The Endogenous Lectin Cerebellar Soluble Lectin and Its Ligands in Central Nervous System Myelin of Myelin-Deficient (mld) Mutant Mice

The myelin-deficient (mld) mutation is autosomal recessive mutation in the murine CNS exhibiting severe hypomyelination. The primary defect results in a drastic reduction of myelin basic protein synthesis caused by a duplication of the myelin basic protein gene with partial inversion of the upstream gene copy. The severe deficit of myelin basic protein is responsible for the absence of the major dense line but cannot explain the heterogeneity of myelin compaction found in mld. We have tested the hypothesis that the endogenous cerebellar soluble lectin (CSL) and/or its endogenous glycoprotein ligands could be involved in myelin abnormalities in the dysmyelinating mutant, mld. Immunocytochemical and immunoblotting techniques showed that the CSL level was not reduced significantly in the mld mutant. Furthermore, two ligands of CSL, the myelin-associated glycoprotein and an axonal glycoprotein, with a relative molecular mass of 31 kDa, were not decreased in level in the purified myelin fraction isolated from mld mice. In contrast, three minor glycoprotein ligands of CSL of relative molecular mass of 23, 18, and 16 kDa were greatly reduced in content. The reduced concentration of these low-molecular-mass glycoproteins in mld myelin suggests that they are constituents of compact myelin. Furthermore, the observation that CSL is specifically localized in vivo in regions where mld myelin is more compact and absent from regions devoid of myelin compaction may suggest that the endogenous CSL lectin, as well as its minor glycoprotein ligands, plays a role in the stabilization of the myelin sheath.     

Endogenous Lectin Cerebellar Soluble Lectin Involved in Myelination Is Absent from Nonmyelinating Schwann Cells

In the sciatic nerve, two major classes of Schwann cells are present which differ in their capability to produce myelin. Myelinating Schwann cells surround most of the axons with the formation of a typical myelin sheath. Nonmyelinating Schwann cells serve to insulate individual axons without formation of myelin. These dissimilarities between the two types of Schwann cells provided an interesting model for studying mechanisms underlying myelination and the formation of contacts between axons and myelinating cells. It is demonstrated here that the endogenous lectin cerebellar soluble lectin (CSL), implicated in myelin stabilization and in formation of contact between axon and myelinating cells in the CNS and in the sciatic nerve, is undetectable in non-myelinating Schwann cells. In contrast, most axons surrounded by these cells contained the major axonal glycoprotein ligand of CSL, a 31-kDa glycoprotein which is present in large amounts. The possible relationship between the presence of CSL in Schwann cells and their capacity to interact with axons and to produce myelin are discussed.     

精神分裂症患者甘露糖结合凝集素基因启动子区多态性及血清浓度的研究

目的:探讨精神分裂症患者甘露糖结合凝集素(MBL)的血清浓度和启动子区基因多态性的相关性及在患者免疫病理机制中的作用。方法:选取2018年6月至2018年12月在我院就诊的精神分裂患者为54例为观察组,另选取同时期本院健康体检中心志愿者56例为正常对照组,采用合酶链反应-序列特异性引物(PCR-SSP)技术分析MBL基因启动子区H/L(-550bp G/C)和X/Y(-221bp C/G)的多态性,酶联免疫吸附法(ELISA)检测血清MBL的浓度。结果:观察组血清MBL浓度(1367.218±1277.429)ng/mL低于正常对照组(1987.781±976.748)ng/mL,差异有统计学意义(P0.05)。观察组HY/HY基因型频率低于对照组(P0.05),HY/LY型频率高于对照组(P0.05)。观察组HY/HY基因型血清MBL浓度明显高于HY/LX、LY/LX型(P0.05);观察组MBL基因启动子区突变型纯合子的血清MBL水平均与对照组差异无统计学意义(P0.05),而MBL基因型杂合子的MBL水平则差异有统计学意义(P0.05)。结论:精神分裂症患者MBL水平低下与患者MBL基因启动子区各基因型的综合调控有关。MBL浓度低下是精神分裂症患者循环免疫复合物(CIC)滞留或清除障碍的分子机制之一。     

Molecular Structure and Properties of Lectin from Tomato Fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro₄ motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.     

一种新型家蝇蛹凝集素结构的初步探讨*

初步分析了分离出的一种分子量为55kDa,具有免疫活性的新型家蝇蛹D-半乳糖结合凝集素的结构,为深入研究其结构与功能之间的关系提供大量的信息。首先通过凝胶电泳染色确定此新型家蝇蛹凝集素具有糖链结构,借助蛋白质测序仪、分光光度计比色、β-消除反应、红外光谱以及原子力显微镜技术对其结构进行初步分析。此种家蝇蛹凝集素是一种蛋白和糖含量分别为97.36% 和2.1% 的球状单体,直径为75nm左右。肽链与糖链的连接方式为O-糖苷键型,肽链的N-末端封闭,糖环为吡喃型,为进一步分析其结构提供了可靠的依据。     

北寄生凝集素的分离和性质研究

采用酸处理Sepharose 6B亲和层析和Sephacryl S-200凝胶过滤,首次从中药北寄生中分得一种有毒凝集素,称为北寄生凝集素(Viscum coloratum lectin,VCL),是一种高毒性的植物毒蛋白,对小鼠静脉给药的LD₅₀为4.4μg/kg,其粗提物也具有很强的毒性,对小鼠腹腔给药的LD₅₀为0.2g/kg,分子量52 000,等电点10.5.     

Epidermal Growth Factor Enhances the Expression of an Endogenous Lectin in Aggregating Fetal Brain Cell Cultures

Aggregating cell cultures prepared from fetal rat telencephalon express the two subunits [cerebellar soluble lectins (CSL) 1 and 2] of a soluble, mannose-specific endogenous lectin (CSL) in a development-dependent manner. Increased CSL synthesis was found at an early postmitotic stage as well as during the period of maximal myelination. Repetitive treatment of early cultures with epidermal growth factor (EGF, 3nM) caused a great stimulation of CSL biosynthesis. Immunocytochemical studies revealed particularly intense CSL-specific staining in small, EGF-responsive cells, presumably glial cells. Large quantities of CSL-immunoreactive material were found also in the extracellular space and on the external side of the plasma membrane, indicating abundant release of CSL. The present findings suggest that EGF or EGF-related factors in the brain are able to regulate the expression of an endogenous lectin, affecting brain ontogeny.     

凝集素芯片对体液细胞进行检测方法的建立和初步应用

建立对体液细胞进行自动捕获的凝集素芯片体系,利用凝集素对糖链的特异亲和作用捕获细胞,提取白血病患者外周血、肺癌胸水和肝腹水中细胞进行荧光标记,凝集素芯片捕获,激光扫描仪检测捕获细胞的荧光信号,常规HE染色后光学显微镜下观察细胞的形态并进行免疫化学反应,流式细胞仪验证凝集素芯片的特异性．结果表明：凝集素芯片可以对体液中的癌细胞进行自动捕获,对癌细胞膜表面糖链进行识别．芯片检测的细胞浓度最少可达每mL10^4个左右．芯片有较好的重复性和特异性．这种凝集素芯片可用于临床体液中癌细胞的检测分析,对癌细胞膜表面凝集素亲和位点进行即时、高通量的检测,为了解细胞膜表面聚糖在癌变过程中的变化提供了一个技术平台．     

长裙竹荪凝集素的分离纯化与部分生化性质

凝集素是一类与糖专一结合的蛋白质或糖蛋白 ,具有众多的生物学性质[1～ 5] ,在细胞凝集、鉴别人类血型物质和分离纯化某些高分子化合物等都有着非常重要的作用 ,已成为生物化学、细胞学、免疫学及医学等领域中有用的科研材料 ,并被应用于临床诊断、治疗和某些工业生产[1] .自 1888年Ｈ .Ｓｔｉｌｌｍａｒｋ首次从蓖麻籽中发现凝集素以来 ,已分离纯化 10 0多种凝集素 ,约有 60种已成为商品 ,其研究开发日益受到人们的重视 .竹荪是一种著名的食、药兼用菌 ,具有许多药用功效 .由于竹荪含有多种生理活性物质 ,从竹荪子实体或菌丝体中分离到的…     

同步纯化人心肌肌钙蛋白T、I

同步纯化人心肌肌钙蛋白Ｔ、Ｉ李志梁付朝平钱学贤陆青王素华黎梅兰（第一军医大学珠江医院心内科,广州５１０２８２）关键词心肌肌钙蛋白Ｔ;心肌肌钙蛋白Ｉ;同步纯化收稿日期：１９９６－０４－１７;接受日期：１９９６－０８－２７。心肌肌钙蛋白包括３种不同的蛋白...     

Isolation of glycoproteins of parainfluenza I (Sendai) by Helix pomatia Lectin column

Glycoproteins of Sendai virus were successfully isolated on a column of Helix pomatia Lectin-Sepharose 6MB following solubilization with Nonidet P-40. This technique can be used as a preparative step for the study of viral glycoproteins.     

海芋凝集素cDNA的分子克隆及其性质预测

用cDNA末端快速扩增-聚合酶链式反应（RACE-PCR）方法克隆了海芋（Alocasia macrorrhiza）凝集素的全长cDNA（GenBank检索号DQ340864）,并用多种生物信息学工具对其性质进行了预测。根据来源于天南星科其他植物的凝集素和类似蛋白的保守区的DNA序列,设计了几个海芋凝集素基因aml特异引物（GSP）。用RNeasy试剂盒从海芋块茎中提取出总RNA,并以此为模板,用SMART^TM RACE cDNA扩增试剂盒提供的经特殊设计的通用引物以及不同的基因特异引物,分别获得海芋凝集素5′-和3′-RACE-PCR扩增片段。这些PCR产物经0．8％琼脂糖凝胶纯化后,分别与T克隆载体pMD 18-T相连,筛选获得阳性克隆并提取质粒,经双酶切和特异引物的PCR验证无误后,进行序列分析。从5′-和3′-RACE-PCR测序结果拼接出全长海芋凝集素cDNA序列,并用新设计自5′-RACE-PCR 5′末端的引物GSP7进行全长3′-RACE-PCR反应,获得全长海芋凝集素cDNA克隆并再次测序验证。这一新克隆的海芋凝集素cDNA的长度为1124核苷酸,分析表明它是一个编码270个氨基酸残基的蛋白质,其等电点为pH 5．7,相对分子量为29．7kD。同源性分析结果表明,海芋凝集素与其他来源于天南星科的甘露糖凝集素以及相似蛋白具有高度同源性。在海芋凝集素序列中发现了2个B型凝集素功能区域和3个甘露糖的结合位点。综合上述信息,认为这一新克隆的海芋凝集素cDNA是一个编码甘露糖识别凝集素的基因序列。     

凝集素芯片技术检测糖蛋白方法的建立及初步应用

建立了凝集素芯片技术检测糖蛋白的方法,对实验条件进行了优化,应用凝集素芯片初步检测分析了Chang?蒺s liver正常肝细胞总蛋白中的糖蛋白糖链构成．将凝集素ConA、GNA固定于环氧化修饰的玻片表面,用Cy3标记标准糖蛋白RNaseB,利用凝集素识别特异糖链的原理建立凝集素芯片检测糖蛋白的方法．摸索出最佳封闭剂是含1% BSA的磷酸缓冲液,最佳孵育时间及温度为3 h和室温,最佳孵育缓冲液为含1% BSA和0.05% Tween-20的磷酸缓冲液,并用甘露糖抑制实验验证了凝集素芯片结合的特异性．用包含10种凝集素的芯片,成功解析了标准糖蛋白RNaseB、Fetuin的糖链构成,证实了凝集素芯片检测糖蛋白糖链的可行性．最后用凝集素芯片初步检测分析了Chang?蒺s liver正常肝细胞总蛋白中的糖蛋白糖链构成,发现 Chang's liver正常肝细胞总蛋白中的糖蛋白可能有多价 Sia或GlcNAc、terminalα-1,3 mannose、GalNAc、Galβ-1,4GlcNAc这些糖链结构的存在．蛋白质糖基化是一种重要的翻译后修饰,它在微生物感染、细胞分化、肿瘤转移、细胞癌变等生命活动中起着重要作用,因此近年来蛋白质的糖基化研究受到广泛的重视,但由于缺乏一种简便、快速、高通量的检测手段,蛋白质糖基化修饰的研究发展缓慢．凝集素芯片技术的出现实现了对糖蛋白的快速、准确、高通量的检测 分析．     

香蕉凝集素基因启动子的分离、序列分析及鉴定

香蕉是世界上最重要的水果之一,由于香蕉果实是利用转基因方法生产重组药用蛋白或有价值的化合物的理想器官,构建能在香蕉果实中高水平表达异源蛋白质的表达载体是非常有意义的。而一个高效表达的载体,启动子则是其最重要元件之一,因此,果实特异性表达启动子的获得是香蕉作为生物反应器的前提。香蕉凝集素是一种在香蕉果实中大量存在的蛋白质,其基因被证明在果肉组织中大量表达。利用染色体步移法克隆到香蕉凝集素基因5′端上游的一段长702bp的序列,经序列测定及软件分析表明,该序列具有典型的启动子结构。此序列置换植物表达载体pBI121的CaMV35S启动子,构建植物表达载体,命名为pBIL2,该启动子下游为gus基因。利用基因枪法转化香蕉的根、叶和果实薄片,对gus基因的瞬时表达进行测定,结果表明所获得的凝集素基因启动子,只在香蕉果肉中瞬时表达,该启动子的表达具有果实特异性,并且表达量较高。     

天花粉凝集素的固定化及其应用

制备了天花粉凝集素ＴＫＬ－Ｓｅｐｈａｒｏｓｅ４Ｂ的亲和柱,用来分离其它来源物质中与ＴＫＬ相互作用的糖复合物,发现从兔红细胞的血影细胞的抽提物中可以得到分子量为６２ｋＤ,４０ｋＤ等糖蛋白,这可能就是ＴＫＬ凝集兔红细胞的分子基础。从人及骆驼、绵羊血浆中也能得到与ＴＫＬ结合的、分子量不等的糖蛋白,进一步鉴定人血浆ＴＫＬ结合物中含有α＿１－抗胰蛋白酶等,为从血浆中分离这些糖蛋白提供了较为便利的方法。在寻找天花粉植物体内此凝集素的内源受体时,探讨了它可能的生理功能。     

家蝇蛹凝集素的分离工艺和性质的初步研究

通过缓冲液浸提、戊二醛固定化红细胞吸附、亲和层析和凝胶过滤等方法,从家蝇蛹中分离纯化出一种对半乳糖专一的凝集素.结果表明,亲和层析后,SDS-聚丙烯酰胺凝胶电泳检测家蝇蛹凝集素(MPL)分子质量为84 ku.SephadexG-200凝胶过滤后,MPL分子质量为86 ku.家蝇蛹凝集素专一性识别D-半乳糖,其血凝活力对热不稳定,不依赖Ca²⁺,在pH 6～9之间稳定.家蝇蛹凝集素对Escherichia coli, Bacillus subtilis和Salmonella typhi有抑制作用.     

苦参凝集素的分离纯化及部分性质研究

从苦参 (SophoraflavescensAit.)根浸出液经硫酸铵分级 ,得苦参凝集素 (SFL)粗品 ,再经DEAE_Sepharose、SephadexG_1 5 0和HPLC层析 ,获得具有强凝集活性的SFL样品 ,用PAGE和SDS_PAGE检测均为单一蛋白染色带。SDS_PAGE显示SFL分子仅有一条肽链 ,SephadexG_1 0 0和SDS_PAGE测得其分子量均为 32kD。当SFL浓度为 0 .97μg/mL时能凝集兔红细胞 ,无血型专一性 ,其凝血活性可被甘露糖和果糖抑制 ,麦芽糖和葡萄糖有弱的抑制作用 ,凝集素分子含有 2 .89%的中性糖 ;当SFL量为 6 2 μg时 ,对棉花枯萎病菌 (FusariumvasinfectumAtk .)、小麦赤霉病菌(Gibberllasaubinetii (Mont.)Sacc .)和水稻稻瘟病菌 (PiriculariaoryzaeCav)菌丝体的生长发育有明显地抑制作用。用Edman法在蛋白测序仪上测出SFL的N端肽链 30个氨基酸的排列顺序为 :T/A/VDXLXFTFSDFDP NGEDLLFQGDAHVTSNN。     

Purification and Characterization of Sophora flavescens Lectin

A lectin with strong hemagglutination activity was isolated from roots of Sophora flavescens Ait. by extraction, fractionation with (NH 4) 2SO 4, ion-exchange chromatography on DEAE-Sepharose and followed by gel filtration on Sephadex G-150 and HPLC assay. The purified lectin showed a single protein band on PAGE and SDS-PAGE . The molecular weight of S. flavescens lectin was 32 kD when SDS-PAGE and Sephadex G-100 was used. The lectin agglutinated rabbit red blood cells at 0.97 μg/mL and showed no specific agglutination with any type of human erythrocytes. The hemagglutination activity could be inhibited by mannose and levulose and slightly by glucose and maltose. The SFL contained 2.89% neutral saccharide. It could inhibit apparently the growth of the mycelium of Gibberlla saubinetii （Mont.） Sacc.,Piricularia oryzae Cav. and Fusarium vasinfectum Atk. at the dosage of 62 μg. It was determined by Edman that the sequence of the N-terminal thirty amino acids was: T/A/VDXLXFTFSDFDPNGEDLLFQGDAHVTSNN.     

植物凝集素的分子生物学研究

植物凝集素是一类具有高度特异性糖结合活性的蛋白,含有一个或多个可与单糖或寡聚糖特异可逆结合的非催化结构域。它的糖结合特异性主要针对外源寡糖,主要生理功能是介异植物的防御反应。到目前为止已克隆了２２２个植物凝集素基因。作者就植物凝集素的分类、性质、功能、凝集素基因的克隆和凝集素的翻译后加工过程作一综述。     

藏红花凝集素分子化学修饰与其活性的关系

对甘露糖专一性结合藏红花凝集素 (Crocussativuslectin ,CSL)分子进行化学修饰 ,测定酿酒酵母 (S .cerevisiae)凝集活性和寡糖专一性结合活性的变化 .实验结果表明 ,Cys的修饰与活性无关 ,Arg、Tyr和His的修饰降低了CSL分子的酵母凝集活性和寡糖结合活性 ,但对CSL的CD光谱无显著影响 ,表明其为凝集素的活性氨基酸残基 .Glu和Asp的化学修饰可使CSL的凝集活性大幅度降低 ,与特异性寡糖的亲和力增大 ,CD光谱变化明显 ,提示CSL分子中的Glu和Asp对其空间结构影响较大 ,氨基酸羧基的修饰导致CSL构象改变 ,蛋白与寡糖的结合位点暴露 ,可有效结合的位点数增加