Precursor genes of Bowman-Birk-type serine proteinase inhibitors comprise multiple inhibitory domains to promote diversity.

BACKGROUND: Proteinase inhibitors are important for the regulation of the activity of enzymes essential for the survival and maintenance of all organisms, and they may hold medicinal and agricultural value. Hyacinthus orientalis L. serine protease inhibitors (HOSPIs), belonging to the Bowman-Birk type inhibitor (BBI) family, have strong inhibitory activities against mammalian serine proteinases. This study explored the relationship between gene structure and multiple isoinhibitor production of these diversified BBIs by analyzing sequences of HOSPI precursor genes. METHODS: Genomic DNA of H. orientalis roots was obtained and fragmented using 13 specific restriction enzymes, which were amplified by inverse and nested polymerase chain reactions, cloned into the pBluescript II SK (+) vector, and directly sequenced using specific primers. HOSPI gene and protein expression were assessed by quantitative real-time PCR and western blot, respectively. Proteinase inhibitory activity of hyacinth bulb extracts was evaluated by fluorescein isothiocyanate-labeled casein. RESULTS: Four distinct HOSPI precursor genes were identified, encoding 2-4 different HOSPI domains that were surrounded by additional sequences (named head, linker, and tail sequences) and some introns. Moreover, 3' splicing of the linker sequence may occur through introns inserted between linker sequences. HOSPI gene and protein expression was higher during the stem elongation and the flowering periods. CONCLUSIONS: These results indicate that gene duplication of the HOSPI precursor as a single set, including tandem repeated HOSPI domains, leads to diversity and effective production of mature HOSPIs by posttranslational processing. GENERAL SIGNIFICANCE: These findings shed light on the diversity of proteinase inhibitors.

Arabidopsis thaliana endonuclease V is a ribonuclease specific for inosine-containing single-stranded RNA.

Endonuclease V is highly conserved, both structurally and functionally, from bacteria to humans, and it cleaves the deoxyinosine-containing double-stranded DNA in Escherichia coli, whereas in Homo sapiens it catalyses the inosine-containing single-stranded RNA. Thus, deoxyinosine and inosine are unexpectedly produced by the deamination reactions of adenine in DNA and RNA, respectively. Moreover, adenosine-to-inosine (A-to-I) RNA editing is carried out by adenosine deaminase acting on dsRNA (ADARs). We focused on Arabidopsis thaliana endonuclease V (AtEndoV) activity exhibiting variations in DNA or RNA substrate specificities. Since no ADAR was observed for A-to-I editing in A. thaliana, the possibility of inosine generation by A-to-I editing can be ruled out. Purified AtEndoV protein cleaved the second and third phosphodiester bonds, 3' to inosine in single-strand RNA, at a low reaction temperature of 20-25°C, whereas the AtEndoV (Y100A) protein bearing a mutation in substrate recognition sites did not cleave these bonds. Furthermore, AtEndoV, similar to human EndoV, prefers RNA substrates over DNA substrates, and it could not cleave the inosine-containing double-stranded RNA. Thus, we propose the possibility that AtEndoV functions as an RNA substrate containing inosine induced by RNA damage, and not by A-to-I RNA editing in vivo.

An Arrhythmic Mutation E7K Facilitates TRPM4 Channel Activation via Enhanced PIP2 Interaction.

A Ca2+-activated monovalent cation-selective TRPM4 channel is abundantly expressed in the heart. Recently, a single gain-of-function mutation identified in the distal N-terminus of the human TRPM4 channel (Glu5 to Lys5; E7K) was found to be arrhythmogenic because of enhanced cell membrane expression. In this study, we conducted detailed analyses of this mutant channel from more functional aspects, in comparison with its wild type (WT). In an expression system, intracellular application of a short soluble PIP2 (diC8PIP2) restored the single-channel activities of both WT and E7K, which had quickly faded after membrane excision. The potency (Kd) of diC8PIP2 for this recovery was stronger in E7K than its WT (1.44 vs. 2.40 µM). FRET-based PIP2 measurements combined with the Danio rerio voltage-sensing phosphatase (DrVSP) and patch clamping revealed that lowering the endogenous PIP2 level by DrVSP activation reduced the TRPM4 channel activity. This effect was less prominent in E7K than its WT (apparent Kd values estimated from DrVSP-mediated PIP2 depletion: 0.97 and 1.06 µM, respectively), being associated with the differential PIP2-mediated modulation of voltage dependence. Moreover, intracellular perfusion of short N-terminal polypeptides containing either the 'WT' or 'E7K' sequences respectively attenuated the TRPM4 channel activation at whole-cell and single-channel levels, but in both configurations, the E7K polypeptide exerted greater inhibitory effects. These results collectively suggest that N-terminal interaction with endogenous PIP2 is essential for the TRPM4 channel to function, the extent of which may be abnormally strengthened by the E7K mutation through modulating voltage-dependent activation. The altered PIP2 interaction may account for the arrhythmogenic potential of this mutation.

Isolation and functional diversity of Bowman-Birk type serine proteinase inhibitors from Hyacinthus orientalis.

Bowman-Birk inhibitors (BBIs) are plant-derived serine proteinase inhibitors. Endogenously, they function as defense molecules against pathogens and insects, but they also have been explored for applications in cancer treatment and inflammatory disorders. Here, we isolated 15 novel BBIs from the bulb of Hyacinthus orientalis (termed HOSPIs). These isoinhibitors consisted of two or three chains, respectively, that are linked by disulfides bonds based on proposed cleavage sites in the canonical BBI reactive site loop. They strongly inhibited trypsin (Ki = 0.22-167 nM) and α-chymotrypsin (Ki = 19-1200 nM). Notably, HOSPI-B4 contains a six-residue reactive loop, which appears to be the smallest such motif discovered in BBIs to date. HOSPI-A6 and -A7 contain an unusual reactive site, i.e. Leu-Met at the P1-P1' position and have strong inhibitory activity against trypsin, α-chymotrypsin, and elastase. Analysis of the cDNA encoding HOSPIs revealed that the precursors have HOSPI-like domains repeated at least twice with a defined linker sequence connecting individual domains. Lastly, mutational analysis of HOSPIs suggested that the linker sequence does not affect the inhibitory activity, and a Thr residue at the P2 site and a Pro at the P3' site are crucial for elastase inhibition. Using mammalian proteases as representative model system, we gain novel insight into the sequence diversity and proteolytic activity of plant BBI. These results may aid the rational design of BBI peptides with potent and distinct inhibitory activity against human, pathogen, or insect serine proteinases.

Unusual quaternary structure of a homodimeric synergistic-type toxin from mamba snake venom defines its molecular evolution.

Snake venoms are complex mixtures of enzymes and nonenzymatic proteins that have evolved to immobilize and kill prey animals or deter predators. Among them, three-finger toxins (3FTxs) belong to the largest superfamily of nonenzymatic proteins. They share a common structure of three ß-stranded loops extending like fingers from a central core containing all four conserved disulfide bonds. Most 3FTxs are monomers and through subtle changes in their amino acid sequences, they interact with different receptors, ion channels and enzymes to exhibit a wide variety of biological effects. The 3FTxs have further expanded their pharmacological space through covalent or noncovalent dimerization. Synergistic-type toxins (SynTxs) isolated from the deadly mamba venoms, although nontoxic, have been known to enhance the toxicity of other venom proteins. However, the details of three-dimensional structure and molecular mechanism of activity of this unusual class of 3FTxs are unclear. We determined the first three-dimensional structure of a SynTx isolated from Dendroaspis jamesoni jamesoni (Jameson's mamba) venom. The SynTx forms a unique homodimer that is held together by an interchain disulfide bond. The dimeric interface is elaborate and encompasses loops II and III. In addition to the inter-subunit disulfide bond, the hydrogen bonds and hydrophobic interactions between the monomers contribute to the dimer formation. Besides, two sulfate ions that mediate interactions between the monomers. This unique quaternary structure is evolved through noncovalent homodimers such as κ-bungarotoxins. This novel dimerization further enhances the diversity in structure and function of 3FTxs.

Cysteine-Rich Secretory Proteins (CRISPs) From Venomous Snakes: An Overview of the Functional Diversity in A Large and Underappreciated Superfamily.

The CAP protein superfamily (Cysteine-rich secretory proteins (CRISPs), Antigen 5 (Ag5), and Pathogenesis-related 1 (PR-1) proteins) is widely distributed, but for toxinologists, snake venom CRISPs are the most familiar members. Although CRISPs are found in the majority of venoms, very few of these proteins have been functionally characterized, but those that have been exhibit diverse activities. Snake venom CRISPs (svCRISPs) inhibit ion channels and the growth of new blood vessels (angiogenesis). They also increase vascular permeability and promote inflammatory responses (leukocyte and neutrophil infiltration). Interestingly, CRISPs in lamprey buccal gland secretions also manifest some of these activities, suggesting an evolutionarily conserved function. As we strive to better understand the functions that CRISPs serve in venoms, it is worth considering the broad range of CRISP physiological activities throughout the animal kingdom. In this review, we summarize those activities, known crystal structures and sequence alignments, and we discuss predicted functional sites. CRISPs may not be lethal or major components of venoms, but given their almost ubiquitous occurrence in venoms and the accelerated evolution of svCRISP genes, these venom proteins are likely to have functions worth investigating.

Effects of acetaldehyde-induced DNA lesions on DNA metabolism.

BACKGROUND: Acetaldehyde, produced upon exposure to alcohol, cigarette smoke, polluted air and sugar, is a highly reactive compound that is carcinogenic to humans and causes a variety of DNA lesions in living human cells. Previously, we reported that acetaldehyde reacts with adjacent deoxyguanosine residues on oligonucleotides, but not with single deoxyguanosine residues or other deoxyadenosine, deoxycytosine, or thymidine residues, and revealed that it forms reversible intrastrand crosslinks with the dGpdG sequence (GG dimer). RESULTS: Here, we show that restriction enzymes that recognize a GG sequence digested acetaldehyde-treated plasmid DNA with low but significant efficiencies, whereas restriction enzymes that recognize other sequences were able to digest such DNA. This suggested that acetaldehyde produced GG dimers in plasmid DNA. Additionally, acetaldehyde-treated oligonucleotides were efficient in preventing digestion by the exonuclease function of T4 DNA polymerase compared to non-treated oligonucleotides, suggesting structural distortions of DNA caused by acetaldehyde-treatment. Neither in vitro DNA synthesis reactions of phi29 DNA polymerase nor in vitro RNA synthesis reactions of T7 RNA polymerase were observed when acetaldehyde-treated plasmid DNA was used, compared to when non-treated plasmid DNA was used, suggesting that acetaldehyde-induced DNA lesions inhibited replication and transcription in DNA metabolism. CONCLUSIONS: Acetaldehyde-induced DNA lesions could affect the relative resistance to endo- and exo-nucleolytic activity and also inhibit in vitro replication and in vitro transcription. Thus, investigating the effects of acetaldehyde-induced DNA lesions may enable a better understanding of the toxicity and carcinogenicity of acetaldehyde.

Omics Technologies for Profiling Toxin Diversity and Evolution in Snake Venom: Impacts on the Discovery of Therapeutic and Diagnostic Agents.

Snake venoms are primarily composed of proteins and peptides, and these toxins have developed high selectivity to their biological targets. This makes venoms interesting for exploration into protein evolution and structure-function relationships. A single venom protein superfamily can exhibit a variety of pharmacological effects; these variations in activity originate from differences in functional sites, domains, posttranslational modifications, and the formations of toxin complexes. In this review, we discuss examples of how the major venom protein superfamilies have diversified, as well as how newer technologies in the omics fields, such as genomics, transcriptomics, and proteomics, can be used to characterize both known and unknown toxins.Because toxins are bioactive molecules with a rich diversity of activities, they can be useful as therapeutic and diagnostic agents, and successful examples of toxin applications in these areas are also reviewed. With the current rapid pace of technology, snake venom research and its applications will only continue to expand.

Crystal structure of the complex between venom toxin and serum inhibitor from Viperidae snake.

Venomous snakes have endogenous proteins that neutralize the toxicity of their venom components. We previously identified five small serum proteins (SSP-1-SSP-5) from a highly venomous snake belonging to the family Viperidae as inhibitors of various toxins from snake venom. The endogenous inhibitors belong to the prostate secretory protein of 94 amino acids (PSP94) family. SSP-2 interacts with triflin, which is a member of the cysteine-rich secretory protein (CRISP) family that blocks smooth muscle contraction. However, the structural basis for the interaction and the biological roles of these inhibitors are largely unknown. Here, we determined the crystal structure of the SSP-2-triflin complex at 2.3 Å resolution. A concave region centrally located in the N-terminal domain of triflin is fully occupied by the terminal ß-strands of SSP-2. SSP-2 does not bind tightly to the C-terminal cysteine-rich domain of triflin; this domain is thought to be responsible for its channel-blocker function. Instead, the cysteine-rich domain is tilted 7.7° upon binding to SSP-2, and the inhibitor appears to sterically hinder triflin binding to calcium channels. These results help explain how an endogenous inhibitor prevents the venomous protein from maintaining homeostasis in the host. Furthermore, this interaction also sheds light on the binding interface between the human homologues PSP94 and CRISP-3, which are up-regulated in prostate and ovarian cancers.

Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

Snake venom metalloproteases (SVMPs) are members of the a disintegrin and metalloprotease (ADAM) family of proteins, as they possess similar domains. SVMPs are known to elicit snake venom-induced haemorrhage; however, the target proteins and cleavage sites are not known. In this work, we identified a target protein of vascular apoptosis-inducing protein 1 (VAP1), an SVMP, relevant to its ability to induce haemorrhage. VAP1 disrupted cell-cell adhesions by relocating VE-cadherin and γ-catenin from the cell-cell junction to the cytosol, without inducing proteolysis of VE-cadherin. The Wnt receptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) are known to promote catenin relocation, and are rendered constitutively active in Wnt signalling by truncation. Thus, we examined whether VAP1 cleaves LRP5/6 to induce catenin relocation. Indeed, we found that VAP1 cleaved the extracellular region of LRP6 and LRP5. This cleavage removes four inhibitory ß-propeller structures, resulting in activation of LRP5/6. Recombinant human ADAM8 and ADAM12 also cleaved LRP6 at the same site. An antibody against a peptide including the LRP6-cleavage site inhibited VAP1-induced VE-cadherin relocation and disruption of cell-cell adhesions in cultured cells, and blocked haemorrhage in mice in vivo. Intriguingly, animals resistant to the effects of haemorrhagic snake venom express variants of LRP5/6 that lack the VAP1-cleavage site, or low-density lipoprotein receptor domain class A domains involved in formation of the constitutively active form. The results validate LRP5/6 as physiological targets of ADAMs. Furthermore, they indicate that SVMP-induced cleavage of LRP5/6 causes disruption of cell-cell adhesion and haemorrhage, potentially opening new avenues for the treatment of snake bites.

Flavorase, a novel non-haemorrhagic metalloproteinase in Protobothrops flavoviridis venom, is a target molecule of small serum protein-3.

Some venomous snakes possess anti-toxic proteins in their sera that may play a role in neutralizing the haemorrhagic factors or toxins in their own venom. Five small serum proteins (SSP-1-SSP-5) were isolated from the serum of Japanese viper (Protobothrops flavoviridis), and were found to act as self-defence proteins against the viper's own toxic components. However, the physiological function of SSP-3 has not been completely elucidated. Affinity chromatography of the venom on an SSP-3-immobilized column identified a novel 55-kDa protein as the target molecule of SSP-3. Sequences of internal fragments of this SSP-3-binding protein showed high homology to those of metalloproteinases from the P. flavoviridis venom. The cDNA sequence revealed that this protein, termed flavorase, is a P-III class metalloproteinase consisting of 423 amino acid residues. The purified protein did not show haemorrhagic and cytotoxic activity. Biacore measurements revealed that SSP-3 was bound to flavorase with a dissociation constant of 6.4 × 10(-9) M. SSP-3 non-competitively inhibited the peptidase activity of flavorase with an inhibition constant of 6.6 × 10(-9) M.

Structural analysis and characterization of new small serum proteins from the serum of a venomous snake (Gloydius blomhoffii).

Some snakes have several anti-toxic proteins in their sera that neutralize their own venom. Five new small serum proteins (SSPs) were isolated from Japanese mamushi (Gloydius blomhoffii) serum by gel-filtration and RP-HPLC, and their N-Terminal sequences were determined. The amino acid sequences of the precursor proteins were deduced from the nucleotide sequences of cDNAs encoding them. Due to the sequence similarity to those of SSPs in habu snake (Protobothrops flavoviridis) serum (>75% identity), these proteins were designated mSSP-1 to mSSP-5 as the homologs of habu proteins. mSSP-1 was stable at 100 °C and in the pH range of 1-10, and inhibited the proteolytic activity of a certain snake venom metalloproteinase. The inhibitory activity was extinguished by modifying the amino groups of mSSP-1. mSSP-1 is the first prostate secretory protein of the 94 amino acid-family protein with a carbohydrate chain in the Asn37 residue.

Accelerated evolution of fetuin family proteins in Protobothrops flavoviridis (habu snake) serum and the discovery of an L1-like genomic element in the intronic sequence of a fetuin-encoding gene.

Habu serum factor (HSF) and HSF-like protein (HLP) are fetuin family proteins isolated from Protobothrops flavoviridis (habu snake) serum with different physiological activities. A comparison of their cDNAs and intronic sequences revealed that nucleotide substitutions were primarily in protein-coding regions, and the substitution patterns indicated accelerated evolution of these proteins. Genomic DNA fragment analysis, including intron 1, revealed a 6.6-kb insertion homologous to the full-length mammalian LINE1 (L1) retrotransposable element (PfL1) only in the HLP gene. This segment retains an open reading frame (ORF) that encodes a reverse transcriptase (RT)-like protein (PfRT). We further found that a large number of homologous segments have dispersed in the habu snake genome, although we could not determine the enzymatic activities of their products. Moreover, an analysis of habu snake liver RNA indicated active transcription of the PfRT genes, suggesting that high levels of RT activity in this snake have driven the evolution of unique phenotypes of venom enzymes and serum inhibitors of them.

Small serum protein-1 changes the susceptibility of an apoptosis-inducing metalloproteinase HV1 to a metalloproteinase inhibitor in habu snake (Trimeresurus flavoviridis).

Viperidae snakes containing various venomous proteins also have several anti-toxic proteins in their sera. However, the physiological function of serum protein has been elucidated incompletely. Small serum protein (SSP)-1 is a major component of the SSPs isolated from the serum of a Japanese viper, the habu snake (Trimeresurus flavoviridis). It exists in the blood as a binary complex with habu serum factor (HSF), a snake venom metalloproteinase inhibitor. Affinity chromatography of the venom on an SSP-1-immobilized column identified HV1, an apoptosis-inducing metalloproteinase, as the target protein of SSP-1. Biacore measurements revealed that SSP-1 was bound to HV1 with a dissociation constant of 8.2 × 10⁻8 M. However, SSP-1 did not inhibit the peptidase activity of HV1. Although HSF alone showed no inhibitory activity or binding affinity to HV1, the SSP-1-HSF binary complex bound to HV1 formed a ternary complex that non-competitively inhibited the peptidase activity of HV1 with a inhibition constant of 5.1 ± 1.3 × 10⁻9 M. The SSP-1-HSF complex also effectively suppressed the apoptosis of vascular endothelial cells and caspase 3 activation induced by HV1. Thus, SSP-1 is a unique protein that non-covalently attaches to HV1 and changes its susceptibility to HSF.