Purification and Amino Acid Sequence of Basic Protein II, a Lysine-49-Phospholipase A2 with Low Activity, from Trimeresurus flavoviridis Venom1

Abstract

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.

Structural Characteristics and Evolution of theProtobothrops elegansPancreatic Phospholipase A2Gene in Contrast with Those ofProtobothropsGenus Venom Phospholipase A2Genes

“…6,7) Group III PLA 2 s are found in bee venoms 8) and ubiquitous tissues. 9 14,17,18) This accords with their branching patterns, in the phylogenetic tree. 19) Mathematical examination of the nucleotide sequences of their cDNAs and genes has revealed that they evolved via Darwinian-type accelerated evolution, acquiring diverse physiological activities.…”

“…Then, in order to obtain an entire fragment harboring the P. elegans pancreatic PLA 2 gene and its 5 0 and 3 0 flanking regions, inverted PCRs were conducted against the liver genomic DNAs following the manufacturer's protocol (Toyobo, Osaka) starting from this 486-nt segment with restriction enzymes, Bal I, Bcl I, EcoR I, Nsi I, Pvu II, Sac I, Sph I, and Ssp I (Nippon Gene, Osaka). The primers employed in PCR against internally ligated fragments were PPP1, 5,6,7,10,11,12,16,17,18,19, and 22, which were based on the known sequences of the previously obtained PCR fragments. The position numbers and sequences of the primers are given in Table 1 0 -CAGCTGCTCCGGAGA-GGAATTGTGAN-3 0 , which can anneal to the 3 0 terminal portion of the 3 0 flanking region of the pancreatic PLA 2 gene, were prepared.…”

Comprehensive snake venomics of the Okinawa Habu pit viper, Protobothrops flavoviridis, by complementary mass spectrometry-guided approaches

“…According to the above findings, from components of the venom the phospholipase A2 class formed the biggest part and includes the three most abundant proteins of the whole venom: The highest protein content belongs to the acidic PLA2 1 (16.6%) eluting as fraction 22. Secondly, the basic PLA2 BP (10.4%) was identified in fraction 16 but could not clearly be assigned as BPI or BPII, because of their high similarity, which differs only in a N58D exchange as a single mutation [45]. The third protein is PLA-N(O) (7.9%) of fraction 17, which is a PLA-N K121N isoform (Uniprot-ID: S6BAM8) previously documented for the P. flavoviridis population at Okinawa Island [15].…”

Accelerated Evolution of Fetuin Family Proteins inProtobothrops flavoviridis(Habu Snake) Serum and the Discovery of an L1-Like Genomic Element in the Intronic Sequence of a Fetuin-Encoding Gene

“…For example, among PLA 2 isozymes of P. flavoviridis (the habu snake), the Asp49 PLA 2 shows phospholipase activity, whereas basic proteins I and II, with potent myotoxicities, show only very weak enzymatic activity. 1,[4][5][6][7] Furthermore, the physiological activities of the enzymes of the MP family of snake venom vary considerably: some induce hypodermic hemorrhage and others trigger apoptotic cell death. 3) Comparisons of the nucleotide sequences of the genes for such snake venom isozymes have revealed a striking feature, that nucleotide substitutions have occurred substantially more in the protein-coding sequences than in the noncoding sequences.…”