小鼠角膜发育期间凝集素受体的分布及变化

用辣根过氧化物酶标记的ConA、WGA和RCA Ⅰ为探针,研究了小鼠发育期间角膜内糖残基的分布和变化。Man残基主要分布在角膜基质和内皮;SA残基主要存在于角膜上皮;Gal残基在角膜上皮和基质中都有分布。Man残基在出生前后的小鼠角膜基质中朝内皮方向呈现递增的梯度。SA和Gal残基随角膜发育最后在成体角膜上皮的外表而密集。胎龄13天是小鼠角膜成纤维细胞合成复合糖的起始时间。     

植物Rubisco活性中心的模拟分析

通过对与不同配基结合的植物Rubisco复合物结构的重叠比较分析 ,发现Rubisco的活性差异是由其中一段Loop6环序列所造成的 ;金属离子与活性中心的结合会造成活性中心巨大的构象变化 .进一步用SwissPDBViewer软件模拟不同配基的植物Rubisco活性中心与此Loop环的氢键相互作用 .结果表明 ,有 3个Lys残基Lys2 0 1、Lys334、Lys175与Rubisco是否处于活性状态密切相关 ,这些残基的结构变化对分子设计可能有重要的参考价值     

野葛葡糖基转移酶PlUGTs的同源建模及其活性位点分析

本文对PIUGTs进行同源建模,并分析其与底物结合的构象及活性位点。通过SWISS-MODEL在线对P1UGTs进行模板预测和选择,运用Swiss—PdbViewer软件显示和优化,利用ACDLABS绘制糖基供体小分子（酶结合底物）,最后通过AutoDock_ADT进行分子对接,并分析PIUGTs酶与不同底物结合的整体构象及分析活性位点。研究结果表明PIUGT1、PIUGT2及PIUGT3均能得到较好的三级构象,并且PIUGT1、PIUGT2与三种底物均可进行较好对接,H18,R278,N359为PIUGTI与三种对接构象活性中心所共有的氨基残基;而G16,H17,V19,T148,N370,E374,E390为PIUGT2与三种对接构象活性中心所共有的氨基残基,但PIUGT3未能得到较好的对接构象。由此推测PIUGT1和PIUGT2均能合成葛根素．而PIUGT3不能催化葛根素的合成。     

酸性木聚糖酶XynⅡ活性中心关键氨基酸残基的鉴定

目的:鉴定来源于宇佐美曲霉(Aspergillus usamii)E001的酸性木聚糖酶XynⅡ活性中心关键氨基酸残基。方法:对XynⅡ进行SWISS-MODEL同源建模和BLAST序列比较,分析XynⅡ中所有可能作为催化残基的保守氨基酸,采用定点突变手段对其进行鉴定研究。结果:只有Glu-79和Glu-170位于酶与底物作用的活性中心,它们分别位于β折叠股B6和B4上,推测Glu-79和Glu-170为XynⅡ活性中心关键氨基酸残基。将Glu-79和Glu-170突变为酸性的Gln,突变酶E79Q,E170Q在大肠杆菌和毕赤酵母中表达后,活性均丧失。结论:79位、170位Glu是木聚糖酶XynⅡ活性中心的关键氨基酸残基,为该酶进一步的结构与功能研究提供了理论基础。     

米曲霉GX0011β-糖基转移酶的化学修饰及活性中心研究

用化学修饰法及其修饰动力学对米曲霉GX0011β-果糖基转移酶的活性中心结构进行了研究。结果表明：NBS、PMSF、EDC能显著抑制酶的活性,底物对这些抑制有明显的保护作用,且残留酶活与修饰剂的浓度相关,抑制均符合拟一级动力学规律,进一步动力学分析,初步认定该酶活性中心包括至少一个丝氨酸（或苏氨酸）、一个色氨酸和一个天冬氨酸（或谷氨酸）残基。pCMB、TNBS能显著抑制酶的活性,但底物对抑制无明显保护作用,推断半胱氨酸和赖氨酸残基可能与维系酶活性中心构象有关,但不是酶活性中心基团。DEPC、AA和NAI对酶的活性抑制作用不明显,排除了组氨酸、精氨酸和酪氨酸残基是该酶活性中心必需基团的可能。     

胎儿发育过程中食管上皮糖复全物的改变

采用十种生物素化凝集素UEA－I、DBA、PSA、BSL、PNA、LCA、RCA－I、SBA、ConA及WGA分别对8W、14W、20W、28W及32W的人胎儿食管上皮进行研究,以确定胎儿食管上皮在发育过程中凝集素的结合位点以及结合方式随年龄的变化。结果提示,BSL、RCA－I、WGA的染色强度及特笥不受年龄影响,而与之相对应,UEA－1在除20W以外的食管上皮显色;含α－D－GlcNAc的糖复合物（DBA受体）只在20W表达;含α-D-man/α-D-Glc的糖复合物（PSA受体）则只出现于32W;ConA体虽在胎儿食管上皮各年龄组都表现阳性反应,但在28W□时受体在上皮各层细胞中的分布方式及染色强度出现了有意义的变化,即腔面细胞染色阴性,中层细胞染色强阳性,基底细胞弱阳性,而在其他时间腔面反应强阳性,中层中等阳性,基底弱阳性;PNA、SBA及LCA在各年龄组均呈阴性反应,这些结果表明,在胎儿食管上皮发育过程中存在着多个素结合位点,含α-L-Fucose、α-D-GalNAc、α-D-Man/α-D-Glc残基的糖复合物在分布方式和／或量上存在着发育相关性改变,即糖复合物的改变具有阶段性,总之,本实验资料提示,胎儿食管上皮在发育过程中表现出不同的结合位点,RCA－Ⅰ、BSL、WGA受体似与年龄变化无关,UEA－Ⅰ、DBA、PSA、ConA与食管上皮结合方式的年龄相关性改变,可能提示含α－L－Fucose、α－D－GalNAc、α－D－Man/α-D-Glc糖复合物的分布的改变是在8－32W胎儿中与胎儿食管上皮的形态学发生和细胞分化有关的特征性事件,细胞表面及细胞周围基质糖复合物的这些改变可能影响细胞与细胞、细胞与细胞基质之间的相互作用,同时修饰靶细胞对支持胎儿食管上皮发育可能是必需的效应分子的反应。     

米曲霉GX0011β-果糖基转移酶的化学修饰及活性中心研究

用化学修饰法及其修饰动力学对米曲霉GX0011β-果糖基转移酶的活性中心结构进行了研究。结果表明：NBS、PMSF、EDC能显著抑制酶的活性,底物对这些抑制有明显的保护作用,且残留酶活与修饰剂的浓度相关,抑制均符合拟一级动力学规律,进一步动力学分析,初步认定该酶活性中心包括至少一个丝氨酸（或苏氨酸）、一个色氨酸和一个天冬氨酸(或谷氨酸)残基。pCMB、TNBS能显著抑制酶的活性,但底物对抑制无明显保护作用,推断半胱氨酸和赖氨酸残基可能与维系酶活性中心构象有关,但不是酶活性中心基团。DEPC、AA和NAI对酶的活性抑制作用不明显,排除了组氨酸、精氨酸和酪氨酸残基是该酶活性中心必需基团的可能。     

纤维二糖抑制外切纤维素酶水解作用机理的分析

纤维二糖是纤维素分子的结构单元, 也是纤维素酶水解纤维素时的主要产物. 它能强烈抑制纤维素酶的水解作用, 但并不影响酶对纤维素的吸附. 红外、荧光、圆二色谱分析表明, 纤维二糖可结合在外切纤维素酶活性部位附近的色氨酸残基上形成“位阻效应”, 从而阻止纤维素分子链进入活性中心. 同时, 结合纤维二糖后, 外切纤维素酶分子构象发生了较大变化, 致使吸附纤维素后不能导致微纤维的分离, 为“无效吸附”.     

应用定点突变与分子模建方法研究蛋白酶抑制剂eglin C突变体对蛋白酶furin和kexin的抑制活力

蛋白质前体加工酶参与许多重要蛋白质闪体的加工成熟过程,哺乳动物来源的furin和酵母中的kexin是该家族的重要成员。首先人工合成了编码枯草杆菌蛋白酶抑制剂eglin C的基因片段,组装后在大肠杆菌中得到表达。以定点突变方法在野生型eglin C抑制活性中心的P1、P2和P4位引入碱性氨基酸残基可以将其改造为很强的furin抑制剂（Ki约10^-9mol/L）,和kexin抑制剂（Ki约10^-11mol/L）。同时根据枯草杆菌蛋白酶和eglin C复合物的晶体结构,计算机同源模建了前体加工酶与eglin C突变体结构之间的相互作用,并结合实验数据得到以下结果：（1）P1位引入的碱性残基是该抑制剂活力的前提;（2）P4位碱性残基的引入可以极大地提高抑制剂活力约两个数量级;（3）P2 的碱性残基将有效提高抑制剂的活力。然而同时可以破坏抑制剂本身的稳定性。（4）野生型P3位的疏水性残基参与抑制剂活性环附近疏水核心的构成。     

分子伴侣GroE系统能量传递机制的研究

用SwissPDBViewer软件对分子伴侣GroE系统与底物的相互作用进行了模拟 ,结果表明 :GroEL顶端结构域在GroES和靶蛋白结合之后发生了明显的变化 ;GroEL的cis环上有与三磷酸腺苷ATP相结合的位点 ,ATP水解之后形成的ADP与活性中心的残基相结合 ,而这种结合除导致残基Thr30的构型发生了变化之外 ,其它残基的空间位置和构型基本保持不变 ,暗示其它残基在能量传递过程中形成了刚性骨架 ,而与ADP分子磷酸键结合的残基Thr30则是能量传递的力点。     

Anaplasma marginale, Eperythrozoon wenyoni: lectin reactions with bovine erythrocytes

Normal bovine erythrocytes were agglutinated with four of five lectins specific for different oligosaccharides. The order of reactivity was wheat germ greater than ricin greater than soybean greater than peanut. Concanavalin A did not agglutinate normal bovine erythrocytes. After neuraminidase treatment of normal bovine erythrocytes, each lectin agglutinated the cells with decreased concentrations of lectin, verifying that partial removal of sialic acid exposes more of each lectin's binding sites or alters the binding site such that fewer molecules of lectin are required to initiate agglutination. A change in agglutination of erythrocytes using soybean agglutinin and peanut agglutinin occurred when cells were obtained from cattle infected with Eperythrozoon wenyoni. The results suggested that an alteration in erythrocyte membranes occurred as a result of this infection as manifested by the increased recognition of both the soybean agglutinin and peanut agglutinin receptor carbohydrates. A similar effect was indicated with erythrocytes obtained during an acute Anaplasma marginale infection; however, an ensuing reticulocytosis masked the effect, requiring the use of fluoresceinated lectins to verify that increased binding of each lectin occurred with infected cells when compared to normal cells.     

Ultrastructural localization of lectin-binding sites and laminin-like immunoreactivity in glial cells and neurites growing out from explant cultures of the central nervous system of embryonic locusts

Summary Lectins with different sugar specificities and labeled with horseradish peroxidase or gold were used to study, at the electron-microscopic level, surface glycoconjugates of glial cells and neurites growing out from explant cultures of the central nervous system of embryonic locusts. Differential binding to differentiating glial cells and to neurites was demonstrated. Concanavalin A (Con A) and wheat-germ agglutinin (WGA) bound to glial and neurite surfaces with different degrees of labeling. The formation of glial processes and junctional complexes was invariably accompanied by a corresponding increase of Con A- and WGA-receptors. Peanut agglutinin (PNA) failed to bind to glial cells but strongly stained the plasma membrane of neurite junctions. Lotus tetragonolobus a. (LTA) did not bind either to glial cells or to neurites. In addition, staining with an antibody against laminin showed labeling in areas of neurite outgrowth and neurite interactions; this resembled the localization of PNA receptors. These findings provide evidence for the presence of different carbohydrates at the surface of neurites and glial cells of locust. Their predominant localization in glial processes and neurite junctions suggests that these carbohydrates constitute part of a group adhesion glycoproteins that also includes laminin.     

Histochemical reactivity of the Geodia cydonium agglutinin (GCA) in human tissues

We applied a peroxidase-antiperoxidase technique to study the distribution pattern and binding characteristics of the lectin from the marine sponge Geodia cydonium (Geodia cydonium agglutinin; GCA) in various human tissues. This lectin has been shown to possess a broad reactivity, but there was a distinct distribution of binding sites within the different organs. In the histochemical system GCA displayed no blood group specificity and labeled red blood cells, the vascular endothelium, and epithelial cells showing blood group antigen expression independent of the ABH blood group status. However, inhibition of GCA reactivity by simple sugars and complex carbohydrates demonstrated tissue-specific differences of lectin binding related to the ABH blood group status of the tissue and revealed information on the structural requirements of the histological lectin binding site. Tissues that totally lacked blood group antigens or that expressed only the H-antigen disclosed a GCA reactivity which was completely inhibited by lactose. In contrast, tissues that expressed blood group A- or blood group B-antigen exhibited a lactose-resistant lectin binding which was inhibited only by water-soluble blood group substance A from peptone A and by bovine glycophorin but not by other complex carbohydrates, including human glycophorin and human asialoglycophorin. Competitive inhibition studies in situ revealed that GCA binding was not inhibited by blood group type I/II carbohydrate sequence-specific lectins or by lectins with other sugar specificities. Inhibition by lactose of GCA binding to some histological sites indicates that the binding site consists of a beta-linked galactose-containing disaccharide. However, periodate oxidation of tissue sections had no effect on lectin binding, pointing to a subterminal location of the relevant sequence. The results obtained from inhibition studies with simple saccharides and complex carbohydrates in relation to the expression of ABH blood group antigens suggest a complex lectin combining site(s) in histological specimens. The lectin may possess either one binding site with a range of affinities for different carbohydrates (besides beta-linked disaccharides the GCA binding site accommodates to carbohydrate determinants carrying the blood group A or blood group B determinant), or may possess two different binding sites. Besides an acceptor site for beta-linked disaccharides, an additional binding site may exist accommodating to extended carbohydrate sequences related to A or B blood group structures. In conclusion, GCA represents a blood group-nonspecific lectin whose binding affinities are determined by the ABH blood group status of the tissue.     

Lectin binding to cystic stages of Taenia taeniaeformis

Studies of membrane glycoconjugates of Taenia taeniaeformis were initiated by assays of the lectin binding characteristics of 35-day-old cysticerci. Parasites fixed in glutaraldehyde were incubated with one of the following FITC-labelled lectins: Concanavalin A (Con A), Lens culinaris agglutinin (LCA), Ricinus communis agglutinin (RCA), peanut agglutinin (PNA), fucose binding protein (FBP) and wheat germ agglutinin (WGA) and either their specific or a nonspecific sugar. Ultraviolet microscopy revealed that only Con A and LCA bound in large amounts to the surface of cysticerci. This binding was partly inhibited by the specific sugar, but the nonspecific sugar had little effect. The lectin not removed by either of the sugars may have been bound nonspecifically to the charged glycocalyx. Lectins were primarily bound on the anterior third of the parasite around the scolex invagination. Kinetic studies of lectin interactions were carried out with LCA and RCA by spectrophotofluorometric analysis of the amount bound specifically or nonspecifically over a range of lectin concentrations. Lens culinaris lectin binding was found to be specific and involve 2 receptors which showed large differences in their affinity for lectin and prevalence on the surface. Ricinus communis lectin did not bind specifically but nonspecific interactions were observed. Adherence of small numbers of host cells was shown to have no measurable effect on the lectin binding characteristics. The results suggest that the major surface carbohydrates exposed are D-mannose and/or D-glucose residues with the other sugar groups poorly represented. This relatively homogeneous surface may have implications for the antigenicity of the parasite in its host.     

Computer modelling studies on the modes of binding of some of theα-glycosidically linked disaccharides to concanavalin A

The possible modes of binding of kojibiose, nigerose, maltose and ManPα(1 → 2)Man to concanavalin A have been investigated using computer modelling studies. While α12 linked disaccharides bind to concanavalin A in two modes,i.e. by placing the reducing as well as non-reducing sugar units in the sugar binding site, nigerose or maltose can bind only in one mode,i.e. by placing the non-reducing sugar unit in the binding site. Though, both the sugar residues in α 12 linked disaccharides can reach the binding site, the preference is high for the non-reducing unit. When the non-reducing residue, in any of these disaccharides, enters the binding site, the allowed orientations and the possible hydrogen bonds with the protein seem to be independent of the glycosidic linkage. However, the number of hydrogen bonds the outward sugar residue forms with the protein are dependent on the type of linkage. Atleast one of the hydroxyl groups adjacent to the glycosidic linkage on the outward sugar residue is involved in the formation of a hydrogen bond with the protein suggesting the presence of an extended binding site. The orientation of the reducing sugar residue in the extended binding site is dependent on the linkage. Its orientation in nigerose is flipped when compared to that found in kojibiose or maltose leading to different non-covalent interactions with the protein which affect their binding affinities.     

Specific binding of sulfated proteoglycans to concanavalin A - Sepharose 4B.

More than 90 % of [³⁵S]proteoglycans isolated from the secretions of human skin fibroblasts bind to Concanavalin A-Sepharose 4B (Con A-Sepharose) in the presence of 1 M NaCl. Above pH 5.0 1 M concentrations of methyl-α-D-mannoside and other haptenic inhibitors for Con A-sugar interaction prevent binding of [³⁵S]proteoglycans, whereas equimolar concentrations of non-haptenic carbohydrates do not effect binding. Below pH 5.0 [³⁵S]proteoglycans bind to Con A-Sepharose in the presence of both methyl-α-D-mannoside and galactose. About 60 % of the proteoglycans bound at pH 4.0 are eluted at pH 7.5 in the presence of 1 M methyl-α-D-mannoside. [³⁵S] Glycosaminoglycans prepared from [³⁵S] proteoglycans do not bind to Con A-Sepharose in the presence of 1 M NaCl.These results indicate a [³⁵S]proteoglycan-Con A interaction via the protein core of the proteoglycan and the sugar binding sites of Con A.     

Fluorimetric studies of tryptophyl exposure in concanavalin A.

Studies of the iodide ion quenching of the intrinsic fluorescence of Concanavalin A indicate that 50% of the tryptophyl fluorescence originates from exposed residues. This agrees with the X-ray crystallographic determination that two of the four tryptophan residues in a Concanavalin A monomer are on the surface. Previous studies have indicated that conformational changes induced by sugar binding alter the environment of aromatic residues. The present investigation finds that neither the specific binding of alpha-methyl-D-mannoside nor alteration of the Concanavalin A quaternary structure changes the number or accessibility of the solvent-exposed tryptophan residues. It therefore appears that the major conformational transitions in Concanavalin A do not affect steric access to the surface tryptophans and the effects previously observed may be ascribed to structurally internal tryptophan residues.     

Porcine pancreatic alpha-amylase shows binding activity toward N-linked oligosaccharides of glycoproteins.

Porcine pancreatic alpha-amylase was shown by interaction analyses using a resonance mirror detector and alpha-amylase-immobilized Sepharose to bind with glycoproteins possessing N-glycans but not O-linked mucin-type glycans. Direct binding of three types of N-glycans to the alpha-amylase was demonstrated by surface plasmon resonance. Binding with biotin-polymer sugar probes revealed that the alpha-amylase has affinity to alpha-mannose, alpha-N-acetylneuraminic acid, and beta-N-acetyllactosamine, which are components of N-glycans. The binding of glycoproteins or carbohydrates enhanced the enzyme activity, indicating that the recognition site for N-glycans is different from its catalytic site. The binding activity was unique to porcine pancreatic alpha-amylase and was not observed for alpha-amylase from saliva, wheat, and fungus.     

Concanavalin A interactions with asparagine-linked glycopeptides. The mechanisms of binding of oligomannose,bisected hybrid,and complex type carbohydrates

The affinity of concanavalin A (Con A) for simple saccharides has been known for over 50 years. However, the specificity of binding of Con A with cell-surface related carbohydrates has only recently been examined in detail. Brewer and coworkers [J Biol Chem (1986) 261:7306–10; J Biol Chem (1987) 262:1288–93; J Biol Chem (1987) 262:1294–99] have recently studied the binding interactions of a series of oligomannose and bisected hybrid type glycopeptides and complex type glycopeptides and oligosaccharides with Con A. The relative affinities of the carbohydrates were determined using hemagglutination inhibition measurements, and their modes of binding to the lectin examined by nuclear magnetic relaxation dispersion (NMRD) spectroscopy and quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves of Con A and the carbohydrates indicate that certain oligomannose and bisected hybrid type glycopeptides are bivalent for lectin binding. From the NMRD and precipitation data, two protein binding sites on each glycopeptide have been identified and characterized. Certain bisected complex type oligosaccharides also bind and precipitate Con A, while the corresponding nonbisected analogs bind but do not precipitate the protein. The precipitation data indicate that the bisected complex type oligosaccharides are also bivalent for lectin binding, while the nonbisected analogs are univalent. The NMRD and precipitation data are consistent with different mechanisms of binding of nonbisected and bisected complex type carbohydrates to Con A, including different conformations of the bound saccharides.Abbreviations Con A Concanavalin A with unspecified metal ion content - CMPL Con A with Mn²⁺ and Ca²⁺ at the S1 and S2 sites respectively, in the locked conformation [12]; trisaccharide1, 3,6-di-O-(-d-mannopyranosyl)-d-mannose - -MDM methyl -d-mannopyranoside - NMRD nuclear magnetic relaxation dispersion, the magnetic field dependence of nuclear magnetic relaxation rates, in the present case, the longitudinal relaxation rate, 1/T₁, of solvent protons     

Interactions of lectins with (Na+ + K+)-ATPase of eel electric organ.

Interaction of lectins with a detergent-solubilized ATPase from eel electric organ was studied. Concanavalin A, which binds to alpha-mannosides, altered the rate of enzyme migration in agar and inhibited the formation of an antigen-antibody precipitate: other lectins had no such effects. Concanavalin A similar amounts partially inhibited (Na+ + K+)-ATPase; this inhibition was reversible by alpha-methylglucoside. There was no corresponding effect of concanavalin A on the potassium p-nitrophenylphosphatase. Concanavalin A also did not interfere with ouabain binding. Thus, concanavalin A binds to an antigenic region also involved in Na+ and/or ATP binding, but does not interact with a K+ site.