Comprehensive Study of the Proteome and Transcriptome of the Venom of the Most Venomous European Viper: Discovery of a New Subclass of Ancestral Snake Venom Metalloproteinase Precursor-Derived Proteins.

The nose-horned viper, its nominotypical subspecies Vipera ammodytes ammodytes ( Vaa), in particular, is, medically, one of the most relevant snakes in Europe. The local and systemic clinical manifestations of poisoning by the venom of this snake are the result of the pathophysiological effects inflicted by enzymatic and nonenzymatic venom components acting, most prominently, on the blood, cardiovascular, and nerve systems. This venom is a very complex mixture of pharmacologically active proteins and peptides. To help improve the current antivenom therapy toward higher specificity and efficiency and to assist drug discovery, we have constructed, by combining transcriptomic and proteomic analyses, the most comprehensive library yet of the Vaa venom proteins and peptides. Sequence analysis of the venom gland cDNA library has revealed the presence of messages encoding 12 types of polypeptide precursors. The most abundant are those for metalloproteinase inhibitors (MPis), bradykinin-potentiating peptides (BPPs), and natriuretic peptides (NPs) (all three on a single precursor), snake C-type lectin-like proteins (snaclecs), serine proteases (SVSPs), P-II and P-III metalloproteinases (SVMPs), secreted phospholipases A2 (sPLA2s), and disintegrins (Dis). These constitute >88% of the venom transcriptome. At the protein level, 57 venom proteins belonging to 16 different protein families have been identified and, with SVSPs, sPLA2s, snaclecs, and SVMPs, comprise ∼80% of all venom proteins. Peptides detected in the venom include NPs, BPPs, and inhibitors of SVSPs and SVMPs. Of particular interest, a transcript coding for a protein similar to P-III SVMPs but lacking the MP domain was also found at the protein level in the venom. The existence of such proteins, also supported by finding similar venom gland transcripts in related snake species, has been demonstrated for the first time, justifying the proposal of a new P-IIIe subclass of ancestral SVMP precursor-derived proteins.

A new era of secreted phospholipase A2.

Among more than 30 members of the phospholipase A2 (PLA2) superfamily, secreted PLA2 (sPLA2) enzymes represent the largest family, being Ca(2+)-dependent low-molecular-weight enzymes with a His-Asp catalytic dyad. Individual sPLA2s exhibit unique tissue and cellular distributions and enzymatic properties, suggesting their distinct biological roles. Recent studies using transgenic and knockout mice for nearly a full set of sPLA2 subtypes, in combination with sophisticated lipidomics as well as biochemical and cell biological studies, have revealed distinct contributions of individual sPLA2s to various pathophysiological events, including production of pro- and anti-inflammatory lipid mediators, regulation of membrane remodeling, degradation of foreign phospholipids in microbes or food, or modification of extracellular noncellular lipid components. In this review, we highlight the current understanding of the in vivo functions of sPLA2s and the underlying lipid pathways as revealed by a series of studies over the last decade.

Recent progress in phospholipase A2 research: from cells to animals to humans.

Mammalian genomes encode genes for more than 30 phospholipase A2s (PLA2s) or related enzymes, which are subdivided into several classes including low-molecular-weight secreted PLA2s (sPLA2s), Ca²+-dependent cytosolic PLA2s (cPLA2s), Ca²+-independent PLA2s (iPLA2s), platelet-activating factor acetylhydrolases (PAF-AHs), lysosomal PLA2s, and a recently identified adipose-specific PLA. Of these, the intracellular cPLA2 and iPLA2 families and the extracellular sPLA2 family are recognized as the "big three". From a general viewpoint, cPLA2α (the prototypic cPLA2 plays a major role in the initiation of arachidonic acid metabolism, the iPLA2 family contributes to membrane homeostasis and energy metabolism, and the sPLA2 family affects various biological events by modulating the extracellular phospholipid milieus. The cPLA2 family evolved along with eicosanoid receptors when vertebrates first appeared, whereas the diverse branching of the iPLA2 and sPLA2 families during earlier eukaryote development suggests that they play fundamental roles in life-related processes. During the past decade, data concerning the unexplored roles of various PLA2 enzymes in pathophysiology have emerged on the basis of studies using knockout and transgenic mice, the use of specific inhibitors, and information obtained from analysis of human diseases caused by mutations in PLA2 genes. This review focuses on current understanding of the emerging biological functions of PLA2s and related enzymes.

Antibacterial actions of secreted phospholipases A2. Review.

Antibacterial properties of secreted phospholipases A2 (PLA2) have emerged gradually. Group (G) IIA PLA2 is the most potent among mammalian secreted (s) PLA2s against Gram-positive bacteria, but additional antibacterial compounds, e.g. the bactericidal/permeability-increasing protein, are needed to kill Gram-negative bacteria. The mechanisms of binding to the bacterial surface and the killing of bacteria by sPLA2s are based on the positive charge of the PLA2 protein and its phospholipolytic enzymatic activity, respectively. The concentration of GIIA PLA2 is highly elevated in serum of patients with bacterial sepsis, and overexpression of GIIA PLA(2) protects transgenic mice against experimental Gram-positive infection. The synthesis and secretion of GIIA PLA2 are stimulated by the cytokines TNF-alpha, IL-1 and IL-6. Secreted PLA2s may be potentially useful new endogenous antibiotics to combat infections including those caused by antibiotic-resistant bacteria such as methicillin-resistant staphylococci and vancomysin-resistant enterococci.

Secreted phospholipase A2 group X overexpression in asthma and bronchial hyperresponsiveness.

RATIONALE: Secreted phospholipase A(2) enzymes (sPLA(2)s) play key regulatory roles in the biosynthesis of eicosanoids, such as the cysteinyl leukotrienes, but the role of these enzymes in the pathogenesis of asthma is not known. OBJECTIVES: To establish if sPLA(2)s are overexpressed in the airways of patients with asthma, and to determine if these enzymes may play a role in the generation of eicosanoids in exercise-induced bronchoconstriction. METHODS: Induced sputum samples were obtained from subjects with asthma with exercise-induced bronchoconstriction and nonasthmatic control subjects at baseline, and on a separate day 30 minutes after exercise challenge. The expression of the PLA(2)s in induced sputum cells and supernatant was determined by quantitative polymerase chain reaction, immunocytochemistry, and Western blot. MEASUREMENTS AND MAIN RESULTS: The sPLA(2)s expressed at the highest levels in airway cells of subjects with asthma were groups X and XIIA. Group X sPLA(2) (sPLA(2)-X) was differentially overexpressed in asthma and localized to airway epithelial cells and bronchial macrophages. The gene expression, immunostaining in airway epithelial cells and bronchial macrophages, and the level of the extracellular sPLA(2)-X protein in the airways increased in response to exercise challenge in the asthma group, whereas the levels were lower and unchanged after challenge in nonasthmatic control subjects. CONCLUSIONS: Increased expression of sPLA(2)-X may play a key role in the dysregulated eicosanoid synthesis in asthma.