Characterization and biological implications of membrane lectins in tumor, lymphoid and myeloid cells

Complex carbohydrates and sugar receptors at the surface of eukaryotic cells are involved in recognition phenomena. Membrane lectins have been characterized, using biochemical, biological and cytological methods. Their biological activities have been assessed using labeled glycoproteins or neoglycoproteins. Specific glycoproteins or neoglycoproteins have been used to inhibit their binding capacity in both in vitro and in vivo experiments. In adults, lymphoid and myeloid cells as well as tumor cells grow in a given organ and eventually migrate and home in another organ; these phenomena are known as the homing process or metastasis, respectively. In specific cases, membrane lectins of endothelial cells recognize cell surface glycoconjugates of lymphocytes or tumor cells, while membrane lectins of lymphocytes and of tumor cells recognize glycoconjugates of extracellular matrices or of non-migrating cells. Therefore, membrane lectins are involved in cell-cell recognition phenomena. Membrane lectins are also involved in endocytosis and intracellular traffic of glycoconjugates. This property has been demonstrated not only in hepatocytes, fibroblasts, macrophages and histiocytes but also in tumor cells, monocytes, thyrocytes, etc. Upon endocytosis, membrane lectins are present in endosomes, whose luminal pH rapidly decreases. In cells such as tumor cells or macrophages, endosomes fuse with lysosomes; it is therefore possible to target cytotoxic drugs or activators, by binding them to specific glycoconjugates or neoglycoproteins through a linkage specifically hydrolyzed by lysosomal enzymes. In cells such as monocytes, the delivery of glycoconjugates to lysosomes is not active; in this case, it would be preferable to use an acid-labile linkage. Cell surface membrane lectins are developmentally regulated; they are present at given stages of differentiation and of malignant transformation. Cell surface membrane lectins usually bind glycoconjugates at neutral pH but not in acidic medium: their ligand is released in acidic specialized organelles; the internalized ligand may be then delivered into lysosomes, while the membrane lectin is recycled. Some membrane lectins, however, do bind their ligand in relatively acidic medium as in the case of thyrocytes. The presence of cell surface membrane lectins which recognize specific sugar moieties opens the way to interesting applications: for instance, isolation of cell subpopulations such as human suppressor T cells, targeting of anti-tumor or anti-viral drugs, targeting of immunomodulators or biological response modifiers.     

Influence of type of linkage and spacer on the interaction of beta-galactoside-binding proteins with immobilized affinity ligands

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.     

Assay and characterization of carbohydrate binding by the lectin discoidin I immobilized on nitrocellulose

Discoidin I is the most abundant galactose binding lectin produced by the cellular slime mold Dictyostelium discoideum and has been implicated in cell-substratum adhesion. We have developed an assay of carbohydrate binding activity utilizing binding of 125I-asialofetuin to discoidin I, or to other lectins, immobilized on nitrocellulose. Among the proteins examined, only lectins exhibited the ability to bind asialofetuin. Specificity of asialofetuin binding was demonstrated by competition with monosaccharides, which inhibited binding consistent with the known sugar specificity of the lectins examined. Experiments with fetuin and derivatives differing in their oligosaccharide structure indicated a requirement for terminal galactosyl residues for probe binding to discoidin I. We have used this assay to characterize the carbohydrate binding behavior of discoidin I. The extent of asialofetuin binding to discoidin I was dependent on the concentrations of both lectin and ligand. Interpretation of equilibrium binding data suggested that, under saturating conditions, 1 mol of oligosaccharide was bound per mole discoidin I monomer. Furthermore, discoidin I in solution and discoidin I on nitrocellulose were equally effective at competing for soluble asialofetuin, suggesting that immobilization had no effect on the carbohydrate binding behavior of discoidin I. Binding was strongly inhibited by ethylenediaminetetraacetic acid; both Ca2+ and Mn2+ could overcome that inhibition, but Mg2+ could not. Preincubation of discoidin I at 60 degrees C stimulated asialofetuin binding 2-fold by increasing the affinity, while preincubation at higher temperatures resulted in a complete loss of activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutaraldehyde cross-linking of lectins to marker enzymes: protection of binding site by specific sugars

The role of bound specific sugars in protecting the sugar binding activity of several galactose binding proteins during their covalent conjugation to horse radish peroxidase by glutaraldehyde-mediated cross-linking was examined by: a) affinity matrix binding of the conjugate, b) enzyme linked lectin assay and c) hemagglutination assay. During conjugation using 1% glutaraldehyde, protection of jack fruit (Artocarpus integrifolia) lectin (jacalin) activity depended on concentration of specific sugar present during conjugation; optimum protection was offered by 50 mM galactose. This indicated the presence of one or more primary groups at the binding site of jacalin, which is (are) essential for sugar binding. On the other hand, such essential amino group(s) was not indicated at the sugar binding site of the peanut lectin, bovine heart galectin or of the human serum anti alpha-galactoside antibody, since exclusion of sugar during their conjugation to HRP did not diminish sugar binding activity. The differential behavior is discussed in the light of reported differences in sugar specificities. Results indicated that sugar mediated blocking of active site may be used in characterization of the latter in lectins.     

Spectroscopical studies on the structural organization of the lectin discoidin I: analysis of sugar- and calcium-binding activities

One of the common characteristics observed in different families of sugar-binding proteins is the presence of aromatic residues in the proximity of the functional sugar-binding site (Quiocho, F. (1986) Annu. Rev. Biochem. 55, 287-315). This general property has made these proteins a very appropriate subject for studies using intrinsic fluorescence assays. In the present report we have studied the sugar binding activity of the lectin discoidin I, using a fluorescence-monitored titration assay. The galactose binding has been estimated, with an affinity constant of 1.8.10(-7) M-1 in the absence of calcium. In the presence of 1 mM Ca2+, the Kd of galactose binding is lowered to 2.7.10(-8) M-1. Calcium binding, by itself, seems to occur as two components with Kd values of 10(-7) and 10(-6) M-1. From these data, and sequence comparison of discoidin I with other lectins, a general model for ligand binding has been proposed in which a sequence from position 176 to 188, together with another region close to an apolar tryptophan residue, most probably Trp-50, would participate in the calcium- and sugar-binding site(s) of this protein.     

Probing the cons and pros of lectin-induced immunomodulation: case studies for the mistletoe lectin and galectin-1

When imagining to monitor animal cells through a microscope with resolution at the molecular level, a salient attribute of their surfaces will be the abundance of glycan chains. They present galactosides at their termini widely extending like tentacles into the extracellular space. Their spatial accessibility and their potential for structural variability endow especially these glycan parts with capacity to act as docking points for molecular sensors (sugar receptors such as lectins). Binding and ligand clustering account for transmission of post-binding signals into the cell interior. The range of triggered activities has turned plant lectins into popular tools in cell biology and immunology. Potential for clinical application has been investigated rigorously only in recent years. As documented in vitro and in vivo for the galactoside-specific mistletoe lectin, its apparent immunomodulatory capacity reflected in upregulation of production of proinflammatory cytokines will not necessarily be clinically favorable but a double-edged sword. In fact, lectin application has been shown to stimulate tumor growth in cell lines, histocultures of human tumors and in two animal models using chemical carcinogenesis or tumor transplantation. When testing immunological effects of the endogenous lectin galectin-1, protection against disorders mediated by activated T cells came up for consideration. Elimination of these cells via CD7-dependent induction of apoptosis, and a shift to the Th2 response by the galectin, are factors to ameliorate disease states. This result encourages further efforts with other galectins. Functional redundancy, synergism, diversity or antagonism among galectins are being explored to understand the actual role of this class of endogenous lectins in inflammation. Regardless of the results of further preclinical testing for galectin-1, these two case studies break new ground in our understanding how glycans as ligands for lectins convey reactivity to immune cells, with impact on the course of a tumor or autoimmune disease.     

Evaluation of the specificity of lectin binding to sections of plant tissue

Hand sections of young corn root tips have been used in a study of problems encountered in the binding of fluorescently-labelled lectins to plant tissues. It was found, surprisingly, that with lectins specific for a sugar known to be present (Lotus and Ulex lectins for L-fucose), with a lectin specific for a sugar thought not to be present (wheat-germ agglutinin for N-acetylglucosamine), with non-lectin glycoprotein and protein (gamma-globulin and bovine serum albumin) and with basophilic dyes (alcian blue and toluidine blue), a coincidental binding pattern similar to the pattern of autofluorescence in the same tissue was obtained. Corn root tissues include cell walls composed of complex polysaccharides esterified with ferulic acid residues, as well as mucilages which are highly hydrated and expanded. In such material, neither standard inhibition controls with haptens nor the use of a wide range of lectin concentrations are adequate to distinguish clearly specific and non-specific binding of fluorescently-labelled lectin. Therefore, lectins are not the simple test probes they have been supposed. Before interpreting results obtained in using fluorescently-labelled lectins on any tissue sections, all available information (biochemical as well as histochemical) about the tissue must be considered.     

Light microscopic localization of glycosyltransferase activities in cells and tissues

We describe an assay for light microscopic visualization of specific glycosyltransferases on tissue sections or on cells. The assay uses a sequence of enzyme reactions that yields two moles of NADH for each mole of the uridine-5'-diphosphate (UDP) released during transfer of a monosaccharide from a UDP sugar to an acceptor. When diaphorase and tetrazolium salts are present in the incubation mixture, the tetrazolium salts are reduced to colored diformazans, which precipitate at the sites of glycosyltransferase activity. The validity of the assay was established by applying the technique to spermatozoa and liver, in which some glycosyltransferases have previously been localized. When suspensions of mouse spermatozoa were assayed for galactosyltransferase (GalTase) activity, diformazan precipitates appeared on the plasma membranes overlying the anterior heads of the spermatozoa, in agreement with immunochemical localizations. In mouse liver slices assayed with bilirubin as acceptor for glucuronyltransferase (GluTase) activity, dense diformazan deposits appeared on the hepatocytes but not on endothelial cells, also in agreement with immunochemical data. In the absence of acceptor or UDP sugar donor, diformazan deposits were minimal and random in all tissues tested. The assay's versatility was tested by incubating tissues with different sugar donors and acceptors to localize other sites of transferase activity. In mouse frozen liver sections, GalTase activity occurred in both hepatocytes and endothelial cells; in sections of rat submaxillary glands, GalTase activity was detected in mast cells. In liver sections, GlcuTase activity with o-aminophenol as acceptor was located primarily on the endothelial cells. With the appropriate sugar donor and acceptor, this assay should detect any transferase, other than the glucosyltransferases, that utilizes UDP sugars.     

Spatial differences of endogenous lectin expression within the cellular organization of the human heart: a glycohistochemical, immunohistochemical, and glycobiochemical study

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.     

Purification and characterization of a β-galactoside-binding soluble lectin from rat and bovine brain

A β-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggragation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the β-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30 000 for the bovine brain lectin and 32 000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15 000 and 16 000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for β-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.     

Biospecific immobilization of mannan-penicillin G acylase neoglycoenzyme on Concanavalin A-bead cellulose

The matter of this work was to evaluate possibilities of biospecific immobilization of synthetic mannan-penicillin G acylase neoglycoconjugate on Concanavalin A support. The conjugate containing 37% (w/w) of yeast mannan was prepared. Significant biospecific interaction of this neoglycoenzyme with Con A was confirmed by precipitation method. The biospecific sorption of conjugate was investigated using Concanavalin A-triazine bead celluloses MT-100 with different content of Con A (from 1.4 to 9.8 mgCon A/gwet support). The results obtained under optimal conditions were compared with those from covalent immobilization of PGA. The sorbent capacity was observed higher for covalent binding of enzyme. On the other hand, the biospecifically immobilized neoglycoenzyme retained a greater amount of initial activity. The maximum amount of 6.6mgimmobilizedneoglycoenzyme/gwet Con A-sorbent (18.1 U/g) was achieved. The amount as well as activity of immobilized mannan-penicillin G acylase was increased by its two multiple layering on surface of sorbent (10.1mg, respectively, 23.5 U/gwet sorbent). Determined storage and operational (using flow calorimetric method) stabilities of biospecifically immobilized enzyme, were similar, possibly somewhat higher that those of covalent bound penicillin G acylase.     

Studies on hormonal modulation of asparagine-linked glycoprotein biosynthesis in explant cultures of rat mammary gland

Glucosidase I has been purified to homogeneity and polyclonal antibodies against the enzyme have been prepared. The anti-glucosidase I antibodies recognized a single band of 85 kDa on western blot at a dilution as high as 1:2000 and also inhibited the enzyme activity, suggesting the specificity of the antibodies. Con A-Sepharose binding experiment indicates that this enzyme itself is a high mannose type N-linked glycoprotein. The increase in the electrophoretic mobility of 85 kDa band following digestion with endoglycosidase H and F strengthened this observation. The presence of any O-linked sugar attached covalently to glucosidase I could not be detected by binding assays with O-linkage specific biotinylated lectins. The studies on developmental regulation suggest that the synthesis of glucosidase I is modulated with the ontogeny of the gland. Lactogenic hormones, viz. insulin, hydrocortisone and prolactin, appeared to regulate the synthesis of glucosidase I. The possible role of these hormones in the overall regulation of protein N-glycosylation has been discussed.     

Nature of the lectin-induced activation of plasma membrane Mg2+ATPase.

The Mg2+ATPase activity of liver plasma membranes decreases markedly with increasing temperature above 30 degrees. This negative temperature dependency is counteracted by the binding of wheat germ agglutinin, concanavalin A, or Ricinus communis agglutinin (at concentrations greater than or equal 0.5 mg/ml) to membranes prior to assay of the enzyme. With one of these lectins bound, the enzyme has a single energy of activation between 20 degrees and 45 degrees. The binding of dimeric succinyl concanavalin A, soybean agglutinin, fucose-binding lectin from Lotus tetragonolobus, or the leucoagglutinin from Phaseolus vulgaris does not alter the temperature dependency of the enzyme. The latter two lectins, however, do prevent the concanavalin A-induced activation of the enzyme at 37 degrees. At saturating substrate concentrations, the enzyme is not inhibited by any of the lectins tested over a wide range of concentrations. Cytochalasin B and colchicine separately or in combination have little influence on the lectin-induced enhancement of enzyme activity. Chlorpromazine and vinblastine sulfate each partially prevent the activation and in combination do so completely. Treatment of the membranes with the detergent Lubrol-PX or phospholipase A prevents activation of the enzyme by concanavalin A. The results are consistent with a restriction by the lectin of an environment which is normally too disordered for maximal enzyme activity above 30 degrees.     

Purification and characterization of a beta-galactoside-binding soluble lectin from rat and bovine brain

A beta-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggregation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the beta-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30,000 for the bovine brain lectin and 32,000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15,000 and 16,000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for beta-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.     

An assay for leukoagglutinating lectins using suspension cultured mouse lymphoma cells (BW5147) stained with neutral red

A sensitive and rapid assay for leukoagglutinating lectins has been developed. This assay utilizes neutral red-stained mouse lymphoma cells from the suspension cultured cell line BW5147. The agglutination of the stained cells can be monitored visually in a manner similar to that for conventional assays for erythroagglutinating lectins using erythrocytes. The activity of lekoagglutinating lectins that are not capable of agglutinating erythrocytes can be quantified by this assay. The utility of the assay was demonstrated using leukoagglutinating and erythroagglutinating lectins from the seeds of Phaseolus vulgaris and Maackia amurensis.     

Correlationships between Enzymatic Activity of Lectins, Putrescine Content and Chloroplast Damage in Xanthoria parietina Phycobionts

Lectins from the lichen Xanthoria parietina develop arginase activity. One of these lectins behaves as a secreted arginase whereas another is an endocellular enzyme. Both enzymes are glycosylated proteins differing in the occurrence of galactose instead of N-acetyl-D-glucosamine in secreted arginase. The affinity for the algal ligand (glycosylated cell wall urease) of secreted arginase is higher than that shown for the endocellular enzyme. When the lectin ligand is absent from the algal cell wall, both endocellular and secreted arginases seem to be able to enter algal cells. This uptake promotes the increase in the amount of algal putrescine, preferently as free polyamine, and the chloroplast is rapidly damaged. Induction of cell wall urease retains lectins outside the cells, on the cell wall, and chloroplast remains healthy.     

Preparation of neoglycoprotein-enzyme conjugate using a heterobifunctional reagent and its use in solid-phase assays and histochemistry

A conjugate of a neoglycoprotein (chemically lactosylated bovine serum albumin) and an enzyme (horseradish peroxidase) has been prepared in solution using a heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)propionate, and has been purified by gel filtration on an Ultrogel AcA-44 column. To preclude any carbohydrate-dependent binding to the sugar residues on the glycoprotein peroxidase, the enzyme has to be treated with sodium periodate and sodium cyanoborohydride prior to coupling, which results in oxidative cleavage of the carbohydrates and reduction of the aldehydes thus formed to primary alcohols. Lactosylated bovine serum albumin-peroxidase conjugate has been employed to detect plastic-bound Ricinus communis agglutinin with dependence of the concentration of the lectin and with dependence of the presence of specific inhibitors. Enzyme-labeled conjugates with unmodified bovine serum albumin are completely ineffective in this assay. Localization of β-galactoside-specific sugar receptors in connective tissue is used to demonstrate the feasibility of application of such neoglycoprotein-enzyme conjugates in histochemistry with a minimum number of steps.     

Evaluation of the specificity of lectin binding to sections of plant tissue

Summary Hand sections of young corn root tips have been used in a study of problems encountered in the binding of fluorescently-labelled lectins to plant tissue. It was found, surprisingly, that with lectins specific for a sugar known to be present (Lotus andUlex lectins forl-fucose), with a lectin specific for a sugar thought not to be present (wheat-germ agglutinin for N-acetylglucosamine), with non-lectin glycoprotein and protein (-globulin and bovine serum albumin) and with basophilic dyes (alcian blue and toluidine blue), a coincidental binding pattern similar to the pattern of autofluorescence in the same tissue was obtained. Corn root tissues include cell walls composed of complex polysaccharides esterified with ferulic acid residues, as well as mucilages which are highly hydrated and expanded. In such material, neither standard inhibition controls with haptens nor the use of a wide range of lectin concentrations are adequate to distinguish clearly specific and non-specific binding of fluorescently-labelled lectin. Therefore, lectins are not the simple test probes they have been supposed. Before interpreting results obtained in using fluorescently-labelled lectins on any tissue sections, all available information (biochemical as well as histochemical) about the tissue must be considered.     

Ferrous human cystathionine beta-synthase loses activity during enzyme assay due to a ligand switch process

Cystathionine beta-synthase (CBS) is a pyridoxal-5'-phosphate-dependent enzyme that catalyzes the condensation of serine and homocysteine to form cystathionine. Mammalian CBS also contains a heme cofactor that has been proposed to allosterically regulate enzyme activity via the heme redox state, with FeII CBS displaying approximately half the activity of FeIII CBS in vitro. The results of this study show that human FeII CBS spontaneously loses enzyme activity over the course of a 20 min enzyme assay. Both the full-length 63-kDa and truncated 45-kDa form of CBS slowly and irreversibly lose activity upon reduction to the FeII form. Additionally, electronic absorption spectroscopy reveals that FeII CBS undergoes a heme ligand exchange to FeII CBS424 when the enzyme is incubated at 37 degrees C and pH 8.6. The addition of enzyme substrates or imidazole has a moderate effect on the rate of the ligand switch, but does not prevent conversion to the inactive species. Time-dependent spectroscopic data describing the conversion of FeII CBS to FeII CBS424 were fitted to a three-state kinetic model. The resultant rate constants were used to fit assay data and to estimate the activity of FeII CBS prior to the ligand switch. Based on this fit it appears that FeII CBS initially has the same enzyme activity as FeIII CBS, but FeII CBS loses activity as the ligand switch proceeds. The slow and irreversible loss of FeII CBS enzyme activity in vitro resembles protein denaturation, and suggests that a simple regulatory mechanism based on the heme redox state is unlikely.     

Leguminous lectins as tools for studying the role of sugar residues in leukocyte recruitment

The natural physiological ligands for selectins are oligosaccharides found in glycoprotein or glycolipid molecules in cell membranes. In order to study the role of sugar residues in the in vivo lectin anti-inflammatory effect, we tested three leguminous lectins with different carbohydrate binding affinities in the peritonitis and paw oedema models induced by carrageenin in rats. L. sericeus lectin was more anti-inflammatory than D. virgata lectin, the effects being reversed by their specific binding sugars (N-acetylglucosamine and alpha-methylmannoside, respectively). However, V. macrocarpa, a galactose-specific lectin, was not anti-inflammatory. The proposed anti-inflammatory activity of lectins could be due to a blockage of neutrophil-selectin carbohydrate ligands. Thus, according to the present data, we suggest an important role for N-acetylglucosamine residue as the major ligand for selectins on rat neutrophil membranes.