CEL-I is a C-type lectin isolated from the Holothuroidea Cucumaria echinata. This lectin shows very high N-acetylgalactosamine-binding specificity. We constructed an artificial gene encoding recombinant CEL-I (rCEL-I) using a combination of synthetic oligonucleotides, and expressed it in Escherichia coli cells. Since the recombinant protein was obtained as inclusion bodies, the latter were solubilized using urea and 2-mercaptoethanol, and the protein was refolded during the purification and dialysis steps. The purified rCEL-I showed comparable hemagglutinating activity to that of native CEL-I at relatively high Ca(2+)-concentrations, whereas it was weaker at lower Ca(2+)-concentrations due to decreased Ca(2+)-binding affinity. rCEL-I exhibited similar carbohydrate-binding specificity to native CEL-I, including strong GalNAc-binding specificity, as examined by hemagglutination inhibition assay. Comparison of the far UV-CD spectra of recombinant and native CEL-I revealed that the two proteins undergo a similar conformational change upon binding of Ca(2+). Single crystals of rCEL-I were also obtained under the same conditions as those used for the native protein, suggesting that they have similar tertiary structures. Although native CEL-I exhibited strong cytotoxicity toward cultured cells, rCEL-I showed low cytotoxicity. These results indicate that rCEL-I has a tertiary structure and carbohydrate-binding specificity similar to those of native CEL-I. Howeger, there is a subtle difference in the properties between the two proteins probably due to the additional methionine residue at the N-terminus of rCEL-I.

A haemagglutinin (lectin) and another protein (inhibitor) purified from the seeds of Momordica charantia inhibited protein and subsequently DNA synthesis in normal (mitogen-stimulated) and leukaemic human peripheral blood lymphocytes. The effect of the lectin was more rapid and more pronounced than that of the inhibitor, probably due to a better penetration of the lectin into cells. Both proteins acted on lymphocytes more markedly and at concentrations much lower than those required to inhibit protein synthesis in Yoshida ascites cells.

Ehrlich ascites tumor cells and ascitic fluid were assayed for glycosidase activity. alpha-Galactosidase and beta-galactosidase, alpha- and beta-mannosidase, alpha-N-acetylgalactosaminidase, and beta-N-acetylglucosaminidase activities were detected using p-nitrophenyl glycosides as substrates. alpha-Galactosidase and alpha-N-acetylgalactosaminidase were isolated from Ehrlich ascites tumor cells on epsilon-aminocaproylgalactosylamine-Sepharose. alpha-Galactosidase was purified 160,000-fold and was free of other glycosidase activities. alpha-N-Acetylgalactosaminidase was also purified 160,000-fold but exhibited a weak alpha-galactosidase activity which appears to be inherent in this enzyme. Substrate specificity of the alpha-galactosidase was investigated with 12 substrates and compared with that of the corresponding coffee bean enzyme. The pH optimum of the Ehrlich cell alpha-galactosidase centered near 4.5, irrespective of substrate, whereas the pH optimum of the coffee bean enzyme for PNP-alpha-Gal was 6.0, which is 1.5 pH units higher than that for other substrates of the coffee bean enzyme. The reverse was found for alpha-N-acetylgalactosaminidase: the pH optimum for the hydrolysis of PNP-alpha-GalNAc was 3.6, lower than the pH 4.5 required for the hydrolysis of GalNAc alpha 1,3Gal. Coffee bean alpha-galactosidase showed a relatively broad substrate specificity, suggesting that it is suited for cleaving many kinds of terminal alpha-galactosyl linkages. On the other hand, the substrate specificity of Ehrlich alpha-galactosidase appears to be quite narrow. This enzyme was highly active toward the terminal alpha-galactosyl linkages of Ehrlich glycoproteins and laminin, both of which possess Gal alpha 1, 3Gal beta 1,4GlcNAc beta-trisaccharide sequences. The alpha-N-acetylgalactosaminidase was found to be active toward the blood group type A disaccharide, and trisaccharide, and glycoproteins with type A-active carbohydrate chains.

The Griffonia simplicifolia-I (GS-I) isolectins have been used to probe the effect of lectin valence on their high-affinity binding to human erythrocytes. These tetrameric lectins are composed of A and B subunits and constitute a series of five isolectins (A4, A3B, A2B2, AB3, B4). The A subunit is specific for alpha-D-GalNAc end groups and binds to the blood type A determinant GalNAcalpha1, as well as to terminal alpha-D-Gal groups found on type B cells. The B subunit is specific for alpha-D-Gal end groups, and binds very specifically to type B erythrocytes. This series of isolectins is tetravalent (A4), trivalent (A3B), divalent (A2B2), and monovalent (AB3) for type A erythrocytes; thus, this system provides the opportunity to examine the effect of lectin valency on the association constants of these GS-I isolectins binding to cells. Cell binding experiments carried out using 125I-labeled GS-I isolectins and type A human erythrocytes allowed us to demonstrate that (1) the association constant of the isolectin monovalent for alpha-D-GalNAc (AB3) is virtually identical to its association constant for the haptenic sugar methyl-N-acetyl-alpha-D-galactosaminide, reported previously, and (2) the association constant of the GS-I isolectins for human type A erythrocytes increases with increasing valency of the isolectin. These results indicate that the increased affinity displayed by the GS-I isolectins for human type A erythrocytes is dependent on their multivalency, and not on an extended binding site nor on nonspecific, or noncarbohydrate, interactions of the lectin with the cell surface. These findings should be of general relevance to understanding the high-affinity interactions observed between other multivalent proteins and multivalent ligands (e.g., cell surfaces).

Lectins are polyvalent proteins of non-immune origin with exquisite carbohydrate binding specificity making them ideal for investigation of cell surface glycoprotein and glycolipid antigens. We examined the cell surface lectin binding phenotypes of 20 UM-SCC squamous cell carcinoma cell lines established from 17 patients with head and neck cancers using a panel of fluorescein-conjugated lectins and inhibition by the appropriate monosaccharide to confirm specificity of using a panel of fluorescein-conjugated lectins and inhibition by the appropriate monosaccharide to confirm specificity of binding. Conconavalin A (Con A) from Canavalia ensiformis and the peanut agglutinin (PNA) from Arachis hypogaea bound all SCC cell lines tested and wheat germ agglutinin (WGA) from Triticum vulgaris bound to 12 of 13 tumor cell lines. The blood group O specific lectin UEA 1 from Ulex europeus also bound to all cell lines regardless of the donor blood type. Lectins of Dolichos biflorus (DBA) and Griffonia simplicifolia (GS I-B4 or BSA I-B4) with binding specificity for glycoproteins associated with blood group A and B respectively, had reactivity that did not directly correlate with blood group antigen expression. In contrast to the other lectins in our panel which exhibited broad reactivity with SCC antigens, the BSA-II lectin from Griffonia simplicfolia,(GS II or BSA II) which has sugar binding specificity for terminal non-reducing GlcNAc, did not bind to any of the screened cell lines. Our results demonstrate a common pattern of lectin-defined carbohydrate expression on the cell surface of squamous cell carcinomas of head and neck that appears promising in defining the malignant cellular phenotype. Lectin binding profile may be useful in differentiating benign from malignant histopathology.

Association constants for the binding of methyl alpha-D-galactopyranoside (methyl alpha-D-Galp) and methyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside (methyl alpha-D-GalNAcp) to three Bandeiraea simplicifolia isolectins (A4, A2B2, B4) were determined by equilibrium dialysis and fluorescence enhancement measurements. The a and B subunits appear to have approximately the same Kassoc for methyl alpha-D-Galp: 1.45 X 10(4), 1.98 X 10(4), and 2.06 X 10(4) M-1 for A4, A2B2, and B4, respectively, as determined by equilibrium dialysis. Fluorescence enhancement measurements on B4 gave an association constant of 2.07 X 10(4) M-1 for methyl alpha-D-Galp and 1.87 X 10(3) M-1 for methyl beta-D-galp. By equilibrium dialysis, we were able to detect 3.3 (theory, 4.0) methyl alpha-D-GalNAcp binding sites for A4 (Kassoc = 1.87 X 10(5) M-1), 1.9 for A2B2 (Kassoc = 1.19 X 10(5) M-1), and were unable to detect any methyl alpha-D-GalNAcp binding sites for B4. However, four very weak methyl alpha-D-GalNAc binding sites for B4 were detected by fluorescence enhancement measurement (Kassoc = 1.26 X 10(2) M-1). Thus, the A subunit has an affinity for methyl alpha-D-GalNAc 3 orders of magnitude greater than the B subunit. Precipitation and hapten inhibition data are in accord with these binding measurements. Toward guaran and type B blood group substance, all isolectins precipitated the same amount of biopolymer. However, AB3, A2B2, and A3B, which are mono-, di-, and trivalent for alpha-D-GalNAcp, were differentially precipitated by type A blood group substance which contains alpha-D-GalNAcp-end groups. A3B precipitated the most, A2B2 less, and AB3 no type A substance. These isolectins should prove useful in studies evaluating the effect of valence on lectin-cell interaction.

BS I-B4, an alpha-D-galactopyranosyl-binding isolectin from Bandeiraea simplicifolia seeds, was found to interact differently with transformed mouse L cells and non-transformed mouse 3T3 cells. The lectin induces detachment of 3T3 cells but increases adhesiveness and clustering of L cells. However, the induced cell aggregation does not lead to cell fusion. A variant clone of L cells, resistant to BS I-B4, which had lost the capacity for agglutination in the presence of the lectin, was isolated. Fluorescence binding studies of this variant suggest a lesion involving alpha and beta-D-galactopyranosyl units on its cell-surface structures. Although the variant cells form colonies in a methylcellulose medium, they do not produce tumours, as do the parental cells, when transplanted in athymic nude mice. The results demonstrate that alterations in cell membrane glyco-conjugates play an important role in tumourigenesis of animal cells, but anchorage-independent growth in vitro, as one of the transformation phenotypes, cannot be correlated absolutely with tumourigenicity in vivo.

Three fluorescein-labeled lectins have been shown to exhibit specificity for the surface of cells in different layers of the epidermis in the newborn rat. An isolectin from seeds of Bandeiraea simplicifolia with specificity for alpha-D-galactosyl end groups labeled the basal and lower spinous cells; a lectin from Ulex europaeus exhibiting specificity for alpha-L-fucosyl units outlines the surface of spinous cells, and a second lectin from B. simplicifolia, with specificity for N-acetyl-D-glucosamine, labels the cornified cells. Appropriate blocking experiments have confirmed the specific nature of the binding.

An alpha-D-galactosyltransferase activity has been detected in membranous fractions (42,000 x g) of Ehrlich ascites cells which transfers galactosyl groups from UDP-galactose to endogenous and exogenous acceptors. The products of the reaction contain alpha-D-galactopyranosyl groups at the nonreducing termini. A solid state assay was developed to follow alpha-D-galactosyltransferase activity in the presence of beta-D-galactosyltransferase. Examination of a variety of insolubilized exogenous acceptors indicated that the most active acceptors for the alpha-D-galactosyltransferase had the structure beta-D-Gal-(1 goes to 4)-beta-D-GlcNAc(1 goes to at their nonreducing termini. Incubation of UDP-[14C]galactose and beta-D-gal-(1 goes to 4)-D-GlcNAc (N-acetyllactosamine) or of UDP-galactose and beta-D-[14C]Gal-(1 goes to 4)-D-GlcNAc in the presence of the alpha-D-galactosyltransferase resulted in the enzymic synthesis of a 14C-labeled trisaccharide. Chemical and enzymic methods of analysis revealed the structure of the trisaccharide to be alpha-D-Gal-(1 goes to 3)-beta-D-Gal-(1 goes to 4)-D-GlcNAc. These data indicate that the alpha-D-galactosyltransferase in Ehrlich ascites cells transfers galactosyl groups to suitable acceptors to form an alpha-(1 goes to 3)-D-galactosidic linkage.

A simple and convenient method for the introduction of radiolabel onto C-6' of N-acetyllactosamine is described. 1-N-Benzyl-3-O-beta-D-galactopyranosyl-D-arabinosylamine (1) was synthesized from 3-O-beta-D-galactopyranosyl-D-arabinose as described by Lee and Lee. Compound 1 was oxidized with D-galactose oxidase, and the product reduced with KB3H4 to introduce the label at C-6'. After dilution with unlabeled material, the N-benzyl-3-O-beta-D-[6-3H]galactopyranosyl-D-arabinosylamine was converted into 2-(benzylamino)-2-deoxy-4-O-D-[6-3H]galactopyranosyl-D-glucononitrile , which was subjected to simultaneous hydrogenolysis of the benzylamino and nitrile groups. N-Acetylation of the amino group as described by Alais and Veyrières afforded the crystalline title compound in 63% yield.

An electrode covered with a lectin/collagen film was constructed to investigate whether the film was usable as a reaction field of binding between the lectin and sugar. The protein-sugar binding on cell surface plays an important role to various physiologic processes. The film is considered to be a cell surface, due to its biocompatibility. The immobilization of concanavalin A (Con A) which is one of proteins was attempted by an electrostatic interaction of the protonated functional groups of film to the negative charged Con A. The merit of this immobilization is that the interaction hardly causes any changes in the protein structure. Because Con A recognizes mannose moiety, the mannose was labeled with an electroactive compound. The binding was estimated from the changes of the electrode response based on the holding of electroactive moiety in the binding site of Con A to the mannose moiety. However, the electrode responses of glucose and galactose labeled with the same substance did not change. The result shows that Con A is immobilized on the film and combines with labeled mannose. Therefore, it is clear that the collagen film is suitable as the reaction field to evaluate the protein-sugar binding.

The highly basic eosinophil major basic protein (MBP), present in the crystalloid core of the eosinophil leukocyte granules, has both cytotoxic and cytostimulatory properties, and is directly implicated in a number of diseases. The crystal structure of MBP resembles that of the C-type lectin (CTL) superfamily, and recent data showed that MBP binds heparan sulfate glycosaminoglycan (GAG), with the CTL ligand-binding region as the binding site. MBP is synthesized as a proform (proMBP) containing an acidic propiece believed to neutralize the basic MBP domain. Using flow cytometry and site-directed mutagenesis, we here demonstrate that the MBP domain of proMBP binds to heparan sulfate GAG on the cell surface, and that this is independent of GAG covalently bound to proMBP. Eight basic residues located in the CTL ligand-binding region of MBP were previously hypothesized to mediate GAG binding, but we find that surface binding was not compromised by the substitution of these residues with alanine. However, the analysis of a series of mutants with surface exposed residues substituted with alanine showed that residues Ser-166, Arg-168, and Arg-171 are involved in surface binding. A binding site formed by these residues is located in the MBP domain between loop 1 and ss-strand 5, outside the CTL ligand-binding region. The binding of a cell-surface heparan sulfate proteoglycan may be important in MBP action, and our findings suggest that two regions, previously shown to contain the cytotoxic and cytostimulatory properties of MBP, are accessible for ligand interaction in cell surface-bound MBP.

Reduced terminal sialylation at the surface of airway epithelial cells from patients with cystic fibrosis may predispose them to bacterial infection. To determine whether a lack of chloride transport or misprocessing of mutant cystic fibrosis transmembrane conductance regulator (CFTR) is critical for the alterations in glycosylation, we studied a normal human tracheal epithelial cell line (9/HTEo(-)) transfected with the regulatory (R) domain of CFTR, which blocks CFTR-mediated chloride transport; DeltaF508 CFTR, which is misprocessed, wild-type CFTR; or empty vector. Reduced cAMP-stimulated chloride transport is seen in the R domain and DeltaF508 transfectants. These two cell lines had consistent, significantly reduced binding of elderberry bark lectin, which recognizes terminal sialic acid in the alpha-2,6 configuration. Binding of other lectins, including Maakia amurensis lectin, which recognizes sialic acid in the alpha-2,3 configuration, was comparable in all cell lines. Because the cell surface change occurred in R domain-transfected cells, which continue to express wild-type CFTR, it cannot be related entirely to misprocessed or overexpressed CFTR. It is associated most closely with reduced CFTR activity.

The enzyme activities involved in O-glycosylation have been studied in three insect cell lines, Spodoptera frugiperda (Sf-9), Mamestra brassicae (Mb) and Trichoplusia ni (Tn) cultured in two different serum-free media. The structural features of O-glycoproteins in these insect cells were investigated using a panel of lectins and the glycosyltransferase activities involved in O-glycan biosynthesis of insect cells were measured (i.e., UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, UDP-Gal:core-1 beta1, 3-galactosyltransferase, CMP-NeuAc:Galbeta1-3GalNAc alpha2, 3-sialyltransferase, and UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase activities). First, we show that O-glycosylation potential depends on cell type. All three lepidopteran cell lines express GalNAcalpha-O-Ser/Thr antigen, which is recognized by soy bean agglutinin and reflects high UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity. Capillary electrophoresis and mass spectrometry studies revealed the presence of at least two different UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases in these insect cells. Only some O-linked GalNAc residues are further processed by the addition of beta1,3-linked Gal residues to form T-antigen, as shown by the binding of peanut agglutinin. This reflects relative low levels of UDP-Gal:core-1 beta1,3-galactosyltransferase in insect cells, as compared to those observed in mammalian control cells. In addition, we detected strong binding of Bandeiraea simplicifolia lectin-I isolectin B4 to Mamestra brassicae endogenous glycoproteins, which suggests a high activity of a UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase. This explains the absence of PNA binding to Mamestra brassicae glycoproteins. Furthermore, our results substantiated that there is no sialyltransferase activity and, therefore, no terminal sialic acid production by these cell lines. Finally, we found that the culture medium influences the O-glycosylation potential of each cell line.

A specific monoclonal antibody against toxin A from Clostridium difficile was generated that did not show thermolabile binding. Nonspecific murine monoclonal antibodies bound toxin A at 4 degrees C, but less effectively at 37 degrees C. Nonspecific human monoclonal antibodies did not bind to toxin A at 4 degrees C. Cytotoxic properties of purified toxin A were not inhibited by Bandeiraea simplicifolia lectin. This points to a carbohydrate moiety on the cell surface and a multivalent nonspecific carbohydrate binding ligand on toxin A.

Retrograde and anterograde degeneration have been reported to be sufficient stimuli to activate glial cells, which, in turn, are involved in phagocytosis of degenerating material. Here we describe a double-fluorescence technique which allows for direct and simultaneous visualization of both labeled incorporated axonal debris and incorporating glial cells in the course of anterograde degeneration. Stereotaxic application of small crystals of biotinylated and tetramethylrhodamine (TRITC)-conjugated dextran amine Mini Ruby into the medial entorhinal cortex resulted in a stable rhodamine fluorescence confined to fibers and terminals in the middle molecular layer of the dentate gyrus, the stratum lacunosum-moleculare, and the crossed temporo-hippocampal pathway. Subsequent stereotaxic lesion of the entorhinal cortex induced transformation of rhodamine-fluorescent fibers and terminals into small granules. Incorporation of these granules by microglial cells [labeled by fluorescein isothiocyanate (FITC)-coupled Bandeiraea simplicifolia isolectin B4] or astrocytes (labeled by FITC-coupled glial fibrillary acidic protein antibodies) resulted in phagocytosis-dependent labeling of these non-neuronal cells, which could be identified by double-fluorescence microscopy. Electron microscopical analysis revealed that, following lesion, the tracer remained confined to entorhinal axons which were found to be incorporated by glial cells. Our data show that TRITC- and biotin-conjugated dextran amines are versatile tracers leading to Phaseolus vulgaris leucoagglutinin-like axonal staining. Lesion-induced phagocytosis of anterogradely degenerating axons by immunocytochemically identified glial cells can be directly observed by this technique on the light and electron microscopical levels.

Lectin histochemical studies were performed on the olfactory system of Scyliorhinus canicula to identify specific glycoconjugates on the cell surface of primary olfactory neurons. The olfactory receptor cells, the olfactory nerve fibers and their terminals in the bulbs were labelled with SBA, BSA-I and BSA-I-B4. The lectin staining patterns indicate that the membranes of small-spotted catshark olfactory neurons had glycoproteins with alpha-galactose residues. This carbohydrate moiety could be related to modulation of the cell-cell interactions in the olfactory system.

Roles of surface sialic acid residues in cell adhesion to substratum were investigated in model systems to clarify their contribution to intercellular interactions. Treatment of cells of a fibroblastic cell line, Swiss 3T3, an epithelial cell line, TES-1, and a T lymphoma cell line, BW5147, with a sialylated oligosaccharide binding lectin enhanced adhesion to plastic plates irrespective of the cell lineage. This enhancement was inhibited by the addition of sialyl lactose. Digestion of the cells with sialidase or endoglycoceramidase also augmented adhesion. On the other hand, adhesion was reduced by pre-coating plastic plates with gangliosides but not with desialylated gangliosides. These findings suggest that sialic acid residues exposed to cell surfaces negatively regulate cell adhesiveness.

BS I-B4, an alpha-D-galactopyranosyl-binding isolectin from Bandeiraea simplicifolia seeds, was found to interact differently with transformed mouse L cells and non-transformed mouse 3T3 cells. The lectin induces detachment of 3T3 cells but increases adhesiveness and clustering of L cells. However, the induced cell aggregation does not lead to cell fusion. A variant clone of L cells, resistant to BS I-B4, which had lost the capacity for agglutination in the presence of the lectin, was isolated. Fluorescence binding studies of this variant suggest a lesion involving alpha and beta-D-galactopyranosyl units on its cell-surface structures. Although the variant cells form colonies in a methylcellulose medium, they do not produce tumours, as do the parental cells, when transplanted in athymic nude mice. The results demonstrate that alterations in cell membrane glyco-conjugates play an important role in tumourigenesis of animal cells, but anchorage-independent growth in vitro, as one of the transformation phenotypes, cannot be correlated absolutely with tumourigenicity in vivo.

The influence of altered carbohydrate structure on the surface number, distribution and turnover of plasma membrane glycoproteins has been studied in Chinese hamster ovary (CHO) cells by comparing three lines that are resistant to the cytotoxic effects of wheat germ agglutinin (WGA) with parental CHO cells. The glycoproteins investigated were members of a group of high molecular weight acidic glycoproteins (HMWAG). On parental cells these represent the major surface components that become labelled by lactoperoxidase-catalysed iodination. They are the only plasma membrane glycoproteins that bind WGA. The mutant lines also possess iodinatable surface polypeptides of high molecular weight, but these were less acidic and electrophoretically less diffuse than those from parental cells. These polypeptides in general did not bind [125I]WGA when two-dimensional polyacrylamide gels were overlaid with iodinated lectin. Mutant cells treated with fluorescein-conjugated WGA showed low surface fluorescence. However, the nuclear envelope and a small region in the perinuclear zone fluoresced strongly. Together, these results confirm that the surface glycoproteins of mutant cells had altered carbohydrate structure. Mouse antiserum prepared against the HMWAG, however, bound equally effectively to the mutant lines as to the parental lines. Indirect immunofluorescence experiments showed that the HMWAG had a fairly uniform distribution over the surface, and that internalization induced by second antibody occurred at a similar rate and in a similar manner in all lines, including the mutants. Electron microscopic observations using immunoperoxidase procedures confirmed the similarities in glycoprotein distribution on mutant and parental cells. Two mouse monoclonal antibodies raised against the HMWAG also revealed no difference in the number or topography of surface glycoproteins. Finally, the half-lives of several HMWAG in a parental and a mutant line (15B) maintained on low-serum medium were compared by means of a 125I/131I double-label technique. Half-lives of HMWAG from the former averaged 12 h and from the latter 11 h. It is concluded that the lack of complex termini on oligosaccharides of this particular group of CHO plasma membrane glycoproteins has no effect on their number, distribution or turnover.