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Beta-defensin 1, pig 是主要在猪舌黏膜中发现的一种抗菌肽。Beta-defensin 1, pig 对大肠杆菌，鼠伤寒沙门氏菌，单核细胞增生性李斯特菌，金黄色葡萄球菌，百日咳博德特氏菌和白色念珠菌等细菌具有活性。
Beta-defensin 1, pig is an antimicrobial peptide found primarily in tongue mucosa of pig. Beta-defensin 1, pig is active against bacteria such as Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Bordetella pertussis and Candida albicans[1][2].

定义
防御素是具有广泛抗菌活性的小型抗菌肽（AMP）。它在宿主防御感染，炎症，伤口修复和获得性免疫中起重要作用

发现
在1960年代研究兔和豚鼠白细胞裂解物的抗菌活性时，发现了防御素。所谓的富含精氨酸的阳离子肽是由其高阴极电泳活性迁移率定义的，并因其分离和详细的化学表征而引起了人们的关注1。

分类

哺乳动物防御素可根据其结构上的差异被细分为三个主要类别：所述一个防御素，b防御素和q防御素。一个防御素具有广泛的 抗革兰氏阴性菌和革兰氏阳性抗菌活性 细菌，真菌和包膜病毒。b-防御素 主要对革兰氏阴性细菌和酵母具有活性。q-防御素是一种 在猕猴白细胞中发现的含有18个氨基酸和3个二硫键的环状肽2。三种防御素（人类嗜中性粒细胞防御素[HNP] -1，HNP-2和HNP-3）构成人类多形核嗜中性粒细胞（PMN）3的嗜铁粒颗粒中总蛋白质的30-50％。

结构特征
防御素是小的半胱氨酸富含阳离子的蛋白质与18-45个氨基酸，并用3.4〜4.5 kDa的分子量。所有防御素共有一个特征性的三个二硫键基序。这些半胱氨酸二硫键对于防御素的生物活性至关重要。

行动方式
抗菌 活性的具体机制涉及细菌膜的透化作用。它 已经假定各个单体低聚以形成 通过阴离子膜的孔径，虽然证据仅仅是 间接4。

防御素的杀微生物活性是通过阴离子脂质双层的透化作用和随后 细胞内含物的释放而实现的。防御素和 细菌膜之间的相互作用主要受静电力控制。透化的一种机理被认为涉及 细菌膜中离子孔的形成。第二种模型也称为地毯模型，它假定防御素被认为聚集成带正电荷的贴剂 ，该贴剂可 在肽周围的较大区域中和膜的阴离子脂质头基。这种中和破坏 了脂质双层的完整性，导致 出现瞬时间隙并允许离子渗透到膜4中。

功能
防御素是免疫细胞响应细菌感染而产生的抗菌肽。它还在阻止人类宿主细胞对HIV 5的侵袭中起作用，并在小鼠6中诱导针对肿瘤的细胞介导的免疫反应。各种防御素对单核细胞，T细胞和树突状细胞具有趋化活性。 细胞在先天宿主防御过程中产生的防御素可作为 启动，动员和增强适应性免疫宿主防御的信号。

参考

1.     Ganz.T (2003). Defensins: Antimicrobial peptides of innate immunity. Nature, 3,710-720.

2.     Schneider JJ, Unholzer A, Schaller M, Schäfer-Korting M, Korting HC (2005). Human defensins. J Mol Med, 83(8), 587-595.

3.     R I Lehrer, A Barton, K A Daher, S S Harwig, T Ganz, and M E Selsted (1989). Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity. J Clin Invest, 84(2), 553–561.

4.     Hoover DM, Rajashankar KR, Blumenthal R, Puri A, Oppenheim JJ, Chertov O, Lubkowski J (2000). The structure of human beta-defensin-2 shows evidence of higher order oligomerization. J Biol Chem, 275(42), 32911-32918.
5.     Zhang L, Yu W, He T, Yu J, Caffrey RE, Dalmasso EA, Fu S, Pham T, Mei J, Ho JJ, Zhang W, Lopez P, Ho DD (2002). Contribution of human alpha-defensin 1, 2, and 3 to the anti-HIV-1 activity of CD8 antiviral factor. Science, 303 (5657), 467.
6.     Biragyn A, Ruffini PA, Leifer CA, Klyushnenkova E, Shakhov A, Chertov O, Shirakawa AK, Farber JM, Segal DM, Oppenheim JJ, Kwak LW. Toll-like receptor 4-dependent activation of dendritic cells by beta-defensin 2. Science, 298(5595), 1025-1029.

Definition
Defensins are small antimicrobial peptide (AMP) with a broad spectrum of antibacterial activity. It plays an important role in host defenses against infections, inflammation, wound repair and acquired immunity

Discovery
Defensins were discovered when the antimicrobial activity of rabbit and guinea-pig leukocyte lysates were studied in the 1960’s. The so-called arginine- rich cationic peptides were defined by their high cathodal electrophorectic activity mobility and attracted attention because of their isolation and detailed chemical characterization1.

Classification

The mammalian defensins can be subdivided into three main classes according to their structural differences: the a-defensins, b-defensins and q-defensins. a-Defensins have broad antimicrobial activity against Gram-negative and Gram-positive bacteria, fungi, and enveloped viruses. b-Defensins are mainly active against Gram-negative bacteria and yeast. q-Defensin is a cyclic peptide containing 18 amino acids with three disulfides discovered in macaque leukocytes2. Three defensins (human neutrophil peptide defensin [HNP]-1, HNP-2, and HNP-3) constitute between 30-50% of the total protein in azurophil granules of human polymorphonuclear neutrophils (PMN)3.

Structural Characteristics
Defensins are small cysteine-rich cationic proteins with 18-45 amino acids and with a molecular weight of 3.4 to 4.5 kDa. All defensins share a characteristic three disulfide bond motif. These cysteine disulfide bonds are essential for the biological activities of defensins.

Mode of action
The specific mechanism of antimicrobial activity involves permeabilization of bacterial membranes. It has been postulated that individual monomers oligomerize to form a pore through anionic membranes, although the evidence is only indirect4.

The microbicidal activity of defensins is brought about by permeabilization of anionic lipid bilayers and the subsequent release of cellular contents. Interactions between defensins and bacterial membranes are governed mainly by electrostatic forces. One mechanism of permeabilization is thought to involve the formation of ion pores in bacterial membranes. The second model also known as carpet model assumes that the defensins are thought to aggregate into positively charged patches that neutralize anionic lipid head groups of the membrane over a wide area around the peptides. This neutralization disrupts the integrity of the lipid bilayer, causing transient gaps to arise and allowing ions to permeate the membrane4.

Functions
Defensins are antimicrobial peptides produced by immune cells in response to bacterial infection. It also functions in blocking of human host cell invasion against HIV5 and the induction of cell-mediated immune responses against tumors in mice6. Various defensins have chemotactic activity for monocytes, T cells and dendritic cells. Defensins produced by cells in the course of innate host defense serve as signals which initiate, mobilize, and amplify adaptive immune host defenses.

References 
1.     Ganz.T (2003). Defensins: Antimicrobial peptides of innate immunity. Nature, 3,710-720.

2.     Schneider JJ, Unholzer A, Schaller M, Schäfer-Korting M, Korting HC (2005). Human defensins. J Mol Med, 83(8), 587-595.

3.     R I Lehrer, A Barton, K A Daher, S S Harwig, T Ganz, and M E Selsted (1989). Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity. J Clin Invest, 84(2), 553–561.

4.     Hoover DM, Rajashankar KR, Blumenthal R, Puri A, Oppenheim JJ, Chertov O, Lubkowski J (2000). The structure of human beta-defensin-2 shows evidence of higher order oligomerization. J Biol Chem, 275(42), 32911-32918.
5.     Zhang L, Yu W, He T, Yu J, Caffrey RE, Dalmasso EA, Fu S, Pham T, Mei J, Ho JJ, Zhang W, Lopez P, Ho DD (2002). Contribution of human alpha-defensin 1, 2, and 3 to the anti-HIV-1 activity of CD8 antiviral factor. Science, 303 (5657), 467.
6.     Biragyn A, Ruffini PA, Leifer CA, Klyushnenkova E, Shakhov A, Chertov O, Shirakawa AK, Farber JM, Segal DM, Oppenheim JJ, Kwak LW. Toll-like receptor 4-dependent activation of dendritic cells by beta-defensin 2. Science, 298(5595), 1025-1029.

抗菌肽介绍一

AMPs是由相对较小的分子组成的异质基团，通常含有不到100个氨基酸。 它们最初是在20世纪60年代由Zeya和Spitznagel 在多形核白细胞溶酶体中描述的。 迄今为止，已在数据库（如数据库）中 确定和登记了2600多个AMP。  它们是由几乎所有的生物群产生的，包括细菌、真菌、植物和动物。 许多脊椎动物AMPs是由上皮表面分泌的，如 哺乳动物的气管、舌、肠粘膜或两栖动物的皮肤。 有些在中性粒细胞、单核 细胞和巨噬细胞中表达。 AMPs参与动物和植物的免疫防御系统。 构成表达或诱导它们在抵御微生物入侵者 的第一道防线中起着关键作用。

结构/分类 AMPs可以根据其氨基酸组成和结构进行分类。 可以区分两大类AMP。

第一类由线性分子组成，它们要么倾向于采用α螺旋结构，要么富含精氨酸、甘氨 酸、组氨酸、脯氨酸和色氨酸等某些氨 基酸。

第二类由含半胱氨酸的肽组成， 可分为单一或多个二硫结构。 在许多情 况下，抗菌活性需要存在二硫桥。 大多数AMPs是阳离子肽，但也有阴离子肽，如真皮素，一种富含天冬氨酸 的人肽和两栖动物的最大蛋白H5皮肤。 其他非阳离子AMPs包括神经肽前体分子的片段，如原啡肽A， 芳香二肽主要从二翅目幼虫中分离出来，或从节肢动物或茴香物种的氧结合 蛋白中提取的肽。

专肽生物可定制合成各类序列的抗菌肽，可标记FITC/FAM/TAMRA等常见荧光素。

Definition

Antimicrobial peptides (AMPs) are as widespread as bacterial inactivator molecules in the innate immune systems of insects, fungi, plants, and mammals. These peptides are also known as host defense peptides (HDPs) as they have other immuno-modulatory functions besides the direct antimicrobial actions and are even capable of killing cancerous cells 1,2. 

Classification

Three broad categories of HDPs have been identified: 1) the linear peptides with helical structures, 2) the cysteine stabilized peptides with beta-sheet, and 3) a group of linear peptides rich in proline and arginine that primarily have been identified in non-mammalian species. 

Structural characteristics

In mammals, cathelicidins and defensins are the two principal AMP families. Cathelicidins are peptides with a conserved proregion and a variable C-terminal antimicrobial domain. Defensins are the best-characterized AMPs, they have six invariant cysteines, forming three intramolecular cystine-disulfide bonds. 

Mode of action

The mode of action of AMPs elucidated to date include inhibition of cell wall formation, formation of pores in the cell membrane resulting in the disruption of membrane potential with eventual lysis of the cell. These peptides also inhibit nuclease activity of both RNase and DNase. 

Functions

They have a broad ability to kill microbes. AMPs form an important means of host defense in eukaryotes. Large AMPs (>100 amino acids), are often lytic, nutrient-binding proteins or specifically target microbial macromolecules. Small AMPs act by disrupting the structure of microbial cell membranes. It plays an active role in wound repair and regulation of the adaptive immune system. They have multiple roles as mediators of inflammation with impact on epithelial and inflammatory cells, influencing diverse processes such as cell proliferation, wound healing, cytokine release, chemotaxis and  immune induction 3. 

References 

1.     Gottlieb CT, Thomsen LE, Ingmer H, Mygind PH, Kristensen HH, Gram L(2008). Antimicrobial peptides effectively kill a broad spectrum of Listeria monocytogenes and Staphylococcus aureus strains independently of origin, sub-type, or virulence factor expression. BMC Microbiol., 8:205.

2.     Yeaman MR and Yount NY (2003). Mechanisms of Antimicrobial Peptide Action and Resistance.  Pharmocological Reviews, 55(1).

3.     Hanna Galkowska H and Olszewski WL (2003). Antimicrobial peptides – their role in immunity and therapeutic potential. Centr Eur J Immunol., 28 (3):138–141.

抗菌肽介绍二

Ribosomally synthesized antimicrobial peptides (AMPs) constitute a structurally diverse group of molecules found virtually in all organisms. Most antimicrobial peptides contain less than 100 amino acid residues, have a net positive charge, and are membrane active. They are major players in the innate immune defense but can also have roles in processes as chemokine induction, chemotaxis, inflammation, and wound healing. In addition to their antimicrobial effects, many of them show antiviral and antineoplastic activities.

INTRODUCTION
AMPs are a heterogeneous group of relatively small molecules usually containing less than a hundred amino acids. They were first described in the 1960’s by Zeya and Spitznagel in polymorphonuclear leukocyte lysosomes.

To date, more than 2600 AMPs have been identified and registered in databases. They are produced by nearly all groups of organisms, including bacteria, fungi, plants, and animals. Many vertebrate AMPs are secreted by epithelial surfaces such as the tracheal, lingual, or intestinal mucosa of mammals or the skin of amphibia. Some are expressed in neutrophils, monocytes, and macrophages.

AMPs are involved in both animal and plant immune defense systems. Constitutively expressed or induced they play a key role in the first line of defense against microbial intruders.

STRUCTURE/CLASSIFICATION
AMPs can be classified on the basis of their amino acid composition and structure. Two major groups of AMPs can be distinguished. The first group consists of linear molecules which either tend to adopt α-helical structure or are enriched in certain amino acids such as arginine, glycine, histidine, proline, and tryptophan. The second group consists of cysteine-containing peptides which can be divided into single or multiple disulfide structures. In many cases, the presence of disulfide bridges is required for antimicrobial activity.

Most AMPs are cationic peptides, but there are also anionic peptides such as dermcidin, an aspartic acid-rich peptide from human and maximin H5 from amphibian skin. Other non-cationic AMPs include fragments from neuropeptide precursor molecules such as proenkephalin A, aromatic dipeptides primarily isolated from dipteran larvae, or peptides derived from oxygen-binding proteins from arthropod or annelid species.

MODE OF ACTION
Most AMPs act by provoking an increase in plasma membrane permeability. They preferentially target microbial versus mammalian cells. Selectivity is influenced by several factors such as differences in membrane composition: membranes of many bacterial pathogens contain negatively charged lipid moieties such as phosphatidylglycerol (PG), cardiolipin, and phosphatidylserine (PS), whereas mammalian membranes, commonly enriched in phosphatidylethanolamine (PE), phosphatidylcholine (PC) and sphingomyelin, are generally neutral in net charge.

The presence of sterols such as cholesterol and ergesterol within the membrane may be a further means by which AMPs can distinguish between mammalian or fungal cells and prokaryotes. A first step in the mechanism of membrane permeabilization is the electrostatic interaction between the positively charged AMP with the negatively charged membrane surface of the microorganism. Subsequent disruption of the membrane by creation of  pores within the microbial membrane ultimately results in cell death of the organism due to leakage of ions, metabolites, cessation of membrane-coupled respiration, and biosynthesis.

Several models for pore formation such as the Barrel-Stave, the Toroidal or Wormhole Model, and the Carpet Model have been proposed (Fig. 1).

FIG. 1. MODE OF ACTION A BARREL-STAVE MODEL B TOROIDAL PORE OR WORMHOLE MODEL C CARPET MODEL

THE BARREL-STAVE MODEL
The Barrel-Stave model describes a mechanism in which AMPs form a barrellike pore within the bacterial membrane with the individual AMPs or AMP complexes being the staves. Arranged in this manner, the hydrophobic regions of the AMPs point outwards towards the acyl chains of the membrane whereas the hydrophilic areas form the pore.

THE TOROIDAL PORE OR WORMHOLE MODEL
The pores described by this model differ from those of the Barrel-Stave model. Primarily, the outer and inner leaflet of the membrane are not intercalated in the transmembrane channel.

THE CARPET MODEL
A different mechanism is proposed in the Carpet model where AMPs first cover the outer surface of the membrane and then disrupt the membrane like detergents by forming micelle-like units. Certain AMPs penetrate the bacterial membrane without channel formation. They act on intracellular targets by e.g. inhibiting nucleic acid and/or protein synthesis.

RESISTANCE
Resistance to AMPs can either be constitutive or inducible. Inherited resistance mechanisms include altered surface charge, active efflux, production of peptidases or trapping proteins, and modification of host cellular processes. For instance, Staphylococcus aureus manages to reduce the overall cell surface charge by esterification of the cell wall component teichoic acid with D-alanine and thereby increases its resistance against human AMPs. Another example for changing the surface net charge is the production of cationic lysine-substituted phosphatidylglycerol (L-PG) found in certain Staphylococcus aureus strains. In Gram-negative bacteria, addition of 4-aminoarabinose (Ara4N) to the phosphate group of the lipid A backbone or increased acylation of lipopolysaccharides (LPS) are important mechanisms of AMP resistance. Exposure to AMPs may also induce stress responses by which microorganisms try to survive. Inducible regulatory mechanisms have been described in a variety of organisms. For instance, the PhoP/PhoQ regulon in Salmonella has been demonstrated to regulate transcriptional activation of surface and secretory proteins, enzymes that modify lipopolysaccharide, lipid and protein constituents of the outer membrane and proteases that likely degrade certain AMPs.

EXAMPLES OF ANTIMICROBIAL PEPTIDES

ADAPTED FROM K.A. BROGDEN, NAT. REV. MICROBIOL. 3, 238-250 (2005)

IMPORTANT FAMILIES OF AMPS
BOMBININS
Bombinins constitute a family of AMPs produced in fire-bellied toads (Bombina species) active against Gram-negative and Gram-positive bacteria and fungi. Bombinins, bombinin-like peptides (BLPs), and Bombinin H molecules are found in the species Bombina bombina, Bombina variegata, and Bombina orientalis, whereas the homologous maximins and maximin H peptides are derived from the giant fire-bellied toad Bombina maxima. Bombinin H peptides contain either 17 or 20 amino acid residues and are more hydrophobic than bombinins, some of them contain D-alloisoleucine at position 2. They exhibit lower antibacterial activity than bombinins but, in contrast to them, they possess haemolytic activity.

CATHELICIDINS
Members of this family are amphipathic, cationic peptides with a broad-spectrum antimicrobial activity. Cathelicidins typically act by disrupting the integrity of bacterial membranes. They are characterized by an evolutionary conserved N-terminal cathelin- like domain of approximately 99-114 amino acid residues linked to a C-terminal antimicrobial domain of 12-100 residues that can be released upon proteolytic processing. Members of this family include linear peptides amongst them a number of proline-rich AMPs that show different types of proline repeat motifs (Bac5, Bac7, PR-39, prophenins) and the tryptophan-rich indolicidin characterized by three regularly spaced proline residues. The protegrins (PG-1 to PG-5) contain two disulfide bridges and an amidated C-terminus. Cathelicidins have been found in every mammalian species examined. In human, LL-37 (Product 4042456) is the only member of the cathelicidin family. The peptide consists of 37 amino acids and contains two leucine residues at the N-terminus. It is proteolytically cleaved from the 18 kDa precursor protein human cathelicidin antimicrobial protein CAP-18. LL-37 is primarily produced by phagocytic leucocytes and epithelial cells, and is involved in various processes such as direct killing of microorganisms, binding and neutralizing LPS, chemotaxis and chemokine induction, regulation of inflammatory responses, and wound healing. Its production is influenced by several factors such as microbial products, host cytokines, vitamin D3, and availability of oxygen. LL-37 orthologues in mouse and rat are CRAMP (mouse) (Product 4056438) and CRAMP (rat), respectively.

CECROPINS
Cecropins were first isolated from the giant silk moth Hyalophora cecropia. They can form amphipathic, α-helical structures and are structurally related to other cecropins as bactericidin, lepidopteran, and sarcotoxin. Cecropin P1 (Product 4039862), found in pig intestine, also belongs to this family. Most cecropins show broad-spectrum antibacterial activity. Cecropin A (Product 4030488) and B (Product 4030477) have also been demonstrated to possess tumoricidal activity against mammalian leukemia, lymphoma, and carcinoma cell lines.

CERATOTOXINS
This family consists of cationic α-helical amphipathic peptides expressed in the female reproductive accessory glands of the Mediterranean fruit fly Ceratitis capitata. The production of the peptides is enhanced by mating. Ceratotoxin A and ceratotoxin B are 29 amino acid peptides differing in two amino acids. Ceratotoxin C and D consist of 32 and 36 amino acids, respectively. The peptides of this family are active against Gram-negative as well as Grampositive bacteria and are supposed to act via the Barrel-Stave model. Ceratotoxin A has been shown to be mainly antibacterial for Gram-negative organisms.

DEFENSINS
Defensins are small cysteine-rich cationic peptides containing three or four disulfide bridges. They have been isolated from molluscs, acari, arachnids, insects, mammals, and plants. They are further divided into families on the basis of the spatial distribution of their cysteine residues. Three families, the α-, β- and θ-defensins, can be distinguished in mammals. α- and β-defensins are characterized by antiparallel β-sheet structures stabilized by three disulfide bonds. The θ-defensins are found in rhesus monkey and some other non-human primates but not in human, chimpanzee and gorilla. They consist of two nine amino acid peptides derived from different precursor proteins joined by head-to-tail cyclization. Invertebrate and plant defensins contain three or four disulfide  bridges, respectively. Insect and mammalian defensins are mainly active against bacteria while most plant defensins possess antifungal activity.

DERMASEPTINS
The peptides of the dermaseptin family are closely related and consist of 28-34 amino acids. They were originally isolated from skin extracts of the South American arboreal frog Phyllomedusa sauvagei and contain a conserved tryptophan residue at position 3. Dermaseptins exhibit broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria.

HISTATINS
Histatins are histidine-rich and mostly cationic peptides found in the saliva of humans and some higher primates. They are active against a broad-spectrum of bacteria and fungi. The antifungal activity of the human salivary peptide histatin-5 has been extensively studied and is supposed to be due to inhibition of mitochondrial respiration and the formation of reactive oxygen species. Histatin-5 has also been shown to inhibit both host-derived and bacterial proteolytc enzymes involved in peridontal diseases. Histatin-8, a peptide from human parotid secretion, has been shown to inhibit hemagglutination activity of Porphyromonas gingivalis 381, a Gram-negative bacterium involved in certain forms of periodontal disease. The peptide may function as a binding domain for the hemagglutinins of Porphyromonas gingivalis during agglutination.

MAGAININS
Magainins constitute a family of linear amphipathic cationic AMPs discovered in the skin of Xenopus laevis. The two closely related members of this family, magainin I (Product 4012844) and magainin II (Product 4013706) differ merely in two positions and are 23 amino acids in length. Magainins exhibit broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria, fungi and protozoa and are also cytotoxic for many murine and human cancer cell lines.

CONCLUSIONS
The structures of AMPs represent a unique source for the targeted exploration of new applications in the therapy of microbial and viral infection, cancer, and sepsis. Modern synthetic methods will allow the relatively cheap and accurate production of lead compounds and peptide candidates. The achievements in peptide library generation, analytical methods as mass spectrometry, and screening and formulation technologies may contribute to solve intrinsic problems associated with the use of AMPs as therapeutic agents such as susceptibility to proteases and host toxicity. Bachem has considerable expertise and long-standing experience in peptide synthesis. With our capacity to upscale the production of simple and modified peptides, we are the partner of choice for the pharmaceutical industries.

Edwin J A Veldhuizen , et al. Expression of Beta-Defensins pBD-1 and pBD-2 Along the Small Intestinal Tract of the Pig: Lack of Upregulation in Vivo Upon Salmonella Typhimurium Infection. Mol Immunol. 2007 Jan;44(4):276-83. : https://pubmed.ncbi.nlm.nih.gov/16620987/
D R Pruthviraj, et al. Identification of a Novel Single Nucleotide Polymorphism in Porcine Beta-Defensin-1 Gene. Asian-Australas J Anim Sci. 2016 Mar;29(3):315-20. : https://pubmed.ncbi.nlm.nih.gov/26950860/
Shokrollah Elahi, et al. The Host Defense Peptide Beta-Defensin 1 Confers Protection Against Bordetella Pertussis in Newborn Piglets. Infect Immun. 2006 Apr;74(4):2338-52. : https://pubmed.ncbi.nlm.nih.gov/16552064/