Figure 5 ALN has differential activity on cells

from various mammalian species. (a) The specific activities of ALN were determined by incubation of dilutions of His-ALN with erythrocytes from different host species. Results are an average of at least three independent experiments conducted in duplicated and error bars represent standard deviation. (b) The species selectivity of ALN was compared to ILY and PLO in hemolysis assays using human (EPZ5676 concentration square), horse (triangle), and pig (inverted triangle) erythrocytes. Representative of two experiments conducted in triplicate and error bars represent standard error of the mean. (c) Dilutions of His-ALN were added to cultured host cells and the amount of ALN required to reduce the cell viability by 50% https://www.selleckchem.com/products/BIBW2992.html was determined using the CellTiter 96® Aqueous AZD5363 solubility dmso One Solution Cell Proliferation Assay (Promega). Error bars indicate one standard deviation from the mean calculated from the averages of at least three independent experiments conducted in triplicate. The highly-conserved Cys residue in the undecapeptide of CDCs is responsible for Thiol activation of this group of toxins [30]. ALN lacks the Cys residue in the undecapeptide (Figure 3a), and like PLO [14], its activity was unaffected by treatment with β-mercaptoethanol

(data not shown). We also determined the effect of recombinant ALN on cultured mammalian cells. His-ALN was applied to human, bovine, canine, hamster, mouse and rabbit cell lines and was highly active on human and rabbit cells (Figure 5c), with low activity on bovine, mouse and canine cells. This toxin had intermediate activity on hamster cells (Figure 5c). This finding mirrors the activity of ALN on blood from different host

species (Figure 5a), and is less species-specific than intermedilysin (ILY) or vaginolysin (VLY) [23, 31]. ILY, VLY, and lectinolysin (LLY) use human CD59 (hCD59) as a membrane receptor [23, 32, 33], leading to host-specificity. Unlike these other CDC toxins ALN hemolysis was not blocked with a monoclonal antibody against hCD59 (data not shown). Consistent with this finding, the predicted ALN amino acid sequence Ponatinib lacks the Tyr-X-Tyr-X14-Ser-Arg signature motif common to all known hCD59-dependent CDCs [33]. The activity of ALN is less sensitive to cholesterol inhibition than PFO Given the more restrictive host species preference of ALN over that of PFO, along with the variant undecapeptide sequence in ALN, we hypothesized that ALN might be less sensitive to inhibition by free cholesterol. As expected, PFO activity was almost completely inhibited by exogenous 0.5 μM cholesterol (7.6%; Figure 6). In contrast, PLO and ALN retained 52.5% and 41.4% activity, respectively, when incubated with 0.5 μM cholesterol and retained ~20% of hemolytic activity at 1 μM cholesterol (Figure 6).