氨肽酶N的表达及其与结石形成的关系(英文)

 为研究大鼠高胆固醇饮食时 ,肝脏氨肽酶N(APN)在实验结石形成中可能的结石发生作用 ,采用 1.2 %胆固醇饮食 4周 ,诱发新西兰兔胆囊结石形成 .根据兔APN基因cDNA序列设计引物 ,提取肝脏总RNA .利用RT PCR检测肝脏APNmRNA水平的变化 ,用组织化学方法观察肝脏毛细胆管膜上APN的表达 .观察新西兰兔胆囊结石形成过程中肝脏APN的mRNA水平的变化、APN表达及胆汁中APN活性、胆脂、总蛋白含量的变化 ,探讨APN在胆石形成中可能的作用 .经成石饲料饲养后 ,随着胆汁饱和度增加和APN活性加强 ,胆囊结石组肝脏APNmRNA水平较对照组明显增高 ,胆囊结石组胆汁中总胆固醇、CSI、总蛋白浓度及APN活性均明显高于对照组 ,且胆汁中APN活性与肝脏APN的表达及胆汁CSI增高呈正相关 .结果提示 ,当存在胆汁过饱和的情况下 ,APN很可能作为促成核因子在胆结石形成早期发挥重要作用     

大螟中肠氨肽酶N基因的克隆及表达谱分析

昆虫中肠氨肽酶N是Bt毒素的重要受体之一, 与Bt毒素的杀虫机制及昆虫Bt抗性的产生密切相关。本研究通过简并引物PCR结合RACE技术克隆并获得大螟 Sesamia inferens Bt受体蛋白--氨肽酶N （aminopeptidase N, APN）基因的cDNA序列全长, 经NCBI同源比对分析, 认为该基因为APN3基因, 并将其命名为SiAPN3（GenBank登录号为HQ636624）。序列分析表明, 该基因的cDNA序列全长为3 411 bp, 开放阅读框为3 018 bp, 编码1 006个氨基酸; 预测蛋白质分子量为114 kD, 等电点为4.95; 其推导的氨基酸序列中具有鳞翅目昆虫氨肽酶N典型的结构特征, 即含有1个N-和12个O-连接的糖基化位点, N-末端具有18个氨基酸的剪切信号肽, 谷氨酸锌化氨肽酶保守结构GAMEN, 锌结合位点HEXXHX18E, C-末端具有22个氨基酸的糖基磷脂酰肌醇(GPI)锚信号肽。采用实时定量PCR研究了SiAPN3在大螟4龄幼虫肠道不同部位和幼虫不同龄期的转录表达谱。结果表明, 该基因在幼虫中肠中的表达量最高, 其次为后肠, 前肠中表达量最低, 且中肠和后肠中的表达量显著高于前肠（P＜0.05）; SiAPN3在3龄幼虫中的表达量最高, 1龄幼虫中最低, 虽然3、 4日龄之间的表达量没有显著差异, 但二者均显著高于其他日龄, 1, 2和5日龄之间不存在显著差异（P>0.05）。该研究为阐明APN基因的功能及大螟对Bt抗性产生的分子机制奠定了基础。     

稻纵卷叶螟氨肽酶N基因的克隆及时空表达分析

【目的】昆虫中肠氨肽酶N(Aminopeptidase N,APN)是Bt杀虫蛋白的重要受体之一,与Bt蛋白的杀虫机制及昆虫对Bt蛋白的抗性密切相关。为阐明稻纵卷叶螟Cnaphalocrocis medinalis(Guenee)APN基因的功能及明确Bt蛋白对稻纵卷叶螟的毒力机制,本研究系统开展了稻纵卷叶螟中肠APN基因的克隆及时空表达分析。【方法】通过简并引物PCR结合RACE技术克隆并获得4条稻纵卷叶螟APN基因的cDNA序列全长,采用实时定量PCR技术研究了APN基因在稻纵卷叶螟不同虫态及幼虫不同组织中的时空表达情况。【结果】经NCBI同源比对分析,认为这4个基因分属于。4PN基因家族的不同类别,分别将其命名为CmAPN1(GenBank登录号:HQ853294)、CmAPN2(GenBank登录号:HQ853295)、CmAPN3(GenBank登录号:KJ143755)、CmAPN4(GenBank登录号:HQ853296)。序列分析表明,CmAPN1-4 cDNA序列全长分别为:3 698、3 478、3 150和3 149 bp,开放阅读框分别为:3 045、2 877、3 045和2 862 bp,分别编码965、958、1 014和952个氨基酸。其推导的氨基酸序列具有鳞翅目昆虫氨肽酶N的典型结构特征,即含有糖基化位点、N-末端信号肽序列、谷氨酸锌化氨肽酶保守结构GAMEN、锌结合位点HEX_2HX_(18)E、C-末端糖基磷脂酰肌醇(GPI)锚信号肽。实时定量研究表明,CmAPNs在幼虫中的表达量显著高于卵、蛹和成虫;在幼虫中,CmAPN7的表达水平明显低于CmAPN22-4,且其在不同龄期中的表达差异显著;CmAPN2-4的表达量随幼虫龄期的增加而增加;CmAPNs在幼虫肠道组织中的表达量显著高于其它组织器官,且CmAPN1和CmAPN2分别在中肠和后肠中呈现高水平表达,CmAPN3在前、中肠内均高水平表达;但CmAPN4在各个组织器官中的表达均保持较低水平。【结论】CmAPNs基因在稻纵卷叶螟的不同虫态和不同组织中呈现了差异显著的时空表达,采用RNA干扰方法进行CmAPNs基因功能研究时,要选择适宜的虫态和虫龄进行干扰。     

褐飞虱中肠两种氨肽酶N的鉴定及蛋白特性分析

[目的]氨肽酶N(aminopeptidase N,APN)是昆虫消化系统中重要的蛋白酶.本研究旨在对在褐飞虱Nilaparvata lugens中肠中两个具有较高转录水平的apn基因(nlapn1和nlapn4)在中肠上皮细胞上的表达及其蛋白功能特性进行鉴定与分析.[方法]利用最大似然法进行褐飞虱NLAPN1和NLA...     

缺失氨肽酶N的变铅青链霉菌的构建和鉴定

用PCR方法扩增变铅青链霉菌(Streptomyces lividans)TK24氨肽酶N基因,体外插入卡那霉素抗性基因进行失活,然后利用不含链霉菌复制起点的重组质粒pPEPN-KAN进行同源重组,获得了氨肽酶N缺失的菌株PEPN-.突变株的胞外氨肽酶N活性与原株基本相同,而胞内氨肽酶N的活性明显低于原株,为原株的42.5±5.7%.     

抗Bt棉棉铃虫幼虫Bt受体氨肽酶N(APN2)基因克隆

通过对Bt棉抗性和敏感棉铃虫幼虫中肠氨肽酶N的克隆和测序 ,鉴定了氨肽酶N基因家族的 1个成员Haapn2 ,其cDNA序列具有 3209个核苷酸 ,含有 3096bp的开放阅读框 ,编码产生1032个氨基酸的蛋白质。其推定氨基酸序列具有锌结合模体HEXXHX₁₈E ,N-末端具有 1 7个氨基酸的疏水性信号序列 ,C-末端还具有 2 2个氨基酸的糖基磷酯酰肌醇 (GPI)添加信号肽。比对抗性和敏感棉铃虫cDNA的开放阅读框 ,抗性品系的开放阅读框中 ,有 5 7个点突变 ,共导致了 1 5个氨基酸的改变 ,其中 2个突变 (谷氨酰胺₁₃₇→谷氨酸、缬氨酸₁₃₇→苏氨酸 )位于Cry1A毒素结合区域 ,可能与棉铃虫对转Bt基因棉产生抗性有关。报道的氨肽酶cDNA序列已提交GenBank ,AY346383和AY2 795 35分别是Bt抗性和敏感品系的Haapn2。     

大肠杆菌高效表达包函体蛋白N－Met的加工

大肠杆菌高效表达包函体蛋白Ｎ－Ｍｅｔ的加工杨新颖,梁镇和,李伯良（中国科学院上海生物化学研究所,２０００３１）关键词牛生长激素;高效表达;Ｎ－Ｍｅｔ氨肽酶基因工程已使许多外源基因或基因片段在大肠杆菌中实现了高效表达,最高的表达蛋白质带可达全菌总蛋白质...     

一种利用荧光标记检测氨肽酶N抑制剂与肿瘤细胞结合的方法

目的：建立利用荧光标记法检测氨肽酶抑制剂和肿瘤细胞结合的方法。方法：以异硫氰酸荧光素（FITC）标记氨肽酶N抑制剂LYRM03和Bestatin,制备荧光探针F1TC—LYRM03、FITC—Bestatin,应用荧光显微成像观察和流式细胞仪检测标记化合物FITC—LYRM03、FITC-Bestatin对肿瘤细胞的结合与氨肽酶N抑制活性的相关性。结果：化合物LYRM03和Bestatin具有肿瘤细胞的氨肽酶N抑制活性,荧光标记化合物FITC—LYRM03、FITC-Bestatin能与肿瘤细胞有不同程度的结合。结论：标记化合物FITC—LYRM03、FITC—Bestatin和肿瘤细胞的结合与对肿瘤细胞的氨肽酶N抑制活性相一致。     

一种利用荧光标记检测氨肽酶N抑制剂与肿瘤细胞结合的方法

目的:建立利用荧光标记法检测氨肽酶抑制剂和肿瘤细胞结合的方法。方法:以异硫氰酸荧光素(FITC)标记氨肽酶N抑制剂LYRM03和Bestatin,制备荧光探针FITC-LYRM03、FITC-Bestatin,应用荧光显微成像观察和流式细胞仪检测标记化合物FITC-LYRM03、FITC-Bestatin对肿瘤细胞的结合与氨肽酶N抑制活性的相关性。结果:化合物LYRM03和Bestatin具有肿瘤细胞的氨肽酶N抑制活性,荧光标记化合物FITC-LYRM03、FITC-Bestatin能与肿瘤细胞有不同程度的结合。结论:标记化合物FITC-LYRM03、FITC-Bestatin和肿瘤细胞的结合与对肿瘤细胞的氨肽酶N抑制活性相一致。     

鳞翅目昆虫氨肽酶N与Bt毒素的结合及其与Bt抗性的关系

随着Bt Cry作物在我国的广泛应用和推广,靶标害虫对其抗性风险已成为Bt Cry作物生态安全研究的重要内容.氨肽酶N(Aminopeptidase N,APN)是位于昆虫中肠刷状缘膜囊泡(Brush Border Membrane Vesicles,BBMV)上Bt Cry毒素重要的受体蛋白之一,它与Bt Cry毒素...     

Purification and characterization of aminopeptidase N from chicken intestine with potential application in debittering

An aminopeptidase with broad substrate specificity was purified to homogeneity (123.7-fold) with a yield of 3.43% from chicken (Gallus gallus) intestine using a combination of chromatographic separation strategies. The enzyme was identified as alanyl aminopeptidase or aminopeptidase N (APN) by Peptide Mass Fingerprinting. The molecular weight of the enzyme was estimated to be ∼180 kDa by SDS-PAGE and gel filtration chromatography. The enzyme was found to be a glycoprotein, having 40% sugar residue and a molecular mass of 108 kDa after deglycosylation. The enzymatic activity was optimal at 60 °C and pH 6.0. The enzyme preferentially hydrolyzed Leu-β-NA (K_m = 0.1 mM) followed by Ala, Phe, Tyr and Gly at N-terminal. The enzyme activity was completely inhibited by 1,10 phenanthroline (1 mM) and bestatin (1 mM) confirming it as a metalloprotease. Potential of this enzyme in combination with other endoproteases for the production of debittered protein hydrolysates has been discussed.     

发酵麸皮多糖酶辅助提取工艺优化及其生物活性研究

通过响应面法优化提取发酵麸皮多糖的工艺,并评价其体外益生和抗氧化活性。以发酵麸皮多糖的得率为响应值,采用纤维素酶酶解与水浴浸提相结合的方法提取发酵麸皮多糖,以纤维素酶添加量、料液比、水浴浸提温度、水浴浸提时间为试验因素建立数学模型,筛选最佳提取工艺条件。通过测定还原力、DPPH和·OH自由基的清除能力对比发酵和未发酵麸皮多糖的体外抗氧化活性,并通过测定嗜酸乳杆菌、植物乳杆菌、两歧双歧杆菌的生长对比发酵和未发酵麸皮多糖的体外益生活性。结果表明,发酵麸皮多糖最佳提取工艺为:料液比1∶16(w/v),酶添加量1 000 U/g,水浴浸提温度90℃,水浴浸提时间60 min,在此条件下发酵麸皮多糖的得率实测值为73. 35%。发酵麸皮多糖具有较强的DPPH和·OH自由基的清除能力,可促进嗜酸乳杆菌、植物乳杆菌和两歧双歧杆菌的生长。     

Expression of a glycosylphosphatidylinositol-linked Manduca sexta aminopeptidase N in insect cells.

Aminopeptidase N (APN; EC 3.4.11.2) is an exopeptidase that is attached to cell membranes by a hydrophobic amino-terminal stalk in vertebrates or a glycosylphosphatidylinositol (GPI) anchor in insects. In this study, we report the cloning, expression, and characterization of an aminopeptidase N from Manduca sexta midgut. The full-length aminopeptidase N cDNA (APN1a) encodes a 995-amino-acid protein. The predicted amino acid sequence differs by 8 amino acids from M. sexta APN1. These different amino acids do not modify any putative glycosylation or glycosylphosphatidylinositol anchor sites. The full-length cDNA was cloned into an expression plasmid, pHSP-HR5, and transiently expressed in an insect cell line derived from Spodoptera frugiperda (Sf21 cells). Immunoblot analysis with anti-APN antiserum showed that APN1a expressed in Sf21 cells is the same size (120 kDa) as APN found in midgut brush border membranes. After treatment with phosphatidylinositol-specific phospholipase C (PIPLC), anti-cross-reacting determinant antibody specific for PIPLC cleavage products recognized the expressed 120-kDa APN1a, but not endogenous Sf21 proteins, indicating that APN1a has an intact glycosylphosphatidylinositol anchor. These results are evidence that Sf21 cells synthesize few, if any, endogenous GPI-linked proteins. Immunofluorescence staining showed that the expressed APN1a was located on the surface of Sf21 cells.     

Design,synthesis and biological evaluation of novel L-isoserine tripeptide derivatives as aminopeptidase N inhibitors

Aminopeptidase N (APN/CD13) is one of the essential proteins for tumour invasion, angiogenesis and metastasis as it is over-expressed on the surface of different tumour cells. Based on our previous work that L-isoserine dipeptide derivatives were potent APN inhibitors, we designed and synthesized L-isoserine tripeptide derivatives as APN inhibitors. Among these compounds, one compound 16l (IC₅₀?=?2.51?±?0.2 µM) showed similar inhibitory effect compared with control compound Bestatin (IC₅₀?=?6.25?±?0.4 µM) and it could be used as novel lead compound for the APN inhibitors development as anticancer agents in the future.     

Design, synthesis and biological evaluation of novel L-lysine ureido derivatives as aminopeptidase N inhibitors

As the exopeptidase over-expressed in the cell surface of endothelial cells, aminopeptidase N (APN/CD13) is an essential target for tumor angiogenesis and metastasis. Based on the previous work of L-lysine amide derivatives in our laboratory, we designed and synthesized two series of L-lysine ureido derivatives as APN inhibitors. Within these compounds, one compound, 5d (IC?? = 4.51 μM), showed similar inhibitory effect compared with Bestatin (IC?? = 5.87 μM).     

ATP依赖的人Lon蛋白酶的表达、纯化及其活性鉴定

ATP依赖的人Lon蛋白酶是一种同质寡聚、环状的蛋白酶,主要位于细胞线粒体基质中。许多研究表明,Lon蛋白酶对于维护细胞的内环境稳定起着重要作用,并参与线粒体蛋白质量控制和代谢调控。将pPROEX1 His6-Lon重组质粒在Escherichia coli Rosetta 2菌株中诱导表达用Ni2＋柱亲和层析法纯化,获得纯度较高的目的蛋白。经纯化后,Lon蛋白酶的比酶活达到0.17 U/mg。通过多肽底物Rhodamine 110、bis-（CBZ-L-alanyl-L-alanine amide）[（Z-AA）2 Rh110]的降解检测显示,Lon蛋白酶具有肽酶活性,并被ATP所刺激。Casein和线粒体转录因子A降解实验表明,纯化的Lon蛋白酶具有蛋白水解活性,而且蛋白水解活性依赖于ATP。     

Purification and properties of glutamyl aminopeptidase from chicken egg-white

Hydrolytic activities characteristic for different aminopeptidases were detected in the egg-white of unfertilized chicken eggs, and one aminopeptidase was isolated in an electrophoretically homogeneous form. The isolated aminopeptidase preferentially hydrolyzed bonds of alpha-glutamyl residue at the NH(2)-end of synthetic substrates and peptides. The enzyme is a dimer with an M(r) of 320,000 and pI of 4.2. Its optimal pH and temperature are 7.6 and 60 degrees C, respectively. EDTA, amastatin, and N-bromosuccinimide are inhibitors, while Ca2++ and Mn2+ are activators of the enzyme Ca2+ also stabilizes the enzyme. According to the observed properties, the isolated chicken egg-white aminopeptidase belongs to the glutamyl aminopeptidases.     

Synthesis and biological activity of [D-Trp6] chicken luteinizing hormone-releasing hormone

An agonist of chicken hypothalamic luteinizing hormone-releasing hormone (cLH-RH). [D-Trp⁶] cLH-RH, was synthesized and tested for luteinizing hormone (LH)-releasing activity using dispersed chicken anterior pituitary cells, as well as for binding to rat anterior pituitary membrane receptors. cLH-RH and mammalian LH-RH (mLH-RH) gave identical dose-response curves in stimulating chicken LH release ($\text{ED}{}_{50}\text{=1.6}$ and $\text{1.8×10}{}^{\mathrm{?9}}\text{M}$ respectively) and similar estimates of potency. The [D-Trp⁶] analogs of cLH-RH and mLH-RH stimulated LH release at lower doses ($\text{ED}{}_{50}\text{=7.0}$ and $\text{～7.0×10}{}^{\mathrm{?11}}\text{M}$ respectively) and were approximately 20-fold more potent. In contrast to the activity in the chicken bioassay, cLH-RH bound to rat anterior pituitary membrane receptors with a much lower affinity than did mLH-RH and had a relative potency of 2%. [D-Trp⁶] cLH-RH was approximately 100-fold more potent than cLH-RH in the rat receptor assay while [D-Trp⁶] mLH-RH was 28-fold more active than mLH-RH. These data demonstrate that substitution of Gly⁶ of LH-RH with D-Trp enhances the LH release from chicken pituitary cells to a similar extent to that observed in mammals, and indicate that the approaches used to produce active LH-RH analogs in mammals are likely to be applicable to birds.     

Biosynthesis of intestinal microvillar proteins. Expression of aminopeptidase N along the crypt-villus axis

The expression of pig small-intestinal aminopeptidase N (EC 3.4.11.2) along the crypt-villus axis was studied in tangential sections of [35S]-methionine-labelled, organ-cultured explants. The only detectable molecular forms of aminopeptidase N along the crypt-villus axis were polypeptides of Mr 140 000 and 166 000, representing the enzyme in a transient and mature form respectively. The synthesis was at a very low level in the crypt region in experiments with labelling periods ranging from 10 min to 3 h. These findings indicate that crypt cells are not fully committed to the expression of aminopeptidase N, either in its mature or in any other immunoreactive molecular form. The expression of aminopeptidase N was markedly stimulated by dexamethasone (1 microgram/ml). During labelling periods of 3 h, dexamethasone caused an approximately threefold increase in the expression of the enzyme in the crypt cells and a moderate increase of about 20% in the villus cells. Whereas the latter can possibly be ascribed to a general protective effect of dexamethasone on villus architecture, these experiments indicate that crypt cells of mucosa from adult individuals exhibit the same sensitivity to glucocorticoids as does the intestinal epithelium during the prenatal and early postnatal phase.