Figure 1.
Deduced aminoacid sequences of light (A) and heavy (B) PcPV2 subunits.
The putative signal sequences are in italics, MACPF domain is marked in red and MACPF signature is boxed. Potential phosphorylation sites are underlined, and potential glycosylation sites are underlined and in bold.
Figure 2.
Bayesian phylogenetic tree of light (A) and heavy (B) PcPV2 subunits of Pomacea canaliculata.
Numbers above branches represent Bayesian posterior probabilities of finding a given clade. Scale bar, 0.2 nucleotide substitution.
Figure 3.
Effect of pH on PcPV2 structure as determined by SAXS (A, B) and Trp fluorescence (C, D).
A: PcPV2 radius of giration Rg as a function of pH. B: Kratky plot of data depicted on Figure 2A that highlights shape variations of PcPV2. C: Trp fluorescence emission spectra at different pH values. D: Mean wavelength of the spectrum (closed circles) and maximum intensity (open circles). Mean wavelength refers to Σ(λ F(λ))/Σ F(λ) where F(λ) corresponds to the fluorescence intensity for a given λ. Error bars in A fall within the size of the points; error bars in B and C never exceeded 1% of the value; error bars in D are 2 nm for wavelengths and 10% for signal intensity.
Figure 4.
In vitro digestibility analyzed by SDS-PAGGE.
A: Gastric digestion. Lanes 1–3, 0, 60 and 120 min of incubation, lanes 4 and 5 positive and negative control, respectively. B: Duodenal digestion. Lanes 1–3, 0, 30 and 120 min incubation; lanes 4 and 5, positive and negative controls, respectively. Positive control: Albumin with enzyme, negative control: albumin without enzyme.
Figure 5.
Binding to cells and presence in circulation of PcPV2.
A,B,C,D: Immunolocalization of PcPV2 at the brush border of rat enterocytes. Rats were fed for 4 days on a diet without (A, B) or with (C, D) egg extracts containing the equivalent of 400 µg PcPV2. Arrows indicate anti-PcPV2 antibody binding to glycocalix. A, C: Bar 100 µm, B, D: Bar 15 µm. E, F: PcPV2 binding to intestinal cells in culture. Fluorescence microscopy of Caco-2 cells incubated for 1 h with Alexa-488 labeled BSA as control (E) or PcPV2 (F). Bar 25 µm. Arrows indicate specific toxin binding to cell surface. G: Hemagglutinating and hemolytic activity. Wells 1–5 two-fold serial dilution of 1.6 µg/ml PcPV2; 6 Control with buffer. H, I: Immunization of rodents by sublethal oral administration of PcPV2. Dot blot analysis of sera from rats orally immunized with PcPV2 (G). Strong positive immunoreactivity can be seen towards PcPV2 antigen. Dots a-c, sera from control or immunized rat diluted 1∶10, 1∶100, 1∶1000, respectively. PcPV2 was blotted onto nitrocellulose (1 µg in 5 µl PBS/spot). H. Effect of lethal i.p. dosis of PcPV2 on control and immunized mice survival. Graph I does not include error bars because all of the 6 immunized animals survived the intraperitoneal injection of a lethal concentration of the toxin, while all of the 6 control animals died after the injection.
Figure 6.
Similarities and differences in structure and function in dichain toxic lectins from bacteria, plants and apple snails.
Question marks indicate unknown steps.