The position of the disulfide bonds in human plasma alpha 2 HS-glycoprotein and the repeating double disulfide bonds in the domain structure

The positions of the inter- and intra-chain disulfide bonds of human plasma alpha 2 HS-glycoprotein were determined. alpha 2 HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)--Cys-14 (A-chain), Cys-71--Cys-82, Cys-96--Cys-114, Cys-128--Cys-131, Cys-190--Cys-201 and Cys-212--Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of alpha 2 HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the S--S bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two S--S bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second S--S loops and the end of domains A and B, and the positions of the ordered structures.     

The position of the disulfide bonds in human plasma α2HS-glycoprotein and the repeating double disulfide bonds in the domain structure

The positions of the inter- and intra-chain disulfide bonds of human plasma α₂HS-glycoprotein were determined. α₂HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)-Cys-14 (A-chain), Cys-71-Cys-82, Cys-96-Cys-114, Cys-128-Cys-131, Cys-190-Cys-201 and Cys-212-Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of α₂HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the SS bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two SS bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second SS loops and the end of domains A and B, and the positions of the ordered structures.     

Isolation and identification of paralytic peptides from hemolymph of the lepidopteran insects Manduca sexta, Spodoptera exigua, and Heliothis virescens

Seven paralytic peptides were isolated and identified from lepidopteran hemolymph. All of these peptides cause rapid, rigid paralysis when injected into Manduca sexta and some other lepidopteran larvae. Each peptide contains 23 amino acid residues including 2 cysteines and the carboxyl termini are acidic. Synthetic peptides in the disulfide or reduced forms, and as carboxyl-terminal acids or amides were equally paralytic. The most potent paralytic peptide, Mas PP I, has the following sequence: H-Glu-Asn-Phe-Ala-Gly-Gly-Cys-Ala-Thr-Gly-Tyr-Leu- Arg-Thr-Ala-Asp-Gly-Arg-Cys-Lys-Pro-Thr-Phe-OH. The two peptides from M. sexta hemolymph are remarkable in that they are autoparalytic (i.e. factors in collected hemolymph that are paralytic when injected into the same larvae).     

A hemolymph major anionic peptide, HemaP, motivates feeding behavior in the sweetpotato hornworm, Agrius convolvuli

We recently identified a novel feeding-modulating peptide, hemolymph major anionic peptide (HemaP), designated Bommo-HemaP (B-HemaP), from hemolymph of the silkworm Bombyx mori. B-HemaP has a unique biological activity in modulating the regular frequency of feeding motivation, which is accompanied by increased foraging behaviors. To confirm the conservation of the HemaP-regulated feeding mechanism in lepidopteran species, we purified and sequenced two candidate peptides from the hemolymph of larvae of the sweet potato hornworm Agrius convolvuli. Unlike B. mori, A. convolvuli had two forms of HemaP, which were designated Agrco-HemaP-1 (A-HemaP-1) and Agrco-HemaP-2 (A-HemaP-2). The amino acid sequence of A-HemaP-2 was identical with that of A-HemaP-1, except for O-glycosylation on the fifth amino acid, threonine, within the N-terminal region. The amino acid sequence of A-HemaP-1/A-HemaP-2 had only 32% identity with B-HemaP. Structural analysis revealed that the carbohydrate moiety of A-HemaP-2 was an α-GalNAc residue. Injection of A-HemaP-1, A-HemaP-2 and recombinant A-HemaP-1 (rA-HemaP-1) individually caused a significant increase in foraging behaviors in A.?convolvuli larvae, and no significant differences were observed among these three A-HemaPs. The CD spectra of these three A-HemaPs were quite similar, and all had α-helix-rich secondary structures. Although A-HemaP-1 and B-HemaP did not exhibit cross-reactivity at any injection doses examined, HemaP might be a conserved molecule among lepidopteran species that can modulate feeding motivation through the fluctuation of peptide levels in hemolymph.     

IN VITRO ANTIVIRAL ACTIVITY OF AN ALKALINE TRYPSIN FROM THE DIGESTIVE JUICE OF Bombyx mori LARVAE AGAINST NUCLEOPOLYHEDROVIRUS

Alkaline trypsin protein of molecular mass 25,436 Da purified from the digestive juice of Bombyx mori larvae indicated strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) under in vitro conditions. Partial N-terminal amino acid sequence of the protein was determined and the cDNA was cloned based on the amino acid sequence. A homology search of the deduced amino acid sequence of the cDNA showed 55% identity with Helicoverpa armigera trypsin and the active site of this protein was completely conserved. Hence, the protein was designated B. mori trypsin (Bmtryp). The results suggest that Bmtryp, an insect digestive enzyme, can be a potential antiviral factor against BmNPV at the initial site of viral infection.     

Separation of Bombyxin from a neuropeptide of Bombyx mori showing Summer-morph-producing Hormone (SMPH) activity in the Asian Comma Butterfly,Polygonia c-aureum L

A neuropeptide from brain-suboesophageal ganglion (Br-SG) complexes of the silkmoth, Bombyx mori, shows summer-morph-producing hormone (SMPH) activity in the Asian comma butterfly, P. c-aureum. The SMPH-active peptide was extracted and demonstrated to be almost the same molecular size as bombyxin (4-5kD), a nueropeptide which shows prothoracicotropic hormone (PTTH) activity when assayed in vitro with prothoracic glands (PGs) of 4th-instar B. mori larvae in vitro. A Sephadex G-50 fraction of 3-8kD molecules prepared from Br-SG complexes of B. mori adults was applied to CM-, SP-, DEAE- or QAE- Toyoperal columns at pH 5.6 (or pH 6.9). The SMPH-activity could be separated from the PTTH-activity (or bombyxin) by subjecting a SMPH- and PTTH-active preparation of B. mori to anion-exchange chromatography at pH 6.9. By reversed-phase HPLC following an anion-exchange chromatography, SMPH-activity was recovered in two fractions of 40-45% acetonitril. Results demonstrate that the B. mori peptide showing the SMPH-activity in P. c-aureum is a different molecule than bombyxin.     

降钙素基因相关肽的合成

降钙素基因相关肽是一个包含有37个氨基酸残基的具有较强降血压生理功能的活性多肽。我们采用常规的固相合成方法,以简单的装置最后经无水氟化氢处理,将肽从树脂上切下,同时脱除所有侧链保护基。粗产物在30％乙酸溶液中用碘作为氧化剂使二个半胱氨酸氧化,形成二硫键。合成的α-人降钙素基因相关肽经反向高效液相层析分离,获得在高效液相层析为单一峰的产物。经酸水解氨基酸分析证明与理论值相符并具有全部生理活性。     

家蚕瘫痪肽结合蛋白基因克隆与分析

ENF肽家族具有保守的N末端结构（Glu—Asn—Phe-）。该家族成员肽大多具有重叠功能活性,在鳞翅目昆虫的免疫反应,生长调控和自体调节等方面都发挥着重要的作用。在昆虫的免疫反应中,血细胞尤其是淋巴液的黏附性是针对外来侵入物的免疫应答过程中的重要因素。家蚕瘫痪肽（paralytic peptide）是ENF肽家族的一种,其具有多种的生物学活性,包括致瘫痪性及在家蚕血细胞免疫反应中的促吞噬细胞扩散活性。ENF肽家族的另一成员,粘虫（Pseudaletia separata）的生长阻抑肽（Growth-blocking peptide）,同家蚕瘫痪肽一样能够在粘虫的血细胞免疫反应中起到调节吞噬细胞的功能。目前,关于昆虫细胞免疫应答的终端调控分子机制的研究还比较少,有文献报道粘虫的生长阻抑肽结合蛋白（GBP—BP）能够起到沉默生长阻抑肽活性的功能,从而可能参与调节细胞免疫应答的终端调控。在本研究中,利用荧光差异显示技术（FDD）分析了家蚕感染BmNPV病毒后基因表达差异情况,在血淋巴中获得了一条差异条带G12782＊通过5＇-RACE技术,首次在家蚕中克隆得到了该基因的全长cDNA序列。通过同源性分析得知,该基因所编码的蛋白质与粘虫的生长阻抑肽结合蛋白具有很大的同源性,并被命名为家蚕瘫痪肽结合蛋白（Bmori paralytic peptide binding protein,PP-BP）。通过RT-PCR研究发现,该蛋白基因在血淋巴中大量表达。同时,利用实时荧光定量PCR（Real-time quantitative PCR）技术分析了该基因在正常饲养家蚕与添食BmNPV病毒的家蚕中的表达差异,结果显示该基因在家蚕添食BmNPV病毒后的表达量大大增强,这就暗示该基因可能与BmNPV病毒刺激后所引起的家蚕血液细胞免疫反应相关。利用生物信息学方法对该基因的结构进行了分析,发现该基因具有两个外显子和一个内含子。这个基因已经登入GenBank数据库,收入号为DQ306881。     

Isolation of a cDNA encoding a CHH-family peptide from the silkworm Bombyx mori

The crustacean hyperglycemic hormone (CHH) peptide family includes four types of neuropeptide in decapod and isopod crustaceans, and the ion-transport peptide in orthopteran insects. To identify a new member of this family in Insecta, a PCR-based search for cDNAs encoding CHH-family peptides was carried out in the silkworm Bombyx mori. A cDNA, named BmCHHL (Bombyx mori CHH-like protein), with an open reading frame of 110 amino acids was isolated. Sequence analyses suggested that the conceptual protein was a precursor of a peptide of 72 amino acids which was amidated at the carboxy terminus. The BmCHHL sequence exhibited significant similarities to members of the CHH family including the orthopteran ion-transport peptide. BmCHHL expression was detected in five or six cells (per hemisphere) in the frontal area of the brain in day 4 fifth instar larvae.     

Structural and functional analysis of the Xestia c-nigrum granulovirus matrix metalloproteinase

Sequence analysis of the Xestia c-nigrum granulovirus (XcGV) genome identified an open reading frame encoding a 469-amino-acid (54-kDa) protein with over 30% amino acid sequence identity to a region of about 150 amino acids that includes the catalytic domains of human stromelysin 1 (Str1)/matrix metalloproteinase 3 (MMP-3) (EC 3.4.24.17) and sea urchin hatching enzyme (HE). Stromelysin homologs have not been reported from baculoviruses or other viruses. Unlike human Str1 and sea urchin HE, the putative XcGV-MMP does not have a signal peptide and lacks the peptide motif involved in the cysteine switch that maintains other MMPs in an inactive form. The putative XcGV-MMP, however, possesses a conserved zinc-binding motif in its putative catalytic domain. The XcGV-MMP homolog was cloned, and a recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) that expresses XcGV-MMP under the polyhedrin promoter was constructed. A distinct pattern of melanization was observed in B. mori larvae infected with MMP-expressing BmNPV. Fat body extracts from larvae overexpressing the 54-kDa recombinant MMP digested dye-impregnated collagen (Azocoll). The enzymatic activity was inhibited by two metalloproteinase inhibitors, EDTA and 1,10-phenanthroline. These results suggest that the XcGV MMP-3 gene homolog encodes a functional metalloproteinase.     

The 30kP protease A responsible for 30-kDa yolk protein degradation of the silkworm, Bombyx mori: cDNA structure, developmental change and regulation by feeding

We have cloned and sequenced the cDNA encoding the major component (43-kDa peptide) of 30kP protease A which selectively hydrolyzes 30-kDa yolk proteins of the silkworm, Bombix mori. The deduced amino acid sequence consisted of 318 amino acids and shared sequences conserved in many serine proteases. Northern blot analysis using the cDNA as probe revealed that 43-kDa peptide mRNA began to rise at the last phase of embryogenesis and reached a maximum level at larval hatching. This level was maintained with some fluctuations throughout post-embryonic development. The concentration of 43-kDa peptide increased greatly toward larval hatching coinciding with the changing pattern of mRNA. When larvae were fed, the peptide concentration abruptly decreased and remained near zero throughout post-embryonic development. The decrease in peptide concentration did not occur, however, when the hatched larvae were starved. Thus, the nutritional shift from endogenous yolk to exogenous food plays a key role in 30kP protease A elimination from neonate larvae.     

Beta-1,3-glucan inducible expression of prophenoloxidase-activating proteinase from eri-silkworm, Samia cynthia ricini

A cDNA clone encoding possible prophenoloxidase-activating serine protease (PAP) was isolated by screening the cDNA library from immunized larval fat body of the wild silkmoth, Samia cynthia ricini. The cDNA encodes a 438 amino acid open reading frame with a predicted 20 residue signal peptide. Samia PAP has high sequence similarity to Bombyx mori and Manduca sexta PAPs, which contain two amino terminal clip domains followed by a carboxyl-terminal catalytic domain. The expression of the gene was barely detectable in the fat body of naive larvae, but induced after injection of the larvae with beta-1,3-glucans or bacterial cells.     

Oxyopinins, large amphipathic peptides isolated from the venom of the wolf spider Oxyopes kitabensis with cytolytic properties and positive insecticidal cooperativity with spider neurotoxins

Five amphipathic peptides with antimicrobial, hemolytic, and insecticidal activity were isolated from the crude venom of the wolf spider Oxyopes kitabensis. The peptides, named oxyopinins, are the largest linear cationic amphipathic peptides from the venom of a spider that have been chemically characterized at present. According to their primary structure Oxyopinin 1 is composed of 48 amino acid residues showing extended sequence similarity to the ant insecticidal peptide ponericinL2 and to the frog antimicrobial peptide dermaseptin. Oxyopinins 2a, 2b, 2c, and 2d have highly similar sequences. At least 27 out of 37 amino acid residues are conserved. They also show a segment of sequence similar to ponericinL2. Circular dichroism analyses showed that the secondary structure of the five peptides is essentially alpha-helical. Oxyopinins showed disrupting activities toward both biological membranes and artificial vesicles, particularly to those rich in phosphatidylcholine. Electrophysiological recordings performed on insect cells (Sf9) showed that the oxyopinins produce a drastic reduction of cell membrane resistance by opening non-selective ion channels. Additionally, a new paralytic neurotoxin named Oxytoxin 1 was purified from the same spider venom. It contains 69 amino acid residue cross-linked by five disulfide bridges. Application of mixtures containing oxyopinins and Oxytoxin 1 to insect larvae showed a potentiation phenomenon, by which an increase lethality effect is observed. These results suggest that the linear amphipathic peptides in spider venoms and neuropeptides cooperate to capture insects efficiently.     

Purification and characterization from human parotid secretion of a peptide which inhibits hemagglutination of Bacteroides gingivalis 381

A peptide from human parotid secretion which inhibited hemagglutination of Bacteroides gingivalis 381 was purified by ultrafiltration followed by DEAE-Sephadex A-25 column chromatography and by gel filtration on Sephadex G-25, and then by reversed-phase HPLC. The complete amino acid sequence of the peptide, determined by automated Edman degradation was as follows; Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr. The peptide contained 12 residues and the charged amino acids predominated with 4 histidine, 2 lysine, 1 arginine and 1 glutamic acid residues, thus being a histidine-rich peptide. The peptide was an active inhibitor of the hemagglutinating activity of B. gingivalis. Specific binding of tritium-labeled peptide to B. gingivalis cells was demonstrated. These results suggest that the histidine-rich peptide may function as a binding domain for the hemagglutinins of B. gingivalis during agglutination.     

家蚕Wnt信号通路下游关键基因Pangolin转录剪接体X3的克隆和分析

【目的】克隆家蚕Bombyx mori Wnt信号通路下游关键基因Pangolin isoforms A/H/I/S转录剪接体X3 (Pangolin X3),分析其序列和表达特征。【方法】从NCBI数据库检索家蚕Pangolin X3,根据其编码序列(coding sequence, CDS)设计引物,利用PCR从家蚕幼虫中肠和血淋巴中进行克隆并测序验证。利用SilkDB 3.0, SMART,多序列比对和系统发育树分析Pangolin X3的序列特征。利用qRT-PCR分析Pangolin X3在家蚕5龄第3天幼虫不同组织(头、血淋巴、体壁、性腺、中肠、前部丝腺、中部丝腺、后部丝腺、脂肪体和马氏管)中的相对表达水平。【结果】从家蚕幼虫中肠和血淋巴克隆了Pangolin X3(GenBank登录号：XM＿038020921)的CDS,其开放阅读框长1 560 bp,编码519个氨基酸残基,预测分子量为55.86 kD,预测等电点为7.53。Pangolin X3蛋白含有保守的β-catenin结合位点和HMG结构域,其氨基酸序列在不同的昆虫中比较保守,特别是与DNA结合的HMG结构域...     

A new cyclolinopeptide containing nonproteinaceous amino acid N-methyl-4-aminoproline

We have found that besides the known cyclolinopeptides A (CLA) and B (CLB), there is a new cyclic peptide in linseed mill cake that we have named CLX. Its composition is very similar to that of CLA, a cyclic peptide with a distinct immunosuppressive activity. The sequence of this peptide has been established as cyclo(PPFFILLX), where X is a non-proteinaceous amino acid, N-methyl-4-aminoproline. This amino acid substitutes for two amino acid residues of CLA, mimicking a dipeptide moiety with a nonplanar cis amide bond. The non-proteinaceous amino acid X may mimic a transition state of the peptide bond which exists in such processes as, e.g., PPIase-catalysed cis/trans amide-Pro bond isomerisation.     

A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus.     

家蚕抗菌肽CMIV基因结构改造及表达产物的研究

参照天然抗菌肽ＣＭＩＶ组分的氨基酸序列,作了近５０％的改动,根据大肠杆菌偏爱的密码子,设计并人工合成了抗菌肽基因片段．将人工合成的抗菌肽类ＣＭＩＶ基因先重组到测序载体ｐＵＣ１１８上,经过序列分析,发现克隆于载体ｐＵＣ１１８上的基因片段与设计的序列完全一致．再将该基因片段重组到表达载体ｐＥＴ２８（ａ）上,抗菌肽以融合蛋白的形式表达．融合蛋白经镍－金属离子胶亲和层析纯化后,再用ＣＮＢｒ裂解,最终产物具有与天然抗菌肽相同的生物学活性