Cloning, expression and biochemical characterization of a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys pallas.
A cDNA encoding a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys Pallas with an N-terminus highly homologous to that of BPLA2 and a C-terminus sequence almost the same as that of APLA2 was inserted into a bacterial expression vector and effectively expressed in Escherichia coli RR1. The protein was produced as insoluble inclusion bodies. After partial purification by washing, the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by FPLC column superose 12. The purified recombinant enzyme with an isoelectric point of pH 6.8 could cross-react with antiserum prepared against acidic phospholipase A2. The enzymatic activity of the expressed basic-acidic hybrid phospholipase A2-II is close to that of denatured-refolded native basic phospholipase A2, and has the same inhibiting effect on platelet aggregation as denatured-refolded acidic phospholipase A2, but lacks the hemolytic activity of denatured-refolded basic phospholipase A2. To study the structural relationships among basic phospholipase A2, acidic phospholipase A2 and basic-acidic hybrid phospholipase A2-II, molecular modeling of basic-acidic hybrid phospholipase A2-II was done. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A2 are discussed. (+info)
cDNA cloning and bacterial expression of phospholipase A(2) inhibitor PLIalpha from the serum of the Chinese mamushi, Agkistrodon blomhoffii siniticus(1).
The cDNA encoding of a phospholipase A(2) inhibitor (PLIalpha) of the Chinese mamushi, Agkistrodon blomhoffii siniticus, was identified from a liver cDNA library by use of a probe prepared by polymerase chain reaction (PCR) on the basis of the amino acid sequence of PLIalpha. It encoded a polypeptide of 166 amino acid residues, including 19 residues of the signal sequence and 147 residues of the complete mature sequence of PLIalpha. The PLIalpha cDNA was subcloned into the expression vector pET-16b and used to transform Escherichia coli strain BL21(DE3)pLysS. The recombinant PLIalpha expressed as a fusion protein was solubilized and purified to homogeneity by use of a metal affinity resin. The purified PLIalpha fusion protein underwent folding to form a trimeric structure like the intact PLIalpha, and showed inhibitory activity against the group II acidic PLA(2) from A. blomhoffii siniticus venom; although its binding constant (1/K(i)) value was 30-fold lower than that of the natural PLIalpha. The elimination of the N-terminal additional peptide from the fusion protein resulted in a marked increase in the inhibition activity with a binding constant comparable to that of the natural PLIalpha against the acidic PLA(2). Furthermore, the carbohydrate chains of the natural PLIalpha were found to play an important role in the inhibitory activity against the basic PLA(2). (+info)
Purification, cloning and sequence analyses for pro-metalloprotease-disintegrin variants from Deinagkistrodon acutus venom and subclassification of the small venom metalloproteases.
Acidic and basic hemorrhagic metalloproteases were purified from the venom of Deinagkistrodon acutus (from Fujian Province, China) using gel filtration and anion exchange on FPLC and reversed-phase HPLC. Their hemorrhagic activities and N-terminal sequences were characterized. Extensive screening of the venom gland cDNA after PCR amplification resulted in the identification and sequencing of a total of seven cDNA clones encoding the multidomain precursors of six acidic and one alkaline low molecular mass metalloproteases. Two of the precursors contain a processable disintegrin domain. Disintegrins of 5 kDa were also purified from the venom. The partial amino-acid sequences and molecular masses determined by electrospray ionization mass spectrometry of the purified proteins specifically match those deduced from two of the cDNA sequences. Moreover, phylogenetic analyses based on 30 complete sequences of low molecular mass venom metalloproteases revealed that they may be classified into three functional subtypes: acidic hemorrhagins, basic and moderate hemorrhagins, and nonhemorrhagic enzymes. Subtype-specific amino-acid substitutions in the C-terminal regions of the enzymes were highlighted to explore the structure-activity relationships of the enzymes. (+info)
Characterization and cDNA cloning of a platelet aggregation inhibitor.
A novel platelet aggregation inhibitor, sal-C, was purified to homogeneity from the venom of Korean snake (Agkistrodon halys brevicaudus). Several lines of experimental evidence clearly indicated that sal-C inhibits not only the collagen-induced platelet aggregation, but also the aggregation mediated by the cell surface glycoprotein IIb-IIIa (GP IIb-IIIa). We have isolated the cDNA encoding sal-C from the cDNA library of the snake venom gland and analyzed its complete nucleotide sequence. Sal-C is a single-chain polypeptide composed of 212 amino acids including 24 cysteines. The deduced polypeptide sequence of sal-C demonstrated considerable homology to previously described protein species of the collagen-induced platelet aggregation inhibitor family. Sal-C does not have the Arg-Gly-Asp (RGD) motif, but contains the Ser-Glu-Cys-Asp sequence. Interestingly, sal-C was found to inhibit GP IIb-IIIa binding to immobilized fibrinogen which is antagonized by the typical RGD motif of disintegrins. (+info)
Ultrastructure of the capillary pericytes and the expression of smooth muscle alpha-actin and desmin in the snake infrared sensory organs.
The infrared sensory membranes of pit organs of pit vipers have an extremely rich capillary vasculature that forms many vascular loops, each serving a small number of infrared nerve terminals. We clarified the ultrastructure of capillary pericytes in the pit membranes by scanning and transmission electron microscopy, and examined the immunoreactivity in their cytoplasm to two contractile proteins: smooth muscle alpha-actin (SM alpha-actin) and desmin. The capillary pericytes had two major cytoplasmic processes: thickened primary processes that radiate to embrace the endothelial tube and flattened secondary processes that are distributed widely on the endothelium. Coexpression of SM alpha-actin and desmin was observed in the pericytes of entire capillary segments, and SM alpha-actin was characterized by prominent filament bundles directed mainly at right angles to the capillary long axis. This expression pattern was different from that of capillary pericytes of the scales, where SM alpha-actin was expressed diffusely in the cytoplasm. In a series of electron microscopic sections, we often observed the pericyte processes depressing the endothelial wall. We also observed a close relationship of the pericytes with inter-endothelial cell junctions, and pericyte processes connected with the endothelial cells via gap junctions. From these findings, we surmised that capillary pericytes in the pit membrane have a close functional relationship with the endothelium, and through their contractile and relaxing activity regulate capillary bloodflow to stabilize production of infrared nerve impulses. (+info)
Toward understanding interfacial activation of secretory phospholipase A2 (PLA2): membrane surface properties and membrane-induced structural changes in the enzyme contribute synergistically to PLA2 activation.
Phospholipase A2 (PLA2) hydrolyzes phospholipids to free fatty acids and lysolipids and thus initiates the biosynthesis of eicosanoids and platelet-activating factor, potent mediators of inflammation, allergy, apoptosis, and tumorigenesis. The relative contributions of the physical properties of membranes and the structural changes in PLA2 to the interfacial activation of PLA2, that is, a strong increase in the lipolytic activity upon binding to the surface of phospholipid membranes or micelles, are not well understood. The present results demonstrate that both binding of PLA2 to phospholipid bilayers and its activity are facilitated by membrane surface electrostatics. Higher PLA2 activity toward negatively charged membranes is shown to result from stronger membrane-enzyme electrostatic interactions rather than selective hydrolysis of the acidic lipid. Phospholipid hydrolysis by PLA2 is followed by preferential removal of the liberated lysolipid and accumulation of the fatty acid in the membrane that may predominantly modulate PLA2 activity by affecting membrane electrostatics and/or morphology. The previously described induction of a flexible helical structure in PLA2 during interfacial activation was more pronounced at higher negative charge densities of membranes. These findings identify a reciprocal relationship between the membrane surface properties, strength of membrane binding of PLA2, membrane-induced structural changes in PLA2, and the enzyme activation. (+info)
Serine protease isoforms of Deinagkistrodon acutus venom: cloning, sequencing and phylogenetic analysis.
The major coagulating fibrinogenase of Deinagkistrdon acutus venom, designated acutobin, was purified by anion-exchange chromatography, gel filtration and reverse-phase HPLC. Approximately 80% of its protein sequence was determined by sequencing the various fragments derived from CNBr cleavage and digestion with endoprotease. Extensive screening of the venom gland cDNA species after amplification by PCR resulted in the isolation of four distinct cDNA clones encoding acutobin and three other serine proteases, designated Dav-PA, Dav-KN and Dav-X. The complete amino acid sequences of these enzymes were deduced from the cDNA sequences. The amino-acid sequence of acutobin contains a single chain of 236 residues including four potential N-glycosylation sites. The purified acutobin (40 kDa) contains approx. 30% carbohydrate by weight, which could be partly removed by N-glycanase. The phylogenetic tree of the complete amino acid sequences of 40 serine proteases from 18 species of Crotalinae shows functional clusters reflecting parallel evolution of the three major venom enzyme subtypes: coagulating enzymes, kininogenases and plasminogen activators. The possible structural elements responsible for the functional specificity of each subtype are discussed. (+info)
Aggretin, a heterodimeric C-type lectin from Calloselasma rhodostoma (Malayan pit viper), stimulates platelets by binding to alpha2beta1 integrin and glycoprotein Ib, activating Syk and phospholipase Cgamma 2, but does not involve the glycoprotein VI/Fc receptor gamma chain collagen receptor.
Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake C-type lectin family and is thought to activate platelets by binding to platelet glycoprotein alpha(2)beta(1). We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to alpha(2)beta(1). Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor gamma chain (Fcgamma)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fcgamma pathway. Platelets from Fcgamma-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including alpha(2)beta(1) or GPIb for the lack of the Fcgamma pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72(SYK), p125(FAK), and PLCgamma2, whereas, in comparison with collagen and convulxin, the Fcgamma subunit neither is phosphorylated nor coprecipitates with p72(SYK). This supports an independent, GPIb- and integrin-based pathway for activation of p72(SYK) not involving the Fcgamma receptor. (+info)