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Abstract The gamma-type inhibitor of snake venom phospholipase A 2 (PLIγ) is expressed extensively in livers of both venomous and non-venomous snakes. It is not clear why PLIγs from different snake species possess diverse activities. To obtain high activity PLIγs and interpret the sequence-function relationships, we used DNA shuffling to hybridize the PLIγs of Sinonatrix annularis (saPLIγ) and Elaphe carinata (ecPLIγ). Chimera PLIγs (cPLIγ) of ∼550 bp were obtained by a series of gene manipulations including DNase I digestion, primer-free PCR, and PCR amplification with PLIγs primer pair. After successful insertion of cPLIγs into pCANTAB5e phage vector, the transformed TG1 strain of Esherichia coli was achieved. The cPLIγ phage library was produced and panned in a five-pace snake venom-coated immune tube. Three high affinity cPLIγ isoforms survived two rounds of panning. Prokaryote expression by the pET28c vector was employed for production of the three cPLIγs and the two parental PLIγs. These all showed anti-hemorrhage activity with cPLIγ 2 demonstrating superior inhibition to the parent PLIγs. Sequence alignment showed that the three kinds of cPLIγ were produced by gene splicing of S. annularis and E. carinata at different sites. Primary sequence changes brought regional changes in secondary and tertiary structure, which may explain the differences in PLIγ activity. Highlights • PLIγ can neutralize snake venom PLA2s, and is regarded as a promising antivenom candidate. • DNA shuffling and phage surface display is an effective way for PLIγ directed evolution. • The chimera PLIγ 2 showed even superior anti-hemorrhage activity than the parent PLIγs. • The α-helix of 150–155aa was believed to be correlative to the activity of PLIγ. [ABSTRACT FROM AUTHOR]

SaPLIγ is a natural phospholipase A 2 (PLA 2 ) inhibitor, isolated from Sinonatrix annularis, that has been demonstrated to protect against envenomation by other venomous snakes. As snake venom PLA 2 s and mammalian secretory PLA 2 s are similar, saPLIγ is thought to have potential to alleviate inflammatory reactions in which PLA 2 s act as a key enzyme for arachidonic acid release. The aim of this study was to investigate the anti-inflammatory effects and mechanisms of action of saPLIγ in an animal model of carrageenan-induced acute inflammation. The results indicated that saPLIγ inhibited PLA 2 subtypes extensively, especially IIA-PLA 2 , in a dose-dependent manner. Paw swelling in mice was reduced markedly by intraperitoneal saPLIγ 2 .5 mg/kg, and the effect was significantly better than observed with dexamethasone at the same dose. Lower neutrophil infiltration and tissue edema was observed in the paws of saPLIγ-treated mice. Additionally, carrageenan-induced cyclooxygenase- 2 (COX- 2 ) and pro-inflammatory cytokines (TNFα and IL-1β) were also significantly down-regulated by saPLIγ in a dose-dependent manner. These results suggested that saPLIγ had effective anti-inflammatory effects in vivo, and these were produced by blocking mammalian IB, IIA, V and X sPLA2 subtypes. [ABSTRACT FROM AUTHOR]

[Display omitted] • A venom affinity column was constructed and adapted for active inhibitors mining. • Venom inhibitors and toxin receptors were identified in the serum and liver of the snake species S. annularis. • Fifteen toxins from D. acutus venom were pooled using venom binding proteins-affinity column Various snake species and snake predators have natural neutralization against snake toxins, which their antidotal abilities are commonly attributed to the intrinsic inhibitors produced by the liver, e.g., phospholipase A 2 inhibitor (PLI) and metalloproteinase inhibitor (SVMPI). Sinonatrix annularis was found to possess broad-spectrum neutralization to different snake venoms in our lab. Although the anti-venom compound PLIγ has been previously characterized in our laboratory, the mechanism of resistance of S. annularis to snake venoms remains obscure. In this research, a venom affinity chromatography was constructed by immobilizing D. acutus venom to NHS-agarose beads and applied for antitoxins mining from S. annularis. The binding capacity of the venom column was validated using a self-prepared rabbit antivenom against D. acutus. Serum and liver homogenate of S. annularis were then applied to the column, the bound components were profiled using SDS-PAGE and mass spectrometry. PLIs, snake venom metalloproteins inhibitor (SVMPI), small serum protein (SSP), heat shock proteins, etc were identified. To identify their toxin targets in D. acutus venom, a reverse separation was conducted by coupling the fractionated S. annularis serum proteins to NHS-agarose beads. Fifteen toxins of five families were captured and identified as follows: PLA 2 s, metalloproteinases, cysteine-rich secretory proteins, snake venom serine proteinases, and C-type lectins. These discoveries increased our understanding of the capacity and mechanism of the natural neutralization of S. annularis to snake venom. These natural inhibitors are medically significant due to their powerful and broad antidotal activities, which may provide alternative and promising drug candidates for snakebite treatment. [ABSTRACT FROM AUTHOR]