植物凝集素的功能

植物凝集素是来源于植物的一类能凝集细胞和沉淀单糖或多糖复合物的非免疫来源的非酶蛋白质。由于其对于单糖或糖复合物特异性结合的能力,使得其在如信号转导、免疫反应、植物防御等诸多信号过程中均具有重要作用。同时植物凝集素具有细胞凝集、抗病毒、抗真菌及诱导细胞凋亡或自噬等多种能力,因此在生命科学、医学及农业方面均有较好的研究价值和应用前景。综述了植物凝集素的研究历史和凝集素的主要功能,并对现阶段凝集素的重点应用做简要介绍。     

植物凝集素的超级家族

凝集素是一类专一、可逆地和糖类结合的蛋白质,迄今已经分离纯化并测定了氨基酸序列的凝集素已有不少,一些凝集素以及它们与配体糖相互结合的复合物的高级结构也已经给出,许多工作已深入到基因水平．就目前已有的知识,说明植物凝集素是一个庞大的蛋白质家族．     

植物凝集素固相亲和层析分离胚胎抗原

在细胞生物学,研究中固相化的植物凝集素是一种较有用的亲和吸附剂。植物凝集素能够识别并结合多糖、糖蛋白或糖脂,类似于酶与底物、抗体与抗原的识别和结合原理。然而,凝集素与它识别的糖以非共价结合,结合力弱且可逆。因此,利用凝集素识别一定顺序的糖链,并与之专一地结合的特性,可用于分离和纯化含糖大分子物质。前几年,我们证明了人体肝癌细胞表面存在着与3—5个月龄胎人体肝细胞起交叉反应的膜抗原,并用3MKCl     

新疆黄精凝集素基因的克隆、序列分析和表达

用百合科黄精属植物凝集素基因的保守序列为引物,从新疆黄精的叶中克隆出黄精凝集素的全长cDNA。序列分析表明, 克隆获得的新疆黄精凝集素(Polygonatum roseum agglutinin, PRA)基因完整的ORF片段大小为550 bp, 编码1 条长159个氨基酸肽链, 没有内含子, 其中N 端的28个氨基酸是信号肽。对新疆黄精凝集素cDNA 序列同源性的分析比较发现, 黄精属植物凝集素基因之间有很高的同源性(92%)。氨基酸序列比对和SWISS-MODEL同源模建分析表明, PRA由12个b-折叠片组成的b-桶结构, 具有与单子叶植物甘露糖结合凝集素相似的空间结构。重组质粒pGEX-4T-1-PRA 和pMAL-p2x-PRA, 分别转化E. coli BL21进行原核表达, 新疆黄精凝集素能够以可溶性融合蛋白形式表达, 分子量约为14 kD。构建真核表达载体pcDNA3-PRA, 免疫小鼠后获得了抗血清。免疫印迹结果显示为单一的条带, 证明该抗血清具有针对PRA抗原的专一性。新疆黄精凝集素基因的克隆、原核和真核的表达以及抗血清的制备, 为进一步研究凝集素蛋白的性质和功能, 并为植物抗病虫基因工程研究提供有用的实验材料。     

新疆黄精凝集素基因的克隆、序列分析和表达

用百合科黄精属植物凝集素基因的保守序列为引物,从新疆黄精的叶中克隆出黄精凝集素的全长cDNA。序列分析表明, 克隆获得的新疆黄精凝集素(Polygonatum roseum agglutinin, PRA)基因完整的ORF片段大小为550 bp, 编码1 条长159个氨基酸肽链, 没有内含子, 其中N 端的28个氨基酸是信号肽。对新疆黄精凝集素cDNA 序列同源性的分析比较发现, 黄精属植物凝集素基因之间有很高的同源性(92%)。氨基酸序列比对和SWISS-MODEL同源模建分析表明, PRA由12个b-折叠片组成的b-桶结构, 具有与单子叶植物甘露糖结合凝集素相似的空间结构。重组质粒pGEX-4T-1-PRA 和pMAL-p2x-PRA, 分别转化E. coli BL21进行原核表达, 新疆黄精凝集素能够以可溶性融合蛋白形式表达, 分子量约为14 kD。构建真核表达载体pcDNA3-PRA, 免疫小鼠后获得了抗血清。免疫印迹结果显示为单一的条带, 证明该抗血清具有针对PRA抗原的专一性。新疆黄精凝集素基因的克隆、原核和真核的表达以及抗血清的制备, 为进一步研究凝集素蛋白的性质和功能, 并为植物抗病虫基因工程研究提供有用的实验材料。     

中国水仙凝集素基因NTA的克隆、序列分析及蛋白结构预测

凝集素是一种糖专一性结合蛋白,它可以识别不同的糖类.植物凝集素是植物防御系统重要的组成部分.本研究采用RT-PCR的方法,从中国水仙花蕾中克隆了凝集素基因NTA,运用生物信息学方法对其核苷酸序列、编码的氨基酸序列进行分析以及对其蛋白结构进行预测.结果表明,得到的NTA基因全长698 bp,包含一个完整的开放阅读框516bp.该基因编码一个含有172个氨基酸的凝集素前体蛋白,该前体蛋白的等电点和分子量分别为5.84和18 615.19 Da.序列比对结果表明该基因编码的蛋白与其他单子叶植物如杂种水仙、雪花莲、君子兰、石蒜花和孤挺花的凝集素蛋白的同源性较高,分别为84%、80%、77%、78%以及82%.蛋白结构预测表明,中国水仙凝集素蛋白与洋水仙凝集素蛋白在结构上非常相似.对该基因编码的蛋白进行分析及蛋白结构模拟可知,该蛋白含有三个特殊的功能结构域和alpha-D卜甘露糖结合表面(QXDXNXVXY).     

黄瓜苋科凝集素基因的表达分析与逆境调控

凝集素是一类具有特异性糖结合活性的蛋白质, 通常具有1个或多个非催化的糖结合结构域。凝集素在植物对病原菌的防御反应中发挥重要作用。由于其抗细菌、真菌、病毒和昆虫等的活性, 凝集素在农业和生物医药领域都具有很大的应用潜力。作为最小的凝集素家族之一, 苋科凝集素的研究较少。该文通过对重要经济作物黄瓜(Cucumis sativus)的基因组进行分析, 对16种苋科凝集素基因在黄瓜基因组中的分布和位置进行研究, 并分析相关基因的外显子/内含子组成。进一步通过启动子分析, 阐明了苋科凝集素基因对非生物胁迫的响应情况。最后, 通过实时荧光定量PCR, 检测了黄瓜中4种苋科凝集素基因对低温、高盐、干旱和ABA处理的响应情况。研究结果可为揭示苋科凝集素的生理功能及其在植物胁迫响应中的作用提供参考。     

植物凝集素及其在抗虫植物基因工程中的应用

植物凝集素是一类具有特异糖结合活性的蛋白,具有一个或多个可以与糖或寡聚糖特异可逆结合的非催化结构域。其糖结合活性是针对外源寡糖,参与植物的防御反应。本文综述了有关植物凝集素分子生物学的研究进展,介绍了植物凝集素的分类、糖结合特性、近年来有关植物凝集素蛋白晶体结构的研究,及其与糖结合能力相关的生物学功能。并对植物凝集素在抗虫植物基因工程中的应用现状及发展前景做了阐述。 Abstract:Plant lectins are proteins possessing at least one non-catalytic domain that binds reversibly to specific mono-or oligosaccharides.They distinguish themselves from other plant proteins by the ability of carbohydrate binding.Most plant lectins are directed to bind foreign polysacchride.Plant lectin is believed to take part in the defense responses against invader.In this paper we presented a review on the classification,characters,functions,crystal structure and,functions related to the carbohydrate binding activity.The status and prospect of plant lectins utilization were also discussed.     

单子叶甘露糖结合凝集素的结构及生物活性

单子叶甘露糖结合凝集素(monocot mannose-binding lectin,MBL)是植物凝集素超家族中具有甘露糖及其衍生物结合专一性的糖结合蛋白,对肿瘤细胞、逆转录病毒具有特异而强烈的抑制作用。作为甘露糖翻译器,MBL在糖组学研究中也扮演着重要的角色。迄今为止,对害虫具有良好抗性的转雪花莲、半夏凝集素基因的水稻、小麦、马铃薯、烟草等农作物均已成功问世,可见MBL在医学和农业应用领域都呈现出巨大的开发价值和广阔的应用前景。本文列举了近20年来新发现的几种典型的MBL,并对其分布、分子结构、糖结合专一性、系统进化、生理作用等方面进行了概述。     

豆科凝集素研究进展

豆科凝集素是植物凝集素中最丰富,也是研究最多的一类凝集素。在生理条件下豆科凝集素大多是以二聚体或四聚体的形式存在,这种低聚物的形式给予豆科凝集素较强的糖专一性和大分子结构的稳定性。豆科凝集素除作为植物储存物质的作用外,还具有识别糖蛋白、糖肽及生物膜中碳水化合物和作为植物与微生物的共生介质等生理功能。现对豆科凝集素的结构、功能及其在生物学、农业和医学方面的应用进行了综述。     

Molecular basis of sugar recognition by the human L-type lectins ERGIC-53, VIPL, and VIP36

ERGIC-53, VIPL, and VIP36 are related type 1 membrane proteins of the mammalian early secretory pathway. They are classified as L-type lectins because of their luminal carbohydrate recognition domain, which exhibits homology to leguminous lectins. These L-type lectins have different intracellular distributions and dynamics in the endoplasmic reticulum-Golgi system of the secretory pathway and interact with N-glycans of glycoproteins in a Ca(2+)-dependent manner, suggesting a role in glycoprotein sorting and trafficking. To understand the function of these lectins, knowledge of their carbohydrate specificity is crucial but only available for VIP36 (Kamiya, Y., Yamaguchi, Y., Takahashi, N., Arata, Y., Kasai, K. I., Ihara, Y., Matsuo, I., Ito, Y., Yamamoto, K., and Kato, K. (2005) J. Biol. Chem. 280, 37178-37182). Here we provide a comprehensive and quantitative analysis of sugar recognition of the carbohydrate recognition domains of ERGIC-53 and VIPL in comparison with VIP36 using a pyridylaminated sugar library in conjunction with frontal affinity chromatography. Frontal affinity chromatography revealed selective interaction of VIPL and VIP36 with the deglucosylated trimannose in the D1 branch of high-mannose-type oligosaccharides but with different pH dependence. ERGIC-53 bound high-mannose-type oligosaccharides with low affinity and broad specificity, not discriminating between monoglucosylated and deglucosylated high-mannosetype oligosaccharides. Based on the sugar-binding properties in conjunction with known features of these proteins, we propose a model for the action of the three lectins in glycoprotein guidance and trafficking. Moreover, structure-based mutagenesis revealed that the sugar-binding properties of these L-type lectins can be switched by single amino acid substitutions.     

Recombinant lectins: an array of tailor-made glycan-interaction biosynthetic tools

Lectins are a heterogeneous group of proteins found in plants, animals and microorganisms, which possess at least one non-catalytic domain that binds reversibly to specific mono- or oligosaccharides. The range of lectins and respective biological activities is unsurprising given the immense diversity and complexity of glycan structures and the multiple modes of interaction with proteins. Recombinant DNA technology has been traditionally used for cloning and characterizing newly discovered lectins. It has also been employed as a means of producing pure and sequence-defined lectins for different biotechnological applications. This review focuses on the production of recombinant lectins in heterologous organisms, and highlighting the Escherichia coli and Pichia pastoris expression systems, which are the most employed. The choice of expression host depends on the lectin. Non-glycosylated recombinant lectins are produced in E. coli and post-translational modified recombinant lectins are produced in eukaryotic organisms, namely P. pastoris and non-microbial hosts such as mammalian cells. Emphasis is given to the applications of the recombinant lectins especially (a) in cancer diagnosis and/or therapeutics, (b) as anti-microbial, anti-viral, and anti-insect molecules or (c) in microarrays for glycome profiling. Most reported applications are from recombinant plant lectins. These applications benefit from the tailor-made design associated with recombinant production and will aid in unraveling the complex biological mechanisms of glycan-interactions, bringing recombinant lectins to the forefront of glycobiology. In conclusion, recombinant lectins are developing into valuable biosynthetic tools for biomedical research.     

Lectin affinity high-performance liquid chromatography: interactions of N-glycanase-released oligosaccharides with leukoagglutinating phytohemagglutinin, concanavalin A, Datura stramonium agglutinin, and Vicia villosa agglutinin

We have developed a lectin affinity high-performance liquid chromatography technique for analysis of oligosaccharides using columns of silica-bound lectins. Purified leukoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), Datura stramonium agglutinin (DSA), and Vicia villosa agglutinin (VVA) were covalently coupled to periodate-oxidized diol-silica by reductive amination. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with the silica-bound lectins. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. The oligosaccharide specificities displayed by silica-bound L-PHA, Con A, and DSA were virtually identical to those established utilizing lectin-agarose conjugates. Analysis of oligosaccharides by lectin affinity HPLC allowed further definition of the specificity of VVA for N-glycanase-released, reduced oligosaccharides. Lectin affinity HPLC is rapid and convenient, providing an important structure-specific dimension to oligosaccharide analysis. This technique is particularly useful when utilized in conjunction with anion-exchange and ion-suppression amine adsorption HPLC methods, which fractionate on the basis of charge and size, respectively. In addition to their utility for oligosaccharide characterization, these affinity columns demonstrate the high degree of oligosaccharide specificity displayed by plant and animal lectins.     

Capillary affinity electrophoresis using lectins for the analysis of milk oligosaccharide structure and its application to bovine colostrum oligosaccharides

Animal colostrum and milk contain complex mixtures of oligosaccharides, which have species-specific profiles. Milk oligosaccharides have various types of structure related to the core structures of glycolipids and N- and O-glycans of glycoproteins and provide a good library to examine the binding of oligosaccharides to various lectins. Recently, we reported a capillary affinity electrophoresis (CAE) method for analyzing the interactions between lectins and complex mixtures of N-linked oligosaccharides prepared from serum glycoproteins. The present paper reports the interactions between 24 milk oligosaccharides and six lectins (PA-I, RCA(120), SBA, WGA, UEA-I, and AAL) analyzed using CAE. Based on the resulting data, we constructed a library that enables us to determine nonreducing terminal monosaccharides, such as Gal, GalNAc, GlcNAc, and Fuc, and to differentiate Gal- or Fuc-linked isomers, such as lacto-N-tetraose, lacto-N-neotetraose, and lacto-N-fucopentaose II and III. In addition, using the library, we show that a combination of the lectins can characterize the neutral oligosaccharides derived from bovine colostrum.     

Carbohydrate microarrays - a new set of technologies at the frontiers of glycomics

Carbohydrate microarray technologies are new developments at the frontiers of glycomics. Results of 'proof of concept' experiments with carbohydrate-binding proteins of the immune system - antibodies, selectins, a cytokine and a chemokine - and several plant lectins indicate that microarrays of carbohydrates (glycoconjugates, oligosaccharides and monosaccharides) will greatly facilitate not only surveys of proteins for carbohydrate-binding activities but also elucidation of their ligands. It is predicted that both naturally occurring and synthetic carbohydrates will be required for the fabrication of microarrays that are sufficiently comprehensive and representative of entire glycomes. New leads to biological pathways that involve carbohydrate-protein interactions and new therapeutic targets are among biomedically important outcomes anticipated from applications of carbohydrate microarrays.     

The X-lectins: A new family with homology to the Xenopus laevis oocyte lectin XL-35

The Xenopus laevis oocyte cortical granule lectin (XL35) has been studied in fertilization and embryonic development. Several nucleic acid sequences that predict proteins homologous to XL35 have since been reported in frog, human, mouse, lamprey, trout, ascidian worm. These proteins also showed high degrees of amino acid sequence homology to a common fibrinogen-like motif that may involve carbohydrate binding. Although their biological functions and carbohydrate binding specificities have not been studied in detail, this new family of lectins has common characteristics. Several independent studies on this new family of lectins strongly suggest that the lectins are expressed and stored in specialized vesicles that may be released upon the infection by pathogens. In addition, some family members have been shown to bind to oligosaccharides from bacterial pathogens. Therefore, this family of lectins likely participates in pathogen surveillance as part of the innate immune system. We propose the name X-lectin family for these homologs of XL35. Published in 2004.     

Oligosaccharide specificities of Phaseolus vulgaris leukoagglutinating and erythroagglutinating phytohemagglutinins. Interactions with N-glycanase-released oligosaccharides

The structural determinants required for interaction of oligosaccharides with leukoagglutinating phytohemagglutinin (L-PHA) and erythroagglutinating phytohemagglutinin (E-PHA) from Phaseolus vulgaris have been studied by immobilized lectin affinity chromatography. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with columns of L- and E-PHA-agarose. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. In virtually all cases, L- and E-PHA yielded identical results, indicating that their specificities for reduced oligosaccharides are similar. Both lectins retarded oligosaccharides bearing alpha 2,3- but not alpha 2,6-linked sialic acid. Desialylated oligosaccharides containing one, two, three, or four peripheral N-acetyllactosamine-type branches were retarded to varying extents by both lectins; however, this interaction was decreased or eliminated by removal of Gal. Desialylated oligosaccharides containing a bisecting GlcNAc residue attached to the beta-linked core Man displayed the greatest interaction with both lectins. Structures containing terminal sulfate or GalNAc did not interact with either lectin. In some instances, the specificities of L- and E-PHA lectins for free, reduced oligosaccharides differed from those established using glycopeptides. Therefore, the structural requirements for interaction with lectins such as L- and E-PHA must be fully and systematically defined using the appropriate authentic standards in order to use lectin affinity chromatography for the fractionation and characterization of free oligosaccharides.     

The role of lectins in plant defence

Summary Recent progress in the search for the physiological role of plant lectins supports the idea that some of these proteins are involved in the defence mechanisms of the plant. To place the evidence in favour of such a defensive role in a broad perspective, a short overview is given of the most important plant pathogens and predators. In addition, the solutions that plants have developed to resist the continuous threat of a hostile environment are briefly discussed in relation to the protective role of proteins in general. The presumed involvement of plant lectins in defence mechanisms is first inferred from an analysis of the biochemical, physiological, cellular biological and molecular biological properties of plant lectins. Subsequently, the available experimental evidence for the involvement of lectins in the plant's defence against viruses, bacteria, fungi and herbivorous invertebrates and vertebrates is discussed in some detail. Since the defensive role of plant lectins is determined largely by their ability to recognize and bind foreign glycans, a brief discussion is given of how the basically protective properties of these proteins can be exploited for histochemical applications in biological and biomedical research.