholesterol, most of the enzyme was Triton X-100-insoluble, indicating that it was localized in rafts, whereas in liposomes prepared without sphingolipids, all of the enzyme was soluble. Aerolysin was no more active against liposomes containing rafts than against those that did not. We conclude that lipid rafts do not promote channel formation by aerolysin.

Section: Discussionsupporting

confidence: 53%

Channel Formation by the Glycosylphosphatidylinositol-anchored Protein Binding Toxin Aerolysin Is Not Promoted by Lipid Rafts

Nelson

Buckley

2000

“…The enzyme-treated cells had greatly reduced sensitivity to the toxin. 3 Liposomes Containing Thy-1 Are More Sensitive to Aerolysin-The results of the experiments comparing wild-type and mutant cell lines were convincing evidence that GPI-anchored proteins confer aerolysin sensitivity to T-lymphocytes. However, since these cells may contain several different proteins anchored in this way, we could not conclude that Thy-1 itself was acting as an aerolysin receptor.…”

Section: Thy-1 Is a Receptor For Aerolysinmentioning

confidence: 90%

“…Among erythrocytes that have been compared, those of the rat are most sensitive (2,3), and we have shown that this is because they contain a 47-kDa glycoprotein that binds both proaerolysin and aerolysin with considerable affinity (4). Presumably the primary function of the receptor is to concentrate the toxin on the cell surface, thereby indirectly promoting oligomerization.…”

mentioning

confidence: 99%

The Glycosylphosphatidylinositol-anchored Surface Glycoprotein Thy-1 Is a Receptor for the Channel-forming Toxin Aerolysin

Nelson

Raja

Buckley

1997

Self Cite

111

103

Aerolysin is a channel-forming protein secreted by virulent Aeromonas spp. Some eucaryotic cells, including T-lymphocytes, are sensitive to very low concentrations of the toxin (<10 ؊9 M). Here we show that aerolysin binds selectively and with high affinity to the glycosylphosphatidylinositol (GPI)-anchored surface protein Thy-1, which is found on T-lymphocyte populations as well as in brain. Less than 1 ng of purified Thy-1 could be detected by probing Western blots with the toxin. Mutant T-cell lines that lack the ability to add GPI anchors to Thy-1 and other surface proteins were much less sensitive to aerolysin, as were wild-type cells that were pretreated with phosphatidylinositol-specific phospholipase C to remove GPI-anchored proteins. Phosphatidylcholine/cholesterol liposomes containing purified Thy-1 in their membranes were much more sensitive to aerolysin than protein-free liposomes.

“…Efficiency of cleavage was analyzed by SDS-PAGE followed by Coomassie staining. Similar cleavages were performed in order to test the hemolytic activity as described (44) and determine the exact cleavage site by mass spectrometry as described (20).…”

Section: Methodsmentioning

confidence: 99%

The Pore-forming Toxin Proaerolysin Is Activated by Furin

Abrami¹,

Fivaz²,

Decroly³

et al. 1998

Self Cite

142

120

Aerolysin is secreted as an inactive dimeric precursor by the bacterium Aeromonas hydrophila. Proteolytic cleavage within a mobile loop near the C terminus of the protoxin is required for oligomerization and channel formation. This loop contains the sequence KVRRAR 432 , which should be recognized by mammalian proprotein convertases such as furin, PACE4, and PC5/6A. Here we show that these three proteases cleave proaerolysin after Arg-432 in vitro, yielding active toxin. We also investigated the potential role of these enzymes in the in vivo activation of the protoxin. We found that Chinese hamster ovary cells were able to convert the protoxin to aerolysin in the absence of exogenous proteases and that activation did not require internalization of the toxin. The furin inhibitor ␣ 1 -antitrypsin Portland reduced the rate of proaerolysin activation in vivo, and proaerolysin processing was even further reduced in furin-deficient FD11 Chinese hamster ovary cells. The cells were also less sensitive to proaerolysin than wild type cells; however, transient transfection of FD11 cells with the cDNA encoding furin conferred normal sensitivity to the protoxin. Together these findings argue that furin catalyzes the cell-surface activation of proaerolysin in vivo.Many toxins are secreted by pathogenic organisms as inactive precursors, presumably to protect the producing cells from self-destruction or to increase the efficiency of delivery to the target cells. Activation of toxin precursors often involves proteolytic processing by enzymes produced either by the pathogen itself or by the host organism. The identification of these proteases may be crucial to our understanding of the pathogenesis of the organism.Aerolysin is a virulence factor secreted by the human pathogen Aeromonas hydrophila (Refs. 1-3, for review see Refs. 4 and 5). The protein is released as a soluble dimeric precursor (6, 7) that can bind to specific receptors on target cells (8 -12). Proaerolysin must be activated by proteolytic cleavage (13), which releases a C-terminal peptide (14) and leads to a change in secondary structure (15). This enables the next step in channel formation, which is the generation of a heptameric oligomer (16,17). Being amphipathic (18), the heptamer can insert into the membrane thereby producing well defined channels (19). In the case of erythrocytes, channel formation leads to cell lysis; however, depending upon the toxin concentration, nucleated cells may undergo a number of changes before death occurs. These include loss of small molecules and ions through the aerolysin channels, vacuolation of the endoplasmic reticulum (12), or even apoptosis. 1 We have shown that activation of proaerolysin with trypsin is due to cleavage at the carboxyl side of Lys-427 2 (20), which is located in an 18-amino acid surface-exposed flexible loop (21). This loop also contains the sequence K 427 VRRAR 432 which corresponds to one of the motifs recognized by furin-like endoproteases, also called proprotein convertases (PC), suggesting that proaero...