小菜蛾PGRP-S2基因的克隆表达及功能分析

肽聚糖识别蛋白(peptidoglycan recognition protein,PGRP)对于昆虫来说是一种高度保守的病原识别蛋白。为阐明PGRP-S2在小菜蛾Plutella xylostella抵抗病原微生物过程中的作用,本研究结合RT-PCR和RACE技术克隆得到小菜蛾PGRP-S2基因的cDNA全长序列,命名为PGRP-S2(GenBank登录号:MG570190)。生物信息学分析结果表明,PGRP-S2的开放阅读框为588 bp,编码195个氨基酸;蛋白质预测分子量为21.46 kDa,理论等电点为8.46;编码蛋白具有PGRP超家族保守结构域和酰胺酶结构域,是典型的肽聚糖识别蛋白,包含一条信号肽,不存在跨膜结构;同源序列比对和系统进化树分析表明PGRP-S2与家蚕Bombyx mori的BmPGRP-S 1进化距离最近。利用大肠杆菌Escherichia coli BL21(DE3)高效表达重组蛋白PxPGRP-S2,利用倒置显微镜及平板涂布观察重组蛋白对大肠杆菌E.coli和金黄色葡萄球菌Staphylococcus aureus的作用,结果表明PxPGRP-S2蛋白能够与两种细菌发生结合并凝集细菌,但不具备直接杀菌功能。本研究为进一步研究基于PGRP-S2介导的小菜蛾免疫防御反应提供基础。     

非洲爪蟾短型肽聚糖识别蛋白基因的克隆与鉴定

采用生物信息学方法首次对非洲爪蟾短型肽聚糖识别蛋白(xePGRP-S)基因进行了克隆,并对其在胚胎发育和成年爪蟾各组织中的表达状况进行了分析。xePGRP-ScDNA全长720bp,开放阅读框为549bp,编码182个氨基酸。序列比对显示xePGRP-S与其他物种PGRP-S的序列相似性在42.4%-50.5%之间。RT-PCR显示在非洲爪蟾胚胎发育至3d时可以明显检测到xePGRP-S的表达,之后呈持续性表达,且在所检测的心、肝、脾、肺、肾、肠和胃这7种组织器官中呈组成型表达。       

大蜡螟中类肽聚糖识别蛋白Gm21的鉴定、基因克隆及序列分析

为了调查毒素蛋白免疫下大蜡螟Galleria mellonella L.产生的免疫相关蛋白,采用双向电泳的方法,从大蜡螟血淋巴中鉴定得到一种昆虫肽聚糖识别蛋白Gm21,通过RT-PCR、cDNA末端快速扩增(RACE)技术克隆得到其全长基因,该基因全长cDNA具有完整的编码框,编码211个氨基酸,序列分析表明该蛋白是一种具有潜在酰胺酶活性的S型肽聚糖识别蛋白.本研究中Gm21蛋白在免疫压力下的上调表达及其cDNA序列的克隆,对进一步深入研究昆虫肽聚糖识别蛋白的功能具有重要的意义.     

光滑鳖甲肽聚糖识别蛋白的生物信息学分析

[目的]预测光滑鳖甲肽聚糖识别蛋白的结构与功能。[方法]采用生物信息学方法对该基因及其编码蛋白的基本理化性质、疏水性、信号肽、二级结构和亚细胞定位等方面进行预测和分析,同时构建其编码产物的系统进化树。[结果]此蛋白的理论分子量为21.882 k D,包含一个由20个氨基酸组成的信号肽,属亲水蛋白,分泌到胞外发挥作用,可能具有信号转导和响应胁迫与免疫反应的功能,与赤拟谷盗的同源性最高,具有能够与肽聚糖结合的保守的PGRP结构域和一个Ⅱ型酰胺酶结构域。[结论]光滑鳖甲肽聚糖识别蛋白Ap PGRP-FD1基因克隆成功,生物信息学分析及结合蛋白质结构与功能预测可为深入研究ApPGRP-FD1提供理论指导。     

大鲵免疫分子CD9的克隆与鉴定

CD9是4次跨膜蛋白家族成员,对机体的免疫调控发挥着重要作用．本文首次报道了大鲵CD9的鉴定及其对免疫刺激的应答．大鲵CD9（Chinese giant salamander CD9, cgsCD9）由232个氨基酸组成,被4个跨膜结构域分割成1个胞内环、2个胞外环和2个短的胞内末端,这是4次跨膜蛋白的主要结构特征．1个糖基化位点NDS、1个CCG模体和7个半胱氨酸残基保守地存在于cgsCD9．cgsCD9与其它脊椎动物CD9具有最高77%氨基酸序列一致性,并与两栖动物CD9归属一族．实时定量RT-PCR结果显示,cgsCD9在大鲵中分布广泛,脾脏组织的表达水平最高,并且脂多糖能显著提高cgsCD9 mRNA的表达．过表达于Jurkat细胞中的cgsCD9能促进核因子κB的活化以及白介素-6的表达,而cgsCD9的作用在CCG模体突变为AAA后几乎丧失．上述结果表明,cgsCD9在大鲵抵抗细菌的免疫应答中发挥重要作用．     

中国大鲵促甲状腺激素受体的克隆及序列分析

本实验以大鲵脑组织为材料,通过RT-PCR和RACE方法成功获得了中国大鲵Andrias davidianus促甲状腺激素受体(TSHR)cDNA全长序列。该序列编码585个氨基酸,具有典型的G蛋白偶联受体家族的特点,有4个N-糖基化位点,7个跨膜α螺旋。同源分析显示,大鲵TSHR与热带爪蟾、变色龙、人的TSHR同源性分别为70%、70%和67%。同时大鲵与其它脊椎动物TSHR在结构上存在一些差异,如缺乏前导序列、胞外部分序列缩短、没有富含亮氨酸的重复单位(LRRs)等,大鲵TSHR结构上的这些差异是否与其不同的生理功能相关有待进一步研究。利用邻接法对部分脊椎动物TSHR氨基酸序列构建分子系统树,结果显示有尾目大鲵为两栖动物中较早分化的一支,表明其进化地位较原始。RT-PCR组织分布分析发现,TSHR基因在大鲵的甲状腺外组织也有表达,其中性腺组织表达量最高,这可能暗示TSHR与大鲵的生殖调节有关。     

三角帆蚌短型肽聚糖识别蛋白S3基因克隆及表达分析

肽聚糖识别蛋白（PGRPs）是机体先天免疫系统中的重要模式识别受体,研究对三角帆蚌的一种短型肽聚糖识别蛋白进行了克隆与表达分析。研究采用5、3 RACE技术,对高通量转录组测序所得三角帆蚌PGRPS3基因（hcPGRPS3）片段分别进行了5,3末端克隆,经拼接得到hcPGRPS3 cDNA全长序列。采用多种分子生物学软件对hcPGRPS3 cDNA全长序列进行了特征分析,实时荧光定量（qRT-PCR）检测了其组织分布,及经肽聚糖（Peptidoglycan,PGN）和脂多糖（Lipopolysaccharide,LPS）刺激后其在肝胰腺中的基因表达变化。hcPGRPS3 cDNA全长1438 bp,开放阅读框为858 bp,编码285个氨基酸,预测分子量大小为32.3 ku,pH 7.0时的理论等点为7.98。序列中不存在信号肽与跨膜结构。氨基酸序列保守性分析表明,其他物种短型PGRP中,以夏威夷短尾鱿PGRP4与hcPGRPS3同源性最高（51%）。qRT-PCR检测结果显示,hcPGRPS3在性腺及肝胰腺中表达水平较高。经PGN或LPS刺激后,肝胰腺中hcPGRPS3表达水平显著上调,表明hcPGRPS3可能在机体抗菌免疫反应中发挥重要作用。     

一种新型酿酒酵母附加型分泌表达载体的构建

用化学法合成克鲁维酵母的菊粉酶基因的信号肽序列(INU),将其嵌入酵母附加型表达质粒pYES2,得到一套新型的分泌表达载体pYES2I,pYES2Ⅱ,pYES2Ⅲ。然后用PCR方法分别扩增大肠杆菌的天冬酰胺酶基因(ASN)和短芽孢杆菌α乙酰乳酸脱羧酶(ALDC)基因,连接到INU下游,得到重组质粒pASN和pALDC。分别将这两个重组质粒转化酿酒酵母菌株INVScⅠ中表达,胞内和胞外的酶活分析表明ASN和ALDC基因都能在酿酒酵母中分泌表达,表明菊粉酶信号肽序列能很好地将酿酒酵母中的重组蛋白分泌到胞外。稳定性分析表明,重组酵母菌株在没有选择压力的条件下连续接种培养100h,未发现重组质粒的不稳定性。     

肽聚糖识别蛋白抗菌活性及参与炎症的免疫作用

哺乳动物肽聚糖识别蛋白(peptidoglycan recognition proteins, PGRPs)是一类可识别肽聚糖的模式识别受体,在先天免疫应答中发挥重要的识别和调节功能。PGRPs通过与肽聚糖结合,诱导激活细菌双组分系统(two-component systems, TCSs)如CssR-CssS、CpxA-CpxR等,诱导氧化应激、硫醇应激和金属应激等反应发挥抗菌活性。现对PGRPs的抗菌活性及其与抗生素的杀菌机制进行比较,旨在为疾病的防治提供理论依据。     

HER-2/neu胞外配体结合区2在大肠杆菌中可溶性表达及纯化

用PCR技术扩增HER 2 neu胞外配体结合区 2 (RLD2 )cDNA ,并将扩增的基因片段克隆于硫氧还蛋白 (TrxA)原核表达载体中 ,获得TrxA RLD2融合蛋白的可溶性表达 .通过插入偶联翻译序列 ,实现TrxA与RLD2蛋白在大肠杆菌中的共表达 .表达产物经免疫印记检测可被抗HER 2 neu特异性抗体识别 .经离子交换层析和钴亲和层析纯化 ,RLD2蛋白的纯度达 90 % .用质谱法分析RLD2蛋白的分子量 ,与预期值相符 .结果表明 ,利用TrxA表达体系在大肠杆菌中获得了HER 2 neuRLD2蛋白高效可溶性表达     

Human peptidoglycan recognition protein-L is an N-acetylmuramoyl-L-alanine amidase

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules coded by up to 13 genes in insects and 4 genes in mammals. In insects PGRPs activate antimicrobial pathways in the hemolymph and cells, or are peptidoglycan (PGN)-lytic amidases. In mammals one PGRP is an antibacterial neutrophil protein. We report that human PGRP-L is a Zn2+-dependent N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28), an enzyme that hydrolyzes the amide bond between MurNAc and l-Ala of bacterial PGN. The minimum PGN fragment hydrolyzed by PGRP-L is MurNAc-tripeptide. PGRP-L has no direct bacteriolytic activity. The other members of the human PGRP family, PGRP-Ialpha, PGRP-Ibeta, and PGRP-S, do not have the amidase activity. The C-terminal region of PGRP-L, homologous to bacteriophage and bacterial amidases, is required and sufficient for the amidase activity of PGRP-L, although its activity (in the N-terminal delta1-343 deletion mutant) is reduced. The Zn2+ binding amino acids (conserved in PGRP-L and T7 amidase) and Cys-419 (not conserved in T7 amidase) are required for the amidase activity of PGRP-L, whereas three other amino acids, needed for the activity of T7 amidase, are not required for the activity of PGRP-L. These amino acids, although required, are not sufficient for the amidase activity, because changing them to the "active" configuration does not convert PGRP-S into an active amidase. In conclusion, human PGRP-L is an N-acetylmuramoyl-l-alanine amidase and this function is conserved in prokaryotes, insects, and mammals.     

Peptidoglycan recognition proteins: a novel family of four human innate immunity pattern recognition molecules.

The innate immune system recognizes microorganisms through a series of pattern recognition receptors that are highly conserved in evolution. Insects have a family of 12 peptidoglycan recognition proteins (PGRPs) that recognize peptidoglycan, a ubiquitous component of bacterial cell walls. We report cloning of three novel human PGRPs (PGRP-L, PGRP-Ialpha, and PGRP-Ibeta) that together with the previously cloned PGRP-S, define a new family of human pattern recognition molecules. PGRP-L, PGRP-Ialpha, and PGRP-Ibeta have 576, 341, and 373 amino acids coded by five, seven, and eight exons on chromosomes 19 and 1, and they all have two predicted transmembrane domains. All mammalian and insect PGRPs have at least three highly conserved C-terminal PGRP domains located either in the extracellular or in the cytoplasmic (or in both) portions of the molecules. PGRP-L is expressed in liver, PGRP-Ialpha and PGRP-Ibeta in esophagus (and to a lesser extent in tonsils and thymus), and PGRP-S in bone marrow (and to a lesser extent in neutrophils and fetal liver). All four human PGRPs bind peptidoglycan and Gram-positive bacteria. Thus, these PGRPs may play a role in recognition of bacteria in these organs.     

Structural basis of heparin binding to camel peptidoglycan recognition protein-S

Short peptidoglycan recognition protein (PGRP-S) is a member of the innate immunity system in mammals. PGRP-S from Camelus dromedarius (CPGRP-S) is found to be highly potent against bacterial infections. It is capable of binding to a wide range of pathogen-associated molecular patterns (PAMPs) including lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). The heparin-like polysaccharides have also been observed in some bacteria such as the capsule of K5 Escherichia coli thus making them relevant for determining the nature of their interactions with CPGRP-S. The binding studies of CPGRP-S with heparin disaccharide in solution using surface plasmon resonance gave a value 3.3×10^-7 M for the dissociation constant (Kd). The structure of the heparin bound CPGRP-S determined at 2.8Å resolution revealed the presence of a bound heparin molecule in the binding pocket of CPGRP-S. It was found anchored tightly to the protein with the help of several ionic and hydrogen bonded interactions. Three sulphate groups of heparin S1, S2 and S3 have been found to interact with residues, Arg-31, Lys-90, Thr- 97, Asn-99 Asn-140, Gln-150 and Arg-170 of CPGRP-S. The binding site includes two subsites, S-I and S-II with cleft-like structures. Heparin disaccharide is bound in subsite S-I. Previously determined structures of the complexes of CPGRP-S with LPS, LTA and PGN also showed that their glycan moieties were also held in subsite S-I indicating that heparin disaccharide also represents an important element for the recognition by CPGRP-S.     

Crystal structure of human peptidoglycan recognition protein S (PGRP-S) at 1.70 A resolution

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind peptidoglycans (PGNs) of bacterial cell walls. These molecules, which are highly conserved from insects to mammals, contribute to host defense against infections by both Gram-positive and Gram-negative bacteria. Here, we present the crystal structure of human PGRP-S at 1.70A resolution. The overall structure of PGRP-S, which participates in intracellular killing of Gram-positive bacteria, is similar to that of other PGRPs, including Drosophila PGRP-LB and PGRP-SA and human PGRP-Ialpha. However, comparison with these PGRPs reveals important differences in both the PGN-binding site and a groove formed by the PGRP-specific segment on the opposite face of the molecule. This groove, which may constitute a binding site for effector or signaling proteins, is less hydrophobic and deeper in PGRP-S than in PGRP-IalphaC, whose PGRP-specific segments vary considerably in amino acid sequence. By docking a PGN ligand into the PGN-binding cleft of PGRP-S based on the known structure of a PGRP-Ialpha-PGN complex, we identified potential PGN-binding residues in PGRP-S. Differences in PGN-contacting residues and interactions suggest that, although PGRPs may engage PGNs in a similar mode, structural differences exist that likely regulate the affinity and fine specificity of PGN recognition.     

Convergent and divergent development among M cell lineages in mouse mucosal epithelium

M cells are specialized epithelial cells mediating immune surveillance of the mucosal lumen by transepithelial delivery of Ags to underlying dendritic cells (DC). At least three M cell phenotypes are known in the airways and intestine, but their developmental relationships are unclear. We used reporter transgenic mouse strains to follow the constitutive development of M cell subsets and their acute induction by cholera toxin (CT). M cells overlying intestinal Peyer's patches (PPs), isolated lymphoid follicles, and nasal-associated lymphoid tissue are induced by distinct settings, yet show convergent phenotypes, such as expression of a peptidoglycan recognition protein-S (PGRP-S) transgene reporter. By contrast, though PP, isolated lymphoid follicle, and villous M cells are all derived from intestinal crypt stem cells, their phenotypes were clearly distinct; for example, PP M cells frequently appeared to form M cell-DC functional units, whereas villous M cells did not consistently engage underlying DC. B lymphocytes are critical to M cell function by forming a basolateral pocket and possible signaling through CD137; however, initial commitment to all M cell lineages is B lymphocyte and CD137 independent. CT causes induction of new M cells in the airway and intestine without cell division, suggesting transdifferentiation from mature epithelial cells. In contrast with intestinal PP M cells, CT-induced nasal-associated lymphoid tissue M cells appear to be generated from ciliated Foxj1(+)PGRP-S(+) cells, indicative of a possible precommitted progenitor. In summary, constitutive and inducible differentiation of M cells is toward strictly defined context-dependent phenotypes, suggesting specialized roles in surveillance of mucosal Ags.     

Peptidoglycan recognition protein expression in mouse Peyer's Patch follicle associated epithelium suggests functional specialization

Mammalian Peyer's Patches possess specialized epithelium, the follicle associated epithelium (FAE), and specialized cells called M cells which mediate transcytosis of antigens to underlying lymphoid tissue. To identify FAE specific genes, we used TOGA gene expression profiling of microdissected mouse Peyer's Patch tissue. We found expression of laminin beta3 across the FAE, and scattered expression of peptidoglycan recognition protein (PGRP)-S. Using the M cell specific lectin Ulex europaeus agglutinin 1 (UEA-1), PGRP-S expression was nearly exclusively co-localized with UEA-1+ M cells. By contrast, the related gene PGRP-L was expressed among a subset of UEA-1 negative FAE cells. Expression of these proteins in transfected cells demonstrated distinct subcellular localization. PGRP-S showed a vesicular pattern and extracellular secretion, while PGRP-L showed localization to both the cytoplasm and the cell surface. The potential function of these PGRP proteins as pattern recognition receptors and their distinctive cellular distribution suggests a complex coordination among specialized cells of the FAE in triggering mucosal immunity and innate immune responses.     

Selective recognition of synthetic lysine and meso-diaminopimelic acid-type peptidoglycan fragments by human peptidoglycan recognition proteins I{alpha} and S

The interactions of a range of synthetic peptidoglycan derivatives with PGRP-Ialpha and PGRP-S have been studied in real-time using surface plasmon resonance. A dissociation constant of K(D) = 62 mum was obtained for the interaction of peptidoglycan recognition protein (PGRP)-Ialpha with the lysine-containing muramyl pentapeptide (compound 6). The normalized data for the lysine-containing muramyl tetra- (compound 5) and pentapeptide (compound 6) showed that these compounds have similar affinities, whereas a much lower affinity for muramyl tripeptide (compound 3) was measured. Similar affinities were obtained when the lysine moiety of the muramyl peptides was replaced by meso-diaminopimelic acid (DAP). Furthermore, the compounds that contained only a stem peptide (pentapeptide, compound 1) and (DAP-PP, compound 2) as well as muramyldipeptide (compound 3) exhibited no binding indicating that the muramyltripeptide (compound 4) is the smallest peptidoglycan fragment that can be recognized by PGRP-Ialpha. Surprisingly, PGRP-S derived significantly higher affinities for the DAP-containing fragments to similar lysine-containing derivatives, and the following dissociation constants were measured: muramylpentapeptide-DAP, K(D) = 104 nm; muramyltetrapeptide-DAP, 92.4 nm; and muramyltripeptide-DAP, 326 nm. The binding profiles were rationalized by using a recently reported x-ray crystal structure of PGRP-Ialpha with the lysine-containing muramyltripeptide (4).     

Tag7 (PGLYRP1) in Complex with Hsp70 Induces Alternative Cytotoxic Processes in Tumor Cells via TNFR1 Receptor

Tag7 (also known as peptidoglycan recognition protein PGRP-S, PGLYRP1), an innate immunity protein, interacts with Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against some tumor cell lines. In this study, we have analyzed the programmed cell death mechanisms that are induced when cells interact with the Tag7-Hsp70 complex, which was previously shown to be released by human lymphocytes and is cytotoxic to cancer cells. We show that this complex induces both apoptotic and necroptotic processes in the cells. Apoptosis follows the classic caspase-8 and caspase-3 activation pathway. Inhibition of apoptosis leads to a switch to the RIP1-dependent necroptosis. Both of these cytotoxic processes are initiated by the involvement of TNFR1, a receptor for TNF-α. Our results suggest that the Tag7-Hsp70 complex is a novel ligand for this receptor. One of its components, the innate immunity protein Tag7, can bind to the TNFR1 receptor, thereby inhibiting the cytotoxic actions of the Tag7-Hsp70 complex and TNF-α, an acquired immunity cytokine.