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1.
Human Brain Lectin: A Soluble Lectin That Binds Actin   总被引:1,自引:0,他引:1  
A biotinylated probe was used for detection of endogenous ligands of a human brain lectin on blotted human brain soluble proteins. Of the various proteins from brain extract resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five reacted with the biotinylated probe. After elimination of saccharidic moieties by periodic treatment of the same extract, a single band with Mr approximately 43,000 was recognized by the lectin. This band was identified as actin using an anti-actin antibody. These results were confirmed by binding of biotinylated lectin to purified actin.  相似文献   

2.
Molecular recognition can be mediated by protein (lectin)-carbohydrate interaction, explaining the interest in this topic. Plant lectins and, more recently, chemically glycosylated neoglycoproteins principally allow to map the occurrence of components of this putative recognition system. Labelled endogenous lectins and the lectin-binding ligands can add to the panel of glycohistochemical tools. They may be helpful to derive physiologically valid conclusions in this field for mammalian tissues. Consequently, experiments were prompted to employ the abundant beta-galactoside-specific lectin of human nerves in affinity chromatography and in histochemistry to purify and to localize its specific glycoprotein ligands. In comparison to the beta-galactoside-specific plant lectins from Ricinus communis and Erythrina cristagalli, notable similarities were especially detectable in the respective profiles of the mammalian and the Erythrina lectin. They appear to account for rather indistinguishable staining patterns in fixed tissue sections. Inhibitory controls within affinity chromatography, within solid-phase assays for each fraction of lectin-binding glycoproteins and within histochemistry as well as the demonstration of crossreactivity of the three fractions of lectin-binding glycoproteins with the biotinylated Erythrina lectin in blotting ascertained the specificity of the lectin-glycoprotein interaction. In addition to monitoring the accessible cellular ligand part by the endogenous lectin as probe, the comparison of immunohistochemical and glycohistochemical detection of the lectin in serial sections proved these methods for receptor analysis to be rather equally effective. The observation that the biotinylated lectin-binding glycoproteins are also appropriate ligands in glycohistochemical analysis warrants emphasis.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

3.
Mammalian endogenous carbohydrate-binding proteins (lectins) play fundamental roles in a variety of mechanisms of interactions both at the molecular and cellular levels. We have investigated the binding of one of them (human brain lectin) to soluble acrylamide copolymerized with derivatives of either lactose (O-beta-lactosyloxyallylallylaminoacrylamide copolymer) or D-mannose (D-alpha-mannosyloxyallylallylaminoacrylamide copolymer) in direct enzyme affinoassays, in an attempt to develop simple procedures for detection and estimation of its carbohydrate-binding activity. Biotinylated plant lectins were utilized as reference standards. Affinoassays employed the polymer dotted on nitrocellulose and the polymer coated on microtiter plates as well as detection of bound biotinylated lectin by streptavidin/horseradish peroxidase reagent. Both assays provided reproducible binding, inhibitable by specific sugars. The microtiter plate assay is well suited to sensitive detection of the negative endogenous lectin by competition with biotinylated brain lectin. We conclude that the use of derivatized acrylamide in dotting and microtiter plate assays may prove practical for detection of endogenous lectins and that such polymers may serve as model substances in the study of biological partners of these carbohydrate-binding proteins.  相似文献   

4.
Summary Molecular recognition can be mediated by protein (lectin)-carbohydrate interaction, explaining the interest in this topic. Plant lectins and, more recently, chemically glycosylated neoglycoproteins principally allow to map the occurrence of components of this putative recognition system. Labelled endogenous lectins and the lectin-binding ligands can add to the panel of glycohistochemical tools. They may be helpful to derive physicologically valid conclusions in this field for mammalian tissues. Consequently, experiments were prompted to employ the abundant -galactoside-specific lectin of human nerves in affinity chromatography and in histochemistry to purify and to localize its specific glycoprotein ligands. In comparison to the -galactoside-specific plant lectins fromRicinus communis andErythrina cristagalli, notable similarities were especially detectable in the respective profiles of the mammalian and the Erythrina lectin. They appear to account for rather indistinguishable staining patterns in fixed tissue sections. Inhibitory controls within affinity chromatography, within solid-phase assays for each fraction of lectin-binding glycoproteins and within histochemistry as well as the demonstration of crossreactivity of the three fractions of lectin-binding glycoproteins with the biotinylated Erythrina lectin in blotting ascertained the specificity of the lectin-glycoprotein interaction. In addition to monitoring the accessible cellular ligand part by the endogenous lectin as probe, the comparison of immunohistochemical and glycohistochemical detection of the lectin in serial sections proved these methods for receptor analysis to be rather equally effective. The observation that the biotinylated lectin-binding glycoproteins are also appropriate ligands in glycohistochemical analysis warrants emphasis. Overall, the introduction of biotinylated mammalian lectins as well as the lectin-binding glycoproteins will aid to critically evaluate the physiological significance of the glycobiological interplay between endogenous lectins and distinct carbohydrate parts of cellular glycoconjugates.  相似文献   

5.
In order to study the distribution of endogenous sugar-binding proteins (lectins) in various areas of the adult bovine heart, we used a battery of biotinylated neoglycoproteins. These tools expose carrier-immobilized carbohydrate moieties as ligands for receptor detection. Characteristic staining patterns depending on the type of carbohydrate ligand were observed in all constituents examined. Comparison to data obtained for lectin distribution in the respective areas of the human heart indicate that the localization of certain types of endogenous sugar receptors can exhibit species-dependent variations.  相似文献   

6.
Biotinylated heparin has been used to detect the presence of specific binding sites in sections of human placenta, which has prompted demonstration of expression of lectin activity for this proteoglycan. Purification of this lectin from full-term placenta facilitates the synthesis of its biotinylated derivative, using biotin-amidocaproyl hydrazide, without affecting its activity. It also enables immunization to obtain antibodies. The labeled lectin is shown to bind specifically to nuclear and cytoplasmic locations in various cell types of human placenta, nuclear expression of lectin binding sites being more pronounced at the full-term stage than after 8 weeks of development. The structurally related histone H2B exhibits obvious differences in its binding pattern. The presence of ligands accessible to the lectin whose binding activity can be inhibited by addition of an excess of heparin correlates in most instances with the level of lectin expression detected immunohistochemically. Biochemical information on the nature of the glycohistochemically inferred lectin-specific ligand(s) is obtained by affinity chromatography on resin-immobilized lectin. It leads to isolation of a proteoglycan with similar electrophoretic mobility in agarose-polyacrylamide gel electrophoresis relative to the independently purified heparan sulfate-containing fibronectin binding proteoglycan from human placenta. Both fractions inhibit binding of heparin to the lectin and contain immunologically detected co-purified lectin, emphasizing their ligand properties. Application of labeled tissue lectins in conjunction with lectin-specific antibodies is proposed to obtain valuable insights into the expression of the receptor as well as the ligand part of protein-carbohydrate recognition.  相似文献   

7.
Protein-carbohydrate recognition may be involved in an array of molecular interactions on the cellular and subcellular levels. To gain insight into the role of proteins in this type of interaction, surgically removed specimens of human endomyocardial tissue were processed for histochemical and biochemical analysis. The inherent capacity of these sections to bind individual sugar moieties, which are constituents of the carbohydrate part of cellular glycoconjugates, was assessed using a panel of biotinylated neoglycoproteins according to a standardized procedure. Together with appropriate controls, it primarily allowed localization of endogenous lectins. Differences in lectin expression were observed between layers of endocardial tissue, myocardial cell constituents, connective-tissue elements, and vascular structures. The endocardium proved to be positive with beta-galactoside-bearing probes; with neoglycoproteins carrying beta-xylosides, alpha-fucosides, and galactose-6-phosphate moieties; and with probes containing a carboxyl group within the carbohydrate structure, namely sialic acid and glucuronic acid. In contrast, only fucose-and maltose-specific receptors were apparent in the elastic layers of the endocardium. Aside from ascertaining the specificity of the protein-carbohydrate interaction by controls, i.e., lack of binding of the probe in the presence of the unlabelled neoglycoprotein and lack of binding of the labelled sugar-free carrier protein, respective sugar receptors were isolated from heart extracts by using histochemically effective carbohydrates as immobilized affinity ligand. Moreover, affinity chromatography using immobilized lactose as affinity ligand as well as the use of polyclonal antibodies against the predominant beta-galactoside-specific lectin of heart demonstrated that the lactose-specific neoglycoprotein binding was due to this lectin. Remarkably, the labelled endogenous lectin, preferred to plant lectins for detecting ligands of the endogenous lectin, localized ligands in tissue parts where the lectin itself was detected glycohistochemically as well as immunohistologically. This demonstration of receptor-ligand presence in the same system is a further step toward functional assignment of the recorded protein-carbohydrate interaction. Overall, the observed patterns of lectin expression may serve as a guideline to elucidate the precise physiological relevance of lectins and to analyze pathological conditions comparatively.  相似文献   

8.
Lectins are ubiquitous proteins of nonimmune origin, present in plants, microorganisms, animals and humans which specifically bind defined monosugars or oligosaccharide structures. Great progress has been made in recent years in understanding crucial roles played by lectins in many biological processes. Elucidation of carbohydrate specificity of human and animal lectins is of great importance for better understanding of these processes. Long before the role of carbohydrate–protein interactions had been explored, many lectins, mostly of plant origin, were identified, characterized and applied as useful tools in studying glycoconjugates. This review focuses on the specificity-based lectin classification and the methods of measuring lectin–carbohydrate interactions, which are used for determination of lectin specificity or for identification and characterization of glycoconjugates with lectins of known specificity. The most frequently used quantitative methods are shortly reviewed and the methods elaborated and used in our laboratories, based on biotinylated lectins, are described. These include the microtiter plate enzyme-linked lectinosorbent assay, lectinoblotting and lectin–glycosphingolipid interaction on thin-layer plates. Some chemical modifications of lectin ligands on the microtiter plates and blots (desialylation, Smith degradation, β-elimination), which extend the applicability of these methods, are also described.  相似文献   

9.
Protein-carbohydrate interactions play crucial roles in numerous biological processes. To study these interactions, we developed a simple and fast procedure for the biotinylation of carbohydrates based on reductive amination. The method allows complete and stable biotinylation of small quantities of oligosaccharides and includes a rapid and simple procedure to remove excess labeling reagent. After biotinylation, the structural and biological integrity of the glycans was intact as determined by HPLC, mass spectrometry, and a plant lectin assay. By using the human C-type lectin DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin), we demonstrate that the biotinylated glycans can be used in a glycan array to determine binding specificities of lectins. Moreover, we show that fluorescent beads coated with selected biotinylated glycans bind to DC-SIGN-expressing dendritic cells in vitro. Finally, by using biotinylated high-mannose N-glycans, we could visualize DC-SIGN-expressing cells in lymph node tissue. The availability of easy biotinylation methods for oligosaccharides such as those described here greatly facilitates the functional analysis of lectins. In addition, the biotinylated glycans will be great tools for investigating functional lectin receptors in situ.  相似文献   

10.
 报道了利用免疫亲和层析法纯化棕尾别麻蝇幼虫血淋巴凝集素的结果.哺乳动物红细胞能够特异地吸附凝集素.用兔红细胞与麻蝇幼虫血淋巴凝集素形成的复合体免疫供血家兔,得到麻蝇幼虫血淋巴凝集素的抗体.再利用抗体制备亲和吸附柱,通过免疫亲和层析一次性纯化了麻蝇幼虫血淋巴凝集素. S D S P A G E结果显示,该凝集素的分子量约为73 k D.这一结果,与用对麻蝇幼虫血淋巴凝集素有抑制作用的糖蛋白—胎球蛋白和甲状腺球蛋白为配基,亲和层析纯化的结果完全相同,表明用这种免疫亲和层析法纯化凝集素是可行的.为不清楚专一性识别糖或专一性识别糖不典型,难于用普通亲和层析纯化的凝集素,提供了一种有效的纯化方法.  相似文献   

11.
Dynamic combinatorial library design exploiting the thiol-disulfide exchange readily affords access to glycosyldisulfides. In order to reveal lectin-binding properties of this type of non-hydrolyzable sugar derivative, libraries originating from a mixture of common building blocks of natural glycans and thiocompounds were tested against three plant agglutinins with specificity to galactose, fucose or N-acetylgalactosamine, respectively, in a solid-phase assay. Extent of lectin binding to matrix-immobilized neoglycoprotein presenting the cognate sugar could be reduced, and evidence for dependence on type of carbohydrate was provided by dynamic deconvolution. Glycosyldisulfides also maintained activity in assays of increased physiological relevance, that is, using native tumor cells and also adding to the test panel an endogenous lectin (galectin-3) involved in tumor spread and cardiac dysfunction. N-Acetylgalactosamine was pinpointed as the most important building block of libraries for the human lectin and the digalactoside as most potent compound acting on the toxic mistletoe agglutinin which is closely related to the biohazard ricin. Because this glycosyldisulfide, which even surpasses lactose in inhibitory capacity, rivals thiodigalactoside as inhibitor, their degrees of intramolecular flexibility were comparatively analyzed by computational calculations. Molecular dynamics runs with explicit consideration of water molecules revealed a conspicuously high degree of potential for shape alterations by the disulfide's three-bond system at the interglycosidic linkage. The presented evidence defines glycosyldisulfides as biologically active ligands for lectins.  相似文献   

12.
Methyl 4-azidobenzoimidate, a photosensitive hetero-bifunctional cross-linking reagent, was synthesized and characterized. This reagent has an imidoester at one end, which reacts spontaneously with primary amines, and an arylazide at the other end, which reacts with a variety of chemical groups upon photolysis by ultraviolet radiation. The reagent molecules were attached to concanavalin A by reactions between imidoester groups of the reagents and free amino groups of the lectin. These activated lectins were purified on a Sephadex G-25 column and showed the binding affinity to an affinity column, glucosylated Sepharose, and to the human erythrocyte ghost membrane. The activated lectins were incubated with the membranes and then unbound lectins were removed by washing. The lectins bound to receptors in the membranes were irradiated with a shortwave ultraviolet lamp to photolyze arylazides attached to the lectins, thus cross-linking the lectins and receptors together. Then the membranes were solubilized and electrophoresed. On gels, the intensity of the lectin receptor band diminished slightly and concomitantly a new band of a higher molecular weight appeared. When 125I-labeled concanavalin A was used, the new band contained the radioactivity. The extent of the appearance of the new band and the decrease of the receptor band were reduced significantly when the ultraviolet irradiation was omitted or the activated lectins were incubated with the membranes in the presence of the lectin inhibitor, alpha-methylmannoside. The irradiation of nonactivated, receptor-bound concanavalin A did not cause those changes. When the activated lectins alone were irradiated with ultraviolet, the band of the lectin dimer appeared whereas nonirradiated lectins appeared mostly as monomers. It is concluded that a small fraction of the activated lectins were cross-linked to receptors in the membrane upon photolysis. In this study, only 8 reagent molecules were attached to a tetramer of the lectin, compared with the presence of approximately 40 available free amino groups. The efficiency of such cross-links of ligands to receptors may be increased by employing longer versions of the hetero-bifunctional cross-linking reagents and also by attaching more of the reagent molecule to ligands.  相似文献   

13.
The Dictyostelium discoideum lectins, discoidin I and discoidin II, and the endogenous ligands to which they bind were immunohistochemically localized in sections of this organism at successive stages of development. For these studies, an axenic strain, AX3, was grown in a macromolecule-depleted medium rather than on bacteria, which themselves contain discoidin-binding ligands. Discoidin I-binding sites (endogenous ligands) in sections of D. discoideum were concentrated in the slime coat around aggregates, whereas discoidin II-binding sites were observed in a vesicle-like distribution in prespore cells and also in spore coats. In contrast, discoidin II did not bind to the slime coat and discoidin I bound relatively poorly to prespore cells and spore coats. The distributions of the endogenous lectins themselves were the same in axenically grown cells as previously reported for cells raised on bacteria. Discoidin I was concentrated in the slime coat and around stalk cells, and discoidin II was prominent in and around prespore cells. The congruent localization of each lectin with its endogenous ligand suggests that discoidin I normally functions in association with glycoconjugates in the slime around aggregates, and discoidin II with the galactose-rich spore coat polysaccharide.  相似文献   

14.
A solubility-insolubility transition assay was used to screen the bark and stems of seven leguminous trees and plants for self-aggregatable lectins. Novel lectins were found in two trees, Robinia pseudoacacia and Wisteria floribunda, but not in the leguminous plants. The Robinia lectin was isolated from coexisting lectin by combined affinity chromatographies on various sugar adsorbents. The purified lectins proved to be differently glycosylated glycoproteins. One lectin exhibited the remarkable characteristics of self-aggregatable lectins: localization in the bark of legume trees, self-aggregation dissociated by N-acetylglucosamine/mannose, and coexistence with N-acetylgalactosamine/galactose-specific lectins, which are potential endogenous receptors. Self-aggregatable lectins are a functional lectin group that can link enhanced photosynthesis to dissociation of glycoproteins.  相似文献   

15.
Cellular activities in the regulation of growth or adhesion/migration involve protein (lectin)–carbohydrate recognition at the cell surface. Members of the galectin family of endogenous lectins additionally bind distinct intracellular ligands. These interactions with protein targets explain the relevance of their nuclear and cytoplasmic presence. Expression profiling for galectins and accessible binding sites is a histochemical approach to link localization with cellular growth properties. Non-cross-reactive antibodies for the homodimeric (proto-type) galectins-1, -2 and -7 and the chimera-type galectin-3 (Gal-3) as well as the biotinylated lectins were tested. This analysis was performed with the FaDu squamous carcinoma cell line and long-term cultured human and porcine epidermal cells as models for malignant and normal cells of squamous cell epithelial origin. A set of antibodies was added for phenotypic cell characterization. Strong nuclear and cytoplasmic signals of galectins and the differential reactivity of labeled galectins support the notion of their individual properties. The length of the period of culture was effective in modulating marker expression. Cytochemical expression profiling is a prerequisite for the selection of distinct proteins for targeted modulation of gene expression as a step toward functional analysis.  相似文献   

16.
Affinity chromatography provides a powerful tool for isolation of carbohydrate-binding proteins. However, the choice of the ligand and spacer has an important impact on effectiveness. The influence of several different ligands on qualitative and quantitative aspects of the purification of two beta-galactoside-specific lectins has been evaluated. Sepharose was modified by coupling four types of neoglycoproteins (galactosylated or lactosylated bovine serum albumin with increasing sugar content) and two naturally occurring asialoglycoproteins at similar densities. Carbohydrate ligands at essentially equal density were made accessible to the lectins by seven commonly used methods. The yield of mistletoe lectin was high when lactosylated neoglycoproteins were used for separation. For these resins the sugar incorporation exceeded 10 sugar groups per protein carrier molecule. The yield was similarly high with the asialoglycoproteins and with lactose; the sugar was coupled to the resin as a p-aminophenyl derivative or by means of divinyl sulfone activation. An epoxy group in linkages of galactose or lactose decreased the binding capacity. A quantitatively similar degree of protein yields was obtained for the beta-galactoside-binding protein of bovine heart, although different proteins were obtained when neoglycoproteins were used as ligand. The nature of the affinity ligand in lectin purification can increase the yield and may also influence the profile of the carbohydrate-binding proteins.  相似文献   

17.
Structural relationships among five beta-galactoside-binding lectins isolated from human, mouse and chick were studied using immunochemical methods. The lectins examined were human placenta lectin with a 14-kDa subunit (human 14K lectin), two types of mouse lectin (mouse 15K and mouse 16K lectin), and two types of chick lectin (chick 14K and chick 16K lectin). Five polyclonal antibodies raised against these lectins were used. Antibody to human 14K lectin cross-reacted with mouse 15K and chick 14K lectins. Antibodies to both mouse 15K and chick 14K lectins cross-reacted with human 14K and chick 16K lectins. Antibody to chick 16K lectin cross-reacted with mouse 15K lectin. An immunological relationship was not found between human 14K and chick 16K lectins, or between mouse 15K and chick 14K lectins. Mouse 16K lectin did not show any immunological relationship with any of the other lectins. A monoclonal antibody raised against chick 14K lectin cross-reacted with chick 16K lectin. These results cannot be explained simply in terms of phylogenic distance but suggest that vertebrate beta-galactoside-binding lectins can be classified into two structural groups on the basis of their antigenicities. One group, which is characterized as a monomer type, includes human 14K and chick 14K lectins. The other group, which is characterized as a dimer type, includes mouse 15K and chick 16K lectins.  相似文献   

18.
Summary Normal human gastric mucosal cells were examined by light and electron microscopy using lectins as a probe. The ABC method was used with biotinylated lectins for light microscopy and HRP-labeled lectins for electron microscopy. The human gastric mucosal cells revealed specific binding patterns for each lectin by light microscopy. Among the lectins tested, in particular, DBA gave a characteristic pattern. It specifically stained the supranuclear region of surface epithelial cells and the perinuclear region of parietal cells. By electron microscopy, the stacked cisternae and the vesicles of the Golgi apparatus of the surface epithelial cells were positive for the DBA staining. These results show that the DBA-positive supranuclear region observed by light microscopy corresponds to the Golgi apparatus. In the parietal cells, DBA, RCA and ConA bound to the intracellular secretory canaliculi which are invaginations of the cell membrane running around the nucleus in the cytoplasm. Therefore, the tubular perinuclear positive region observed by light microscopy corresponds to the membranes of the intracellular secretory canaliculi. In addition, the ConA reagent stained the endoplasmic reticulum, Golgi apparatus, nuclear envelope, and cell membrane of the parietal cell, which explains the diffuse cytoplasmic staining observed at the light microscopic level with this lectin. Lectins have proved to be very useful for the evaluation of in situ cytochemical aspects of the glycoconjugates characteristic to human gastric mucosal cells.  相似文献   

19.
Cell surface complex carbohydrates have emerged as key recognition molecules, mediating physiological interactions between cells. Typically, glycans on one cell surface are engaged by complementary carbohydrate binding proteins (lectins) on an apposing cell, initiating appropriate cellular responses. Although many cell surface lectins have been identified in vertebrates, only a few of their endogenous carbohydrate ligands have been established. Each major class of cell surface glycans-glycoproteins, glycolipids, and proteoglycans-has been implicated as physiologically relevant lectin ligands. The current minireview focuses on findings that implicate glycosphingolipids as especially important molecules in cell-cell recognition in two different systems: the recognition of human leukocytes by E-selectin on the vascular endothelium during inflammation and the recognition of nerve cell axons by myelin-associated glycoprotein in myelin-axon stabilization and the regulation of axon regeneration.  相似文献   

20.
Monosaccharides on the apical processes of the retinal pigment epithelium were examined using lectin-affinity cytochemical methods. Lectin receptor sugars were localized with lectin-horseradish peroxidase (HRP) and lectin-ferritin conjugates as well as with biotinylated lectins, avidin, and biotinylated HRP. In contrast, only wheat germ agglutinin (WGA) receptors were identified with biotinylated WGA followed by avidin-ferritin or free avidin and biotinylated ferritin. Labeling with avidin-ferritin subsequent to biotinylated lectin treatment was dependent upon the source and lot of the reagent. These findings are similar to those reported for the endothelium of bone marrow sinusoids (Pino RM: Am J Anat, 169:259, 1984). Since both the retinal pigment epithelial and bone marrow sinusoidal surfaces are highly anionic (negative), we investigated the possibility that the charge of the lectin reagents and cell surfaces might affect the localization of monosaccharides on cell surfaces. Analytical isoelectric focusing revealed that biotinylated ferritin and some avidin-ferritins are highly anionic, while the other lectin reagents have more cationic (positive) components. Based on this information, a less charged biotinylated ferritin marker was made that made it possible to localize biotinylated lectins bound to the cell surface.  相似文献   

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