Snake venom metalloproteinase (SVMP) family is one of main component in venom of Japanese vipers, which contribute to serious local tissue damage of victims. While, venomous snakes have resistance proteins to neutralize the toxicity of their toxins. In previous research, we isolated small serum proteins (SSP-1 to -5) as a new group of natural resistance comportments. SSPs belong to the prostatic secretory protein of 94 amino acids (PSP94) protein family. SSP-1, SSP-2, and SSP-5 have a molecular weight of about 10 kDa, while SSP-3 and SSP-4 have a molecular weight of 6 kDa consisting of only N-terminal domain of the other SSPs.
Interestingly, these inhibitors target different toxins as follows; SSP-1 is suppressed apoptosis of vascular endothelial cells induced by snake venom metalloprotease, HV1. SSP-2 and SSP-5 have high affinity for a CRISP family protein, triflin found as an ion channel blocker. SSP-3 inhibits a novel P-III type snake venom metalloproteinase, flavorase.
In this study, we addressed to establish of protein expression system of SSPs to identify interaction sites with target toxins. Initially, we tried to identify a target toxin of SSP-4 due to difficult isolation of the native as a minor component of the sera P.f.. The recombinant SSP-4 wild type synthesized by E. coli protein expression system showed specific binding to the SVMP (HV1) and Habu serum factor (HSF). The binding affinity between SSP-4 and HV1 was evaluated by using surface plasmon resonance (SPR), and the results showed that KD=1.14×10-5 M. Moreover, mutations of SSP-3N4C and SSP-4N3C designed based on N-terminal domain and C-terminal domain of SSP-3 or SSP4 which have high sequence identity. Here, we also show results of their binding affinity against HV1 and their inhibitory activities against the toxicity of HV1 on endothelial cells.