Binding Sites For Concanavalin Research Articles

Proton Positions in the Mn2+ Binding Site of Concanavalin A as Determined by Single-Crystal High-Field ENDOR Spectroscopy [J. Am. Chem. Soc. 2001, 123, 8378−8386

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionNEXTORIGINAL ARTICLEThis notice is a correctionProton Positions in the Mn2+ Binding Site of Concanavalin A as Determined by Single-Crystal High-Field ENDOR Spectroscopy [J. Am. Chem. Soc.2001, 123, 8378−8386].Raanan Carmieli, Palanichamy Manikandan, A. Joseph Kalb (Gilbo, and Daniella GoldfarbCite this: J. Am. Chem. Soc. 2001, 123, 49, 12438Publication Date (Web):November 15, 2001Publication History Published online15 November 2001Published inissue 1 December 2001https://doi.org/10.1021/ja015136rCopyright © 2001 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views233Altmetric-Citations3LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (9 KB) Get e-Alerts Get e-Alerts

Proton positions in the Mn(2+) binding site of concanavalin A as determined by single-crystal high-field ENDOR spectroscopy.

High-field (95 GHz) pulsed EPR and electron-nuclear double resonance (ENDOR) techniques have been used for the first time to determine coordinates of ligand protons of a high-spin metal center in a protein single crystal. The protein concanavalin A contains a Mn(2+) ion which is coordinated to two water molecules, a histidine residue, and three carboxylates. Single crystals of concanavalin A were grown in H(2)O and in D(2)O to distinguish the exchangeable water protons from the nonexchangeable protons of the imidazole group. Distinct EPR transitions were selected by performing the ENDOR measurements at different magnetic fields within the EPR spectrum. This selection, combined with the large thermal polarization achieved at 4.5 K and a magnetic field of approximately 3.4 T allowed us to assign the ENDOR signals to their respective M(S) manifolds, thus providing the signs of the hyperfine couplings. Rotation patterns were acquired in the ac and ab crystallographic planes. Two distinct crystallographic sites were identified in each plane, and the hyperfine tensors of two of the imidazole protons and the four water protons were determined by simulations of the rotation patterns. All protons have axially symmetric hyperfine tensors and, by applying the point-dipole approximation, the positions of the various protons relative to the Mn(2+) ion were determined. Likewise, the water protons involved in H-bonding to neighboring residues were identified using the published, ultrahigh-resolution X-ray crystallographic coordinates of the protein (Deacon et al. J. Chem. Soc., Faraday Trans. 1997, 93(24), 4305-4312).

Histochemical characterization of primary capillary endothelial cells from porcine brains using monoclonal antibodies and fluorescein isothiocyanate-labelled lectins: implications for drug delivery

Primary endothelial cells isolated from cerebral microvessels by combined mechanical and enzymatic treatment from porcine brains were characterized with regard to identity, purity and membrane surface characteristics. Cells were grown in culture to adherent monolayers and characterized morphologically and histochemically by their binding for fluorescently-labelled lectins and monoclonal antibodies detected by indirect immunofluorescence. The binding patterns of the cells were compared with the affinity of frozen tissue sections of porcine brain cortex for the markers. Endothelial cells in culture were characterized by the binding of von Willebrand factor, vimentin and fibronectin antibodies. They failed to react with anti-glial fibrillary acid protein, anti-galactocerebroside C and anti-neurofilament 160 antibodies characteristic for astrocytes, oligodendrocytes and neurons, respectively. Cell cultures were stained by the lectins, wheat germ agglutinin, horse gram agglutinin and soybean agglutinin, demonstrating the presence of N-acetylglucosamine and N-acetylgalactosamine residues on membrane surface. Binding sites for concanavalin A and peanut agglutinin characteristic for mannose and galactose could not be detected. Cell age and differentiation had no effect on lectin and antibody staining. Cell cultures gave staining results similar to those of microvessels in frozen tissue sections. The results of morphology, antibody and lectin staining pattern indicate that our in vitro endothelial cell culture model retained many histological characteristics observed for capillary microvessels in vivo and appears to be suitable for studying uptake and targeting properties of drug carrier systems with regard to the blood–brain barrier.

Lectin binding patterns in two cultured endothelial cell types derived from bovine corpus luteum.

Epithelial cells of different phenotypes derived from bovine corpus luteum have been studied intensively in our laboratory. In this study, specific lectin binding was examined for cells of type 1 and 3, which were defined as endothelial cells. In order to confirm differences in their glycocalyx at the light microscopic level, five biotinylated lectins were applied to postconfluent cultures which had been fixed with buffered paraformaldehyde or glutaraldehyde. Cells were not permeabilized with any detergent. Lectin binding was localized with a streptavidin-peroxidase complex which was visualized with two different techniques. The DAB technique detected peroxidase histochemically, while the immunogold technique used an anti-peroxidase gold complex together with silver amplification. Neither cell type 1 nor cell type 3 bound a particular lectin selectively, yet each cell type expressed a particular lectin binding pattern. With the DAB technique, diverse lectin binding patterns were seen, probably indicating either "outside" binding, i.e., a diffuse pattern, a lateral-cell-side pattern and a microvillus-like pattern, or "inside" binding, i.e., a diffuse pattern, and a granule-like pattern. With the immunogold technique, only "outside" binding was observed. In addition, the patterns of single cilia or of single circles were detected, the latter roughly representing 3-micron-sized binding sites for concanavalin A. When localizing them at the ultrastructural level, single circles corresponded with micron-sized discontinuities of the plasma membrane. Shedding vesicles were detected whose outer membrane was labelled with concanavalin A. Our results confirm the diversity of the two cell types under study. The "inside" lectin binding may be caused by way of transient plasma membrane openings and related to shedding of right-side out vesicles ("ectocytosis").

Molecular modelling of protein-carbohydrate interactions. Docking of monosaccharides in the binding site of concanavalin A

A general procedure is described for addressing the computer simulation of protein-carbohydrate interactions. First, a molecular mechanical force field capable of performing conformational analysis of oligosaccharides has been derived by the addition of new parameters to the Tripos force field; it is also compatible with the simulation of protein. Second, a docking procedure which allows for a systematic exploration of the orientations and positions of a ligand into a protein cavity has been designed. This so-called 'crankshaft' method uses rotations and variations about/of virtual bonds connecting, via dummy atoms, the ligand to the protein binding site. Third, calculation of the relative stability of protein ligand complexes is performed. This strategy has been applied to search for all favourable interactions occurring between a lectin [concanavalin A (ConA)] and methyl alpha-D-mannopyranoside or methyl alpha-D-glucopyranoside. For each monosaccharide, different stable orientations and positions within the binding site can be distinguished. Among them, one corresponds to very favourable interactions, not only in terms of hydrogen bonding, but also in terms of van der Waals interactions. It corresponds precisely to the binding mode of methyl alpha-D-mannopyranoside into ConA as revealed by the 2.9 A resolution of the crystalline complex (Derewenda et al., 1989). Some implications of the present modelling study with respect to the molecular basis of the specificity of the interaction of lectins with various monosaccharides are presented.

Ultrastructural detection of carbohydrates in the pellicle of Pneumocystis carinii

The presence of carbohydrates in the pellicle of Pneumocystis carinii was demonstrated by methenamine silver (MS) and thiocarbohydrazide-silver proteinate (TCH-SP) staining techniques. An intense, positive reaction with MS was observed on both the electron-dense outer layer and the electron-lucent middle layer of the pellicle, whereas in the case of TCH-SP only the outer layer stained well. The middle layer did not stain as intensely with TCH-SP as with MS. This observation indicates that the pellicle of P. carinii contains carbohydrates but that the outer and middle layers differ in carbohydrate composition. The binding sites of concanavalin A (Con A) and Macura pomifera (MPA) lectins were elucidated on the pellicle of P. carinii using colloidal gold as a marker. In a preembedding staining method using Con A and MPA lectins, the adherence of gold particles was observed on the surface of all stages of the parasite. The homogeneous distribution of the gold particles indicates that the outer electron-dense layer contains both Con A- and MPA-specific carbohydrates. In postembedding staining with Con A, the adherence of gold particles was found on both the outer and middle layers, whereas in the case of MPA only the outer layer was labeled with gold particles. These results indicate that the electron-dense outer layer contains a considerable amount of carbohydrates specific for these lectins, whereas the electron-lucent middle layer seems to lack MPA-specific carbohydrates.

Lectin-binding in normal and osteoarthrotic articular cartilage from STR/1N-mouse knee joints.

Fluorescein-isothiocyanate (FITC) labeled lectins were used to study the distribution pattern of specific binding-sites in histological sections of normal and osteoarthrotic articular cartilage from the mouse knee joint. Male inbred mice of the STR/1N-strain develop spontaneous arthrotic articular cartilage lesions on the medial condyle of tibia and femur. The varus-deformity of the knee joint leads to a recurrent medial patellar luxation with osteoarthrotic defects on the medial part of the facies patellaris femoris. It was demonstrated that the lectin staining pattern of osteoarthrotic articular cartilage, especially on the facies patellaris femoris, was different from that of normal articular cartilage. The differences in lectin staining corresponded to those observed between normal and fibrillated articular cartilage from human patellae. The normal articular cartilage of the mouse knee joint possessed lectin binding-sites for Concanavalin A (ConA) and wheat germ agglutinin (WGA), but not for Ulex europaeus agglutinin (UEA), soy bean agglutinin (SBA) and peanut agglutinin (PNA). In addition to the completely changed distribution pattern of ConA and WGA in osteoarthrotic cartilage, SBA, PNA and UEA developed distinct staining patterns particular to the fibrillated areas of arthrotic cartilage. The increased lectin-binding to arthrotic articular cartilage may be due to unmasking of sugars in the course of bondage breakdown in fibrillated cartilage or the production of pathological glycoproteins. It is evident that lectins can demonstrate minute differences between normal and arthrotic cartilage and it is concluded, therefore, that lectins are sensitive and specific tools for the study of degenerative joint diseases.

Lectin-binding in normal and fibrillated articular cartilage of human patellae.

Fluorescein-isothiocyanate (FITC) labeled lectins were used to study the distribution of specific binding-sites in histological sections of normal and fibrillated articular cartilage of human patellae. It has been shown that normal articular cartilage reveals lectin binding-sites for Concanavalin A (Con A) and wheat germ agglutinin (WGA), but not for soybean agglutinin (SBA), peanut agglutinin (PNA) and Ulex europaeus agglutinin (UEA). In fibrillated cartilage the distribution pattern of Con A and WGA is completely changed. SBA, PNA and UEA show a distinct staining pattern in particular in the fibrillated areas of degenerated cartilage. Lectin-staining of the extracellular matrix and the chondrocytes in both normal and fibrillated cartilage did not show any correlation with material that was either PAS- or Alcian blue-positive. In comparison with the conventional PAS- and Alcian blue reaction lectin-staining proved to be superior. Visualization of intra- and extracellular glycoconjugate-changes in normal and fibrillated cartilage in areas with no PAS and/or Alcian blue staining indicates that all layers of the cartilage are involved in the pathological process. It is evident that lectins can demonstrate minute differences between normal and arthrotic cartilage and we therefore conclude that lectins are sensitive and specific tools for the study of degenerative joint diseases.

Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of 125I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant ( K d) as the controls. The binding of 125I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity ( K d ≈ 6.6 · 10 −8 M). At high lectin concentrations, low affinity ( K d ≈ 6.7 · 10 −5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone. Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized 125I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl- D-mannopyranoside.

Dynamic redistribution of concanavalin A binding sites on isolated guinea pig keratinocytes.

Modifications in the distribution of the binding sites for concanavalin A (Con A) were studied on trypsin isolated living guinea pig keratinocytes. Fluorescein-labelled Con A was used and the in vitro procedure has included short-term cultures, experiments at 37 degrees C and 4 degrees C, the study of colchicine and vincaleucoblastine effects. It was possible to induce different patterns of staining corresponding to distinct redistribution of Con A binding sites; the distinct redistribution was correlated to the effects of Con A, colchicine and vincaleucoblastine. These findings demonstrated that the system used was appropriate to the study of some dynamic events on the keratinocytes membranes.

Lectin Binding by Trypanosoma equiperdum

Agglutination studies with 6 plant lectins indicated that the unaltered surface coat of Trypanosoma equiperdum isolated from rat blood lacks the carbohydrate molecules preferentially bound by these proteins. However, trypsin, pronase, chymopapain, or papain treatments exposed the binding sites for Concanavalin A and the phytohemagglutinins M and P and trypsinized cells were attached to Concanavalin A immobilized on agarose beads. Lipolytic, amylytic, and other proteolytic enzymes or other agents did not reduce or induce lectin agglutination and wheat germ, Anti A, and Anti H lectins did not clump the trypanosomes under any of the conditions employed. Carbohydrate residues resembling D-mannose or n-acetyl-D-galactosamine are therefore within the surface coat of T. equiperdum or on the cell membrane underneath it. The results are contrasted with the lectin induced agglutination of other parasite species and mammalian cells.

Fine structural localization of Concanavalin A binding sites on hamster spermatozoa.

The plasma membrane of epididymal spermatozoa of the golden hamster (Mesocricetus auratus) exhibits morphological differences over various parts of the head and tail as detected by air-dried replicas and freeze-etching techniques. In an attempt to ascertain whether any topographical differences exist in the number or distribution of carbohydrate moieties associated with the cell surface, cells were labeled with Concanavalin A and marked with hemocyanin. It was found that while the plasma membrane over the acrosomal region differed from that of the postacrosomal region in membrane components revealed by freeze fracturing, there was no apparent difference in the distribution or density of Con A binding sites detectable by hemocyanin localization. The tail regions exhibited differences in both fracture face appearance and the distribution of detectable carbohydrate moieties. It was also found that binding sites for Concanavalin A exist on the inner and outer acrosomal membranes in addition to those on the plasma membrane.

Epidermal intercellular binding of concanavalin a and pemphigus antibody

The binding sites of Concanavalin A (Con A) and pemphigus antibody in the intercellular spaces of the human epidermis have been investigated. Extraction of human skin with phosphate-buffered saline (PBS) and 70% ethanol resulted in complete loss of the binding sites of the pemphigus antibody, whereas the extracted skin still retained its reactivity to Con A. Preincubation with more than 0.25 mg/ml of Con A with normal skin caused a reduction in the binding of pemphigus sera. However, preincubation of pemphigus sera with human skin caused no inhibition of the subsequent binding of fluorescein-labelled Con A. These results indicate that Con A binding sites might be related to, but not identical with those of pemphigus antibody.

Structural difference in sites on surface membrane of mature and immature erythrocytes.

MAMMALIAN cells transformed by oncogenic viruses or chemical carcinogens undergo characteristic changes in their surface properties such as lectin-induced cell agglutination1,2. Normal cells show similar changes at their mitotic phase3 or after mild protease digestion of their surface membrane4,5. This suggests that cells change their surface properties under various conditions and during the cell cycle. With this concept in mind, we studied the changes on the membrane surface during the maturation of rabbit erythrocytes. We describe here the changes in cell agglutinability and in the binding sites of concanavalin A (con A) on the cell surface during erythrocyte maturation.

Electron spin resonance study of the transition metal-binding site of concanavalin A

The ESR spectrum of Mn 2+ bound to the transition metal-binding site of concanavalin A is characteristic of bound Mn 2+ with low coordination symmetry. When Ca 2+ is bound to the Ca 2+-binding site of concanavalin A, the spectrum of bound Mn 2+ is modified. Binding of methyl-α- d-glucopyranoside to the saccharide-binding site causes no change in the spectrum.