刺参甘露糖结合凝集素的生物信息学分析

甘露糖结合凝集素(mannan-binding lectin,MBL)属于C型动物凝集素超家族,特异地结合甘露糖。MBL是宿主起始免疫系统一个重要成分,因此近年来逐渐成为研究的热点。采用生物信息学技术对刺参(Apostichopus japonicus)MBL(AJ-MBL)的两个基因编码的蛋白质结构和功能进行了分析,包括信号肽、分子量、等电点、跨膜结构域、糖基化和磷酸化位点、二级结构和三级结构等,旨在了解其结构特征,为动物免疫反应方面研究提供理论基础。     

半乳糖凝集素糖结合的新模式：一个糖结合域的双重配体结合

半乳糖凝集素家族通过糖识别结构域(CRD)可以专一性识别和结合含β-半乳糖的多糖配体来发挥其生物学功能.到目前发现的CRD对β-半乳糖的识别模式是非常保守的,在结构已知的半乳糖凝集素结构中,一个CRD只能结合一个多糖配体分子.最近,通过对人源半乳糖凝聚素-3 CRD与对硝基TF二糖(TFN)复合物的晶体结构解析首次发现,一个CRD可以同时结合2个TFN分子.与这2个TFN分子有双向结合的残基突变体E165A结构分析显示,一个残基的突变引起的结构上的微小变化会使结合位点2丧失结合糖底物的能力,而位点1的配体结合却不受影响.这表明,结合位点1对糖底物保守的识别和结合是基本的、主要的,而结合位点2对于糖有条件的结合,是额外的、次要的.序列比对和立体化学分析显示,参与新位点2结合的关键残基在其他半乳糖凝集素分子中都是保守的,而它们参与糖配体结合并不常见,表明它们作用的发挥是有条件的.可能在复杂寡聚结构的情况下,如有多重分支结构,双重结合位点将有利于对这类配体分子的辨识和结合,已有一系列研究报道,具有分支结构的寡糖与半乳糖分子的亲和势明显高于单价糖配体,与上述分析相一致.对这类双重位点糖结合的可能生物学意义进行了讨论.     

用SDSL-EPR技术测量LSECtin-CRD与甘露糖结合过程中的位点运动性变化

目的:应用定点自旋标记-电子顺磁共振(SDSL-EPR)技术检测肝脏及淋巴结窦内皮细胞C型凝集素(LSECtin)的糖基识别结构域CRD上不同位点氨基酸的运动特性及其与甘露糖结合过程中的构象变化。方法:通过SOE-PCR方法对LSECtin-CRD引入半胱氨酸点突变,构建p ET28b-Tat-LSECtin-CRD载体,可溶性表达LSECtin-CRD突变体蛋白;对突变位点进行自旋标记;EPR检测标记后样品的EPR波谱;用SS-FDDM软件对EPR波谱进行模拟和解析,获得旋转相关时间τc,研究其构象特点及变化。结果:在CRD的Ca~(2+)结合位点和配体结合功能区构建了5个突变体蛋白;LSECtin-CRD与Ca~(2+)和甘露糖结合后,4个位点的τc值明显升高,表明CRD结构域整体运动性降低;5个突变位点运动性差异较大,其中A258位点运动性变化最大,可能是参与糖基结合的关键位点。结论:SDSL-EPR技术能够有效研究LSECtin-CRD参与糖基结合过程中的构象运动性,研究结果证明了LSECtin-CRD在与糖基结合后整体运动性受限。     

一种新的甘露糖结合凝集素——朱顶兰凝集素基因的克隆及序列分析

运用同源克隆的方法设计简并引物,通过3′和5′RACE技术,从石蒜科植物朱顶兰(Amaryllis vittata Ait)总RNA中克隆了编码此凝集素(AVA)的全长cDNA序列。该基因全长686 bp,起始密码子位于第41～43 bp,终止密码子位于515～517bp处,开放阅读框长474 bp,编码158个氨基酸,包含信号肽序列、成熟蛋白序列和C-末端剪切序列的前体蛋白。成熟蛋白由109个氨基酸残基组成,分子量为11.9kD。成熟蛋白在氨基酸水平上与雪花莲凝集素、水仙凝集素、石蒜凝集素和君子兰凝集素分别有73.4％、85.3％、80.7％和83.5％的同源性;朱顶兰凝集素的分子模式显示其与雪花莲凝集素有极其相似的三维结构;在Blocks数据库中检索AVA蛋白氨基酸序列的结构域,发现有3个凝集素功能结构域,并具有3个典型的甘露糖专一结合位点盒(QDNY)。     

一种新的甘露糖结合凝集素--朱顶兰凝集素基因的克隆及序列分析

运用同源克隆的方法设计简并引物,通过3′和5′RACE技术,从石蒜科植物朱顶兰(Amaryllis vittata Ait)总RNA中克隆了编码此凝集素(AVA)的全长cDNA序列.该基因全长686 bp,起始密码子位于第41～43 bp,终止密码子位于515～517 bp处,开放阅读框长474 bp,编码158个氨基酸,包含信号肽序列、成熟蛋白序列和C-末端剪切序列的前体蛋白.成熟蛋白由109个氨基酸残基组成,分子量为11.9kD.成熟蛋白在氨基酸水平上与雪花莲凝集素、水仙凝集素、石蒜凝集素和君子兰凝集素分别有73.4%、85.3%、80.7%和83.5%的同源性;朱顶兰凝集素的分子模式显示其与雪花莲凝集素有极其相似的三维结构;在Blocks数据库中检索AVA蛋白氨基酸序列的结构域,发现有3个凝集素功能结构域,并具有3个典型的甘露糖专一结合位点盒(QDNY).     

若干外源凝集素对胰岛素与其受体相互作用的影响

本文研究了若干外源凝集素对胰岛素与其脂肪细胞胰岛素受体相互作用的影响。结果表明与D-甘露糖和D-葡萄糖专一结合的外源凝集素,如ConA(伴刀豆球蛋白A)、豌豆及蚕豆凝集素可抑制~(125)Ⅰ-胰岛素与其受体的结合。与D-半乳糖专一结合的外源凝集素,如蓖麻凝集素Ⅰ和天花粉凝集素基本上不抑制~(125)Ⅰ-胰岛素与其受体的结合,在低浓度时有一定程度的促进胰岛素与受体结合的能力。另外两个和低聚糖专一结合的半夏凝集素和PHA(菜豆凝集素)的效应和蓖麻凝集素Ⅰ等类似。这些结果说明在胰岛素受体分子表面可能存在着与ConA等专一结合的糖,而且它们在胰岛素受体与胰岛素结合部位的附近。而和其他几个外源凝集素专一结合的糖或低聚糖链则和胰岛素受体与胰岛素结合的部位相距较远,并就这些凝集素对胰岛素与受体结合的促进作用进行了讨论。     

掌叶半夏凝集素基因PPA2抗蚜功能分析

掌叶半夏凝集素(Pinellia pedatisecta Agglutinin,PPA)基因属于单子叶甘露糖结合凝集素家族,具有抗虫活性。采用反转录聚合酶链式反应(RT-PCR)技术从掌叶半夏幼叶中克隆到1个新的凝集素基因,命名为PPA2,其开放阅读框长408 bp,编码135个氨基酸,有一个甘露糖保守结合域,包含3个甘露糖专一结合位点。分别构建了由35S启动子和韧皮部特异性启动子At PP2驱动的植物表达载体,农杆菌介导的叶盘法转化烟草后,经鉴定获得了单拷贝插入高表达量的纯合转基因烟草株系。通过Western-blot分析,将获得的转基因纯合株系按蛋白表达水平分成高、中、低3类并进行抗蚜试验,结果表明这些转基因烟草均具有明显抗蚜效果,35S-PPA2转基因植株平均抑蚜率极高,而3种蛋白表达类型的At PP2-PPA2转基因植株普遍低于35S启动子驱动下的转基因植株。PPA2可以作为高效抗蚜候选基因,具有重要的应用价值。     

应用生物素标记的凝集素分析登革Ⅱ型病毒糖蛋白

应用十二种生物素标记的凝集素,分析了经SDS-PAGE和电转印分离的登革Ⅱ型病毒(D_2V)糖蛋白。结果表明:D_2V的包膜糖蛋白E可与三种D-甘露糖特异的凝集素(conAPSA及LCA)结合,提示E蛋白的寡糖链为高甘露糖型。D_2V的糖基化非结构蛋白NS_1可与两种D-甘露糖特异的凝集素conA及LCA结合,提示NS_1的寡糖链亦为高甘露糖型。实验结果还显示了一些可能为C6/36细胞感染D_2V后新合成、合成量增加或减少以至完全受抑制的糖蛋白成分,有关这些糖蛋白的性质、功能、意义尚不清楚。在本实验条件下,这种生物素标记凝集素印迹法证明了其敏感性和糖基特异性,可以作为分析各种微量含糖大分子的一种实用方法。     

若干外源凝集素对胰岛素与其受体相互作用的影响

本文研究了若干外源凝集素对胰岛素与其脂肪细胞胰岛素受体相互作用的影响。结果表明与D-甘露糖和D-葡萄糖专一结合的外源凝集素,如ConA (伴刀豆球蛋白A)、豌豆及蚕豆凝集素可抑制~(125)I-胰岛素与其受体的结合。与D-半乳糖专一结合的外源凝集素,如蓖麻凝集素I 和天花粉凝集素基本上不抑制~(125)I-胰岛素与其受体的结合,在低浓度时有一定程度的促进胰岛素与受体结合的能力。另外两个和低聚糖专一结合的半夏凝集素和PHA (菜豆凝集素)的效应和蓖麻凝集素I 等类似。这些结果说明在胰岛素受体分子表面可能存在着与ConA等专一结合的糖,而且它们在胰岛素受体与胰岛素结合部位的附近。而和其他几个外源凝集素专一结合的糖或低聚糖链则和胰岛素受体与胰岛素结合的部位相距较远,并就这些凝集素对胰岛素与受体结合的促进作用进行了讨论。     

植物凝集素研究进展

植物凝集素广泛分布于植物界,它可以根据不同性质进行分类,按进化及结构相关性可以分为七个家族;豆科凝集素,单子叶植物甘露糖结构凝集素,含橡胶素结构域的几丁质结合凝集素,2型核糖体失活蛋白,葫芦科韧皮部凝集素,木菠萝素相关凝集素和苋科凝集素,在长期的进化过程中,它们形成几种不同的结合模体来识别一些外源多糖,在植物中未发现合适的内源性多糖受体。植物凝集素在生物学研究,农业和医学上有广泛的应用。     

2010年舟山某医院拉米夫定无良好应答乙肝患者耐药监测分析

目的观察2010年舟山某医院拉米夫定单药治疗无良好应答的乙肝患者其基因型和P区耐药位点突变的关系。方法随机选取舟山某医院就诊的124例拉米夫定单药治疗无良好应答的本地乙肝患者,男64例,女60例,平均年龄(46.92±15.16)岁,用实时荧光定量PCR检测其基因型和HBV-DNA,并用毛细管电泳测序技术对P区进行突变位点检测。结果 124例患者35例(28.23%)B型和89例(71.77%)C基因型。C型HBV-DNA为(5.83±0.91)log10 copies/mL,高于B型的(5.07±1.13)log10 copies/mL,差异具有统计学意义(t=3.55,P<0.01)。拉米夫定在B基因型中的耐药率为45.71%(16/35),低于C基因型的65.17%(58/89),差异具有统计学意义(2χ=3.951,P<0.05)。拉米夫定耐药株在B基因型中rtM204 I/V/S位点突变率为68.75%(11/16),高于C基因型的37.93%(22/58),差异具有统计学意义(2χ=4.821,P<0.05)。结论对于本地区拉米夫定治疗无良好应答的C基因乙肝患者,应加强拉米夫定的耐药位点筛查,而拉米夫定治疗无良好应答的B基因乙肝患者应加强rtM204 I/V/S位点检测。     

条石鲷仔、稚、幼鱼脂肪酸组成变化研究

通过采用GC/MS法研究了条石鲷(Oplegnathus fasciatus)仔鱼、稚鱼及幼鱼阶段的脂肪酸组成和变化特点。共检测到28种脂肪酸,其中饱和脂肪酸(SFA)13种,单不饱和脂肪酸(MUFA)7种,多不饱和脂肪酸(PUFA)8种。结果表明:条石鲷鱼苗内源性营养阶段以饱和脂肪酸C16∶0、C20∶0及单不饱和脂肪酸C16∶1、C18∶1作为能量代谢的主要来源;必需脂肪酸C20∶4(n-6)(AA)、C22∶5(n-3)(DPA)和C22∶6(n-3)(DHA)在稚鱼期含量较低,∑EPA+DHA仅为6.89%,认为是发生稚鱼"胀鳔病"的主要原因;仔鱼开口前体内的DHA和EPA是由母体卵黄提供的。     

Point mutations abolishing the mannose-binding capability of boar spermadhesin AQN-1

The mannose-binding capability of recombinant wild-type boar spermadhesin AQN-1 and of its site-directed mutants in the highly-conserved region around of the single glycosylation site (asparagine 50) of some spermadhesins, where the carbohydrate binding site has been proposed to be located, was checked using a solid-phase assay and a biotinylated mannose ligand. Substitution of glycine 54 by amino acids bearing an unipolar side chain did not cause significant decrease in the mannose-binding activity. However, amino acids with uncharged polar side chains or having a charged polar side chain abolished the binding of biotinylated mannose to the corresponding AQN-1 mutants. The results suggest that the higher surface accessibility of amino acids possessing polar side chains compared to those bearing nonpolar groups may sterically interfere with monosaccharide binding. The location of the mannose-binding site in AQN-1 appears to be topologically conserved in other heparin-binding boar spermadhesins, i.e., AQN-3 and AWN, but departs from the location of the mannose-6-phosphate-recognition site of PSP-II. This indicates that different spermadhesin molecules have evolved non-equivalent carbohydrate-binding capabilities, which may underlie their distinct patterns of biological activities.     

Detection of mannose-binding proteins on mouse lymphocytes

D-Mannose influences both the allogeneic stimulation of mouse spleen cells and the induction of cytotoxic T-lymphocytes. At low concentrations (10(-3) -4 x 10(-3) M), D-mannose increases [3H]thymidine incorporation and induction of cytotoxic cells. Higher concentrations lead to an inhibition of the allogeneic response. To test the involvement of mannose receptors, unstimulated and allogeneically stimulated spleen cells were lysed and the proteins electrophoretically separated. After blotting, the D-mannose-binding structures were identified by means of a biotinylated mannose-neoglycoprotein and avidin peroxidase. Two mannose-binding proteins of unstimulated mouse spleen lymphocytes with a molecular mass of 29 and 31 kDa were detected. In vitro allogeneically stimulated spleen lymphocytes show three other mannose-binding proteins with a molecular mass of 53, 65, and 76 kDa.     

AIDS合并马尔尼菲青霉病36例分析

目的探讨艾滋病合并马尔尼菲青霉病的临床表现,治疗与转归。方法回顾分析本院2008年8月～2009年8月收治的艾滋病合并马尔尼菲青霉36例。结果 36例艾滋病合并马尔尼菲青霉病平均年龄35.8岁。马尔尼菲青霉感染临床表现呈现非特异性,其中发热86.11%,贫血94.44%,GGT升高69.44%,AST升高63.89%,淋巴结肿大88.89%,脾大63.89%,低蛋白血症83.33%,咳嗽36.11%,皮损30.56%(其中典型改变仅5例,占13.89%),CD4+50cells/mm388.89%,骨髓培养(27/27)及皮损活检培养(2/2)阳性率100%,血液培养阳性率69.44%(25/36)。36例经抗真菌治疗,其中29例给予HAART治疗,28例治愈,7例好转,1例死亡。结论马尔尼菲青霉是艾滋病常见的机会性感染,早发现,早治疗,长程敏感抗真菌药物联合治疗可提高治愈率,减少复发。     

Helianthus tuberosus lectin reveals a widespread scaffold for mannose-binding lectins

BACKGROUND: Heltuba, a tuber lectin from the Jerusalem artichoke Helianthus tuberosus, belongs to the mannose-binding subgroup of the family of jacalin-related plant lectins. Heltuba is highly specific for the disaccharides Man alpha 1-3Man or Man alpha 1-2Man, two carbohydrates that are particularly abundant in the glycoconjugates exposed on the surface of viruses, bacteria and fungi, and on the epithelial cells along the gastrointestinal tract of lower animals. Heltuba is therefore a good candidate as a defense protein against plant pathogens or predators. RESULTS: The 2.0 A resolution structure of Heltuba exhibits a threefold symmetric beta-prism fold made up of three four-stranded beta sheets. The crystal structures of Heltuba in complex with Man alpha 1-3Man and Man alpha 1-2Man, solved at 2.35 A and 2.45 A resolution respectively, reveal the carbohydrate-binding site and the residues required for the specificity towards alpha 1-3 or alpha 1-2 mannose linkages. In addition, the crystal packing reveals a remarkable, donut-shaped, octahedral assembly of subunits with the mannose moieties at the periphery, suggesting possible cross-linking interactions with branched oligomannosides. CONCLUSIONS: The structure of Heltuba, which is the prototype for an extended family of mannose-binding agglutinins, shares the carbohydrate-binding site and beta-prism topology of its galactose-binding counterparts jacalin and Maclura pomifera lectin. However, the beta-prism elements recruited to form the octameric interface of Heltuba, and the strategy used to forge the mannose-binding site, are unique and markedly dissimilar to those described for jacalin. The present structure highlights a hitherto unrecognized adaptability of the beta-prism building block in the evolution of plant proteins.     

Essential tyrosine residues in 3-ketosteroid-delta(1)-dehydrogenase from Rhodococcus rhodochrous.

Tetranitromethane treatment of 3-ketosteroid-Delta(1)-dehydrogenase of Rhodococcus rhodochrous caused loss of the catalytic activity in a time- and concentration-dependent manner. Peptides (P-81) and (PN-83) were isolated from tryptic digests of the native and tetranitromethane-treated enzyme proteins, respectively. PN-83 was the nitrated form of P-81. The amino acid sequence was GGAPLIDYLESDDDLEFMVYPWPDYFGK (positions 97-124 of the dehydrogenase sequence). PN-83 showed a low yield of PTH-Tyr of position 116, i.e. less than 5% of that of P-81, and instead a high yield of PTH-3-nitrotyrosine. This indicated that tetranitromethane modifies Y-116 under the experimental conditions used. Mutation of Y-104, Y-116, and Y-121 to smaller amino acid residues, Phe, Ser, or Ala, significantly changed the catalytic activity of the dehydrogenase. All of the mutants contained FAD and exhibited the same spectrophotometric properties as those of the wild type enzyme. The K(m) values for 4-androstene-3,17-dione of the Y-104, Y-116, and Y-121 mutants changed to large values. The most drastic change was observed for Y116A. The K(d) values for 1,4-androstadiene-3,17-dione of the Y116 mutants changed to 1.5-2.6-fold larger values than that of the recombinant enzyme. The Y-121 mutant enzymes exhibited catalytic activities like those of the recombinant enzyme, but the catalytic efficiencies of Y121F and Y121A drastically decreased to 0. 014-0.054% of that of the recombinant enzyme. The present results indicate that Y-121 plays an important role in the catalytic function, and that Y-116 and Y-104 act on binding of the substrate steroid.     

Nucleotide sequence, genomic organization and cell-cycle-dependent expression of a Chlamydomonas 14-3-3 gene

Members of the 14-3-3 protein family have been identified as regulatory elements in intracellular signalling pathways and cell cycle control. Previously we reported the nucleotide sequence of a 14-3-3 cDNA cloned from the unicellular green alga Chlamydomonas reinhardtii. In this communication, we describe the nucleotide sequence, the genomic organization and the cell-cycle-dependent expression of the corresponding gene. The coding sequence of this gene was found to be interrupted by four introns of 124, 116, 81, and 659 bp, respectively. Introns 2-4 were found in conserved positions as compared to the Arabidopsis 14-3-3 genes. A counterpart to intron 1 absent in the Arabidopsis 14-3-3 genes was found in the human 14-3-3 epsilon gene.     

Curculin,a sweet-tasting and taste-modifying protein,is a non-functional mannose-binding lectin

A three-dimensional model of curculin, a sweet-tasting and taste-modifying protein from the fruits of Curculigo latifolia, was built from the X-ray coordinates of GNA, a mannose-binding lectin from snowdrop (Galanthus nivalis). The three mannose-binding sites present in GNA were found in curculin but are devoid of mannose-binding activity as shown by docking experiments performed with mannose. Some regions well exposed on the surface of the three-dimensional model of curculin could act as epitopes responsible for the sweet-tasting properties of this protein.     

四种生物农药及其与二种化学农药混配对稻飞虱混合种群的防治效果

试验以喷雾法研究4种生物农药及其与2种化学农药组合混用对水稻褐飞虱Nilaparvata lugens(Stal)和白背飞虱Sogatella furcifera(Horvath)混合种群的防治效果。结果表明:0.36%苦参碱AS、0.5%藜芦碱WP、1.8%阿维菌素EC、400亿孢子/克白僵菌WP,分别以1500mL/hm2、3000g/hm2、900mL/hm2、1800g/hm2的制剂用量单独使用,只有阿维菌素效果较好,药后7、14d的防效分别达到了73.3%和82.3%,但与对照药剂48%毒死蜱EC1500mL/hm2和25%噻嗪酮WP750g/hm2的防效差异极显著。而4种生物农药与2种化学农药分别以单用剂量减半混合使用的8个处理中,①苦参碱+毒死蜱、②藜芦碱+毒死蜱、③阿维菌素+毒死蜱、④球孢白僵菌+毒死蜱、⑤苦参碱+噻嗪酮、⑥藜芦碱+噻嗪酮6种组配,药后1d的校正防效为81.8%～89.2%,3d为88.2%～96.5%,7d为95.6%～98.1%,14d为86.0%～97.8%。因此,以上生物农药与化学农药的6种组合混用可以有效防治稻飞虱低龄若虫。