A new low molecular-weight protein effector of human complement from the venom of the Central Asian cobra Naja naja oxiana

Six protein effectors of human complement were isolated from the whole venom of the Central Asian cobra Naja naja oxiana. Three of them have acidic properties and molecular weights of 61 000, 5000 and 3000, and the rest are basic proteins with molecular weights of 54 000, 9000 and 7000. Two low molecular weight basic proteins CFB-II and CFB-III are isolated in as high amounts as 115 and 85 mg per g of dry venom. All the effectors inhibit the classical pathway of complement activation and, with the exception of CFB-II and CFB-III, the alternative pathway. The latter, on the contrary, enhances the alternative pathway of activation. N-Terminal sequence determination for CFB-III demonstrated its identity to the earlier characterized cytotoxin II. The action of CFB-III on the classical pathway of complement activation consists in the component C4 inactivation. A mechanism for the CFB-III activation of the alternative pathway is proposed implying the CFB-III induced transformation of the C3 component into a C3b-like one producing a soluble C3 convertase.     

Complement-inhibiting acidic factors from the venom of Central Asian cobra Naja naja oxiana

Two anticomplementic factors isolated from the venom of the Central Asian cobra Naja naja oxiana by chromatography on DEAE-Sepharose CL-6B and subsequent gel filtration on Sephacryl S-200 were studied. Of these, five factors (CFA-Ia, CFA-Ib, CFA-Ic, CFA-IIa and CFA-IIb), CFA-Ib had been characterized earlier, while CFA-Ia was assigned to a previously identified H-CoF factor. It was shown that CFA-Ic has a molecular mass of 3900 Da; its content in the venom amounts to 2.6 mg/g of dry venom. This factor inhibits the classical pathway of C3 convertase formation abrogating the C2 component activation by subcomponent C1s [Ki = (2.5 +/- 0.8).10(-7) M]. CFA-IIa and CFA-IIb are present in the venom in very low amounts (2 mg/g) and have Mr of 5700 and 3200 Da, respectively. The complement-inhibiting action was studied for a more active CFA-IIa. Factor CFA-IIa was shown to inactivate the native component of C2 with a rate constant, k, of (2.7 +/- 0.2).10(3) s-1M-1 (37 degrees C, pH 7.4). CFA-IIa had no effect on C2 and C2a within their complexes with C4b.     

A factor from the venom of the Central Asiatic cobra Naja naja oxiana, which inactivates component C4 of human complement

An acid glycoprotein (mol. m. 60 kDa) containing 6 sialic acid residues and N-terminal Thr was isolated from the venom of the central asian cobra Naja naja oxiana. The protein has an anticomplementary activity selectively inactivating of the C4 component of the human complement. This factor (CFA-Ib) binds C4 with Ki = 0.27 +/- 0.13 microM and then irreversible inactivates it with a rate constant k = 0.75 +/- 0.25 min-1. Membrane bound C4b restores its ability of CFA-Ib binding. This binding hinders component C2 sorption on C4b and C3 convertase formation.     

Primary structure and functional properties of cobra (Naja naja naja) venom Kunitz-type trypsin inhibitor.

A trypsin inhibitor from the venom of the cobra Naja naja naja has been isolated by a single step of reverse-phase high-performance liquid chromatography. The protein strongly inhibits trypsin (Ki = 3.5 pM). The primary structure was determined by peptide analysis of the [14C]carboxymethylated inhibitor. The 57-residue polypeptide chain belongs to the family of Kunitz-type inhibitors, and exhibits 42% residue identity with bovine pancreatic trypsin inhibitor. The structure shows only 70% identity with the corresponding peptide from the Capa cobra (Naja nevia), establishing that the inhibitor molecule exhibits extensive variations. Functionally, a basic residue at position P3' correlates with strong inhibition.     

Purification, characterization and primary structure of a chymotrypsin inhibitor from Naja atra venom

A chymotrypsin inhibitor, designated NA-CI, was isolated from the venom of the Chinese cobra Naja atra by three-step chromatography. It inhibited bovine alpha-chymotrypsin with a Ki of 25 nM. The molecular mass of NA-CI was determined to be 6403.8 Da by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) analysis. The complete amino acid sequence was determined after digestion of S-carboxymethylated inhibitor with Staphylococcus aureus V8 protease and porcine trypsin. NA-CI was a single polypeptide chain composed of 57 amino acid residues. The main contact site with the protease (P1) has a Phe, showing the specificity of the inhibitor. NA-CI shared great similarity with the chymotrypsin inhibitor from Naja naja venom (identities=89.5%) and other snake venom protease inhibitors.     

Sequence characterization of venom toxins from Thailand cobra

Several toxins with distinct pharmacological properties were isolated from the venom of Thailand cobra (Naja naja siamensis) by cation-exchange chromatography. Two neurotoxins and one basic toxin with cardiotoxic activity were further purified and sequenced. The neurotoxins characterized were closely similar to the previously reported long- and short-chain neutrotoxins. The complete sequences of one minor neurotoxin and one cardiotoxin analogue were determined with the automatic protein sequencer in non-stop single runs of Edman degradation coupled with C-terminal sequence determination with carboxypeptidase digestion. The minor neurotoxin consists of 62 amino-acid residues with 8 cysteine residues and is found to be almost identical to cobrotoxin, a major toxic component of Formosa cobra (Naja naja atra). The sequence comparison of the 60-residue cardiotoxin with other reported cytotoxins of snake venoms indicates that 8 cysteine residues at the positions 3, 14, 21, 38, 42, 53, 54, and 59 are invariant among all sequences, with only two conservative changes at other positions along the sequence. The upshot of this report exemplified the facile sequence analysis of venom toxins by the application of pulsed-liquid phase protein sequencer and also revealed new analogues of a minor neurotoxin and one major cardiotoxin reported previously on the same species of Thailand cobra.     

Affinity adsorbent based on combinatorial phage display peptides that bind alpha-cobratoxin

Combinatorial phage display was used to discover peptides that selectively bind to the alpha-cobratoxin (neurotoxin) component of the multi-component venom of the Thai cobra, Naja kaouthia. Peptide sequences determined in this way were synthesized chemically and were covalently attached to agarose through the alpha-amino terminus. Such affinity chromatography supports selectively bound the alpha-cobratoxin component from crude venom, while passage of the crude venom over the support selectively depleted the venom of this component. The selective binding of alpha-cobratoxin to peptide-based solid-phase supports suggests that a limitless variety of peptides similarly obtained by combinatorial phage display can be used to craft specific analytical and preparative tools.     

Purification, characterization, and partial amino acid sequence of nerve growth factor from cobra venom.

The nerve growth factor (NGF) from Naja naja (cobra) venom has been purified and its structure compared to the NGF from mouse submaxillary gland. A two-step purification procedure has been devised, consisting of a gel filtration step in 1 M acetic acid followed by chromatography of the active pool on carboxymethylcellulose at pH 5. The molecular weight of the native protein was found to be 28000, and this value was reduced by approximately one-half under denaturing conditions. These values are comparable to those obtained for mouse 2.5S or betaNGF. Tryptic peptide maps of S-[14C]carboxymethyl NGF gave the number of labeled peptides expected for a structure composed of two identical or very similar subunits. Thus, the quaternary structures of mouse and cobra NGF are the same. Cyanogen bromide (CNBr) treatment of Naja naja NGF produced three fragments, of which two were purified to homogeneity. These fragments and the whole protein were analyzed in the automated protein Sequencer. The amino-terminal CNBr fragment of the protein was also subjected to digestion by thermolysin and the resultant peptides were purified and characterized. These data plus those from the characterization of the tryptic peptides provided the basis of the construction of a tentative primary structure of Naja naja NGF which is approximately 60% identical with mouse NGF.     

Identification of serotonin-sensitive aryl acylamidase activity with cobra venom acetylcholinesterase

Acetylcholinesterase purified from cobra (Naja naja) venom exhibits a serotonin-sensitive aryl acylamidase activity. Both acetylcholinesterase and aryl acylamidase activities co-eluted in column chromatographic procedures (Sephadex G-75 and Zinc-Sepharose), co-migrated on polyacrylamide gel electrophoresis, co-immunoprecipitated by anti-snake venom antibody and showed the same heat denaturation profile at 40 degrees C. Further, several potent acetylcholinesterase inhibitors at different concentrations inhibited the cholinesterase and aryl acylamidase activities to the same extent. It is concluded that in cobra venom, acetylcholinesterase is associated with a serotonin-sensitive aryl acylamidase activity similar to earlier observations made with acetylcholinesterase from different sources.     

Characterization of a cytotoxin-like basic protein from the cobra (Naja naja naja) venom

A cytotoxin-like basic protein has been isolated from the venom of the nominate race of cobra (Naja naja naja from Pakistan) by a single step of high-performance liquid chromatography. The primary structure was determined and consists of 62 amino acid residues in a single polypeptide chain. It is highly similar to that of the cytotoxin-like basic proteins isolated from other Naja species, but differs in two of the SS-loop structures from that of cytotoxins.     

Purification of a Class B1 platelet aggregation inhibitor phospholipase A2 from Indian cobra (Naja Naja) venom

A platelet aggregation inhibitor phospholipase A(2) (NND-IV-PLA(2)) was isolated from Naja naja (Eastern India) venom by a combination of cation and anion exchange chromatography. NND-IV-PLA(2) is the most catalytically active enzyme isolated from the Indian cobra venom. The acidic PLA(2) profile of Eastern regional Indian cobra venom is distinctly different from that of the western regional venom. However the acidic PLA(2)s from both the regions follow the pattern of increasing catalytic activity with increase in acidic nature of the PLA(2) isoform. NND-IV-PLA(2) is a Class B1 platelet aggregation inhibitor and inhibits platelet aggregation induced by ADP, collagen and epinephrine. Modification of active site histidine abolishes both catalytic activity and platelet aggregation inhibition activities while aristolochic acid, a phospholipase A(2) inhibitor has only partial effect on the two activities.     

Cobra venom contains a pool of cysteine-rich secretory proteins

A large family of cysteine-rich secretory proteins (CRISPs) includes proteins of different origin, the function of the majority of CRISPs being unknown. For CRISPs isolated from snake venom, two types of activities were found: two proteins blocked cyclic nucleotide-gated ion channels, several others blocked potassium-stimulated smooth muscle contraction. Thus, snake CRISPs represent potentially valuable tools for studies of ion channels, which makes promising a search for new CRISPs. Here we report on the isolation of several novel CRISPs from the venoms of Asian cobra Naja kaouthia and African cobra Naja haje using a combination of different types of liquid chromatography. Four CRISP variants were identified in N. kaouthia venom and three proteins, one of them acidic, were found in N. haje venom. Acidic CRISP was found in a reptilian venom for the first time. Our data suggest that each cobra venom contains a pool of different CRISPs.     

Antigenic relationships between human and cobra complement factors C3 and cobra venom factor (CVF) from the Indian cobra (Naja naja)

The presence of a factor immunologically related to cobra venom factor (CVF) was demonstrated in serum and plasma from the Indian cobra (Naja naja kaoutia). The factor was purified from cobra plasma by affinity chromatography on an anti-CVF gel and was found to consist of a protein composed of two polypeptide chains similar in size to those of human C3. With use of immunoblotting technique, common antigenic determinants were found in the smaller chain of the prepared material and the beta-chain of human C3; the larger chain may display antigenic determinants present in the alpha-chain of human C3. These findings suggest that this molecule represents the C3 of the cobra complement system. Common antigenic determinants were also demonstrated in the alpha-chain of CVF and the beta-chains of human and cobra C3. No reactions were observed between the beta- and gamma-chains of CVF and any antiserum against human C3 or its subunits. Upon immunodiffusion analysis, cobra serum was found to contain a factor besides C3 sharing antigens specific for CVF, while cobra C3 was antigenically deficient compared to CVF. This suggests that cobra C3 physiologically is degraded to a molecule very similar to or identical with CVF.     

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。     

New biological activity against phospholipase A2 by Turmerin, a protein from Curcuma longa L

Turmerin is a protein from Turmeric (Curcuma longa L.) with a relative molecular mass of 14 kDa. The protein inhibits the enzymatic activity and neutralizes the pharmacological properties, such as cytotoxicity, oedema and myotoxicity of multitoxic phospholipase A2 (NV-PLA2) of cobra (Naja naja) venom at a 1:2.5 molar ratio of NV-PLA2:Turmerin. A Lineweaver-Burk plot indicates that Turmerin follows a linear mixed type of inhibition.     

Expression of neurotoxin II from Naja oxiana cobra venom in Escherichia coli in a hybrid form with thioredoxin

Neurotoxin II from the venom of cobra Naja oxiana is a short type alpha-neurotoxin, which competitively inhibits nicotinic acetylcholine receptor. The toxin gene was expressed as a construct fused with the thioredoxin gene and the linker encoding the enteropeptidase recognition site and a Met residue between the genes. The fusion protein was mainly cleaved by cyanogen bromide, since enteropeptidase was less effective. The yield of neurotoxin II was 6 mg/l of the bacterial culture. The resulting recombinant protein was identified with native neurotoxin II by its N-terminal analysis, mass spectrometry, and NMR spectroscopy. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.     

鸡血清与卵黄中抗中华眼镜蛇毒IgY动态变化研究

目的探索特异性IgY的产生和变化规律。方法用眼镜蛇毒原毒免疫产蛋母鸡,ELISA定期检测卵黄中的抗体效价变化,小鼠体外中和实验检测其生物活性。第1次免疫40周后,眼镜蛇毒攻击已免疫母鸡,检测攻击前后鸡血清中抗体效价变化情况,未经眼镜蛇毒免疫的母鸡作阴性对照。结果经免疫后第7天蛋黄中即可检测到抗体,经多次加强免疫,40周时蛋黄中还能保持高效价的抗体,通过分离纯化,此抗体可保护实验小鼠免受4 LD50眼镜蛇毒的攻击;同时,鸡血清中也保留着较高效价的抗体,可中和4 LD50以上的眼镜蛇毒。结论用眼镜蛇毒免疫鸡,经多次加强免疫,卵黄和鸡血清中可持久保持高效价的特异性抗体,初步检测此抗体可中和4 LD50的蛇毒。     

Production of potent antivenin against cobra venom with conjugated-cobrotoxin.

Cobra venom (Naja naja atra) and its fractions obtained by ammonium sulfate precipitation were subjected to chromatography on CM-Cellulose colum. A highly purified cobrotoxin obtained by the repeated chromatography on preparative CM-Cellulose column was 6.7 times more toxic than the original cobra venom. The toxin was detoxified by a bifunctional reagent, glutaraldehyde, to about 99.8% and utilized for immunization in animals. Mice received 4 weekly immunization with detoxified cobrotoxin and challenged one week after the last injection showed 60% protection in rabbits by immunization with detoxified cobrotoxin reached 360 LD50 neutralizing level against the cobra venom within 30 days. The results indicate that it is feasible and promising to prepare potent antivenin in animals by glutaraldehyde-treated cobrotoxin.     

Amino-acid sequences of four cytotoxins (cytotoxins I, II, III and IV) purified from the venom of the Thailand cobra, Naja naja siamensis

Four cytotoxins, designated as cytotoxins I, II, III and IV, were isolated from the venom of the Thailand cobra (Naja naja siamensis) by gel filtration on Sephadex G-75 followed by CM-cellulose chromatography. The amino-acid sequences were determined by a combination of conventional methods. Cytotoxins I, II, III and IV were each composed of 60 amino-acid residues and their molecular weights were calculated to be 6693, 6646, 6709 and 6739, respectively. The amino-acid sequences were compared with those of cytotoxins from other cobra venoms already sequenced.