Ohanin, a novel protein from king cobra venom, induces hypolocomotion and hyperalgesia in mice

We have identified, purified, and determined the complete amino acid sequence of a novel protein, ohanin from Ophiophagus hannah (king cobra) venom. It is a small protein containing 107 amino acid residues with a molecular mass of 11951.47 +/- 0.67 Da as assessed by electrospray ionization-mass spectrometry. It does not show similarity to any known families of snake venom proteins and hence is the first member of a new family of snake venom proteins. It shows similarity to PRY and SPRY domain proteins. It is nontoxic up to 10 mg/kg when injected intraperitoneally in mice. Ohanin produced statistically significant and dose-dependent hypolocomotion in mice. In a pain threshold assay, it showed dose-dependent hyperalgesic effect. The ability of the protein to elicit a response at greatly reduced doses when injected intracerebroventricularly as compared with intraperitoneal administration in both the locomotion and hot plate experiments strongly suggests that ohanin acts on the central nervous system. Since the natural abundance of the protein in the venom is low (approximately 1 mg/g), a synthetic gene was constructed and expressed. The recombinant protein, which was obtained in the insoluble fraction in Escherichia coli, was purified under denaturing condition and was refolded. Recombinant ohanin is structurally and functionally similar to native protein as determined by circular dichroism and hot plate assay, suggesting that it will be useful in future structure-function relationship studies.     

眼镜王蛇泛素融合蛋白基因和核糖体蛋白L30基因的克隆及分析

从广西产眼镜王蛇（Ophiophagus hannah）毒腺中抽提总RNA,经mRNA纯化后构建眼镜王蛇毒腺cDNA文库。从所构建的cDNA文库中,随机筛选200个克隆测序,得到两个在进化上高度保守的基因：泛素融合蛋白基因（GenBank登录号为AF297036）和核糖体蛋白L30基因（GenBank登录号是AF297033）。前者cDNA的开放阅读框为387bp,后者为348bp。前者编码128个氨基酸残基组成的泛素融合蛋白前体;后者编码115个氨基酸残基组成的核糖体蛋白L30前体。由cDNA序列推导出的氨基酸序列分析表明,泛素融合蛋白前体包括N-末端的泛素结构域（76个氨基酸残基）和C-末端的核糖体蛋白L40结构域（52个氨基酸残基）。该蛋白为一高碱性蛋白,C末端含有一个“锌指”模式结构。与16个物种比较的结果表明,眼镜王蛇与脊椎动物的泛素融合蛋白氨基酸序列相似度较高,具有高度的保守性。     

Isolation of a cyp2b10-like cDNA and of a clone derived from a cyp2b10-like pseudogene

By screening Balb/c male mouse liver cDNA library with a rat CYP2B1 cDNA probe, we have isolated a 1795 bp cyp2b10-like clone, referred to as P16. Its sequence exhibited 34 base differences (98% similarity) with the cyp2b10 published sequence, together with a 97% identity at the amino acid sequence level. By RT-PCR and PCR analyses with Balb/c female and male liver RNA and genomic DNA, using a region showing 8 base differences between the P16 and the cyp2b10 sequences, we have confirmed the identity of our cloned cDNA, and failed in finding a PCR product exhibiting a sequence 100% identical with that of cyp2b10. Our results therefore suggest that the P16 sequence is the authentic cyp2b10 sequence. We have also isolated a partial clone, P21, which 1609 bp sequence overlapped with that of P16, except for a T-->G transversion, giving rise to a premature TGA stop codon, indicating that it was derived from a pseudogene.     

Primary structure of cobra complement component C3.

Complement component C3 is a multifunctional protein known to interact specifically with more than 10 different plasma proteins or cell surface receptors. Cobra venom contains cobra venom factor, a structural analogue of C3 that shares some properties with C3 (e.g., formation of a C3/C5 convertase) but differs in others (e.g., susceptibility to regulation by factors H and I). The elucidation of structural differences between C3 and cobra venom factor can be expected to help identify functionally important regions of C3 molecules. To that end we have undertaken the molecular cloning of both cobra C3 and cobra venom factor to take advantage of the unique biologic system where both proteins are produced by the same species. We report the primary structure of cobra C3 mRNA and the derived protein structure. Cobra C3 mRNA is 5211 bp in length. It contains an open reading frame of 4953 bp coding for a single pre-pro-C3 molecule, consisting of a 22-amino acid signal sequence, a 633-amino acid beta-chain (70 kDa), and a 992-amino acid alpha-chain (112 kDa) which is separated from the beta-chain by four arginine residues. There are no N-glycosylation sites in cobra C3. Cobra C3 exhibits approximately 58% nucleotide sequence identity with C3 from mammalian species. At the protein level, sequence identity is approximately 52% and sequence similarity approximately 71%. All 27 cysteine residues are highly conserved as are the C3 convertase cleavage site, the thioester site, and the factor B binding site. Cobra C3 also seems to have homologous binding sites for factor H and properdin, as well as a conserved sequence in the functionally important region of the C3a anaphylatoxin. The sequence homology at the CR2 and CR3 binding sites does not exceed the overall sequence homology. Accordingly, the existence of CR2 and CR3 binding sites can neither be deduced nor excluded.     

蛇神经毒素的表达和鉴定

抽提中华眼镜蛇毒腺总RNA,通过反转录PCR扩增Cobrotoxin cDNA,克隆并测序。该cDNA编码83个氨基酸,包括21个氨基酸的信号肽和62个氨基酸的成熟蛋白。该成熟蛋白的氨基酸序列和通过蛋白测序从台湾眼镜蛇鉴定的Cobrotoxin完全一致。PCR扩增编码Cobrotoxin的DNA,并亚克隆到表达载体pMAL-P₂。此外,通过合成寡核苷酸片段,拼接成完整的CM11基因,并将其克隆至pMAL-P₂。经IPTG诱导,两种神经毒素基因在大肠杆菌中都得到高效的可溶性融合表达。表达产物通过SDS-PAGE和蛋白印迹杂交加以鉴定。表达的融合蛋白经过Sepharose 6Bamylose亲和色谱和DEAESepharose FF离子交换色谱得到有效纯化。经Xa因子酶切后得到的两种重组神经毒素都具有小白鼠体内毒性。     

Protein fold analysis of the B30.2-like domain

The B30.2-like domain occurs in some members of a diverse and growing family of proteins containing zinc-binding B-box motifs, whose functions include regulation of cell growth and differentiation. The B30.2-like domain is also found in proteins without the zinc-binding motifs, such as butyrophilin (a transmembrane glycoprotein) and stonustoxin (a secreted cytolytic toxin). Currently, the function for the B30.2-like domain is not clear and the structure of a protein containing this domain has not been solved. The secondary structure prediction methods indicate that the B30.2-like domain consists of fifteen or fewer beta-strands. Fold recognition methods identified different structural topologies for the B30.2-like domains. Secondary structure prediction, deletion and lack of local sequence identity at the C-terminal region for certain members of the family, and packing of known core structures suggest that a structure containing two beta domains is the most probable of these folds. The most C-terminal sequence motif predicted to be a beta-strand in all B30.2-like domains is a potential subdomain boundary based on the sequence-structure alignments. Models of the B30.2-like domains were built based on immunoglobulin-like folds identified by the fold recognition methods to evaluate the possibility of the B30.2 domain adopting known folds and infer putative functional sites. The SPRY domain has been identified as a subdomain within the B30.2-like domain. If the B30.2-like domain is a subclass of the SPRY domain family, then this analysis would suggest that the SPRY domains are members of the immunoglobulin superfamily.     

Sequence of a cDNA clone encoding the zinc metalloproteinase hemorrhagic toxin e from Crotalus atrox: evidence for signal, zymogen, and disintegrin-like structures.

The sequence of two overlapping cDNA clones for the zinc metalloproteinase hemorrhagic toxin e (also known as atrolysin e, EC 3.4.24.44) from the venom gland of Crotalus atrox, the Western diamondback rattlesnake, is presented. The assembled cDNA sequence is 1975 nucleotides in length and encodes an open reading frame of 478 amino acids. The mature hemorrhagic toxin e protein as isolated from the crude venom has a molecular weight of approximately 24,000 and thus represents the processed product of this open reading frame. From the deduced amino acid sequence, it can be hypothesized that the enzyme is translated with a signal sequence of 18 amino acids, an amino-terminal propeptide of 169 amino acids, a central hemorrhagic proteinase domain of 202 amino acids, and a carboxy-terminal sequence of 89 amino acids. The propeptide has a short region similar to the region involved in the activation of matrix metalloproteinase zymogens. The proteinase domain is similar to other snake venom metalloproteinases, with over 57% identity to the low molecular weight proteinases HR2a and H2-proteinase from the Habu snake Trimeresurus flavoviridis. The carboxy-terminal region, which is not observed in the mature protein, strongly resembles the protein sequence immediately following the proteinase domain of HR1B (a high molecular weight hemorrhagic proteinase from the venom of T. flavoviridis) and the members of a different family of snake venom polypeptides known for their platelet aggregation inhibitory activity, the disintegrins. The cDNA sequence bears striking similarity to a previously reported sequence for a disintegrin cDNA. This report is evidence that this subfamily of venom metalloproteinases is synthesized in a proenzyme form which must be proteolytically activated.(ABSTRACT TRUNCATED AT 250 WORDS)     

中国长白山乌苏里蝮蛇金属蛋白酶解整合蛋白Ussurin基因克隆和序列分析

采用Clontech链转换建库试剂盒 ,建立了中国长白山乌苏里蝮蛇毒腺cDNA文库 ,从中克隆了金属蛋白酶 解整合蛋白Ussurin ,并进行了序列分析。结果显示 ,Ussurin开框读码序列由 14 34bp组成 ,编码 4 78个氨基酸。由核苷酸顺序推导的氨基酸序列可以看出 ,Ussurin最初的翻译产物是酶原前体 ;依次含有 18氨基酸组成的信号肽 ,171氨基酸组成的酶原区和由 2 89氨基酸组成的Ussurin(2 0 0氨基酸组成的金属蛋白酶结构域、16氨基酸组成的间隔区和 73氨基酸组成的解整合蛋白结构域 )。Ussurin的金属蛋白酶结构域含有 3对二硫键 ;解整合蛋白结构域含有 6对二硫键和特征性RGD(精氨酸 甘氨酸 天冬氨酸 )结构。其基因序列和结构域组成与GenBank中蛇毒金属蛋白酶 解整合蛋白呈现高度同源性属于P Ⅱ。氨基酸序列blast比对发现 ,酶原区和解整链蛋白结构域呈现极高的同源性 ,而金属蛋白酶结构域却出现了极高的变异 ,推测这些变异结构区是为了适应不同的底物、不同受体或同一受体的不同结构域     

Amino acid sequence of nerve growth factor purified from the venom of the Formosan cobra Naja naja atra

Nerve growth factor (NGF) was isolated from the venom of the Formosan cobra (Naja naja atra). The amino acid sequence was determined by a combination of conventional methods. The total number of amino acid residues was 116, giving a molecular mass of 13,057 Da. The sequence was identical with that deduced from the nucleotide sequence of an NGF cDNA from the venom gland of Naja naja siamensis, reported by Selby et al. [J. Neurosci. Res., 18, 293-298 (1987)].     

Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom

The major l-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78μg/mL (0.006μM) and 1.56μg/mL (0.012μM) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50μg/mL (0.2-0.4μM). Catalase at the concentration of 1mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme.     

Analysis of Candida utilis genomic DNA,homologous to cDNA of chicken A1 (1) collagen gene

Genomic fragments, homologous to chicken A1(1) collagen cDNA encoding triple-helical domain, were revealed by Southern analysis in various fungi. Such a genomic fragment from Candida utilis was cloned and sequenced. Analysis of the obtained DNA sequence revealed the 119 bp segment, which has possibly originated from the 54bp module common for the fibrillar collagen genes of higher eukaryotes.     

Cloning, localization, and structure of new members of the butyrophilin gene family in the juxta-telomeric region of the major histocompatibility complex

 New members of the butyrophilin (BT) gene family have been identified by cDNA and genomic cloning. Six genes are described: BT2.1, 2.2, 2.3, and BT3.1, 3.2, and 3.3. BT2, BT3, and BT form three distinct subfamilies sharing about 95% amino acid identity at the intra subfamily level and 50% identity at the interfamily level. All the BT2 and BT3 subfamily members map close to BT in the juxta-telomeric region of the major histocompatibility complex. The BT2 members have the canonical structural organization of BT, i.e., two immunoglobulin domains followed by a transmembrane anchor and a B30.2 intracytoplasmic domain. In the BT3 subfamily, only BT3.3 has the structural organization of BT. The two other genes, BT3.1 and BT3.2, code for putative protein without the B30.2 domain. In the case of BT3.2, this is due to an Alu insertion in the B30.2 coding exon, leading to a new polyadenylation site. Received: 21 April 1997 / Revised: 13 June 1997     

Human septin 3 on chromosome 22q13.2 is upregulated by neuronal differentiation

An expression sequence tag identified in a screen for genes upregulated by retinoic acid induced neuronal differentiation of the human teratocarcinoma cell line Ntera2/D1 was found in close genomic proximity to a region of high sequence homology to the septin subfamily of GTPase genes. We could show that the tag corresponds to the 3' untranslated region of this novel gene named septin 3 and cloned three isoforms A (2191 bp), B (4378 bp), and C (1896 bp) from human Ntera2/D1 cDNA. We present the genomic localization and organization on chromosome 22q13.2, a chromosomal hot spot for translocations implicated in leukemia. Interestingly, MSF the closest paralog of septin 3 is a fusion partner in a therapy-related acute myeloid leukemia. Quantitative PCR confirmed the upregulation of the putative septin by neuronal differentiation and northern blotting showed only one band corresponding to sep3B with a neurospecific expression pattern in adult human tissues.     

Taiwan cobra chymotrypsin inhibitor: cloning, functional expression and gene organization

A cDNA encoding chymotrypsin inhibitor was constructed from the cellular RNA isolated from the venom glands of Naja atra (Taiwan cobra). The resultant amino acid sequence showed that the mature protein is comprised of 57 amino acid residues with six cysteine residues. Cloned protein was expressed and isolated from the inclusion bodies of E. coli and refolded into a functional protein in vitro. Deleting the first three residues at its N-terminus caused a moderate increase in the inhibitory constant (K(i)) against chymotrypsin. The genomic DNA encoding the chymotrypsin inhibitor was amplified by PCR. The gene shares virtually an identical structural organization with the beta-bungarotoxin B1 chain (a snake Kunitz/BPTI neurotoxic homolog) gene. Moreover, the overall sequence identity of the N. atra chymotrypsin inhibitor and beta-bungarotoxin B1 chain genes was up to 83%. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.     

Escherichia coli O157:H7 Typing Phage V7 Is a T4-Like Virus

The complete genome sequence of the Escherichia coli O157:H7 typing phage V7 was determined. Its double-stranded DNA genome is 166,452 bp long, encoding 273 proteins and including 11 tRNAs. This virus belongs to the genus T4-like viruses within the subfamily Tevenvirinae, family Myoviridae.     

Identification and molecular characterization of a β-1,4-endoglucanase gene (Rr-eng-1) from Rotylenchulus reniformis

β-1,4-endoglucanses, a.k.a. cellulases, are parasitism genes that facilitate root penetration and migration by plant-parasitic nematodes. Rotylenchulus reniformis is a sedentary semi-endoparasite for which little molecular data has been collected. In this report, we describe the isolation and characterization of a predicted glycosyl hydrolase family 5 cellulase from R. reniformis that we have named Rr-eng-1. The Rr-eng-1 cDNA was 1,341 bp long and was comprised of a 19 bp 5'-untranslated region (UTR), a 1,245 bp open reading frame (ORF), and an 80 bp 3'-UTR. The Rr-eng-1 genomic sequence was 2,325 bp. Alignment of the cDNA and genomic sequences revealed seven introns and eight exons for Rr-eng-1. BLASTN analysis showed the Rr-eng-1 cDNA was most homologous to the Hg-eng-6 mRNA from Heterodera glycines. Southern blot analysis indicated that at least three Rr-eng-1-like sequences were present in the R. reniformis genome. Translation of the Rr-eng-1 ORF yielded a 414 amino acid peptide (Rr-ENG-1) having an N-terminal signal sequence for secretion. No cellulose binding module (CBM) was detected in Rr-ENG-1; however, a putative CBM linker sequence N-terminal to the catalytic domain was present. Rr-ENG-1 was most homologous to Hg-ENG-6 but also shared a number of intron splice positions with Mi-ENG-2. Quantitative RT-PCR indicated that Rr-eng-1 was highly expressed in the J2 and adult vermiform life-stages with a sharp decline in expression detected in sedentary females.     

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.