Fig 1.
General morphology of scorpion digestive system and its location.
Schematic ventral (A) and lateral (B) views of scorpion digestive system and its divisions. Right picture (A) represents ventral view of Tityus serrulatus MMG. PMG, prosomal midgut. Fig 1B was modified from [2].
Table 1.
Possible digestive enzymes identified after the transcriptomic analysis in the midgut and midgut glands of the scorpion Tityus serrulatus.
Fig 2.
Quantitative analysis of selected proteins likely involved in digestion.
Values are means and SEM from relative normalized spectra counting (NSC) calculated based on possible digestive enzymes identified. Shotgun proteomics experiment on triplicate samples with MMG of fasting and fed scorpions.
Fig 3.
Effect of pH on endopeptidase activities using different substrates.
Sample source was either crude MMG extracts (A and B) or chromatographically separated (C and D). (A) Hemoglobin 2%. (B) Casein-FITC 0.2%. C) Activated (✱) and non-activated (●) C1 samples. (D) Effect of pH on isolated cysp1 (✱) and cysp2 (●) samples. Buffers used (100 mM): Gly-HCl, pHs 1.5 and 2; Citrate-phosphate, pHs 2.6–7; MES, pH 7; TRIS-HCl, pHs 7.5–9; Gly-HCl 9.5–10. Buffers used in A, C and D contain 3 mM cysteine and 3 mM EDTA.
Table 2.
Peptidase absolute and specific activities involved in protein digestion in MMG from the scorpion Tityus serrulatus using different substrates.
Fig 4.
Hydrophobic chromatographic fractioning of Tityus serrulatus MMG homogenate.
MMG homogenate was fractioned with 50% ammonium sulfate on a HiTrap Butyl column (GE) equilibrated in 50 mM phosphate buffer (pH 6.0). Elution was performed using a gradient of 1.7–0 M ammonium sulfate in the same buffer. (A) Activity of each fraction against 10 μM Z-FR-MCA was measured in 100 mM Tris-HCl buffer (pH 8.0) containing 10 mM CaCl2 (●) or in the presence of 5.0 mM benzamidine (○). (B) The activity of each fraction against 10 μM Z-FR-MCA was measured in 100 mM CP-buffer (pH 5.5) containing 3.0 mM cysteine and 3.0 mM EDTA in the absence (●) and presence of different peptidase inhibitors: (□) 10 μM E-64; (○) 1.0 mM PMSF; (Δ) 10 μM pepstatin.
Fig 5.
Acid activation of cysteine endopeptidases from Tityus serrulatus´ MMG.
Effect of incubating MMG homogenate (A) at 30°C for 60 minutes under different pH conditions. (B) Effect of time on acidic activation of cysteine peptidases from Tityus serrulatus MMG homogenate. After incubation in acidic buffer (pH 2.6), 2 μl of each enzyme preparation was assayed in 200 μl of 0.1 M CP buffer (pH 5.5) with Z-FR-MCA to measure activity at constant pH. Activity increase was calculated as ratio of incubated enzyme activity over non-incubated control activity. All buffers used for activation (0.1 M CP, pH 2.6–7.0) and activity assays contained 3.0 mM cysteine and 3.0 mM EDTA.
Fig 6.
Phylogenetic relationships among endopeptidases present in MMG of the scorpion Tityus serrulatus using maximum likelihood algorithm.
Sequences displayed in red were identified by proteomics.
Fig 7.
Section of Cathepsin-L Bayesian phylogeny including only clade Arachnida 3.
For whole topology see S8 Fig Red squares indicate duplications (15 in total) as inferred by Notung v2.7.
Fig 8.
Schematic representation of midgut and midgut glands secretory (SC) and digestive cells (DC).
Figure displays enzymes present in secretory vesicles (SV) and lysosome-like (LL) organelles. Lysosomes probably fuse or exchange contents with pinocytic vesicles to end up in digestive vacuoles. DC: digestive cells, DV: digestive vacuoles, F: pre digested food, M: mitochondria, P: pinocytosis, RER: rough endoplasmic reticulum, S: spherites, SC: secretory cells.