Two Immunoregulatory Peptides with Antioxidant Activity from Tick Salivary Glands

Ticks are blood-feeding arthropods that may secrete immunosuppressant molecules, which inhibit host inflammatory and immune responses and provide survival advantages to pathogens at tick bleeding sites in hosts. In the current work, two families of immunoregulatory peptides, hyalomin-A and -B, were first identified from salivary glands of hard tick Hyalomma asiaticum asiaticum. Three copies of hyalomin-A are encoded by an identical gene and released from the same protein precursor. Both hyalomin-A and -B can exert significant anti-inflammatory functions, either by directly inhibiting host secretion of inflammatory factors such as tumor necrosis factor-alpha, monocyte chemotectic protein-1, and interferon-gamma or by indirectly increasing the secretion of immunosuppressant cytokine of interleukin-10. Hyalomin-A and -B were both found to potently scavenge free radical in vitro in a rapid manner and inhibited adjuvant-induced inflammation in mouse models in vivo. The JNK/SAPK subgroup of the MAPK signaling pathway was involved in such immunoregulatory functions of hyalomin-A and -B. These results showed that immunoregulatory peptides of tick salivary glands suppress host inflammatory response by modulating cytokine secretion and detoxifying reactive oxygen species.

Frog skins keep redox homeostasis by antioxidant peptides with rapid radical scavenging ability

The question of how amphibians can protect themselves from reactive oxygen species when exposed to the sun in an oxygen-rich atmosphere is important and interesting, not only from an evolutionary viewpoint, but also as a primer for researchers interested in mammalian skin biology, in which such peptide systems for antioxidant defense are not well studied. The identification of an antioxidant peptide named antioxidin-RL from frog (Odorrana livida) skin in this report supports the idea that a peptide antioxidant system may be a widespread antioxidant strategy among amphibian skins. Its ability to eliminate most of the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical tested within 2 s, which is much faster than the commercial antioxidant factor butylated hydroxytoluene, suggests that it has a potentially large impact on redox homeostasis in amphibian skins. Cys10 is proven to be responsible for its rapid radical scavenging function and tyrosines take part in the binding of antioxidin-RL to radicals according to our nuclear magnetic resonance assay. (C) 2010 Elsevier Inc. All rights reserved.

The application of in vitro sperm competition test to evaluate the impact of ZP-derived peptides on fertilization capacity of cat sperm

The present study aimed to establish a sensitive in vitro assay to assess the binding capacity of cat spermatozoa. Cat oocytes and epididymal sperm cells were isolated from gonads and cultured for in vitro fertilization. Before fertilization, the sperm ce

In vitro activities of small peptides from snake venom against clinical isolates of drug-resistant Mycobacterium tuberculosis

The recent re-emergence of tuberculosis, especially the multidrug-resistant cases, has highlighted the importance of screening effective novel drugs against Mycobacterium tuberculosis. In this study, the in vitro activities of small peptides isolated from snake venom were investigated against multidrug-resistant M. tuberculosis. Minimum inhibitory concentrations (MICs) were determined by the Bactec TB-460 radiometric method. A small peptide with the amino acid sequence ECYRKSDIVTCEPWQKFCYREVTFFPNHPVYLSGCASECTETNSKWCCTTDKCNRARGG (designated as vgf-1) from Naja atra (isolated from Yunnan province of China) venom had in vitro activity against clinically isolated multidrug-resistant strains of M. tuberculosis. The MIC was 8.5 mg/l. The antimycobacterial domain of this 60aa peptide is under investigation. (C) 2003 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved.

Zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) mediates multiple intracellular signaling pathways

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-kappa B activity with and without lipopolysacharide (LPS) stimulation. (C) 2008 Elsevier Ltd. All rights reserved.

Cloning, characterization and expression analysis of SIMP (source of immunodominant MHC-associated peptides) in grass carp Ctenopharyngodon idella

SIMP (source of immunodominant MHC-associated peptides) plays a key rote in N-linked glycosylation with the active site of oligosaccharyltransferase, being the source of MHC-peptides in the MHC I presentation pathway. In the present study, the SIMP gene has been cloned from grass carp Ctenopharyngodon idella by rapid amplification of cDNA ends (RACE). The full length of the cDNA sequence is 4384 bp, including a 1117 bp 5' UTR (untranslated region), a 2418 bp open reading frame, and a 849 bp 3' UTR. The deduced amino acids of the grass carp SIMP (gcSIMP) are a highly conserved protein with a STT3 domain and 11 transmembrane regions. The gcSIMP spans over more than 24,212 bp in length, containing 16 exons and 15 introns. Most encoding exons, except the first and the 15th, have the same length as those in human and mouse. The gcSIMP promoter contains many putative transcription factor binding sites, such as Oct-1, GCN4, YY1, Sp1, Palpha, TBP, GATA-1, C/EBP beta, and five C/EBP alpha binding sites. The mRNA expression of gcSIMP in different organs was examined by real-time PCR. The gcSIMP was distributed in all the organs examined, with the highest level in brain, followed by the level in the heart, liver, gill, trunk kidney, muscle, head kidney, thymus, and the lowest level in spleen. Furthermore, the recombinant gcSIMP has been constructed successfully and expressed in Escherichia coli by using pQE-40 vector, and the polyclonal antibody for rabbit has been successfully obtained, which was verified to be specific. Identification of gcSIMP will help to explore the function in fish innate immunity. (c) 2007 Elsevier Ltd. All rights reserved.

NK-lysin of channel catfish: Gene triplication, sequence variation, and expression analysis

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. In addition to the previously known four classes of antimicrobial peptides, a fifth class of antimicrobial peptides has been recently identified to include NK-lysins that have a globular three-dimensional structure and are larger with 74-78 amino acid residues. NK-lysin has been shown to harbor antimicrobial activities against a wide spectrum of microorganisms including bacteria, fungi, protozoa, and parasites. To date, NK-lysin genes have been reported from only a limited number of organisms. We previously identified a NK-lysin cDNA in channel catfish. Here we report the identification of two noveltypes of NK-lysin transcripts in channel catfish. Altogether, three distinct NK-lysin transcripts exist in channel catfish. In this work, their encoding genes were identified, sequenced, and characterized. We provide strong evidence that the catfish NK-lysin gene is tripled in the same genomic neighborhood. All three catfish NK-lysin genes are present in the same genomic region and are tightly linked on the same chromosome, as the same BAC clones harbor all three copies of the NK-lysin genes. All three NK-lysin genes are expressed, but exhibit distinct expression profiles in various tissues. In spite of the existence of a single copy of NK-lysin gene in the human genome, and only a single hit from the pufferfish,genome, there are two tripled clusters of NK-lysin genes on chromosome 17 of zebrafish in addition to one more copy on its chromosome 5. The similarity in the genomic arrangement of the tripled NK-lysin genes in channel catfish and zebrafish suggest similar evolution of NK-lysin genes. (c) 2005 Elsevier Ltd. All rights reserved.

Two cathelicidin genes are present in both rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

Further to the previous finding of the rainbow trout rtCATH_1 gene, this paper describes three more cathelicidin genes found in salmonids: two in Atlantic salmon, named asCATH_1 and asCATH_2, and one in rainbow trout, named rtCATH_2. All the three new salmonid cathelicidin genes share the common characteristics of mammalian cathelicidin genes, such as consisting of four exons and possessing a highly conserved preproregion and four invariant cysteines clustered in the C-terminal region of the cathelin-like domain. The asCATH_1 gene is homologous to the rainbow trout rtCATH_1 gene, in that it possesses three repeat motifs of TGGGGGTGGC in exon IV and two cysteine residues in the predicted mature peptide, while the asCATH_2 gene and rtCATH_2 gene are homologues of each other, with 96% nucleotide identity. Salmonid cathelicidins possess the same elastase-sensitive residue, threonine, as hagfish cathelicidins and the rabbit CAP18 molecule. The cleavage site of the four salmonid cathelicidins is within a conserved amino acid motif of QKIRTRR, which is at the beginning of the sequence encoded by exon W. Two 36-residue peptides corresponding to the core part of rtCATH_1 and rtCATH_2 were chemically synthesized and shown to exhibit potent antimicrobial activity. rtCATH_2 was expressed constitutively in gill, head kidney, intestine, skin and spleen, while the expression of rtCATH_1 was inducible in gill, head kidney, and spleen after bacterial challenge. Four cathelicidin genes have now been characterized in salmonids and two were identified in hagfish, confirming that cathelicidin genes evolved early and are likely present in all vertebrates.

Discovery and characterization of two types of liver-expressed antimicrobial peptide 2 (LEAP-2) genes in rainbow trout

The sequences and gene organisation of two LEAP-2 molecules (LEAP-2A and LEAP-2B) from rainbow trout, Oncorhynchus mykiss are presented. Both genes consist of a 3 exon/2 intron structure, with exon sizes comparable to known mammalian genes. LEAP-2A notably differs from LEAP-2B in having larger introns and a larger 3'UTR. The predicted proteins contain a signal peptide and prodomain, followed by a mature peptide of 41 aa containing four conserved cysteines. The RXXR cleavage site to release the mature peptide was also conserved. Both genes were found to be constitutively expressed in the liver, with expression in the intestine, and to a lesser extent the skin, evident after bacterial challenge. (C) 2004 Elsevier B.V. All rights reserved.

Immune Responses to Six Synthetic Peptides of Capsid Protein Immune Responses to Six Synthetic Peptides of Capsid Protein

Many B cell epitopes within p24 of human immunodeficiency virus type 1 (HIV-1) were identified, while most of them were determined by using murine monoclonal antibodies reacting with overlapping peptides of p24. Therefore these epitopes may not represent the actual epitopes recognized by the HIV-1 infected individuals. In the present study, immune responses of 67 HIV-1 positive sera from Yunnan Province, China to five peptides on p24 of HIV-1 and one of HIV-2 were analyzed. All of 67 sera did not recognize peptide GA-12 on HIV-1 and peptide AG-23 on HIV-2, which indicated that GA-12 was not human B cell epitope and AG-23 did not cross-react with HIV-1 positive serum. Except 13 sera (19.4%), all remaining sera did not recognize peptides NI-15, DR-16, DC-22 and PS-18, which indicated that these four peptides represented B cell linear epitopes of HIV-1 p24 in some HIV-1 infected individuals but not the immuno-dominant epitopes in most individuals. Cellular & Molecular Immunology. 2005;2(4):289-293.

三种两栖类动物皮肤和脑中活性多肽结构、功能与多样性分析

两栖动物是最原始的陆生脊椎动物，分布比较广泛。无尾目两栖动物现有3000 多种，它们皮肤裸露、光滑，为适应广泛的栖息地和生态条件，已进化出各种有效的皮肤防御系统。抗菌肽（Antimicrobial peptides，AMPs）作为两栖类先天防御系统的重要组成部分，在皮肤分泌液中含量异常丰富。我们以来源于云南省普洱市景东县的铃蟾科微蹼铃蟾（Bombina microdeladigitora）和楚雄州双柏县雨蛙科华西雨蛙（Hyla annectans）为实验材料，对其皮肤分泌液中抗菌肽的分子多样性并对其结构和功能进行研究。微蹼铃蟾皮肤抗菌肽多样性非常丰富，我们从单一个体中克隆得到了64 条编码不同抗菌肽的cDNA 序列，其中有两条序列只编码Maximin 一种抗菌肽，其余 62 条均编码Maximin 和Maximin H 两类抗菌肽。这64 条cDNA 序列共编码44 种Maximins 和30 种Maximin Hs，其中有32 种Maximins 和20 种Maximin Hs 为新鉴定的抗菌肽，其余和铃蟾属其它种中发现的抗菌肽具有相同的序列。除了皮肤外，两栖动物的脑也是抗菌肽的丰富资源库。我们分别从微蹼铃蟾和大蹼铃蟾（B. maxima）脑中得到了大量新的抗菌肽cDNA 序列。其中从微蹼铃蟾脑中克隆到21 条新的cDNA序列，共编码16 种Maximins 和10 种Maximin Hs，其中7 种Maximins 和4 种Maximin Hs 为新鉴定的抗菌肽。从大蹼铃蟾脑中克隆到39 条新的cDNA 序列，编码27 种Maximins 和20 种Maximin Hs，其中16 种 Maximins 和12 种Maximin Hs 为新鉴定的抗菌肽。在以上新鉴定的抗菌肽中，Maximins 均为阳离子抗菌肽，Maximin Hs 中除以前鉴定的Maximin H5 外，尚有十余种阴离子抗菌肽。抗菌肽碱基转换/颠换（R=s/v）分析表明，RMaximin<1 而RMaximin H>1，说明这两种抗菌肽碱基转换和颠换发生的几率并不相同，Maximin 间差异主要由碱基颠换引起，而Maximin H 则主要由碱基转换引起。种间进化分析表明，大蹼铃蟾和微蹼铃蟾的遗传距离较近，而它们与欧洲花铃蟾（B. variegata）的遗传距离均较远。种内各部分遗传距离差异较大。与信号肽和酸性间隔肽相比，成熟肽的遗传距离明显增大，其中Maximin 的进化速度比Maximin H 更快。抗菌肽Maximin 和Maximin H 种内、种间均存在正选择（ω>1），而信号肽和酸性间隔肽在分化过程中没有正选择（ω<1），说明Maximin 和Maximin H 经受着达尔文正选择驱动的快速进化，是抗菌肽多样性产生的根本原因。这与抗菌肽参与最终的生物防御功能，从而增加物种对环境的适应是一致的。功能研究发现，有些微蹼铃蟾Maximins 抗菌肽是多功能分子，不但对革兰氏阴性菌、革兰氏阳性菌和真菌起抗菌作用，而且还具有很强的抗氧化功能。脑中抗菌肽基因的大量表达也预示着抗菌肽可能在神经信号传导中起一定作用。用基因克隆方法我们从微蹼铃蟾皮肤得到大量缓激肽前体序列，由1-4 个拷贝的Bombinakinin 或1-4 个拷贝的Bombinakinin 和1 个拷贝的Bombinakinin-GAP 组成。这与大蹼铃蟾皮肤中缓激肽前体由1-8 个拷贝的Bombinakinin 或1-8 个拷贝的Bombinakinin 和1 个拷贝的Bombinakinin-GAP 组成有所不同。按同样方法，我们从大蹼铃蟾脑中也得到了三条缓激肽前体序列，其中两条含有6 个 Bombinakinin 拷贝，另一条含2 个Bombinakinin 拷贝。通过比较只含Bombinakinin 和同时含有Bombinakinin 和Bombinakinin-GAP 的前体cDNA 序列后发现， 前者序列中缺失了一段碱基序列TGCGGGTA， 从而导致移码突变， 终止了 Bombinakinin-GAP 的表达。通过生物化学的手段从微蹼铃蟾皮肤分泌液中分离到一种丝氨酸蛋白酶抑制剂BMSI1，与铃蟾属其它种中的胰蛋白酶抑制剂具有很高的相似性。根据已有铃蟾属丝氨酸蛋白酶抑制剂cDNA 序列设计引物，以皮肤cDNA 为模板，扩增丝氨酸蛋白酶抑制剂的基因序列，结果得到两条不同的序列。这两条前体序列与铃蟾属其它两栖动物皮肤中的丝氨酸蛋白酶抑制剂具有高度相似性（>70%），而且它们都含有10 个半胱氨酸残基。BMSI1 对五种丝氨酸蛋白水解发色底物的抑制活性测定表明，BMSI 1 能抑制胰蛋白酶和凝血酶的水解活性，其K(i)分别为0.02 μM 和0.15 μM。通过随机筛选cDNA 文库的方法，我们从大蹼铃蟾脑中得到了一条完整的 Somatostatin（SST）序列，根据该序列，我们在大蹼铃蟾和微蹼铃蟾脑cDNA 文库中筛选到两条变异体序列SST-L(Leu11-SST-14)和SST-R(Arg14-SST-14)。功能研究表明，这两种变异体具有和SST 相似的生物学功能，可抑制肿瘤细胞增殖、抑制细胞因子释放以及具有一定的镇痛作用。从大蹼铃蟾和微蹼铃蟾脑中得到了阿片肽前体POMC 和Proenkephalin 的 cDNA 序列，序列比对发现与东方铃蟾具有较高的同源性。从华西雨蛙皮肤cDNA 中克隆得到两类活性多肽，命名为Annins。其中一类为抗菌肽类似肽，共11 条序列，编码单一的成熟肽序列，其信号肽与雨蛙科信号肽具有很高的同源性，但酸性间隔肽和成熟肽相差较大。其成熟肽由15-17 个氨基酸残基组成，活性分析表明无抗菌和抗氧化作用，但在较高浓度时对部分细菌和多种细胞有促进生长作用，推测可能在使伤口快速愈合方面起重要作用；另一类编码具有2 个拷贝的成熟肽序列，成熟肽由5 个氨基酸残基组成，具有一定的镇痛活性，其镇痛机理可能是拮抗bradykinin 作用。

微生物对抗菌肽耐受的诱导及耐受机理初步研究

抗菌肽是一类具有强大杀菌能力的肽类分子，同时还具有离子调节、免疫调节、蛋白酶抑制剂和自由基清除等其他生物活性。现已鉴定的抗菌肽超过1,200 种，几乎存在于所有生物种类中。在抗生素耐受严重的今天，抗菌肽极有潜力成为新型的有效抗菌药物，许多抗菌肽已进入临床前研究或临床研究。在本论文中，我们选择了无指盘臭蛙（Odorrana grahami）来源的三种抗菌肽（Brevinin 2E-OG1、Nigrocin-OG4 和Palustrin-OG1），单独或组合使用，以藤黄微球菌（Micrococcus luteus）、枯草芽孢杆菌（Bacillus subtilis）和白假丝酵母菌（Candida albicans）为研究对象，进行微生物对抗菌肽耐受性的实验诱导；并通过检测胞外蛋白酶活性、蛋白质组学等方法对微生物耐受抗菌肽机制进行初步的研究。将微生物培养于含有低浓度抗菌肽（单独使用或组合使用）培养基中，每日转接一次，每十次酌情提高抗菌肽浓度。80 次转接后，除藤黄微球菌未对 Palustrin-OG1 产生耐受外，其余所有的实验菌株均表现出对所用三种抗菌肽的耐受。但是Palustrin-OG1 与Brevinin 2E-OG1 或Nigrocin-OG4 联合使用能在一定程度上降低耐受性。将诱导后细菌于不含抗菌肽条件下培养，转接5 次后，对耐受现象无影响，说明这种耐受是可以稳定遗传的。抗菌肽耐受机制之一是分泌蛋白酶水解胞外抗菌肽，我们通过两种方式检测胞外蛋白酶活性，一种是检测发酵液的酪蛋白水解活性，另一种是检测发酵液处理抗菌肽后对抗菌活性的影响。结果发现枯草芽孢杆菌和藤黄微球菌发酵液存在着蛋白酶活性，推测胞外蛋白酶可能与二者对抗菌肽的耐受有关；而白假丝酵母菌发酵液中未检测到蛋白酶活性。另外，我们还通过蛋白质组学的手段对枯草芽孢杆菌耐受机制进行了初步的研究，鉴定了5 个差异表达的蛋白，表达上调的蛋白有yraA（功能未知）、Tpx （巯基过氧化物酶，Thiol peroxidase）、pdhD（二氢硫辛酰胺脱氢酶，dihydrolipoamide dehydrogenase），表达下调的有cotN/TasA（芽孢膜相关蛋白，spore coat-associated protein）和gapA（三磷酸甘油醛脱氢酶，Glyceraldehyde 3-phosphate dehydrogenase 1 ，GAPDH）。yraA 和Tpx 都由Spx 调控，yraA 可以水解小肽增加自由氨基酸，而自由氨基酸增多时gapA、tasA 表达水平会下降，Spx 是由sigma-M 因子调控的，所以我们推测sigma-M 因子在B. subtiis 对抗菌肽耐受中起到了重要的作用。总之，本研究发现抗菌肽的联合作用会减缓微生物对其耐受的程度，为抗菌肽类药物研发提供了一种新思路；同时对抗菌肽耐受机制的初步研究也为今后的深入研究打下了基础。另外，我们还设计了一种新型的抗菌肽系统命名方法，并构建了昆明动物研究所抗菌肽数据库。

抗菌肽Shiva-1和CP10A的表达

抗菌肽( Antimicrobial Peptides, AMPs)是一类小分子活性肽的统称，具有抗细菌、真菌、病毒、肿瘤、内毒素和原生动物的作用，并且具有刺激单核细胞和嗜中性粒细胞的趋化、促进创伤愈合等生物学活性。目前几乎在所有的生物物种中都分离到了抗菌肽，而其主要产生于昆虫和两栖类动物体的分泌物。除了天然分离之外，也有许多人工合成的抗菌肽。抗菌肽的结构和作用机制独特，以两亲性α-螺旋和疏水性结构为主，大多通过引起质膜的透化而溶解微生物细胞。抗菌肽因其分子量小、抗菌谱广、抗菌活性高、耐热性强、不易产生抗药性、无致畸以及对高等生物正常细胞无毒害等优点而在新药开发等方面具有很大的优势和潜力。如今一些抗菌肽已经应用于临床、食品、农业等领域或者正处在临床实验和研究开发阶段。但抗菌肽通过天然分离或化学合成，目前尚不能满足人们的需求。因此本研究利用基因工程手段构建抗菌肽的表达载体，并尝试利用大肠杆菌和毕赤酵母表达对其表达，以期为抗菌肽的基因工程研究和大规模生产应用奠定一定的基础和进行一定的探索。 本论文根据文献已经报道的抗菌肽Shiva-1和CP10A，自行设计并通过PCR技术获得了其DNA序列并克隆于表达载体中，构建了毕赤酵母表达载体pPICZαA-Shiva和大肠杆菌表达载体pET-32a(+)-CP10A、pET-28a(+)-CP10A。在此基础之上，本论文进一步研究探索了这些重组表达载体的表达，成功实现了抗菌肽CP10A在大肠杆菌AD494（λDE3）菌株中的融合表达，表达产物以包涵体形式存在。在本论文的研究中，对表达的包涵体形式的融合蛋白进行了变性、Ni-NTA亲和柱层析纯化和复性研究，首次成功实现了融合蛋白TrxA-CP10A在变性条件下的纯化和复性。CP10A在大肠杆菌中的融合表达为其后续的切割和活性研究奠定了一定的基础。 本论文的研究结果为进一步探索抗菌肽的结构功能关系提供一定前提条件，也为抗菌肽的基因工程化及其开发应用奠定一定理论依据和方法学参考。