蜜蜂毒主要过敏原磷脂酶A_2研究概述

本文综述了国内外蜜蜂毒主要过敏原磷脂酶Ａ_２的研究进展,主要包括一般的蛋白质特性、作用机制、免疫学及生物学作用等。     

α1–6(α1–3)-Difucosylation of the asparagine-boundN-acetylglucosamine in honeybee venom phospholipase A2

Chymotryptic glycopeptides were prepared from a honeybee (Apis mellifica) venom phospholipase A2 (E.C. 3.1.1.4) fraction, with high affinity towards lentil (Lens culinaris) lectin. Treatment of the glycopeptide mixture with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A, followed by HPLC fractionation, yielded two oligosaccharides, which were analysed by 500 MHz 1H-NMR spectroscopy to give the following structures [formula: see text] This is the first report on a naturally occurring glycoprotein N-glycan with two fucose residues linked to the asparagine-bound N-acetylglucosamine.     

Alteration of the tertiary structure of the major bee venom allergen Api m 1 by multiple mutations is concomitant with low IgE reactivity

We have engineered a recombinant form of the major bee venom allergen (Api m 1) with the final goal of reducing its IgE reactivity. This molecule (Api mut) contains 24 mutations and one deletion of 10 amino acids. The successive introduction of these sequence modifications led to a progressive loss of specific IgE and IgG reactivity and did not reveal any immunodominant epitopes. However, Api mut exhibited a clear loss of reactivity for Api m 1-specific IgE and IgG. Injection of Api mut into mice induced specific antibody production. This humoral response was as high as that induced by the Api m 1 but the cross-reactivity of the antibodies was weak. As inferred by far UV circular dichroism, this mutant was correctly folded. However, near UV circular dichroism and denaturation curves of Api mut showed that it exhibits a dynamic tertiary structure and that it is a highly flexible molecule. Finally, as all the sequence modifications have been introduced outside the human and murine T cell epitope regions, we investigated its T cell properties in mice. We showed that Api mut-specific T lymphocytes induced in vivo were stimulated in vitro by both proteins. These data provide new insights in the design of hypoallergenic molecules.     

Characterization of the isoforms of phospholipase A2 from honeybee venom

Phospholipase A₂ from the venom of the European honeybee (Apis mellifera) consists of three isoforms with approximate molecular masses of 16, 18, and 20 kDa, respectively, as deduced from SDS-PAGE. These variants, termed PLA-16, PLA-18, and PLA-20, were isolated by lectin affinity chromatography and preparative polyacrylamide gel electrophoresis. The amino acid sequences of the N-terminal peptide portions of all three isoforms, as assessed by automated Edman degradation, were identical with that expected for honeybee phospholipase A₂. Sequencing data suggest that, while PLA-18 and PLA-20 carry oligosaccharide residues at asparagine-13, PLA-16 has escaped glycosylation during biosynthesis. Release of the carbohydrate from PLA-18 and PLA-20 with peptide: N-glycosidase F abolished the molecular mass differences between the three isoforms of phospholipase. Differences in sensitivity to α-mannosidase and monosaccharide composition of PLA-18 and PLA-20 further indicate that their electrophoretic separation is based on structural features of the N-glycosidically linked oligosaccharide. Noticeably, PLA-20 contains N-acetylgalactosamine, a sugar not having yet been described as a constituent of insect glycoproteins.     

Antiapoptotic role of major royal jelly protein 8 of honeybee (Apis mellifera) venom

The dominant protein components of honeybee royal jelly (RJ) are major royal jelly proteins (MRJPs), which exhibit various biological properties. However, the biological basis of why bee venom contains MRJPs and what role MRJPs play in bee venom remains to be elucidated. This study reports the antiapoptotic role of MRJP 8 of Apis mellifera venom (AmMRJP 8) in melittin-treated mammalian cells. Recombinant AmMRJP 8 reduced caspase-3 activity and melittin-induced cell apoptosis. Additionally, recombinant AmMRJP 8 decreased the production levels of H₂O₂ and proinflammatory molecules. These results indicate that MRJP in bee venom plays a role in cell protection in bee venom-induced inflammatory responses.     

Therapeutic application of natural inhibitors against snake venom phospholipase A(2)

Natural inhibitors occupy an important place in the potential to neutralize the toxic effects caused by snake venom proteins and enzymes. It has been well recognized for several years that animal sera, some of the plant and marine extracts are the most potent in neutralizing snake venom phospholipase A(2) (svPLA(2)). The implication of this review to update the latest research work which has been accomplished with svPLA(2) inhibitors from various natural sources like animal, marine organisms presents a compilation of research in this field over the past decade and revisiting the previous research report including those found in plants. In addition to that the bioactive compounds/inhibitor molecules from diverse sources like aristolochic alkaloid, flavonoids and neoflavonoids from plants, hydrocarbones -2, 4 dimethyl hexane, 2 methylnonane, and 2, 6 dimethyl heptane obtained from traditional medicinal plants Tragia involucrata (Euphorbiaceae) member of natural products involved for the inhibitory potential of phospholipase A(2) (PLA(2)) enzymes in vitro and also decrease both oedema induced by snake venom as well as human synovial fluid PLA(2). Besides marine natural products that inhibit PLA(2) are manoalide and its derivatives such as scalaradial and related compounds, pseudopterosins and vidalols, tetracylne from synthetic chemicals etc. There is an overview of the role of PLA(2) in inflammation that provides a rationale for seeking inhibitors of PLA(2) as anti-inflammatory agents. However, more studies should be considered to evaluate antivenom efficiency of sera and other agents against a variety of snake venoms found in various parts of the world. The implications of these new groups of svPLA(2) toxin inhibitors in the context of our current understanding of snake biology as well as in the development of new novel antivenoms therapeutics agents in the efficient treatment of snake envenomations are discussed.     

Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity

Background

Eosinophils are involved in various inflammatory processes including allergic inflammation during which angiogenesis has been documented. Angiogenesis is most likely connected to the hypoxia which characterizes inflamed tissues. Eosinophils produce VEGF and are pro-angiogenic. However, to the best of our knowledge no study has been performed to verify the existence of a direct link between eosinophils, hypoxia and angiogenesis in allergic inflammation.

Objective

To characterize eosinophil function and angiogenic potential under hypoxic conditions.

Methods

Human peripheral blood eosinophils were cultured in normoxic or hypoxic conditions with or without cytokines. Viability and apoptosis were assessed by Annexin V/PI staining. Anti- or pro-apoptotic protein levels, HIF-1α levels and MAPK phosphorylation were analyzed by immunoblot analysis. Angiogenic mediator release was evaluated by ELISA.

Results

Hypoxic eosinophils were more viable than normoxic ones after up to three days. In addition in hypoxia, anti-apoptotic Bcl-XL protein levels increased more than pro-apoptotic Bax levels. Hypoxia increased VEGF and IL-8 release. In hypoxic eosinophils high levels of HIF-1α were observed, particularly in the presence of GM-CSF. MAPK, particularly ERK1/2 inhibitors, decreased hypoxia-mediated VEGF release and HIF-1α expression.

Conclusion

Eosinophils respond to hypoxia by up-regulation of survival and of some of their pro-angiogenic functions indicating a correlation between eosinophilic inflammation and angiogenesis.     

The anticoagulant activity of Malayan cobra (Naja naja sputatrix) venom and venom phospholipase A2 enzymes

Malayan cobra (Naja naja sputatrix) venom was found to exhibit an in vitro anticoagulant activity that was much stronger than most common cobra (genus Naja) venoms. The most potent anticoagulants of the venom are two lethal phospholipase A2 enzymes with pI's of 6.15 and 6.20, respectively. The anticoagulant activity of the venom is due to the synergistic effect of the venom phospholipase A2 enzymes and polypeptide anticoagulants. Bromophenacylation of the two phospholipase A2 enzymes reduced their enzymatic activity with a concomitant drop in both the lethal and anticoagulant activities.     

Structure and phylogeny of the venom group I phospholipase A(2) gene

Phospholipases A(2) (PLA(2)s) catalyzing the hydrolysis of phospholipids form a family of proteins with diverse physiological and pharmacological properties. While there have been several reports on the cloning of PLA(2) cDNAs, very few studies have been carried out on the PLA(2) genes and, most importantly, no information has been available on the gene structure and function of group I venom PLA(2). This study, on the PLA(2) gene from a spitting cobra, besides being the very first report on any venom group I PLA(2) gene, constitutes the missing link in the biology and evolution of phospholipases. The 4-kb gene consists of four exons and three introns and resembles the human pancreatic PLA(2) gene. However, the size of intron 3 in particular is much smaller than that in the pancreatic gene. Interestingly, the information for the toxic and most of the pharmacological properties of the venom PLA(2) can be attributed to the end of exon 3 and the whole of exon 4 of the gene. This functional delineation fits in well with the theory of adaptive evolution exhibited by the venom PLA(2)s. We also show that the mammalian pancreatic and elapid PLA(2)s have similar paths of evolution (probably following gene duplication) from a common ancestral gene. Venom group II phospholipases, although evolved from the same ancestor, diverged early in evolution from the group I PLA(2) genes. Intriguingly, CAT reporter gene assays and DNase 1 footprinting studies on the promoter and its deletion constructs using CHO and HepG2 cell lines identified the possible involvement of cis elements such as Sp1, AP2, gamma-IRE, and (TG)(12) repeats in the expression of the gene in a tissue-specific manner.     

Antimicrobial activity of major royal jelly protein 8 and 9 of honeybee (Apis mellifera) venom

Honeybee venom is a complex mixture of toxic components, including major royal jelly protein (MRJP) 8 and 9. MRJP 8 and MRJP 9 are allergens, and MRJP 8 reduces melittin-induced cell apoptosis. However, their functional roles are poorly understood, and their antimicrobial activities have not been determined. In this study, the antimicrobial role of MRJP 8 and MRJP 9 of honeybee (Apis mellifera) venom (AmMRJP 8 and AmMRJP 9) was demonstrated. The presence of AmMRJP 8 and AmMRJP 9 in the secreted venom was observed using antibodies against recombinant AmMRJP 8 and AmMRJP 9 produced in baculovirus-infected insect cells. Recombinant AmMRJP 8 and AmMRJP 9 exhibited an inhibitory activity against microbial serine proteases. Consistent with their inhibitory activity, they induced structural damage by binding to microbial surfaces, resulting in a broad-spectrum antimicrobial activity against bacteria and fungi. They had little effect on hemolysis. Therefore, AmMRJP 8 and AmMRJP 9 could function as antimicrobial agents in honeybee venom.     

Characterization of phospholipase A2 from the venom of Horned viper (Cerastes cerastes)

Phospholipase A2 has been purified from the venom of Horned viper (Cerastes cerastes) by gel permeation chromatography followed by reverse-phase HPLC. The primary structure was established by sequence analysis of the intact protein and its enzymic peptides. The structure has 120 residues, properties like other group IIB phospholipases, but only 45-55% identity with the enzyme from other viperid species, and large variations even within the species (26% residue differences at known positions in another form).     

Isolation and characterization of the major phospholipase a2 from the venom of trimeresurus purpureomaculatus (shore pit viper)

• 1.1. The major phospholipase A₂ (PLA-DE4) of the venom of Trimeresurus purpureomaculatus (shore pit viper) has been purified to electrophoretic homogeneity.
• 2.2. The isoelectric point of the purified enzyme was determined to be 4.20, and the mol. wt was 31,700 as estimated by Sephadex G-75 gel filtration chromatography; and 14.000 as estimated by SDS-polyacrylamide gel electrophoresis.
• 3.3. The purified enzyme hydrolyzed phosphatidylcholine (PC) faster than phosphatidylethanolamine (PE), whereas phosphatidylserine (PS) was not hydrolyzed at all (PC > PE > PS = 0). However, in reaction system consisted of mixtures of PC and PS, phosphatidylserine was effectively hydrolyzed by the enzyme.
• 4.4. The phospholipase A₂ exhibited edema-forming activity but not hemolytic, hemorrhagic or anticoagulant activities. It was not lethal to mice at a dosage of 10 μg/g by i.v. route.

     

Purification of a Class B1 platelet aggregation inhibitor phospholipase A2 from Indian cobra (Naja Naja) venom

A platelet aggregation inhibitor phospholipase A(2) (NND-IV-PLA(2)) was isolated from Naja naja (Eastern India) venom by a combination of cation and anion exchange chromatography. NND-IV-PLA(2) is the most catalytically active enzyme isolated from the Indian cobra venom. The acidic PLA(2) profile of Eastern regional Indian cobra venom is distinctly different from that of the western regional venom. However the acidic PLA(2)s from both the regions follow the pattern of increasing catalytic activity with increase in acidic nature of the PLA(2) isoform. NND-IV-PLA(2) is a Class B1 platelet aggregation inhibitor and inhibits platelet aggregation induced by ADP, collagen and epinephrine. Modification of active site histidine abolishes both catalytic activity and platelet aggregation inhibition activities while aristolochic acid, a phospholipase A(2) inhibitor has only partial effect on the two activities.     

Structural analysis of trimeric phospholipase A(2) neurotoxin from the Australian taipan snake venom

Snake pre-synaptic neurotoxins endowed with phospholipase A(2) activity are potent inducers of paralysis through the specific disruption of the neuromuscular junction pre-synaptic membrane and represent a valuable tool for investigating neuronal degeneration and recovery. They have different structural complexity and a wide range of lethal potency and enzymatic activity, although they share a similar mechanism of action. Although no correlation has been reported between neurotoxicity and enzymatic activity, toxicity increases with structural complexity and phospholipase A(2) oligomers show 10-fold lower LD(50) values compared to their monomeric counterparts. To date, no structural study has been performed on multimeric SPANs with the aim of shedding light on the correlation between structural complexity and neurotoxicity. In the present study, we investigated the structure of taipoxin, a trimeric phospholipase A(2) neurotoxin, as well as that of its subunits, by X-ray crystallography and small angle X-ray scattering analysis. We present the high-resolution structure of two isoforms of the taipoxin β subunit, which show no neurotoxic activity but enhance the activity of the other subunits in the complex. One isoform shows no structural change that could justify the lack of activity. The other displays three point mutations in critical positions for the catalytic activity. Moreover, we designed a model for the quaternary structure of taipoxin under physiological conditions, in which the three subunits are organized into a flat holotoxin with the substrate binding sockets exposed on the same side of the complex, which suggests a role for this interface in the toxin-membrane interaction. Database The coordinates and structure factors have been deposited in the RCSB Protein Data Bank (http://www.rcsb.org) under accession numbers 3VBZ and 3VC0, corresponding to β isoforms 1 and 2 respectively Structure digital abstract ? taipoxin beta isoform 2 and?taipoxin beta isoform 2?bind?by?x-ray crystallography?(View interaction).     

Isolation and characterization of phospholipase A2, from Agkistrodon bilineatus (common cantil) venom

• 1.1. Phospholipase A₂ was isolated from Agkistrodon bilineatus venom by Sephadex G-75 and CM-Cellulose column chromatographies.
• 2.2. The purified phospholipase A₂-I gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
• 3.3. The enzyme preparation had a molecular weight of 14,000, isoelectric point of pH 8.77 and possessed 123 amino acid residues.
• 4.4. The purified phospholipase A₂ possessed lethal, indirect hemolytic and anticoagulant activities.
• 5.5. The enzyme hydrolyzed the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS).
• 6.6. The concentration of mouse diaphragm was inhibited and the contraction of guinea pig left atrium was increased by phospholipase A₂-I.
• 7.7. Phospholipase A₂ activity of this preparation was inhibited by ethylenediamine tetraacetic acid, p-bromo phenacyl bromide, n-bromo succinimide or dithiothreitol, but not by diisopropyl fluorophosphate or benzamidine.

     

Isolation and characterization of a lethal phospholipase A2 (NN-IVb1-PLA2) from the Indian cobra (Naja naja naja) venom.

Indian cobra (Naja naja naja) venom is reported to contain multiple forms of phospholipase A2. Only a couple of them have been isolated and characterized. A lethal phospholipase A2 (NN-IVb1-PLA2) from Naja naja naja venom has been purified in three steps involving CM-Sephadex C-25, Sephadex G-50 and rechromatography on CM-Sephadex C-25 columns. It is a basic protein with pl value between 7-7.5 and has molecular weight between 11,000-11,500. The LD50 of NN-IVb1-PLA2 is 1.2 mg/K g body weight. It induces neurotoxic symptoms in the experimental mice and is devoid of myotoxic, anticoagulant, edema inducing and direct hemolytic activities.     

Purification and characterization of two highly different group II phospholipase A2 isozymes from a single viperid (Eristocophis macmahoni) venom

Two phospholipase A2 isozymes have been purified from leaf-nosed viper by gel permeation chromatography followed by reverse-phase HPLC and cation-exchange FPLC. Both enzymes contain seven pairs of half-cystine, typical of group II phospholipase A2. Surprisingly large differences, affecting both N- and C-terminal regions, exist between the two isozymes purified from the same snake venom. Exchanges occur at no less than 27 of 121 positions (22%), suggesting the possible existence of two genes for phospholipase A2. The residue identity with the enzymes from other Viperidae species is also low, only 44-48%, indicating extensive variations of this protein structure at large. Functionally, the present isozymes do not possess the cationic regions ascribed to myotoxicity and anti-coagulant effects of the enzyme.     

Myotoxin II from Bothrops asper (Terciopelo) venom is a lysine-49 phospholipase A2

A basic, dimeric myotoxic protein, myotoxin II, purified from Bothrops asper venom has a similar molecular weight and is immunologically cross-reactive with antibodies raised to previously isolated B. asper phospholipases A2, except that it shows only 0.1% of the phospholipase activity against L-alpha-phosphatidylcholine in the presence of Triton X-100. Its 121 amino acid sequence, determined by automated Edman degradation, clearly identifies it as a Lys-49 phospholipase A2. Key amino acid differences between myotoxin II and phospholipase active proteins in the Ca2(+)-binding loop region, include Lys for Asp-49, Asn for Tyr-28, and Leu for Gly-32. The latter substitution has not previously been seen in Lys-49 proteins. Other substitutions near the amino terminus (Leu for Phe-5 and Gln for several different amino acids at position 11) may prove useful for identifying other Lys-49 proteins in viperid and crotalid venoms. Myotoxin II shows greater sequence identity with other Lys-49 proteins from different snake venoms (Agkistrodon piscivorus piscivorus, Bothrops atrox, and Trimeresurus flavoviridis) than with another phospholipase A2 active Asp-49 molecule isolated from the same B. asper venom. This work demonstrates that phospholipase activity per se, is not required in phospholipase molecules for either myotoxicity or edema inducing activities.