Studies On The Novel Phospholipase A₂ Homologue From Agkistrodon Blomhoffii Ussurensis

Agkistrodon blomhoffii ussurensis, a member of the Agkistrodon blomhoffii ussurensis Emelianof, Crotalinae Viperidae, popular named as Manushi, distribute mainly over northeast China, Korean and parts of Russia. Phospholipase A₂ homologue from snake venom, for its phospholipase activity and other varied biological activities, has been used as natural protein to study relationship of structure and function.A neurotoxin was purified to homogeneity and characterized from the snake venom of Agkistrodon blomhoffii Ussuriensis. By three steps method, HiTrap SP (5ml) cation exchange, Superdex 75 gel-filtration (700x16), Source Q (5ml) anion exchange chromatography, yield 3.25%, and by one step affinity chromatography of anti-toxin immunoglobulin yolk (IgY) ligands, yield 12.9%, 4 times to three steps method, the neurotoxin was obtained, which molecular mass is 13881 kDa assayed by Q-TOF â…¡/MS, isoelectric point is 8.56 analyzed by IEF and shows a single band in SDS-PAGE.Based on amino acid composition analysis, N-terminal sequencing, peptide prints assayed by Q-TOF â…¡ /MS and cDNA sequencing of the purified neurotoxin from Agkistrodon blomhoffii ussurensis, the amino acid sequence of the neurotoxin was determined, which is composed of 122 amino acid residue, 14 cysteine (Cys) maybe formed 7 disulfide bond. Amino acids sequence analyzing and molecular modeling show the neurotoxin is a new phospholipase A₂, named Gln49-PLA2, with a Gln at position 49. Comparing with other PLA₂s, the amino acid sequence of the Gln49-PLA₂ shows higher similarity to Asp49-PLA₂ variants (93% to 61%) than that of Lys49-PLA₂ variants (60% to 56%), and the phylogenetic tree indicates that Gln49-PLA₂ is more closely related to the Asp49-PLA₂ variants than the Lys49-PLA₂ variants.The biological activities of Gln49-PLA₂ were detected, including phospholipase A₂ activity on egg yolk phospholipids, hemolytic activity on washed erythrocytes, anticoagulant efrect on pig platelet-rich plasma, bacteriostasis Escherichia coli, and the results show that it has no phospholipase A₂ activity hemolytic activity and bacteriostasis,but has obvious anticoagulant effect in vitro, with the concentration up lmg/ml, clotting time extended, and indicates that anticoagulant activity is not necessarily dependent on the phospholipase A2 activity. Amino acid sequence variation of Gln49-PLA2, especially Gin replacing Asp at position 49, tailing to bind the co-factor Ca2+, is the main reason for lack of phospholipase A2 activity.Gln49-PLA2 induced a dose-dependent myonecrosis upon i.m. injection in 615 mice. At the dose of O.lmg/mice, 3 hours later, the plasma CK level showed an increase up to 20 times in comparison to the control value. Histological evaluation of formalin-fixed muscle tissue confirmed the myotoxic effect. The myotoxic effect could be inhibited by heparin, with Gln49-PLA2 (50ug) pre-incubated with heparin (50ug) for 20min, the plasma CK level showed a decrease 58% in comparison to the only Gln49-PLA2 (50ug) injection.Gln49-PLA2 induced a dose-dependent death of K562, Hela, HLF cell, the LD50 are 28.6 umol/L, 1- 3 umol/L, 0. 6 umol/L respectively, assayed by MTT method. When Gln49-PLA2 pre-incubated with the same concentration heparin for 15min, 4.35 umol/L final dose adding, the Hela live cell number showed an increase 39.6% in comparison to the only Gln49-PLA2 adding, suggesting cytotoxicity of Gln49-PLA2 could be inhibited by heparin.The neurotoxic activity of Gln49-PLA2 was detected, and the LD50 is 18.2mg/kg. Its analgesic effect was assayed by the hot plate test. When injected (i.p.) in adult 615 mice, it induced a time-dose dependent analgesic effect which was inhibited by naloxone, suggesting an opioid action mechanism relating to analgesic effect. When compared with pethidine hydrochloride (30mg/kg), the Gln49-PLA2 in a dose of 1.25mg/kg (i.p.) is approximate 20-fold more potent than pethidine hydrochloride as an analgesic, on a molar basis about 880-fold more potent than pethidine hydrochloride.