Unique structure (construction and configuration) and evolution of the array of small serum protein genes of Protobothrops flavoviridis snake.

The nucleotide sequence of Protobothrops flavoviridis (Pf) 30534 bp genome segment which contains genes encoding small serum proteins (SSPs) was deciphered. The genome segment contained five SSP genes (PfSSPs), PfSSP-4, PfSSP-5, PfSSP-1, PfSSP-2, and PfSSP-3 in this order and had characteristic configuration and constructions of the particular nucleotide sequences inserted. Comparison between the configurations of the inserted chicken repeat-1 (CR1) fragments of P. flavoviridis and Ophiophagus hannah (Oh) showed that the nucleotide segment encompassing from PfSSP-1 to PfSSP-2 was inverted. The inactive form of PfSSP-1, named PfSSP-1δ(Ψ), found in the intergenic region (I-Reg) between PfSSP-5 and PfSSP-1 had also been destroyed by insertions of the plural long interspersed nuclear elements (LINEs) and DNA transposons. The L2 LINE inserted into the third intron or the particular repetitive sequences inserted into the second intron structurally divided five PfSSPs into two subgroups, the Long SSP subgroup of PfSSP-1, PfSSP-2 and PfSSP-5 or the Short SSP subgroup of PfSSP-3 and PfSSP-4 The mathematical analysis also showed that PfSSPs of the Long SSP subgroup evolved alternately in an accelerated and neutral manner, whereas those of the Short SSP subgroup evolved in an accelerated manner. Moreover, the ortholog analysis of SSPs of various snakes showed that the evolutionary emerging order of SSPs was as follows: SSP-5, SSP-4, SSP-2, SSP-1, and SSP-3 The unique interpretation about accelerated evolution and the novel idea that the transposable elements such as LINEs and DNA transposons are involved in maintaining the host genome besides its own transposition natures were proposed.

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.

Identification and evolution of venom phospholipase A2 inhibitors from Protobothrops elegans serum.

The cDNAs encoding venom phospholipase A(2) (PLA(2)) inhibitors (PLIs), named Protobothrops elegans (Pe)γPLI-A, PeγPLI-B, PeαPLI-A, and PeαPLI-B, were cloned from the P. elegans liver cDNA library. They were further divided into several constituents due to nucleotide substitutions in their open reading frames. For PeαPLI-A, two constituents, PeαPLI-A(a) and PeαPLI-A(b), were identified due to three nonsynonymous substitutions in exon 3. Far-western blot and mass-spectrometry analysis of the P. elegans serum proteins showed the presence of γPLIs, and αPLIs, which can bind venom PLA(2)s. In αPLIs from Protobothrops sera, A or B subtype-specific amino acid substitutions are concentrated only in exon 3. A comparison of γPLIs showed that γPLI-As are conserved and γPLI-Bs diversified. Mathematical analysis of the nucleotide sequences of Protobothrops γPLI-B cDNAs revealed that the particular loops in the three-finger motifs diversified by accelerated evolution. Such evolutionary features should have made serum PLIs acquire their respective inhibitory activities to adapt to venom PLA(2) isozymes.

Identification of novel serum proteins in a Japanese viper: homologs of mammalian PSP94.

Three small serum proteins (SSP-1, -2, and -3), with molecular masses of 6.5-10kDa, were isolated from Habu (Trimeresurus flavoviridis) serum, and the amino acid sequences were determined by protein and cDNA analysis. Despite only limited sequence identity to any mammalian prostatic secretory protein of 94 amino acids (PSP94), all of the Cys residues in these SSPs were well conserved. SSPs are the first PSP94 family proteins to be identified in reptiles. SSP-1 and -3 weakly inhibited the proteolytic activity of a snake venom metalloproteinase. On the other hand, SSP-2 formed a tight complex with triflin, a snake venom-derived Ca(2+) channel blocker that suppresses the smooth muscle contraction. This suggests a role for SSP-2 in the self defense system of venomous snakes.

Active fragments of the antihemorrhagic protein HSF from serum of habu (Trimeresurus flavoviridis).

Certain snakes have antihemorrhagic proteins in their sera. Habu serum factor (HSF), an antihemorrhagic protein isolated from the serum of the Japanese habu snake (Trimeresurus flavoviridis) is composed of two cystatin-like domains (D1 and D2) and a His-rich domain, and it inhibits several snake venom metalloproteinases (SVMPs). The activity of HSF can be abolished by trinitrophenylation of Lys residues with 2,4,6-trinitrobenzene sulphonic acid. Upon complex formation of HSF with SVMP, however, the loss of its inhibitory activity by the chemical modification was suppressed, and Lys(15), Lys(41), and Lys(103) residues in HSF were not trinitrophenylated. In order to identify the domain that is critical to the inhibitory activity on SVMPs, native HSF was digested with papain followed by cleavage with cyanogen bromide, yielding a low-molecular mass fragment that was composed of two peptide chains (residues 5-89 and 312-317) linked by a disulfide bond. This fragment inhibited several SVMPs and showed significant antihemorrhagic activity. This indicates that the N-terminal half of D1 is indispensable for the antihemorrhagic activity of HSF. Furthermore, a three-dimensional model of two cystatin-like domains constructed by the homology modeling has indicated that three Lys residues (15, 41, and 103) are exposed to the same surface of HSF molecule.

Snake infrared receptors respond to dimethylsulfoxide in the blood stream.

1. We used extracellular recording of the infrared (IR)-sensitive trigeminal ganglion (TG) neurons (primary neurons) of a crotaline snake, Trimeresurus flavoviridis, which has very sensitive thermoreceptors, to examine changes in the IR response induced by dimethylsulfoxide (DMSO), in vivo. 2. The responses in the TG were recorded after each concentration of DMSO (1, 10, and 25%) was administered in the bloodstream. 3. At a constant temperature, DMSO dose-dependently potentiated the IR-triggered discharges of IR-sensitive TG neurons in this snake. 4. It is suggested that the increased IR response to DMSO is due to its chemical effect, or to an indirect effect via its vasoactive role in the thermoreceptors of IR-sensitive snakes.

Isolation and characterization of a novel proteinase inhibitor from the snake serum of Taiwan habu (Trimeresurus mucrosquamatus).

A proteinase inhibitor (designated as TMI) was isolated and purified from the snake serum of Taiwan habu (Trimeresurus mucrosquamatus) by using successive chromatographies which included Sephadex G-100, DEAE-Sephacel chromatographies, and C(4) reverse-phase HPLC. The purified inhibitor was shown to be a homogeneous protein with a molecular mass of about 47 or 36 kDa in the presence or absence of a reducing agent, beta-mercaptoethanol. The inhibitor decreases in molecular mass by about 23% with N-linked neuraminidase treatment, suggesting that it is a glycoprotein. Further enzymatic analyses indicated that this inhibitor possesses strong inhibitory activities toward three zinc-dependent metalloproteinases and not fibrinogenolytic serine proteases previously isolated from the venom of the same snake species with an IC(50) of about 0.2-1.1 microM. Its IC(50) value was approximately three orders of magnitude more effective than those of the tripeptide inhibitors we previously purified from the crude venom of the same snake (Biochem. Biophys. Res. Commun. 248, 562-568 (1998)). The purified inhibitor showed stronger inhibitory action against caseinolytic activities of crude venoms from closely related species of Taiwan habu than those from unrelated species. N-terminal sequence analysis showed that its sequence is distinctly different from sequences of those serum inhibitors reported for other snake species in the literature. Based on inhibition susceptibility and primary structures of various snake protease inhibitors, it is suggested that this novel inhibitor isolated from the serum of Taiwan habu may be a unique self-defense protein factor mainly for protection against envenomation from snakes of the same genus.