Distribution and nature of saccharides of plasma membranes of rat ascites hepatoma cells as detected by ferritin-conjugated lectins and dialysed iron

Electron microscopic cytochemical studies were made on saccharides involved in the plasma membranes of rat ascites hepatoma cells (AH7974F) using ferritin-conjugated lectins and dialysed iron (DI). In the rat hepatoma cells, saccharide receptors for each of the three lectins used (concanavalin A (ConA), wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA)) were shown to be distributed homogeneously throughout the plasma membranes. When the cells were agglutinated, however, the saccharide receptors for each lectin appeared to form clusters on the plasma membranes. The cluster formation induced by one lectin was found to lead to a changed distribution of saccharide receptors for another lectin. None of the cluster formation types induced by lectins yield any noticeable effects upon the distribution of DI reactive acidic saccharides on the plasma membranes.     

138 Molecular Evolution of Glutamine Synthetase In Protists

The binding of fluorescein isothiocyanate (FITC) conjugated lectins to gametes of Aglaothamnion byssoides Itono during the fertilization was studied by the use of confocal microscope. The physiological effects of lectins and carbohydrates on gamete binding were also examined. Three lectins, concanavalin A (ConA), Soybean agglutinin (SBA) and wheat germ agglutinin (WGA) bound to the surface of spermatia, but each lectin labeled different region of the spermatium. SBA bound only to the spermatial appendages but ConA bound to the whole spermatial surface except spermatial appendages. WGA labeled narrow region which connects spermatial body and appendages. During fertilization, ConA and WGA specific substances on the spermatial surface moved towards the area contacting with trichogyne and accumulated on the surface of fertilization canal. Spermatial binding to trichogynes was inhibited by pre-incubation of spermatia with SBA, while trichogyne receptors were blocked by the complementary carbohydrate, N-acetyl-D-galactosamine. WGA and its complementary carbohydrate had little effect on gamete binding. For searching the step of sexual isolation, crossing experiment was performed between Aglaothamnion byssoides and twelve other red algal species. Results showed that the gamete recognition was genus-specific: the gametes bound freely with their partners of the same genus. When two species from same genus were crossed, sexual isolation occurs gradually during the fertilization process. Therefore, sexual isolation in red algae appears to be determined by multi-step process and gamete binding is the initial step.     

Ricin D-saccharide interaction as studied by ultraviolet difference spectroscopy

The interaction of ricin D with specific saccharides was investigated by ultraviolet difference spectroscopy. Upon binding to saccharides, ricin D displayed ultraviolet difference spectra with maxima at 280 nm and 288 nm. Such difference spectra suggest that the environment of a tyrosine residue(s) located at or near the saccharide-binding site is changed by the binding of saccharide. In addition to the two positive peaks, a small trough was observed around 300 nm in the complexes with galactose-containing saccharides but not in the complex with N-acetylgalactosamine or galactosamine, suggesting the participation of tryptophan in the binding with galactose-containing saccharides. The magnitude of the difference maxima increased with increasing concentration of saccharides until the binding site was saturated. From the variation of the maximum at 288 nm as a function of saccharide concentration, the association constants were obtained for the binding of saccharides to ricin D at various temperatures and pH's. The saccharide binding of ricin D decreased with increasing temperature and with decreasing pH below pH 6.0. It was suggested that difference maximum at 288 nm observed in the ricin D-saccharide interaction reflects the binding of saccharides to the high-affinity saccharide-binding site of ricin D.     

137 Gamete Recognition and Sexual Isolation in a Red Alga,Aglaothamnion Byssoides (Rhdophyta)

The binding of fluorescein isothiocyanate (FITC) conjugated lectins to gametes of Aglaothamnion byssoides Itono during the fertilization was studied by the use of confocal microscope. The physiological effects of lectins and carbohydrates on gamete binding were also examined. Three lectins, concanavalin A (ConA), Soybean agglutinin (SBA) and wheat germ agglutinin (WGA) bound to the surface of spermatia, but each lectin labeled different region of the spermatium. SBA bound only to the spermatial appendages but ConA bound to the whole spermatial surface except spermatial appendages. WGA labeled narrow region which connects spermatial body and appendages. During fertilization, ConA and WGA specific substances on the spermatial surface moved towards the area contacting with trichogyne and accumulated on the surface of fertilization canal. Spermatial binding to trichogynes was inhibited by pre‐incubation of spermatia with SBA, while trichogyne receptors were blocked by the complementary carbohydrate, N‐acetyl‐D‐galactosamine. WGA and its complementary carbohydrate had little effect on gamete binding. For searching the step of sexual isolation, crossing experiment was performed between Aglaothamnion byssoides and twelve other red algal species. Results showed that the gamete recognition was genus‐specific: the gametes bound freely with their partners of the same genus. When two species from same genus were crossed, sexual isolation occurs gradually during the fertilization process. Therefore, sexual isolation in red algae appears to be determined by multi‐step process and gamete binding is the initial step.     

Role of conserved and nonconserved residues in the Ca(2+)-dependent carbohydrate-recognition domain of a rat mannose-binding protein. Analysis by random cassette mutagenesis.

The carbohydrate-recognition domain of rat serum mannose-binding protein A has been subjected to random cassette mutagenesis. Mutant domains, expressed in bacteria, were initially screened for binding to invertase-coated nitrocellulose and then analyzed further for Ca2+ affinity, saccharide binding, resistance to proteolysis, and oligomerization. The results are consistent with previous evolutionary and structural studies. Six out of seven completely inactive mutants have changes in residues directly involved in ligating Ca2+. Most changes in conserved residues which form part of the hydrophobic core characteristic of Ca(2+)-dependent (C-type) animal lectins result in decreased affinity for Ca2+, even though these residues are distant from the Ca2+ sites. Changes can be made in large portions of the surface without affecting saccharide binding. The results indicate that the precise arrangement of the regular portion of the domain containing the hydrophobic core is necessary for formation of a stable Ca(2+)-ligated structure under physiological conditions. The data also suggest that the saccharide-binding site is likely to be in close proximity to the bound Ca2+.     

Nuclear magnetic resonance investigation of cadmium 113 substituted pea and lentil lectins

The lentil (LcH) and pea (PSA) lectins, which are members of the class of D-glucose/D-mannose binding lectins, are Ca2+ X Mn2+ metalloproteins that require the metal ions for their saccharide binding and biological activities. We have prepared a variety of Cd2+ derivatives of PSA and LcH, with Cd2+ in either the transition metal (S1) or calcium (S2) sites, or in both. Thus, Cd2+ X Zn2+, Cd2+ X Mn2+, and Ca2+ X Cd2+ derivatives were prepared, in addition to the Cd2+ X Cd2+ derivatives which we have recently reported. This is the first report of stable mixed metal Cd2+ complexes of lectins. The physical and saccharide binding properties of the Cd2+ derivatives of both lectins were characterized by a variety of physiochemical techniques and found to be the same as those of the corresponding native proteins. 113Cd NMR spectra of mono- and disubstituted 113Cd2+ complexes of LcH and PSA were recorded and compared with 113Cd NMR data for concanavalin A (ConA) (Palmer, A.R., Bailey, D.B., Behnke, W.D., Cardin, A.D., Yang, P.P., and Ellis, P.D. (1980) Biochemistry 19, 5063-5070). The data for the PSA and LcH derivatives were found to be very similar, indicating close homology of their metal ion binding sites. 113Cd resonances at 44.6 ppm and -129.4 ppm for 113Cd2+ X 113Cd2+ X LcH, and at 46.6 and -130.4 for the corresponding PSA derivative, are chemical shifts very similar to those observed for 113Cd2+ X 113Cd2+ X ConA. Assignment of the resonances to the transition metal (S1) and calcium (S2) sites were unambiguous since the Ca2+ X 113Cd2+ and 113Cd2+ X Zn2+ derivatives of both lectins showed single resonances characteristic of the S1 and S2 sites, respectively. The results indicate that, unlike ConA, 113Cd2+ binds tightly to PSA and LcH. Binding of monosaccharide to both lectins induce small (2 ppm) upfield shifts in their S2 113Cd resonances, in contrast to the larger shift (8 ppm) observed in ConA. The 113Cd2+ X Mn2+ complexes of PSA and LcH fail to show a 113Cd resonance characteristic of these derivatives, which provides evidence for the close proximity of the metal ions in the two proteins. The present findings indicate that the coordinating ligand atoms to the metal ions at the S1 and S2 sites in LcH, PSA, and ConA are the same.     

Mechanism and biological role of nitric oxide binding to cytochrome c'

The binding of nitric oxide to ferric and ferrous Chromatium vinosum cytochrome c' was studied. The extinction coefficients for the ferric and ferrous nitric oxide complexes were measured. A binding model that included both a conformational change and dissociation of the dimer into subunits provided the best fit for the ferric cytochrome c' data. The NO (nitric oxide) binding affinity of the WT ferric form was found to be comparable to the affinities displayed by the ferric myoglobins and hemoglobins. Using an improved fitting model, positive cooperativity was found for the binding of NO to the WT ferric and ferrous forms, while anticooperativity was the case for the Y16F mutant. Structural explanations accounting for the binding are proposed. The NO affinity of ferrous cytochrome c' was found to be much lower than the affinities of myoglobins, hemoglobins, and pentacoordinate heme models. Structural factors accounting for the difference in affinities were analyzed. The NO affinity of ferrous cytochrome c' was found to be in the range typical of receptors and carriers. In addition, cytochrome c' was found to react with cytosolic light-irradiated membranes in the presence of succinate and carbon monoxide. With these results, a biochemical model of cytochrome c' functioning as a nitric oxide carrier was proposed.     

Negative cooperativity associated with binding of multivalent carbohydrates to lectins. Thermodynamic analysis of the "multivalency effect".

Our previous study demonstrated that isothermal titration microcalorimetry (ITC) could be used to determine the thermodynamics of binding of a series of synthetic multivalent carbohydrates to the Man/Glc-specific lectins concanavalin A (ConA) and Dioclea grandiflora lectin (DGL) [Dam, T. K., Roy, R., Das, S. K., Oscarson, S. and Brewer, C. F. (2000) J. Biol. Chem. 275, 14223-14230]. The higher affinities of the multivalent carbohydrates for the two lectins were shown to be due to their greater positive entropy of binding contributions relative to monovalent analogues. In the present study, ITC data from our previous report for binding of di-, tri-, and tetravalent carbohydrate analogues possessing terminal 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside residues to ConA and DGL were subjected to Hill plot analysis. Hill plots of the binding of monovalent methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside to ConA and DGL are linear with slopes near 1.0, demonstrating a lack of binding cooperativity and allosteric transitions in the proteins. However, Hill plots for the binding of the di-, tri-, and tetravalent trimannoside analogues to both lectins are curvilinear with decreasing tangent slopes below 1.0, indicating increasing negative cooperativity upon binding of the analogues to the lectins. The curvilinear Hill plots are consistent with decreasing affinity and functional valencies of the multivalent analogues upon sequential binding of lectin molecules to the carbohydrate epitopes of the analogues. The following paper [Dam, T. K., Roy, R., Pagé, D., and Brewer, C. F. (2002) Biochemistry 41, 1359-1363] provides direct evidence of the decreasing affinity constants of multivalent carbohydrates upon sequential binding of lectin molecules.     

Thermodynamic, kinetic, and electron microscopy studies of concanavalin A and Dioclea grandiflora lectin cross-linked with synthetic divalent carbohydrates

The jack bean lectin concanavalin A (ConA) and the Dioclea grandiflora lectin (DGL) are highly homologous Man/Glc-specific members of the Diocleinae subtribe. Both lectins bind, cross-link, and precipitate with carbohydrates possessing multiple terminal nonreducing Man residues. The present study investigates the binding and cross-linking interactions of ConA and DGL with a series of synthetic divalent carbohydrates that possess spacer groups with increasing flexibility and length between terminal alpha-mannopyranoside residues. Isothermal titration microcalorimetry was used to determine the thermodynamics of binding of the two lectins to the divalent analogs, and kinetic light scattering and electron microscopy studies were used to characterize the cross-linking interactions of the lectins with the carbohydrates. The results demonstrated that divalent analogs with flexible spacer groups between the two terminal Man residues possess higher affinities for the two lectins as compared with those with inflexible spacer groups. Furthermore, despite their high degree of homology, ConA and DGL exhibit differences in their kinetics of cross-linking and precipitation with the divalent analogs. Electron microscopy shows the loss of organized cross-linked lattices of the two lectins with analogs possessing increased distance between the terminal Man residues. The loss of lattice patterns with the analogs is distinct for each lectin. These results have important implications for the interactions of lectins with multivalent carbohydrate receptors in biological systems.     

1H-NMR study on the chitotrisaccharide binding to hen egg white lysozyme.

Interaction between hen egg white lysozyme and chitotrisaccharide was investigated by 1H-NMR spectroscopy using partially acetylated chitotrisaccharides and chemically modified lysozyme. Monoacetyl (GlcN-GlcN-GlcNAc), diacetyl (GlcN-GlcNAc-GlcNAc), or triacetyl chitotrisaccharide [(GlcNAc)3] was added to the lysozyme solution, and the changes in the 1H-NMR signals of the lysozyme were analyzed. Although many of the resonances were affected by addition of the saccharide, the most remarkable effect was seen on the signal of Trp28 C5H which is in a hydrophobic box adjacent to the saccharide-binding site. The signal shifted upfield by 0.2 ppm upon (GlcNAc)3 binding, whereas the chemical shift change of the signal resulting from binding of GlcN-GlcNAc-GlcNAc or GlcN-GlcN-GlcNAc was smaller than that resulting from (GlcNAc)3 binding. When the Asp101-modified lysozyme was used instead of the native lysozyme, the chemical shift change of the Trp28 C5H signal resulting from (GlcNAc)3 binding was also smaller than that for the native lysozyme. The chemical shift change of the signal reflects the conformational change of the hydrophobic box region which should synchronize with the movement of the binding site resulting from the saccharide binding. Therefore, the conformational change resulting from the saccharide binding might be reduced when the sugar residues located at binding subsites A and B of the lysozyme are deacetylated, as well as when Asp101 interacting with the sugar residues at the same subsites is modified.     

Ultraviolet difference spectroscopic analysis of the saccharide-binding properties of Ricinus communis agglutinin

The nature of the binding of saccharides to Ricinus communis agglutinin was studied by ultraviolet difference spectroscopy. Upon binding of galactose and galactose-containing saccharides, R. communis agglutinin displayed difference spectra with an extreme maximum at 291-293 nm and a smaller maximum at 284-285 nm. Such difference spectra suggest that the environment of a tryptophan residue located at or near the saccharide-binding site of R. communis agglutinin is being changed by an interaction between a tryptophan residue and the bound saccharides. The value of the difference spectra (delta epsilon) increased upon progressive addition of saccharide until the saccharide binding site was saturated with ligand. From the increase in delta epsilon at 291-293 nm, the association constants were obtained for the R. communis agglutinin-saccharide interaction over the temperature range 5-35 degrees C and various pH values. The results clearly demonstrate that the association constants are nearly equal in the range of pH 5-8, but decrease beyond the above pH range and with elevation of temperature. From the thermodynamic parameters for the binding of various saccharides to R. communis agglutinin, we suggest that there exists a subsite structure in the saccharide-binding site of the R. communis agglutinin molecule.     

Nonexistence of Exo-cellobiohydrolase (CBH) in the Cellulase System of Trichoderma viride

Chemical modification of histidine residues in ricin E was studied with regard to saccharide binding. The analytical data indicate that 6 out of 7 histidine residues in ricin E are eventually modified with diethylpyrocarbonate (DEP) at pH 6.0 and 25°C in the absence of specific saccharides. Modification of histidine residues greatly decreased the cytoagglutinating activity of ricin E, and only 10% of the residual activity was found after modification of 6 histidine residues/mol. The data of affinity chromatography using lactamyl- and galactosamine-cellulofine columns suggest that modification of histidine residues does not have much effect on the binding ability at the low affinity saccharide-binding site of ricin E but abolishes the binding ability at the high affinity saccharide-binding site. In the presence of lactose, one histidine residue/mol was protected from the DEP modification with retention of a fairly high cytoagglutinating activity. Such a protective effect was also observed for specific saccharides such as galactose and A^-acetylgalactosamine, but not for glucose, a non-specific saccharide. On treatment with hydroxylamine, the modified ricin E restored 67 % of the cytoagglutinating activity. Based on these findings, it is suggested that in the high affinity saccharide- binding site of ricin E there exists one histidine residue responsible for saccharide binding.     

Glycoconjugates in normal human kidney. A histochemical study using 13 biotinylated lectins

The avidin-biotin-peroxidase complex technique was used with 13 lectins to study the glycoconjugates of normal human renal tissue. The evaluated lectins included Triticum vulgaris (WGA), Concanavalin ensiformis (ConA), Phaseolus vulgaris leukoagglutinin and erythroagglutinin (PHA-L and PHA-E), Lens culinaris (LCA), Pisum sativum (PSA), Dolichos biflorus (DBA), Glycine max (SBA), Arachis hypogaea (PNA), Sophora japonica (SJA), Bandeiraea simplicifolia I (BSL-I), Ulex europaeus I (UEA-I) and Ricinus communis I (RCA-I). Characteristic and reproducible staining patterns were observed. WGA and ConA stained all tubules; PHA-L, PHA-E, LCA, PSA stained predominantly proximal tubules; DBA, SBA, PNA, SJA and BSL-I stained predominantly distal portions of nephrons. In glomeruli, WGA and PHA-L stained predominantly visceral epithelial cells; ConA stained predominantly basement membranes and UEA-I stained exclusively endothelial cells. UEA-I also stained endothelial cells of other blood vessels and medullary collecting ducts. Sialidase treatment before staining caused marked changes of the binding patterns of several lectins including a focal loss of glomerular and tubular staining by WGA; an acquired staining of endothelium by PNA and SBA; and of glomeruli by PNA, SBA, PHA-E, LCA, PSA and RCA-I. The known saccharide specificities and binding patterns of the lectins employed in this study allowed some conclusions about the nature and the distribution of the sugar residues in the oligosaccharide chains of renal glycoconjugates. The technique used in this report may be applicable to other studies such as evaluation of normal renal maturation, classification of renal cysts and pathogenesis of nephrotic syndrome. The observations herein reported may serve as a reference for these studies.     

Surface carbohydrates of Eudiplozoon nipponicum pre- and post-fusion

The development of the monogenean Diplozoon (Nordmann, 1832) (Diplozoidae) necessitates fusion of two larval stages (diporpae) into one double organism. How diporpae find, distinguish and contact each other is unclear, nor is the nature of the stimuli responsible for the dedifferentiation of cells and the formation of new tissues at the site of somatic fusion. Previous studies have implied a role for carbohydrates and glycoproteins in the interactions between helminth parasites and their hosts. Hypothetically, glycoconjugates may also be involved in the establishment of parasite-parasite associations. Changes in the surface saccharide residues during the development of Eudiplozoon nipponicum, a gill ectoparasite of carp (Cyprinus carpio) are described. Flat-fixed specimens and sections of diporpae, juveniles (just-fused) and adult worms were examined following exposure to a panel of 12 FITC-conjugated lectins. All developmental stages exhibited a specific surface binding pattern with ten lectins, indicating that Man/Glc, GlcNAc, Gal and GalNAc are probably present on their surfaces. No reaction was observed with Fuc-specific lectins (UEA-I and LTA). There is evidence that parasite development is accompanied by both qualitative and quantitative changes in the saccharide pattern distribution. The diporpa sucker reacted with nine lectins, excluding BS-II. A very strong binding of PNA, LCA and ConA (Gal and Man/Glc-specific lectins) was observed with the papilla glands of juvenile worms. The role of glandular secretions in this unique fusion process is discussed.     

Fluorescence spectroscopic studies on tryptophan at the saccharide-binding site of castor bean hemagglutinin

The environment of tryptophan in castor bean hemagglutinin (CBH) was analyzed by fluorescence spectroscopy with regard to saccharide binding. Upon binding of specific saccharides, the fluorescence maximum of 333 nm of CBH shifted to a wavelength 2 nm shorter, owing to the change in the environment of tryptophan at the saccharide-binding site. By analyzing the change in the fluorescence intensity at 320 nm as a function of concentration of saccharides, the association constants for binding of saccharides to CBH were determined. The results suggest that the saccharide-binding site on each B-chain is actually composed of a subsite with which the saccharide residue linked to galactopyranoside at the non-reducing end can interact, and another site which recognizes the galactopyranoside moiety. Quenching data indicated that five out of 22 tryptophans in CBH are surface-localized and are available for quenching with both KI and acrylamide, and three other tryptophans are buried and are available only to acrylamide. Binding of raffinose to CBH decreased by 2 the number of tryptophan residues accessible to quenchers in the CBH molecule. We speculate that raffinose binds to CBH in such a manner as to shield the tryptophan located at the subsite from quenching by KI and acrylamide. The results also suggest that the tryptophan residue at the saccharide-binding site on each B-chain is localized near the surface, and present in the positively charged environment.     

Evaluation of cellulose-based biospecific adsorbents as a stationary phase for lectin affinity chromatography

Macroporous cellulose Granocel was evaluated as a matrix for the immobilization of two lectins Concanavalin A (ConA) (108 kDa) and Wheat Germ Agglutinin (WGA) (36 kDa). Two different methods were employed for the immobilization of the lectins via their protein moieties by a Schiff's bases reaction. One of them results in covalent coupling of the lectin directly to the support and the other gives the attachment through a long spacer arm which benefits the immobilization of voluminous ConA molecules. The adsorbents were characterized by the glycoproteins sorption recording adsorption kinetic data and isotherms. The adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg/ml support and a high recovery (up to 93%). The adsorption isotherms of glucose oxidase (GOD) onto ConA adsorbents reveals an adsorption behavior with high and low affinity binding sites. The dissociation constant K(d) of the ligand-sorbate complex is approximately 1 x 10(-6) and 0.4 x 10(-5)M, respectively. It was supposed that the second step is related to the sorption of solvated GOD onto already adsorbed GOD forming sorbate dimers.     

Lectin binding sites as markers of neonatal porcine uterine development.

To identify lectin binding sites and to determine if lectin binding patterns change with age in developing neonatal porcine uterine tissues, gilts (n = 3/day) were hysterectomized on Day 0 (birth), 7, 14, 28, 42, or 56. Lectin binding was visualized in Bouin's-fixed uterine tissues with seven biotinylated lectins (ConA, DBA, PNA, RCA-I, SBA, UEA-I, and WGA) and avidin-peroxidase staining procedures. Lectin specificities were demonstrated by pre-incubating lectins with appropriate inhibitory sugars (0.2 M). Staining intensity was evaluated visually (absent, weak, moderate, or strong) for three endometrial tissues; luminal epithelium, glandular epithelium, and stroma. Staining intensities for DBA, PNA, SBA, and WGA were not affected by neonatal age. Staining with these lectins was greater in uterine epithelium (moderate or strong) than in stroma (weak). In contrast, binding patterns for ConA, UEA-I, and RCA-I were affected by neonatal age. Strong epithelial staining associated with ConA binding was observed on all days, whereas stromal ConA staining decreased in intensity from moderate to weak after Day 14. Epithelial staining with UEA-I increased from moderate to strong after Day 28, whereas stromal UEA-I staining decreased from moderate to weak after day 28. Staining with RCA-I was homogeneous for luminal epithelium and stroma but variegated for glandular epithelium on and after Day 7. These observations indicate that a variety of lectin binding sites are present in developing neonatal porcine endometrial tissues and that developmentally related alterations in the distribution and/or orientation of glycoconjugates containing alpha-D-mannose, beta-D-galactose, beta-D-acetyl-N-galactosamine, and alpha-L-fucose residues occur between birth and Day 56 as these tissues mature.     

Lectin receptors on the peri- and early post-implantation mouse embryo

Summary Mouse embryos at the blastocyst, blastocyst outgrowth, and primitive streak (day 7.5) stages of development were analysed for expression of lectin receptors using a panel of six FITC-conjugated lectins with affinities for five distinct saccharides (BSL, ConA, DBA, LTL, UEA and WGA). Blastocyst trophoblast expressed receptors for all the lectins but later tissues of the trophectoderm lineage lost receptors for distinct but overlapping subsets of the lectin panel. The inner cell mass (ICM) of the early blastocyst lacked receptors only for UEA. Differentiation of primary endoderm was accompanied by the aquisition of UEA receptors but subsequent differentiation into visceral and parietal endoderm involved the loss of receptors for both fucose binding lectins (UEA and LTL). Embryonic ectoderm in the day 7.5 egg cylinder retained receptors only for ConA and WGA. Thus, in general, differentiation during the peri- and early post-implantation period was associated with a differential loss of lectin receptors in all cell lineages of the mouse conceptus.     

The role of metal ions in substrate recognition and stability of concanavalin A: a molecular dynamics study

The binding of carbohydrate substrates to concanavalin A (Canavalia ensiformis agglutinin (ConA)) is essential for its interaction with various glycoproteins. Even though metal ions are known to control the sugar binding ability of legume lectins, the interplay between sugar and metal ion binding to ConA has not been elucidated in a detailed manner at the atomic level. We have carried out long, explicit solvent molecular dynamics simulations for tetrameric, dimeric, and monomeric forms of ConA in both the presence and absence of trimannoside and metal ions. Detailed analyses of these trajectories for various oligomeric forms under different environmental conditions have revealed dynamic conformational changes associated with the demetalization of ConA. We found that demetalization of ConA leads to large conformational changes in the ion binding loop, with some of the loop residues moving as far as 17 Å with respect to their positions in the native trimannoside and metal ion-bound crystal structure. However, the β-sheet core of the protein remains relatively unperturbed. In addition, the high mobility of the ion binding loop results in drifting of the substrates in the absence of bound metal ions. These simulations provide a theoretical rationale for previous experimental observations regarding the abolition of the sugar binding ability upon demetalization. We also found that the amino acid stretches of ConA, having high B-factor values in the crystal structure, show relatively greater mobility in the simulations. The overall agreement of the results of our simulations with various experimental studies suggests that the force field parameters and length of simulations used in our study are adequate to mimic the dynamic structural changes in the ConA protein.     

Distribution of cell surface saccharides on pancreatic cells. II. Lectin-labeling patterns on mature guinea pig and rat pancreatic cells

The surface saccharide composition of collagenase-dispersed pancreatic cells from adult guinea pig and rat glands was examined by using eight lectins and their ferritin conjugates: Concanavalin A (ConA); Lens culinaris (LCL); Lotus tetragonolobus (LTL); Ricinus communis agglutinins I and II (RCA I, RCA II); Soybean agglutinin (SBA); Ulex europeus lectin (UEL); and wheat germ agglutinin (WGA). Binding studies of iodinated lectins and lectin-ferritin conjugates both revealed one population of saturable, high-affinity receptor sites on the total cell population (approximately 95% acinar cells). Electron microscopy, however, revealed differences in lectin-ferritin binding to the plasmalemma of acinar, centroacinar, and endocrine cells. Whereas acinar cells bound heavily all lectin conjugates, endocrine and centroacinar cells were densely labeled only by ConA, LCL, WGA, and RCA I, and possessed few receptors for LTL, UEL, and SBA. Endocrine and centroacinar cells could be differentiated from each other by using RCA II, which binds to centroacinar cells but not to endocrine cells. Some RCA II receptors appeared to be glycolipids because they were extracted by ethanol and chloroform-methanol in contrast to WGA receptors which resisted solvent treatment but were partly removed by papain digestion. RCA I receptors were affected by neither treatment. The apparent absence of receptors for SBA on endocrine and centroacinar cells, and for RCA II on endocrine cells, was reversed by neuraminidase digestion, which suggested masking of lectin receptors by sialic acid. The absence of LTL and UEL receptors on endocrine and centroacinar cells was not reversed by neuraminidase. We suggest that the differential lectin-binding patterns observed on acinar, centroacinar, and endocrine cells from the adult pancreas surface-carbohydrate-developmental programs expressed during morphogenesis and cytodifferentiation of the gland.