Amino acid sequence of Trimeresurus flavoviridis phospholipase A2

The amino acid sequence of phospholipase A2 from the venom of Trimeresurus flavoviridis (the Habu snake) was determined. The enzyme subunit has a molecular weight of 13,764 and consists of a single polypeptide chain of 122 amino acids and seven disulfide bonds. The fragmentation was conducted by digesting the reduced and S-carboxymethylated derivative of the protein with Achromobacter protease I, chymotrypsin, and trypsin, respectively. Achromobacter protease I peptides were used for alignment and to establish overlaps over chymotryptic and tryptic peptides. The automated Edman degradation of the S-carboxymethylated protein, which was extended to the N-terminal 30 amino acid residues, supplemented the deletions found with the enzymatic peptides alone. T. flavoviridis phospholipase A2 was found to be highly (65-67%) homologous in sequence to the enzymes from T. okinavensis, Crotalus adamanteus, and Crotalus atrox (viperid family) and less (35-44%) homologous to those from elapid snakes and mammalian pancreas. The T. flavoviridis enzyme appears to be similar in secondary structure composition to the C. atrox enzyme.     

Acquisition of native structure from reduced Trimeresurus flavoviridis phospholipase A2

Oxidation of reduced T. flavoviridis phospholipase A2 under suitable conditions resulted in recovery of its active, native structure. The oxidized product was eluted at the same positions as native phospholipase A2 on reserved phase column and DEAE-Toyopearl 650M column chromatographies. The native and regenerated proteins were also identical in mobility on polyacrylamide gel electrophoresis and in circular dichroism spectra. Des-octapeptide(1-8)-phospholipase A2 (L-fragment), which shows only greatly reduced activity, was reduced and oxidized but no effective reformation of the native structure was found, indicating that the entire sequence is required for efficient reorganization.     

Contractile activity of Trimeresurus flavoviridis phospholipase A2 on guinea pig ileum and artery.

Trimeresurus flavoviridis phospholipase A2 (PLA2) induced strong contractions of the smooth muscles of guinea pig ileum and artery in a concentration-dependent manner (10(-10)-10(-6) M). When the same dose of PLA2 was administered in repetition to the ileal preparation, the contraction diminished progressively and was no longer recovered even by consecutive washings. The enzymatically inactive derivative of PLA2, in which His-47 was p-bromophenacylated, was unable to elicit contraction. Also, no activity was observed when the Ca(2+)-free medium was used. The contraction induced by PLA2 was inhibited completely by 1.0 x 10(-6) M indomethacin, but not by nordihydroguaiaretic acid. These results imply that the PLA2-induced contraction is due essentially to the hydrolytic action of the enzyme against phospholipid membranes to liberate arachidonic acid that is then converted to pharmacologically active prostaglandins. In guinea pig artery, PLA2 caused both contraction and relaxation.     

Interaction of Trimeresurus flavoviridis phospholipase A2 and its fragment with calcium ion

Dimeric T. flavoviridis phospholipase A2 has been studied in terms of the interaction with essential Ca2+ by equilibrium gel filtration, ultraviolet difference spectroscopy, fluorescence measurements, and chemical modifications with p-bromophenacyl bromide. The subunit bound to Ca2+ with a 1:1 molar ratio and no cooperative binding was observed. The hypochromic effect produced upon the binding of Ca2+ is due to perturbation of (a) specific tryptophan residue(s) located in the vicinity of the active site and appears to be characteristic of this enzyme. On the basis of the pH dependence of the dissociation constants, it has been found that the alpha-amino group (pKa 8.7) controls the binding of Ca2+. Deprotonation of the alpha-amino group is possibly accompanied by conformational transition to the active form which is able to bind Ca2+. This is in contrast to the case of bovine pancreatic phospholipase A2 in which Asp-49 (pKa 5.2) is responsible for the metal ion binding (Fleer et al. (1981) Eur. J. Biochem. 113, 283-288). Des-octapeptide(1-8)-phospholipase A2 (L-fragment) was found to be capable of binding Ca2+ under the control of a group with a pKa of 7.6. This pKa value was similar to an apparent pKa of 7.5 determined for the histidine residue in the active site of the native enzyme by way of p-bromophenacyl bromide modification. It appears that the N-terminal (octapeptide) sequence affects the binding mode of Ca2+, possibly because of conformational transition arising from its removal. The reinvestigation showed that the N-terminal octapeptide sequence is Gly-Leu-Trp-Gln-Phe-Glu-Asn-Met.     

Purification and amino acid sequence of basic protein II, a lysine-49-phospholipase A2 with low activity, from Trimeresurus flavoviridis venom

A basic protein (pI 10.3), named basic protein II, was purified to homogeneity from the venom of Trimeresurus flavoviridis (Habu snake) after four chromatographic steps. The amino acid sequence of this protein was determined by sequencing the S-pyridylethylated derivative and its peptides produced by chemical (cyanogen bromide) and enzymatic (chymotrypsin, clostripain, and Staphylococcus aureus V8 protease) cleavages. The protein consisted of 122 amino acid residues and was found to be identical in sequence to basic protein I from the same source except that Asp-58 of basic protein I is replaced by asparagine. Like basic protein I, the structural feature of basic protein II is that Tyr-28 and Asp-49 common in phospholipases A2 from snake venoms and mammalian pancreas are replaced by asparagine and lysine, respectively. Thus, basic protein II belongs to the category of lysine-49-phospholipase A2. The action of basic protein II on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine released only oleic acid, indicating that it has phospholipase A2 activity. Its molar activity toward 1,2-dilauroyl-sn-glycero-3-phosphorylcholine, however, was only 1.7% of that of T. flavoviridis phospholipase A2 isolated previously. Affinity for Ca2+ and reactivity toward p-bromophenacyl bromide of basic protein II were 8 and 5.3 times, respectively, smaller than those of phospholipase A2 from the same source, substantiating the low phospholipase A2 activity of basic protein II.     

Investigation of an active site histidine-aspartate couple in Trimeresurus flavoviridis phospholipase A2

When Trimeresurus flavoviridis phospholipase A2 was reacted with methyl p-nitrobenzenesulfonate, its activity decreased following first-order kinetics. The pH dependence of the rate constants of inactivation showed that His-48 with an apparent pKa of 6.5 controls the reaction. In the pH region below 6.5, N1-methylhistidine was predominantly formed. On the other hand, N1,N3-dimethylhistidine was almost exclusively produced in the pH region above 6.5. No N3-methylhistidine was detected at any pH tested. Such observations suggested that the first methylation occurred at the N1-position of the imidazole ring followed by a second methylation at the N3-position, and that His-48 couples the carboxylate of Asp-99 at the N3-position of the imidazole ring, in accord with the interaction observed in the crystal structure of homologous Crotalus atrox phospholipase A2. As it has been reported that, in the reaction of chymotrypsin with methyl p-nitrobenzenesulfonate at pH 7.8, only monomethylation occurred at the N1-position of the His-57 imidazole group (Nakagawa, Y. & Bender, M.L. (1970) Biochemistry 9, 259-267), the nature of the active site histidine-aspartate couple of T. flavoviridis phospholipase A2 seems not to be identical with that of chymotrypsin.     

Interactions of monodispersed and micellar substrates with a phospholipase A2 from Trimeresurus flavoviridis

Bindings of the phospholipase A2 from Trimeresurus flavoviridis to the monodispersed and micellar n-alkylphosphorylcholines (n-CnPC) were studied at 25 degrees C and ionic strength 0.2 by the aromatic CD and tryptophyl fluorescence methods, respectively. The bindings to micelles of the substrate analog were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and that these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of the substrate (monomer) molecules, N = 9-13. The binding constant to the micelle was about 40 times greater than it was to the monodispersed substrate. The binding constant to the micellar substrate analog increased on the binding of Ca2+ to the enzyme and decreased on modification of the N-terminal alpha-NH2 group, whereas the binding to the monodispersed substrate analog was independent of pH, of the Ca2+ binding, and of the chemical modification of the alpha-NH2 group. The kinetics of the hydrolyses of monodispersed and micellar dihexanoylphosphatidylcholines (diC6PC) were studied at 25 degrees C and ionic strength 0.2 by the pH-stat method in the presence of saturating amounts of Ca2+. The catalytic center activity, kappa cat, as well as the binding constant, 1/Km, for the micellar substrate, were found to be much greater than those for the monodispersed substrate. The binding constant, 1/Km, of the monodispersed substrate was independent of pH; this was in good agreement with that of the substrate analog described above. The pH-dependence curve of kappa cat for the monodispersed substrate exhibited two transitions, one below pH 6.5 and the other above pH 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structures of genes encoding phospholipase A2 inhibitors from the serum of Trimeresurus flavoviridis snake

Inhibitors (PLIs) against snake venom gland phospholipases A₂ (PLA₂s) have been found in their sera. A cDNA encoding a PLI from Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) serum, cPLI-A, was isolated from the Tf liver cDNA library and sequenced. Northern blot analysis with cPLI-A showed that PLIs are expressed only in liver. Genes for PLIs, gPLI-A and gPLI-B, were isolated from the Tf genomic DNA library and their nucleotide (nt) sequences were determined. The genes consisted of four exons and three introns, and exon 4 encoded the carbohydrate recognition domain (CRD)-like motif. Comparison of the nt sequences between gPLI-A and gPLI-B showed that these genes are highly homologous, including introns, except that exon 3 is rich in nonsynonymous nt substitutions which are almost four times as frequent as synonymous nt substitutions. This evolutionary feature of PLI genes is different from that of venom gland PLA₂ isozyme genes in which nonsynonymous nt substitutions are spread over the entire mature protein-coding region.     

The amino acid sequence and properties of an edema-inducing Lys-49 phospholipase A2 homolog from the venom of Trimeresurus mucrosquamatus

Three phospholipase A2 enzymes or homologs were purified from the venom of Trimeresurus mucrosquamatus (Taiwan habu). The most abundant one was found to be a phospholipase homolog without enzyme activity, and its complete amino acid sequence was determined using oligopeptide fragments derived from digestion by endopeptidases Glu-C, Asp-N, Lys-C and alpha-chymotrypsin, and by means of gas-phase sequencing. The sequence revealed that the protein belonged to the Lys-49 family of snake venom phospholipase A2. This protein's function was characterized as edema-inducing. The Lys-49 protein has the potential to bind membrane phospholipid and Ca2+ (Kd = 1.6 x 10(-4) M) as shown by ultraviolet difference spectra; however, the catalytic site appeared to be inactive and the edematous response was independent of the protein's hydrolytic activity. Mast cells and platelets were shown to be subject to activation by the Lys-49 protein.     

Isolation and fundamental properties of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis

Phospholipase A2 inhibitor was purified from the blood plasma of Habu, Trimeresurus flavoviridis, by Sephadex G-200 gel filtration, DEAE-cellulose chromatography, and Blue-Sepharose CL-6B column chromatography. The purified inhibitor was shown to be a glycoprotein with a molecular weight of about 100K. It was found to consist of four subunits whose molecular weights were around 20-24K. In order to examine the inhibition mechanism of the inhibitor, the interaction of the inhibitor with a phospholipase A2 from T. flavoviridis venom was examined by Sephadex G-100 gel filtration. One inhibitor molecule was found to bind directly to one phospholipase A2 molecule in both the presence and absence of Ca2+. The inhibitor inhibited the phospholipase A2 from T. flavoviridis venom with an apparent dissociation constant, Ki, of 1.7 X 10(-10) M, but not the porcine pancreas enzyme or the Agkistrodon halys blomhoffii enzyme belonging to the same family, Crotalidae, as T. flavoviridis, or the phospholipase C from Bacillus cereus.     

Identification of the tryptophan residue located in the calcium binding site of Trimeresurus flavoviridis phospholipase A2

When phospholipase A2 from the venom of Trimeresurus flavoviridis (the Habu snake) was oxidized with N-bromosuccinimide at pH 4.0, its activity decreased linearly with increase in the extent of oxidation of tryptophan residues. Oxidation of two of the four tryptophan residues caused an apparent loss of activity. The accessibilities of the tryptophan residues were analyzed with differently oxidized phospholipase A2 preparations and were determined to be in the following order: Trp-3 approximately Trp-30 greater than Trp-68 greater than Trp-108. The magnitude of the difference spectrum with a negative peak at 292 nm which is produced upon the binding of Ca2+ in the vicinity of tryptophan residue(s) decreased in a concave manner with increase in the extent of oxidation of tryptophan residues and was greatly diminished when 2 mol of tryptophan residues were oxidized. The activity and Ca2+-induced difference spectrum are thus related to either Trp-3 or Trp-30 or both. Des-octapeptide(1-8)-phospholipase A2 (L-fragment) is 14% as active as phospholipase A2 and is able to give a Ca2+-induced difference spectrum which is smaller than, but similar to, that of phospholipase A2. Its activity and the magnitude of the Ca2+-induced difference spectrum decreased along similar paths with increase in the amount of tryptophan residues oxidized, but in a manner indicating that two tryptophan residues are apparently responsible for the activity and the Ca2+-induced difference spectrum. The order of accessibility of the tryptophan residues of L-fragment was Trp-30 approximately Trp-108 greater than Trp-68. Trp-108, however, could be excluded from the residues located in the active site by reference to the tertiary structure of homologous Crotalus atrox phospholipase A2. Thus, Trp-30 is located in the Ca2+ binding site and is responsible for the activity of L-fragment. It is thus concluded that in phospholipase A2 Trp-30 is located in the Ca2+ binding site. From the concave decrease of relative magnitude of the Ca2+-induced difference spectrum and the linear decrease of relative activity upon oxidation of phospholipase A2, it may be assumed that both Trp-3 and Trp-30 are required to produce the Ca2+-induced difference spectrum, while only Trp-30 need be intact for activity. Anomalous binding of Ca2+ was observed for oxidized phospholipase A2.     

Preliminary crystallographic study of phospholipase A2 from the venom of Trimeresurus flavoviridis (habu snake)

Crystallization and a preliminary crystallographic study of Trimeresurus flavoviridis (habu snake) phospholipase A2 (PLA2) were carried out. Although crystals were obtained from various solutions, crystals suitable for X-ray analysis could be obtained from polyethylene glycol solutions only when a repeated seeding technique was applied starting from twinned crystals. The crystal is monoclinic with space group P21, with a = 44.1, b = 55.7, c = 48.8 A, and beta = 92.4 degrees. An asymmetric unit contains a dimer consisting of two identical subunits made of 122 amino acids. The crystal reflects X-rays beyond 2.5 A. A Pt derivative gave a good isomorphous crystal.     

Purification, cDNA cloning and characterization of the vascular apoptosis-inducing protein, HV1, from Trimeresurus flavoviridis.

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification and cDNA cloning from Crotalus atrox of a vascular apoptosis-inducing protein (VAP1) that specifically induces apoptosis in vascular endothelial cells. We report here the purification and cDNA cloning of another vascular apoptosis-inducing protein, HV1, from crude venom of Trimeresurus flavoviridis. The protein, namely HV1, was purified as an inducer of apoptosis in cultured vascular endothelial cells. HV1 was a homodimeric protein with a molecular mass of 110 kDa. HV1 cDNA encoded a protein with 612 amino-acid residues. The amino-acid sequence predicted from the cDNA was highly homologous to VAP1. The amino-acid sequence of HV1 indicated that HV1 belongs to the metalloprotease/disintegrin family, and that it is a multidomain polypeptide with a proprotein domain, a metalloprotease domain, a disintegrin-like domain and a cysteine-rich domain. In the disintegrin-like domain, the sequence DECD, replaces the RGD sequence that has frequently been found in such domains. This replacement also occurs in VAP1. Our results indicate HV1 as the first identified homolog of VAP1.     

The complete amino acid sequence of the high molecular mass hemorrhagic protein HR1B isolated from the venom of Trimeresurus flavoviridis

Hemorrhage is a common occurrence in a victim bitten by crotalid and viperid snakes, and hemorrhagic components in these various venoms have been isolated and characterized. Previously, we have shown that a low molecular weight hemorrhagic protein (HR2a, 202 amino acid residues) isolated from the venom of Trimeresurus flavoviridis is a member of a new subfamily of metalloproteinases. We now report the complete amino acid sequence of a high molecular mass hemorrhagic protein isolated from the same venom. This protein, HR1B, is a mosaic protein composed of 416 residues containing four asparagine-linked oligosaccharide chains. The amino-terminal half (residues 1-203) of HR1B contains a metalloproteinase domain, the sequence of which is 62% identical to that of HR2a and 52% identical to that of hemorrhagic toxin d isolated from Crotalus atrox venom. The most interesting finding is that the middle region (residues 204-300) of HR1B shows a striking similarity to disintegrins, Arg-Gly-Asp-containing platelet aggregation inhibitors, recently found in several viper venoms. Interestingly, however, this region of HR1B does not contain the Arg-Gly-Asp sequence which is known to be a putative binding site in the disintegrins for the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. We also found that the carboxyl-terminal region (residues 213-336) of the middle part of HR1B shows 30% identity to residues 1543-1656 of von Willebrand factor and that the remaining region at the carboxyl-terminal end is unique and has a cysteine-rich sequence. These results suggest that the middle portion of HR1B, which shows structural similarities to the disintegrins and von Willebrand factor, may be important in synergistically stimulating hemorrhagic activity in the NH2-terminal metalloproteinase domain.     

Prostatic spermine-binding protein. Cloning and nucleotide sequence of cDNA, amino acid sequence, and androgenic control of mRNA level

cDNA for mRNA of an androgen-dependent spermine-binding protein (SBP) of rat ventral prostate was cloned by inserting cDNA into a dG-tailed expression vector, pUC8, and screening the expression library with anti-SBP antibodies. Hybrid-selected translation using plasmid DNA from positive clones yielded a 34-kDa protein which was immunoprecipitated by affinity-purified anti-SBP antibodies. SBP mRNA is about 1260 bases long as measured by Northern blot hybridization. An amino acid sequence deduced from the nucleotide sequence of the cDNA was identical to an amino acid sequence found in SBP. SBP is extremely rich in acidic residues. Aspartic and glutamic acids, which make up about 33% of the total sequence, comprise 89 of a stretch of 126 amino acids at the carboxyl-terminal end. By dot hybridization analysis, SBP mRNA was not detected in rat liver, kidney, brain, submaxillary gland, or uterus. The prostate levels of SBP mRNA were measured by mRNA translation and dot hybridization. SBP mRNA level decreased to less than 20% of normal 2 days after castration of rats, and this decrease was reversed by 5 alpha-dihydrotestosterone injection into castrated rats.     

Interactions of Trimeresurus flavoviridis phospholipase A2 and its N-terminal octapeptide-removed and p-bromophenacylated derivatives with acridine and anilinonaphthalene dyes

Interactions of dimeric Trimeresurus flavoviridis (the Habu snake) phospholipase A2 (PLA2), des-octapeptide(1-8)-PLA2 (L-fragment) (14% of PLA2 activity), and p-bromophenacyl bromide (BPB)-inactivated PLA2 (BP-PLA2) with dyes, namely, proflavine, 1-anilinonaphthalene-8-sulfonate (Ans), and 2-toluidinylnaphthalene-6-sulfonate (Tns), were investigated. All dyes were bound in a 1:1 molar ratio to the subunit of the proteins. Proflavine was bound most strongly to PLA2 and Ans and Tns were bound to the three proteins with comparable affinities. Capabilities of the dyes for inhibiting alkylation of His-47 of PLA2 with BPB were in the following order: Ans greater than proflavine greater than Tns. Fluorescences of Ans and Tns that were increased in the presence of PLA2 were further greatly enhanced upon the addition of Ca2+, with concomitant formation of the ternary complexes. Ca2+, however, inhibited, competitively or noncompetitively, the bindings of the dyes to PLA2. All dyes were bound to the active site of PLA2 but with different orientations. Inactivation of L-fragment with BPB was inhibited by the dyes in the following order: Tns greater than proflavine approximately Ans. Addition of Ca2+ to the binary complexes formed from L-fragment and Ans or Tns caused no additional enhancement of fluorescence in spite of the formation of the ternary complexes. The active site structures are different between PLA2 and L-fragment, and the N-terminal octapeptide moiety of PLA2 possibly plays a role in maintaining the optimally arranged active site structure of the molecule. Comparison of the data suggests that the N-terminal moieties of PLA2S from snakes of an elapid family and from mammalian pancreas are essential for catalysis of a micellar substrate, whereas those of PLA2S from snakes of a viperid family, such as T. flavoviridis, are not. BP-PLA2 bound Ca2+ and was similar to L-fragment in terms of the fluorescence measurements. It appears that the active site of PLA2 has a space large enough to accommodate p-bromophenacyl, Ans or Tns, and Ca2+ together. Comparison of the emission maxima of Ans and Tns complexed with the three proteins indicated that Tns could be a useful fluorescent probe informing us of the state (disorder) of the active site of PLA2.     

Nucleotide sequence of a cDNA encoding a common precursor of disintegrin flavostatin and hemorrhagic factor HR2a from the venom of Trimeresurus flavoviridis.

The venom of Trimeresurus flavoviridis has three disintegrins that act as platelet aggregation inhibitors by binding to integrin alphaIIb beta3 on platelets through its Arg-Gly-Asp sequence. We isolated the cDNA encoding the flavostatin precursor that is one of the disintegrins in T. flavoviridis venom. The open reading frame consisted of four regions, a pre-peptide region, a metalloprotease region, a spacer region and a disintegrin region, indicating that the flavostatin precursor belongs to the metalloprotease/disintegrin family. Surprisingly, the deduced amino acid sequence of the metalloprotease region was completely consistent with that of hemorrhagic metalloprotease HR2a, which indicated that this metalloprotease released from the flavostatin precursor functions as a hemorrhagic factor. These observations indicated that a disintegrin and a hemorrhagic metalloprotease were synthesized as a common precursor. Thus, our results support the hypothesis that a disintegrin is synthesized as a metalloprotease/disintegrin precursor and matures by cleavage from the precursor molecule.     

Transition of environmental polarity of tyrosine-76 of Trimeresurus flavoviridis phospholipase A2 upon active site ligand binding

Modification of Trimeresurus flavoviridis phospholipase A2 with a 5-fold molar excess of tetranitromethane produced 40% active mononitrotyrosyl phospholipase A2 in which Tyr-76 was specifically nitrated. This is in contrast to the case of mammalian pancreatic phospholipases A2 where Tyr-70 but not Tyr-76 was nitrated. When Ca2+ was bound to T. flavoviridis mononitrotyrosyl phospholipase A2, nitrated tyrosine (Tyr(NO2))-76 moved from a less polar site to a polar site with the decrease of the pKa value of its hydroxyl group. Nitration of Tyr-76 did not influence the binding affinity to Ca2+. Addition of laurylphosphorylcholine to mononitrotyrosyl phospholipase A2 in the presence of Ca2+ caused the movement of Tyr(NO2)-76 from a polar environment to a less polar environment with the rise in the pKa value. Tyrosine-76 is located in the site whose environmental polarity is affected by the binding of the ligands to the active site. As Tyr-76 is located in the site not proximal to the active site, it could be assumed that the conformational change induced by the binding of the ligands extends to the region remote from the active site in T. flavoviridis phospholipase A2. This might provide evidence of long-range diffusional coupling between remote sites in the noncooperative globular protein.     

The amino acid sequence of the phospholipase A2 isoenzyme from porcine pancreas

The primary structure of porcine pancreatic isophospholipase A2 (EC 3.1.1.4) has been investigated. The sequence of procine isophospholipase differs from the sequence of porcine phospholipasy by four substitutions; viz. Ala12 leads to Thr; His17 leads to Asp leads to; Met20 leads to Leu and Glu71 leads to Asn.