Joannsin, a novel Kunitz-type FXa inhibitor from the venom of Prospirobolus joannsi

Abstract

The repugnatorial glands of millipedes release various defensive chemical secretions. Although varieties of such defensive secretions have been studied, none of them is protein or peptide. Herein, a novel factor Xa (FXa) inhibitor named joannsin was identified and characterised from repugnatorial glands of Prospirobolus joannsi. Joannsin is composed of 72 amino acid residues including six cysteines, which form three intra-molecular disulfide bridges. It is a member of Kunitz-type protease inhibitor family, members of which are also found in the secretory glands of other arthropods. Recombinant joannsin exhibited remarkable inhibitory activity against trypsin and FXa with a Ki of 182.7 ± 14.6 and 29.5 ± 4.7 nM, respectively. Joannsin showed strong anti-thrombosis functions in vitro and in vivo. Joannsin is the first peptide component in millipede repugnatorial glands to be identified and is a potential candidate and/or template for the development of anti-thrombotic agents. These results also indicated that there is Kunitz-type protease inhibitor toxin in millipede repugnatorial glands as in other arthropods secretory glands.

Keywords: Kunitz-type inhibitor; Millipede; factor Xa; repugnatorial gland; thrombosis.

Figure 1: Repugnatorial glands in P. joannsi and FXa-inhibiting activity in its secretions

. A) Defensive secretions from the millipede repugnatorial glands. B) Pairs of defensive glands in each body somite of the millipede are indicated by the red arrow. C) The components containing FXa-inhibiting activity. At the concentration of ∼ 0.1 mg/ml, the mixture components significantly inhibited FXa.

Figure 2: Primary structure of joannsin (A) and sequence alignment with other Kunitz-type serine protease inhibitors (B)

. Mature joannsin is boxed, and the stop codon is indicated by a bar (-). The identical amino acid residues are indicated by the symbol (#). Cysteine residues are highlighted in grey and the trypsin interaction sites are shown in red.

Figure 3: Recombinant expression of joannsin

. Recombinant joannsin was purified by Sephadex G-50 gel filtration (A) and C4 RP-HPLC (B), indicated by an arrow. SDS-PAGE of samples after each purification (inserted in B): collected fusion protein purified with affinity (line 1); after chemical cleavage, recombinant joannsin was purified by a Sephadex G-50 (line 2); recombinant joannsin purified by a C4 RP-HPLC (line 3). The purified recombinant joannsin was subjected to MALDI-TOF mass spectrometry analysis (C).

Figure 4: Coagulation effect of joannsin

. Compared with the control group, the recalcification time (A), APTT (B) and PT (C) were prolonged.

Figure 5: Protease inhibiting activity of joannsin

. The effect of joannsin on FXa, trypsin, thrombin, plasmin, FXIIa, chymotrypsin and kallikrein with the chromogenic substrate (A). SDS-PAGE after the reaction of joannsin and FXa (B): human prothrombin (line 1); FXa (line 2); joannsin inhibited the activity of FXa to human prothrombin with different concentrations (0 nM: line 3; 10 nM: line 4; 20 nM: line5; 50 nM: line 6), black arrow and red arrow indicated human prothrombin and thrombin, respectively. According to the Dixon method, the Ki of joannsin towards FXa and trypsin is 29.5 ± 4.7 nM (C) and 182.7 ± 14.6 nM (D), respectively.

Figure 6: Joannsin inhibited thrombus formation in vivo

. Joannsin inhibited carrageenan-induced thrombosis in mouse tail at 12 (A) and 24 (B) h after injection in a dose-dependent manner. (C) The representative thrombus in the mouse tail at 24 h. (a) The control group, (b, c, d) the joannsin groups with different concentrations (2 mg/kg, 4 mg/kg and 6 mg/kg, respectively), (e) the group of apixaban (0.2 mg/kg). Data were presented as mean ±SE. * P< 0.05, ** P< 0.01, *** P<0.001 compared with CK, # P< 0.05, ## P< 0.01, compared with Apixaban. Analysed by student’s t test for two-sample comparison.