Location of intrinsic and inducible phenoloxidase activity in molluscan hemocyanin

The phenoloxidase (PO) activity of the hemocyanins (Hcs) from two molluscan species, the gastropod Helix pomatia (Hp) and the cephalopod Sepia officinalis (So), was studied. With catechol as substrate the Hcs showed a weak o-diPO activity, which was moderately enhanced on limited proteolysis with subtilisin. The sites in the Hc molecules mainly responsible for this activity were identified. The highest intrinsic o-diPO activity and also by far the highest level of induction were found in the functional units (FUs) Hp f and So g, isolated from Hp beta-Hc and So Hc (subunit 2), respectively. The results thus support the earlier conclusion, made on the basis of sequence homology between molluscan Hcs, that Hp f and So g are functional and structural analogues. The subtilisin treatment of Hp f also induced monoPO activity, considered to be at the origin of browning of the sample.     

Processing of crayfish hemocyanin subunits into phenoloxidase

Hemocyanin and phenoloxidase are both copper-binding proteins involved in the immune system for a wide range of animal species. In crayfish, these proteins were purified and characterized from plasma and hemocytes, respectively. Recently, we have reported that the processing of one of the hemocyanin subunits occurs by a proteolytic cleavage under acidic conditions which results in the release of an antibacterial peptide designated as astacidin 1 from the C-terminus [J. Biol. Chem. 278 (2003) 7927]. In the present paper, we show that cleavage of crayfish hemocyanin subunit 2 at the N-terminal part results in that the processed hemocyanin exhibits phenoloxidase activity. The calculated mass of the cloned hemocyanin 2 is 78,372Da, which corresponds to the size obtained after SDS-PAGE under reducing conditions of the purified hemocyanin and pI is estimated to be 5.70. The complete hemocyanin 2 sequence shows 74% and 44% similarity with hemocyanin 1 and prophenoloxidase of crayfish, respectively. Crayfish hemocyanin exhibited phenoloxidase activity in presence of trypsin, but no activity could be detected if treated with sodium dodecyl sulfate. These results show that hemocyanin of crayfish is involved in several immune responses such as an oxygen carrier protein, as a precursor for an antibacterial peptide, and a molecule with phenoloxidase function.     

Amino acid sequence and glycosylation of functional unit RtH2-e from Rapana thomasiana (gastropod) hemocyanin

The complete amino acid sequence of Rapana thomasiana hemocyanin functional unit RtH2-e was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of peptides obtained by cleavage with EndoLysC proteinase, chymotrypsin, and trypsin. The single-polypeptide chain of RtH2-e consists of 413 amino acid residues and contains two consensus sequences NXS/T (positions 11-19 and 127-129), potential sites for N-glycosylation. Monosaccharide analysis of RtH2-e revealed a carbohydrate content of about 1.1% and the presence of xylose, fucose, mannose, and N-acetylglucosamine, demonstrating that only N-linked carbohydrate chains of high-mannose type seem to be present. On basis of the monosaccharide composition and MALDI-MS analysis of native and PNGase-F-treated chymotryptic glycopeptide fragment of RtH2-e the oligosaccharide Man(5)GlcNAc(2), attached to Asn(127), is suggested. Multiple sequence alignments with other molluscan hemocyanin e functional units revealed an identity of 63% to the cephalopod Octopus dofleini and of 69% to the gastropod Haliotis tuberculata. The present results are discussed in view of the recently determined X-ray structure of the functional unit g of the O. dofleini hemocyanin.     

Lignans from the roots of Echinops giganteus

Two new lignans, (+)-4-(3-methylbutanoyl)-2,6-di(3,4-dimethoxy)phenyl-3,7-dioxabicyclo[3.3.0]octane and (+)-4-hydroxy-2,6-di(3,4-dimethoxy)phenyl-3,7-dioxabicyclo[3.3.0]octane, together with the known lupeol and sitosteryl beta-D-glucopyranoside, have been isolated from the roots of Echinops giganteus var. lelyi C. D. Adams (Compositae). The structures were elucidated on the basis of spectral studies and comparison with published data.     

987P fimbriae from porcine enterotoxigenic Escherichia coli: Characterization, N-terminal amino acid sequence and immunization with purified antigen

Abstract The 987P fimbrial antigen was purified from a spontaneous overproducing variant. The protein was characterized with respect to M _r, amino acid sequence and partial covalent structure. The purified protein was used for vaccination trials, and excellent protection of piglets upon challenge with 987P positive enterotoxigenic strains was seen.     

Purification, amino acid sequence and characterization of the class IIa bacteriocin weissellin A, produced by Weissella paramesenteroides DX

Weissella paramesenteroides DX has been shown to produce a 4450-Da class IIa bacteriocin, weissellin A, composed of 43 amino acids with the sequence KNYGNGVYCNKHKCSVDWATFSANIANNSVAMAGLTGGNAGN. The bacteriocin shares 68% similarity with leucocin C from Leuconostoc mesenteroides. Computational analyses predict that the bacteriocin is a hydrophobic molecule with a beta-sheet type conformation. Weissellin A exhibited various levels of activity against all gram-positive bacteria tested, but was not active against Salmonella enterica Enteritidis. The antimicrobial activity was not associated with target-cell lysis. The bacteriocin retained activity after exposure to 121 °C for 60 min or to −20 °C for 6 months, and to pH 2.0-10.0. It was not sensitive to trypsin, α-chymotrypsin, pepsin and papain, but was inactivated by proteinase K. At a dissolved oxygen concentration of 50%, weissellin A was produced with growth-associated kinetics. The properties of weissellin A make this bacteriocin a potentially suitable agent for food and feed preservation.     

Hemocyanin-derived phenoloxidase activity with broad temperature stability extending into the cold environment in hemocytes of the hair crab Erimacrus isenbeckii

Phenoloxidase (PO) activity is a major component of the innate immune response in arthropods. In this study, we characterized PO activity from the hair crab Erimacrus isenbeckii, which inhabits very cold regions (2.4-3.4°C) of the Bering Sea. Hemocyte lysate supernatant (HLS) prepared from E. isenbeckii was inactive HLS until activated by nonspecific agents such as sodium dodecyl sulfate and trypsin, and elicitors such as lipopolysaccharide and lipoteichoic acid from the cell wall constituent of bacteria. The PO activity was maximal at 4°C, decreased slightly at temperatures up to 60°C, and fell rapidly at 80°C. Both L-DOPA and catechol were efficient substrates for the PO (EC 1.10.3.1), with K(m) values of 0.96 and 1.15mM, respectively, whereas tyrosine and hydroquinone were not. We isolated a protein fraction from HLS as a hexamer of 75kDa units with 216.7-fold higher PO activity than that of the HLS. The N-terminal amino acid analysis of an isolated protein revealed 80% sequence identity to hemocyanins from other crabs. These results suggest that cold-adapted hemocyanin-derived PO activity is important to the survival of these crabs. This is the first report of a crab PO activity with broad temperature stability extending into the cold environment.     

Functional properties of hemocyanin from Oncomelania hupensis, the intermediate host of Schistosoma japonicum

The gastropod mollusc, Oncomelania hupensis is a unique intermediate host for the human parasite Schistosoma japonicum. It is a primary factor for the epidemic of schistosomiasis and its distribution is consistent with the epidemic area of schistosomiasis. Here we report the functional properties of hemocyanin of O. hupensis (OhH), a copper-containing respiratory protein which was isolated from its hemolymph and purified by ammonium sulfate fractionation and ultracentrifugation. We identified the protein characters including UV absorption at 340 nm, copper content and quaternary structure. Furthermore, by induction of phenoloxidase and enzyme-linked immunosorbent assay we show that OhH exhibited o-diphenoloxidase activity after limited proteolysis, and shared carbohydrate epitopes with glycoconjugates of S. japonicum.     

Isolation, complete amino acid sequence and characterization of a previously unreported post-synaptic neurotoxin - AlphaN3, from the venom of Bungarus candidus

The Malayan krait (Bungarus candidus) is one of the medically most important snake species in Southeast Asia. The venom from this snake has been shown to posses both presynaptic and post-synaptic neurotoxins. We have isolated a previously uncharacterized post-synaptic neurotoxin - alphaN3 from the venom of B. candidus. Isolation of the toxin was achieved in three successive chromatography steps - gel filtration on a Sephadex G75 column, followed by ion exchange chromatography (Mono-S strong cationic exchanger) and a final reverse-phase chromatography step (PRO-RPC C18 column). Purified toxin alphaN3 was shown to have an apparent molecular weight of ∼7 to 8 kDa on SDS-PAGE. The complete amino acid sequence of toxin alphaN3 was determined by Edman degradation and was found to share a high degree of homology with known post-synaptic neurotoxins (93% with alpha-bungarotoxin from Bungarus multicinctus, 50% with alpha cobratoxin from Naja kaouthia). The intravenous LD₅₀ of toxin alphaN3 was determined to be 0.16 ± 0.09 μg/g in mice which is comparable to alpha-bungarotoxin from B. multicinctus. Experiments with isolated nerve-muscle preparations suggested that toxin alphaN3 was a post-synaptic neurotoxin that produced complete blockade of neuromuscular transmission by binding to nicotinic acetylcholine receptors.     

Large differences in amino acid sequences among ferredoxins from several species of genus Solanum

The complete amino acid sequences of [2Fe-2S] ferredoxins from four species of genus Solanum (S. nigrum, S. lyratum, S. indicum, and S. abutiloides) were determined by automated Edman degradation of the entire S-carboxymethylcysteinyl proteins and of the peptides obtained by enzymatic digestion. The amino acid sequences of these four ferredoxins differed from each other by 12-19, whereas 0-4 differences have been observed among ferredoxins from plants in the same genus and 14-40 differences were seen between different families. This suggests that these Solanum plants are distantly related to each other taxonomically.     

豇豆几丁质酶N端序列测定及与其它植物的比较

通过自动 Edman降解程序 ,测定了经诱导、纯化的豇豆几丁质酶 N端 1 0个氨基酸的序列 ,并将该序列与其它植物几丁质酶 N端相应部分的氨基酸序列进行了比较分析。结果表明 ,该豇豆 ( Vigna sesquipedalis)几丁质酶 N端 1 0个氨基酸的序列为 EQCGSQAGGA,与 类几丁质酶同一部分同感序列同源性高达 1 0 0 % ;而与 、 及 类几丁质酶的相应序列均无同源性。结合考虑此酶的等电点 ( 8.3)及分子量 ( 33k D) ,可推测该豇豆几丁质酶属于 类几丁质酶。其 N端序列的高度保守性提示 ,该段序列可作为 类几丁质酶的一段主要特征序列 ,并可据其合成核酸探针 ,以分离、克隆其它 类几丁质酶编码基因。     

The amino acid sequence of plastocyanin from Rumex obtusifolius

The amino acid sequence of plastocyanin from dock has been completed. It is a single polypeptide chain of 99 residues which is closely related to other plant plastocyanins. Compared to a preliminary sequence presented earlier, the completed sequence now shows two changes, at positions 53 and 92.     

The amino acid sequence of ferredoxin from Sambucus nigra

The amino acid sequence of the ferredoxin from Sambucus nigra consists of a single polypeptide chain of 97 amino acid residues, 5 of which are cysteine. The positions of the 4 cysteine residues which bind the iron atoms of the active centre are identical to those of other ferredoxins. Due to difficulties of obtaining pure protein, residues 87–90 have only been identified from the amino acid analysis of peptide C 10 and by homology with other higher plant ferredoxins.     

The amino acid sequence of plastocyanin from Cucumis sativus

The amino acid sequence of plastocyanin from cucumber (Cucumis sativus) has been determined. Analysis was by the dansyl—phenylisothiocyanate meth     

红螯螯虾酚氧化酶原及其特性研究

采用同源克隆策略和RACE技术, 从红螯螯虾Cherax quadricarinatus血细胞中克隆得到酚氧化酶原基因的全长cDNA序列, 共2951 bp, 开放读码框为1995 bp, 编码665个氨基酸. 预测的分子量和等电点分别为75.7 kD和6.23. 酚氧化酶原含有两个推测的tyrosinase copper-binding motifs (带有六个组氨酸残基)和一个thiol-ester-like motif, 这些特征和其他甲壳动物的酚氧化酶原特征相同. 红螯螯虾酚氧化酶原氨基酸序列与通讯螯虾Pacifastacus leniusculus、欧洲龙虾Homarus gammarus、美洲龙虾Homarus americanus 和克氏原螯虾Procambarus clarkii 酚氧化酶原的相似率分别为68%、63%、63%和59%. 酚氧化酶原基因双酶切后连接入pET-28a原核表达载体, 转化到大肠杆菌BL21后重组表达酚氧化酶原蛋白. 在重组蛋白纯化后, 免疫新西兰大耳兔制备得到的酚氧化酶原多克隆抗体, 其效价大于1:12800. 红螯螯虾血淋巴、肝和鳃组织中的酚氧化酶原mRNA表达和酚氧化酶活性较高, 而神经、心、肠和肌肉中较低. 中华绒螯蟹螺原体和嗜水气单胞菌免疫红螯螯虾后, 血淋巴细胞、肝和鳃组织中的酚氧化酶原和酚氧化酶活性在免疫后的不同时间均出现了显著性的增加, 此结果表明酚氧化酶原和酚氧化酶在红螯螯虾对抗细菌感染的过程中起到重要的免疫作用. 此结果为进一步深入研究酚氧化酶原基因和酚氧化酶的功能及其调控机理奠定基础.       

Predicting membrane protein types by fusing composite protein sequence features into pseudo amino acid composition

Membrane proteins are vital type of proteins that serve as channels, receptors, and energy transducers in a cell. Prediction of membrane protein types is an important research area in bioinformatics. Knowledge of membrane protein types provides some valuable information for predicting novel example of the membrane protein types. However, classification of membrane protein types can be both time consuming and susceptible to errors due to the inherent similarity of membrane protein types. In this paper, neural networks based membrane protein type prediction system is proposed. Composite protein sequence representation (CPSR) is used to extract the features of a protein sequence, which includes seven feature sets; amino acid composition, sequence length, 2 gram exchange group frequency, hydrophobic group, electronic group, sum of hydrophobicity, and R-group. Principal component analysis is then employed to reduce the dimensionality of the feature vector. The probabilistic neural network (PNN), generalized regression neural network, and support vector machine (SVM) are used as classifiers. A high success rate of 86.01% is obtained using SVM for the jackknife test. In case of independent dataset test, PNN yields the highest accuracy of 95.73%. These classifiers exhibit improved performance using other performance measures such as sensitivity, specificity, Mathew's correlation coefficient, and F-measure. The experimental results show that the prediction performance of the proposed scheme for classifying membrane protein types is the best reported, so far. This performance improvement may largely be credited to the learning capabilities of neural networks and the composite feature extraction strategy, which exploits seven different properties of protein sequences. The proposed Mem-Predictor can be accessed at http://111.68.99.218/Mem-Predictor.     

The amino acid sequence of cytochrome c from niger-seed, Guizotia abyssinica

The amino acid sequence of cytochrome c from niger-seed has been determined by sequence analysis of chymotryptic and tryptic peptides using the dansyl-phenylisothiocyanate method and by qualitative analysis of peptide composition by the dansyl method. Although the spectral ratios indicated the protein was not completely pure, no indication of impurity was found during the sequence analysis and no peptides in addition to those given here were obtained. In certain cases the alignment of peptides was by homology with other cytochromes c. Four residues in the proposed sequence, alanine-1, cysteine-25, histidine-26 and lysine-61 were identified only from peptide compositions. The amino-terminus of the protein is acetylated. The sequence contains two residues of ϵ-N-trimethyllysine.     

Complete amino acid sequence of three reptile lysozymes

To study the structure and function of reptile lysozymes, we have reported their purification, and in this study we have established the amino acid sequence of three egg white lysozymes in soft-shelled turtle eggs (SSTL A and SSTL B from Trionyx sinensis, ASTL from Amyda cartilaginea) by using the rapid peptide mapping method. The established amino acid sequence of SSTL A, SSTL B, and ASTL showed substitutions of 43, 42, and 44 residues respectively when compared with the HEWL (hen egg white lysozyme) sequence. In these reptile lysozymes, SSTL A had one substitution compared with SSTL B (Gly126Asp) and had an N-terminal extra Gly and 11 substitutions compared with ASTL. SSTL B had an N-terminal extra Gly and 10 residues different from ASTL. The sequence of SSTL B was identical to soft-shelled turtle lysozyme from STL (Trionyx sinensis japonicus). The Ile residue at position 93 of ASTL is the first report in all C-type lysozymes. Furthermore, amino acid substitutions (Phe34His, Arg45Tyr, Thr47Arg, and Arg114Tyr) were also found at subsites E and F when compared with HEWL. The time course using N-acetylglucosamine pentamer as a substrate exhibited a reduction of the rate constant of glycosidic cleavage and increase of binding free energy for subsites E and F, which proved the contribution for amino acids mentioned above for substrate binding at subsites E and F. Interestingly, the variable binding free energy values occurred on ASTL, may be contributed from substitutions at outside of subsites E and F.