Ikuo Nobuhisa

Accelerated evolution of crotalinae snake venom gland serine proteases

Eight cDNAs encoding serine proteases isolated from Trimeresurus flavoviridis (habu snake) and T. gramineus (green habu snake) venom gland cDNA libraries showed that nonsynonymous nucleotide substitutions have accumulated in the mature protein‐coding regions to cause amino acid changes. Southern blot analysis of T. flavoviridis genomic DNAs using two proper probes indicated that venom gland serine protease genes form a multigene family in the genome. These observations suggest that venom gland serine proteases have diversified their amino acid sequences in an accelerating manner. Since a similar feature has been previously discovered in crotalinae snake venom gland phospholipase A2 (PLA2) isozyme genes, accelerated evolution appears to be universal in plural isozyme families of crotalinae snake venom gland.

NMR Solution Structure of the Tandem Src Homology 3 Domains of p47phox Complexed with a p22phox-derived Proline-rich Peptide

The phagocyte NADPH oxidase plays a crucial role in host defense against microbial infections by generating reactive oxygen species. It is a multisubunit enzyme composed of membrane-bound flavocytochromeb558 as well as cytosolic components, including p47phox, which is essential for assembly of the complex. When phagocytes are activated, the cytosolic components of the NADPH oxidase translocate to flavocytochrome b558 due to binding of the tandem Src homology 3 (SH3) domains of p47phox to a proline-rich region in p22phox, a subunit of flavocytochrome b558. Using NMR titration, we first identified the proline-rich region of p22phox that is essential for binding to the tandem SH3 domains of p47phox. We subsequently determined the solution structure of the p47phox tandem SH3 domains complexed with the proline-rich peptide of p22phox using NMR spectroscopy. In contrast to the intertwined dimer reported for the crystal state, the solution structure is a monomer. The central region of the p22phox peptide forms a polyproline type II helix that is sandwiched by the N- and C-terminal SH3 domains, as was observed in the crystal structure, whereas the C-terminal region of the peptide takes on a short α-helical conformation that provides an additional binding site with the N-terminal SH3 domain. Thus, the C-terminal α-helical region of the p22phox peptide increases the binding affinity for the tandem SH3 domains of p47phox more than 10-fold.

Accelerated evolution of snake venom phospholipase A2 isozymes for acquisition of diverse physiological functions

The nucleotide sequences of two cDNAs and four genes encoding Trimeresurus gramineus venom gland phospholipase A2 (PLA2) isozymes were determined and compared internally and externally with those encoding Trimeresurus flavoviridis venom gland PLA2 isozymes. It was revealed that the protein-coding regions are much more diversified than the 5 and 3 untranslated regions (UTRs) and the introns except for the signal peptide domain. The numbers of nucleotide substitutions per site (KN) for the UTRs and the introns were approximately one-quarter of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions and were at the same level as the KN value of T. gramineus and T. flavoviridis TATA box-binding protein (TBP) genes, indicating that the protein-coding regions of PLA2 isozyme genes are unusually variable and that the UTRs including the introns of venom gland PLA2 isozyme genes have evolved at similar rate to those of non-venomous genes. The numbers of nucleotide substitutions per non-synonymous site (KA) values were close to or larger than the KS values for the protein-coding regions in venom gland PLA2 isozyme genes, indicating that the protein-coding regions of snake venom gland PLA2 isozyme genes have evolved via accelerated evolution. Furthermore, the evolutionary trees derived from the combined sequences of the 5 and 3 UTRs and the signal peptide domain of cDNAs were in accord with the consequences from taxonomy. In contrast, the evolutionary trees from the mature protein-coding region sequences of cDNAs and from the amino acid sequences showed random patterns. Estimations of nucleotide divergence of genes and the phylogenetic analysis reveal that snake venom group IJ PLA2 isozyme genes have been evolving under adaptive pressure to acquire new physiological activities.

Accelerated evolution of Trimeresurus okinavensis venom gland phospholipase A2 isozyme-encoding genes.

Three Trimeresurus okinavensis (To; himehabu snake, Crotalinae) venom gland phospholipase A2 (PLA2) isozymeencoding genes, gPLA2-o1, gPLA2-o2 and gPLA2-o3, were isolated from its genomic DNA library. The nucleotide (nt) sequence analysis revealed that two of the three genes (gPLA2-o2 and gPLA2-o3) occasionally have been converted to inactivated genes by introduction of one base insertion or substitution. It was confirmed from Southern blot analysis that the To haploid genome contains only three venom gland PLA2 isozyme genes herein isolated. Comparison of these genes showed that nonsynonymous nt substitutions have occurred more frequently than synonymous nt substitutions in the protein-coding regions, except for the signal-peptide coding domain, implying that To venom gland PLA2 isozyme genes have evolved via accelerated evolution. Such an evolutionary feature of To venom gland PLA2 isozyme genes proves the general universality of accelerated evolution previously drawn for venom gland PLA2 isozyme genes of other crotalinae snakes. The variability in the mature protein-coding regions of three To venom gland PLA2 isozyme genes appears to have been brought about by natural selection for point mutations.

Regional evolution of venom-gland phospholipase A2 isoenzymes of Trimeresurus flavoviridis snakes in the southwestern islands of Japan.

Conventional chromatographic analysis showed that phospholipase A(2) (PLA(2)) isoenzymes of the venom of Trimeresurus flavoviridis (Habu snake) of Okinawa island are profoundly different in composition from those of T. flavoviridis of Amami-Oshima and Tokunoshima islands. The most striking feature was that myotoxic [Lys(49)]PLA(2) isoenzymes, called BPI and BPII, which are expressed abundantly in the venoms of Amami-Oshima and Tokunoshima T. flavoviridis, are missing from the venom of Okinawa T. flavoviridis. Northern blot analysis of Okinawa T. flavoviridis venom-gland mRNA species showed the absence of BPI and BPII mRNA species. Analysis by single-stranded conformational polymorphism-PCR of venom-gland mRNA species of T. flavoviridis from three islands, with reference to five DNA species each encoding different PLA(2) isoenzymes from Tokunoshima T. flavoviridis venom gland, also suggested that BPI and BPII mRNA species are not expressed in Okinawa T. flavoviridis venom gland. In contrast, genomic Southern blot analysis with a variety of probes showed that only the bands corresponding to the upstream and downstream regions of the genes for BPI and/or BPII can be detected in Okinawa T. flavoviridis. These results suggested that the genes for BPI and BPII in Okinawa T. flavoviridis genome had been inactivated to form pseudogenes. Differently from Amami-Oshima and Tokunoshima T. flavovirdis genomic DNAs, PCR amplification of the segments of BPI and BPII genes between the 5 moiety of second exon and the middle portion of second intron failed for Okinawa T. flavoviridis genomic DNAs. In sequence analysis of the two segments involving polymorphism between BPI and BPII genes, which are located in first exon and third exon, respectively, only one base was detected at the polymorphic positions for pseudogene in Okinawa T. flavoviridis genome. Based on these facts, it became evident for pseudogene that the upstream region of BPI gene down to the 5 moiety of second exon and the downstream region of BPII gene starting from the middle portion of second intron are in a linked form with a possible insertion. Such observations suggest that venom-gland genes for PLA(2) isoenzymes in T. flavoviridis snakes isolated for one to two million years have evolved independently. Their evolution is regional and seems, from several lines of consideration and observation, to be adaptive to the environment.

Interisland Evolution of Trimeresurus flavoviridis Venom Phospholipase A2 Isozymes

AbstractnTrimeresurus flavoviridis snakes inhabit the southwestern islands of Japan. A phospholipase A2 (PLA2), named PL-Y, was isolated from Okinawa T. flavoviridis venom and its amino acid sequence was determined from both protein and cDNA. PL-Y was unable to induce edema. In contrast, PLA-B, a PLA2 from Tokunoshima T. flavoviridis venom, which is different at only three positions from PL-Y, is known to induce edema. A new PLA2, named PLA-B′, which is similar to PLA-B, was cloned from Amami-Oshima T. flavoviridis venom gland. Three T. flavoviridis venom basic [Asp49]PLA2 isozymes, PL-Y (Okinawa), PLA-B (Tokunoshima), and PLA-B′ (Amami-Oshima), are identical in the N-terminal half but have one to four amino acid substitutions in the β1-sheet and its vicinity. Such interisland sequence diversities among them are due to isolation in the different environments over 1 to 2 million years and appear to have been brought about by natural selection for point mutation in their genes. Otherwise, a major PLA2, named PLA2, ubiquitously exists in the venoms of T. flavoviridis snakes from the three islands with one to three synonymous substitutions in their cDNAs. It is assumed that the PLA2 gene is a prototype among T. flavoviridis venom PLA2 isozyme genes and has hardly undergone nonsynonymous mutation as a principal toxic component. Phylogenetic analysis based on the amino acid sequences revealed that T. flavoviridis PLA2 isozymes are clearly separated into three groups, PLA2 type, basic [Asp49]PLA2 type, and [Lys49]PLA2 type. Basic [Asp49]PLA2-type isozymes may manifest their own particular toxic functions different from those of the isozymes of the PLA2 type and [Lys49]PLA2 type.

Activation of the superoxide-producing phagocyte NADPH oxidase requires co-operation between the tandem SH3 domains of p47phox in recognition of a polyproline type II helix and an adjacent α-helix of p22phox

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial for host defence, requires an SH3 (Src homology 3)-domain-mediated interaction of the regulatory protein p47phox with p22phox, a subunit of the oxidase catalytic core flavocytochrome b558. Although previous analysis of a crystal structure has demonstrated that the tandem SH3 domains of p47phox sandwich a short PRR (proline-rich region) of p22phox (amino acids 151-160), containing a polyproline II helix, it has remained unknown whether this model is indeed functional in activation of the oxidase. In the present paper we show that the co-operativity between the two SH3 domains of p47phox, as expected from the model, is required for oxidase activation. Deletion of the linker between the p47phox SH3 domains results not only in a defective binding to p22phox but also in a loss of the activity to support superoxide production. The present analysis using alanine-scanning mutagenesis identifies Pro152, Pro156 and Arg158 in the p22phox PRR as residues indispensable for the interaction with p47phox. Pro152 and Pro156 are recognized by the N-terminal SH3 domain, whereas Arg158 contacts with the C-terminal SH3 domain. Amino acid substitution for any of the three residues in the p22phox PRR abrogates the superoxide-producing activity of the oxidase reconstituted in intact cells. The bis-SH3-mediated interaction of p47phox with p22phox thus functions to activate the phagocyte oxidase. Furthermore, we provide evidence that a region C-terminal to the PRR of p22phox (amino acids 161-164), adopting an a-helical conformation, participates in full activation of the phagocyte oxidase by fortifying the association with the p47phox SH3 domains.

Regional and accelerated molecular evolution in group I snake venom gland phospholipase A2 isozymes.

In accordance with detection of a few phospholipase A2 (PLA2) isozyme genes by Southern blot analysis, only two cDNAs, named NnkPLA-I , and NnkPLA-II, encoding group I PLA2s, NnkPLA-I and NnkPLA-II, respectively, were isolated from the venom gland cDNA library of Elapinae Naja naja kaouthia of Malaysia. NnkPLA-I and NnkPLA-II showed four amino acid substitutions, all of which were brought about by single nucleotide substitution. No existence of clones encoding CM-II and CM-III, PLA2 isozymes which had been isolated from the venom of N. naja kaouthia of Thailand, in Malaysian N. naja kaouthia venom gland cDNA library was verified by dot blot hybridization analysis with particular probes. NnkPLA-I and NnkPLA-II differed from CM-II and CM-III with four and two amino acid substitutions, respectively, suggesting that their molecular evolution is regional. The comparison of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s indicated that the 5- and 3-untranslated regions are more conserved than the mature protein-coding region and that the number of nucleotide substitutions per nonsynonymous site is almost equal to that per synonymous site in the protein-coding region, suggesting that accelerated evolution has occurred in group I venom gland PLA2s possibly to acquire new physiological functions.

Structural elements of Trimeresurus flavoviridis serum inhibitors for recognition of its venom phospholipase A2 isozymes

Five inhibitors (PLI‐I–V) against Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) venom phospholipase A2 (PLA2) isozymes have been isolated from its serum. PLI‐I, which is composed of two repeated three‐finger motifs, and PLI‐IV and PLI‐V, which contain a sequence similar to the carbohydrate recognition domain (CRD) of C‐type lectins, were expressed in the forms fused with glutathione S‐transferase (GST). The resulting GST‐PLIs showed ability to bind to three Tf venom PLA2 isozymes. The binding study with the truncated forms indicated that one of two three‐finger motifs of PLI‐I was able to bind to PLA2 isozymes. The N‐terminal 37‐amino acid fragment and the CRD‐like domain of PLI‐IV and PLI‐V were bound to PLA2 isozymes. On the other hand, their C‐terminal 12‐amino acid segment also associated with PLA2 isozymes. When either of two units of a hydrophobic tripeptide in this sequence was replaced by trialanine, the binding was completely abolished, indicating that the C‐terminal hydrophobic cores of PLI‐IV and PLI‐V were critically responsible for the binding to venom PLA2 isozymes.

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.