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PubMed1999

Flow injection analysis of binding reaction between fluorescent lectin and cells.

A fluorometric binding assay for lectin and yeast cells using the avidin-biotin system was previously reported (Y. Oda, M. Kinoshita, and K. Kakehi, Anal. Biochem. 254, 41-48, 1997). However, the true amount of bound lectin could not be determined by this method due to difficulty in determination of the number of bound biotin molecules. In the present study, we have developed a method for assaying the binding reaction between fluorescent lectin and cells using a flow injection technique, which allows estimation of the amount of lectin bound to cells. An aliquot of the cell suspension was directly analyzed by injection into a flow injection system after the binding between the fluorescently labeled lectin and cells. The labeled lectins showed good linearity, at least over a range of 20-1000 ng as the injected amount. The intrinsic fluorescence of the labeled lectins did not change upon the binding. The binding reaction of the hydroxycoumarin-labeled lectins with yeast cells was rapid and reached an equilibrium state within 10 min. Scatchard analysis showed that Saccharomyces cerevisiae cells contained approximately 1. 3-1.6 x 10(8) binding sites per cell for Concanavalin A, Lycoris radiata agglutinin, and Tulipa gesneriana lectin with affinity constants of 3.2-4.7 x 10(6) M-1. The present method was applied to the study of binding between lectins and bacteria and mouse spleen cells. The assay method described here is highly sensitive and will be an alternative to assays using lectins labeled with radioisotopes. The procedure is quite simple and can be completed within 1 h.

Agglutination Tests↗
PubMed1999

Distribution of lectin binding sites in Xenopus laevis egg jelly.

Eggs from the anuran Xenopus laevis are surrounded by a thick jelly coat that is required during fertilization. The jelly coat contains three morphologically distinct layers, designated J1, J2, and J3. We examined the lectin binding properties of the individual jelly coat layers as a step in identifying jelly glycoproteins that may be essential in fertilization. The reactivity of 31 lectins with isolated jelly coat layers was examined with enzyme-linked lectin-assays (ELLAs). Using ELLA we found that most of the lectins tested showed some reactivity to all three jelly layers; however, two lectins showed jelly layer selectivity. The lectin Maackia amurensis (MAA) reacted only with J1 and J2, while the lectin Trichosanthes kirilowii (TKA) reacted only with J2 and J3. Some lectins were localized in the jelly coat using confocal microscopy, which revealed substantial heterogeneity in lectin binding site distribution among and within jelly coat layers. Wheat germ agglutinin (WGA) bound only to the outermost region of J3 and produced a thin, but very intense, band of fluorescence at the J1/J2 interface while the remainder of J2 stained lightly. The lectin MAA produced an intense fluorescence-staining pattern only at the J1/J2 interface. Several lectins were also tested for the ability to inhibit fertilization. WGA, MAA, and concanavalin A significantly inhibited fertilization and WGA was found to block fertilization by preventing sperm from penetrating the jelly. Using Western blotting, we identified high-molecular-weight components in J1 and J2 that may be important in fertilization.

Animals↗
PubMed2006

Vatairea macrocarpa (Leguminosae) lectin activates cultured macrophages to release chemotactic mediators.

The lectin from the legume Vatairea macrocarpa is a galactose/N-acetylgalactosamine binding protein that induced cellular inflammatory response mediated by resident cells. This study investigated which inflammatory mediators would be released from lectin-activated cells. The intraperitoneal injection in rats of the supernatant from cultured macrophages, but not from mast cells, stimulated with lectin induced a time- and dose-dependent release of a neutrophil chemotactic factor, termed MNCF-VML. Pharmacological modulation with dexamethasone inhibited both the lectin-induced chemotactic activity in vivo and also the lectin-induced release of MNCF-VML into the supernatant of cultured macrophages. Cyclooxygenase and lipoxygenase metabolites are apparently not involved in the action of this factor or its release, since indomethacin or MK886 were unable to affect the lectin response. The molecular weight of MNCF-VML was found to be greater than 5 kDa, which led to the investigation of which cytokine(s) could be involved by the following approaches: (a) treatment of animals with antiserum to tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1, or IL-8 before intraperitoneal injection of lectin and (b) addition of antiserum to TNF-alpha, IL-1, or IL-8 to the supernatant of lectin-stimulated macrophages before intraperitoneal administration. Antiserum to TNF-alpha, but not IL-1 nor IL-8, inhibited the neutrophil migration induced either by lectin or MNCF-VML. Our data suggest that neutrophil migration induced by V. macrocarpa lectin occurs via the release of cytokines such as TNF-alpha by macrophages. Thus, this lectin may represent an important tool to better understand pathological situations where an excess of leukocytes at inflammatory sites causes tissue injury.

Animals↗
PubMed1992

Determination of lectin characteristics by a novel agglutination technique.

A technique generally applicable for the determination of lectin characteristics is described. A sensitive light transmission/scattering method was adapted for the determination of lectin levels and lectin activity. Applying this procedure Geodia cydonium lectin-mediated agglutination was studied in an agglutimeter device using erythrocytes and even T-lymphocytes. In the Geodia lectin/T-lymphocyte system chosen, (i) a lectin concentration as low as 0.57 micrograms/ml could be measured accurately, (ii) the observed cell agglutination velocity constant with a maximal value of 0.75 min-1 was calculated, and (iii) the size of the agglutinates at a given lectin concentration and time period was estimated. The Geodia lectin activity was determined in parallel also in the erythrocyte system. Here, compared to the lectin/T-lymphocyte system the agglutination efficiency of the Geodia lectin-mediated agglutination was more than 10-fold higher and the lowest detectable lectin concentration was 0.06 micrograms/ml. Compared to the hemagglutination assay the lectin/erythrocyte system turns out to be more sensitive and to give much more information on agglutination behavior; this conclusion is supported by additional data using a second lectin isolated from Pellina semitubulosa. The superiority of the agglutination method described here over other known methods must be seen in its accuracy; moreover more lectin characteristics can be determined.

Agglutination↗
PubMed1978

Specific modifications of hepatoma cell-surface glycoproteins with enzymes. Effects on in vitro growth as investigated by the use of lectins.

The effects of enzymic treatment on the interactions between Zajdela's tumor cells and various lectins. Concanavalin A (ConA); Wheat Germ Agglutinin (WGA); Robinia lectin; have been studied. (1) The number of lectin-binding sites and the affinity constants were investigated. (2) The effects of the lectins on cell growth and [3H]thymidine incorporation were studied on untreated and enzyme-treated cells. It was observed that treatment of tumor cells with neuraminidase resulted in a change in the binding characteristics of each lectin. However, additional treatment of the cells with galactose oxidase had no further effect on lectin binding. ConA and Robinia lectin induced a decrease of the untreated tumor cell growth and a stimulation of the [3H]thymidine incorporation. This paradoxal result may be explained as a consequence of the stimulation of the [3H]thymidine uptake observed in the presence of lectins. The enzymatic treatments themselves did not change the cell growth although they did induce a change in the effect of ConA and Robinia lectin on cell growth and [3H]thymidine incorporation. As a result of neuraminidase treatment, the effects of ConA were totally suppressed but those of Robinia lectin only partially. Although WGA interacted with untreated and enzyme-treated cell surfaces, it had no effect on tumor cell growth nor [3H]thymidine incorporation. The results are discussed in terms of lectin transport.

Animals↗
PubMed1988

Distribution of lectin-binding sites in oxyntic and chief cells of isolated rabbit gastric glands.

The distribution of lectin-binding sites in oxyntic and chief cells of isolated rabbit gastric glands was determined with seven fluoresceinated lectins, to ascertain which lectins might best be used in the biochemical characterization of cell membranes and glycoproteins of these two cell types. Oxyntic cell canaliculi were labeled by wheat germ, Helix pomatia, and peanut lectins, suggesting a predominance of N-acetylhexosamines. Tubulovesicles were heavily stained by wheat germ, Helix pomatia, and Ricinus communis I lectins, indicative of N-acetylhexosamine- and galactose-containing glycoconjugates. Diffuse oxyntic cell cytoplasmic staining was observed with the mannose-binding lectin concanavalin A. This lectin, along with wheat germ, soybean, Helix pomatia, and Ricinus communis I lectins, bound to oxyntic cell basolateral membranes, indicating mannose, N-acetylhexosamine, and galactose residues. Chief cell apical membranes were labeled with peanut, Ricinus communis I, Helix pomatia, and Ulex europaeus lectins, suggesting a predominance of N-acetylhexosamine, galactose, and fucose residues. None of the lectins demonstrated any significant affinity for chief cell cytoplasm or basolateral membrane. Ulex europaeus agglutinin-binding sites were additionally concentrated in lateral intercellular spaces. The results of this study indicate that the range of utility of isolated rabbit gastric glands can be expanded to include histochemical work. In addition, the data suggest the applicability of lectin affinity chromatography in the isolation and characterization of oxyntic and chief cell membranes and glycoproteins.

Animals↗
PubMed2005

Rapid identification of Mycobacterium species by lectin agglutination.

The purpose of the present study is to explore the possibility that plant lectins can be used for the development of rapid and inexpensive technique for differentiation of mycobacterial species. The method is based on interaction between mycobacteria and lectins as visualized by agglutination in a microtiter plate. We employed 18 mycobacterium species and determined the minimal lectin concentration (MLC) of 23 different lectins. For some of the bacteria as a high as 1000 microg/ml of one or more lectins were required to induce agglutination, while for other strains as low as 1.95 microg/ml of the lectin were needed. A unique pattern of agglutination was observed for each species over a range of 62-1000 microg/ml lectin concentrations. There were little or no variations in MLC within strains (intraspecies) of each of two species tested. In contrast, there were marked interspecies variations in MLC. Analysis of the MLC showed that the highest score of interspecies differences with 23 lectins was obtained at 125 microg/ml lectin concentration. At this concentration it was found that the pattern of agglutinations with only two lectins was sufficient to differentiate mycobacterium species from each other. Because the bacteria-lectin interaction is adaptable to various methods of visualization, our findings may set the stage for developing a rapid and reliable tool to differentiate mycobacterium species.

Agglutination↗
PubMed2001

Potential of lectin-N-(2-hydroxypropyl)methacrylamide copolymer-drug conjugates for the treatment of pre-cancerous conditions.

N-(2-Hydroxypropyl)methacrylamide (HPMA)-lectin (wheat germ agglutinin (WGA), peanut agglutinin (PNA)) drug conjugates for treatment of the pre-cancerous conditions ulcerative colitis and Barrett's esophagus are being developed. Cell-surface glycoproteins that are altered in disease and development bind lectins. PNA binds alpha-lactose and the Thomsen-Friedenreich (TF) antigen, a disease- and development-associated glycoprotein. PNA incorporation in conjugates may allow for preferential delivery to diseased over healthy tissues. Conjugates were prepared by attaching lectins to HPMA copolymers via an amide linkage. Frontal affinity chromatography was used to measure dissociation constants (K(d)) of free and conjugated lectins. Animal models of colitis (DSS, TNBS/EtOH) were developed. Human biopsy specimens were obtained. Free and HPMA copolymer-conjugated FITC-labeled lectin and anti-TF antigen antibody binding patterns were examined in normal neonatal, adult and diseased rodent tissues and normal and diseased human tissues. K(d) values of free and conjugated lectins were similar ( approximately 10(-5) M(-1)). Free and conjugated lectins had comparable binding patterns. In health, strong WGA binding was seen in goblet cells; PNA binding was minimal, occurring only in the supranuclear goblet cell region. In disease, WGA binding was not altered, but PNA binding was increased in both human and rodent tissues; entire goblets bound the lectin. Anti-TF antigen antibody binding was minimal, but did overlap with PNA binding patterns both in normal and diseased tissues. Conjugation of lectins to HPMA copolymers does not affect binding affinity. Alterations in glycoprotein structures in development and disease resulted in modified lectin binding patterns. In development and disease, the PNA binding seen was to the TF antigen and other lactose-containing glycoproteins. The results suggest that site-specific delivery of therapeutic agents such as cyclosporin A (CsA) for ulcerative colitis and mesochlorin e(6) for Barrett's esophagus may be achieved. P(HPMA)-lectin-CsA conjugates have been prepared and preliminary in vivo studies are underway.

Algorithms↗
PubMed2002

Use of lectin binding characteristics to identify gastrointestinal parasite eggs in faeces.

Identification of the presence and abundance of species is important when choosing therapies and control strategies for internal parasitism of livestock. Here we examine lectin binding characteristics of eggs isolated from sheep faeces as a means for identifying the parasite genera contributing to infection. The intensity of lectin staining varied with incubation time, incubation volume, concentration of lectin and concentration of eggs. Formalin fixed eggs had greater autofluorescence but exhibited the same lectin staining pattern as fresh eggs. The stage of egg development did not influence staining. Eggs from Haemonchus contortus, H. placei, Trichostrongylus colubriformis, T. vitrinus, Ostertagia circumcincta, Nematodirus spathiger and the cestode Monezia expansa were incubated with a panel of fluoroscein isothiocyanate (FITC)-labelled lectins. Lectin binding exhibited a genus specific pattern. Haemonchus spp. stained strongly positive with peanut agglutinin (PNA), and were positive for concanavalin A (ConA), Ricinus communis agglutinin (RCA) and Maclura pomifera lectin (MPA). Trichostrongylus spp. were PNA-, ConA-, RCA- and strongly MPA+. O. circumcincta were weakly positive for PNA, MPA, ConA and negative for RCA. N. spathiger were weakly positive for the four lectins, and M. expansa were weakly positive for PNA, RCA and MPA and were strongly ConA+. The genus specificity of lectin staining was used to identify the presence of Trichostrongylus and Haemonchus eggs in faeces from sheep with mixed field infections, however correspondence between lectin staining and larval differentiation for identifying a low prevalence of Ostertagia in the field infection was poor. Refinements in methods for rapid egg isolation may improve egg differentiation on the basis of lectin staining, which could be undertaken by flow cytometry or microscopy.

Animals↗
PubMed1999

A unique primary structure, cDNA cloning and function of a galactose-specific lectin from ascidian plasma.

The complete amino acid sequence of a galactose-specific lectin from the plasma of the ascidian Halocynthia roretzi has been determined by sequential Edman degradation analysis of peptide fragments derived by proteolytic fragmentation and chemical cleavage of the reductive S-pyridylethylated lectin. Peptide fragments were separated by reverse-phase HPLC. The N-terminal and C-terminal amino acid sequences were determined by Edman degradation and enzymatic digestion. The H. roretzi plasma lectin is a single-chain protein consisting of 327 amino acids and four disulfide bonds, one of which was found to be cross-linked intramolecularly. A comparison of the amino acid sequence of the H. roretzi plasma lectin with the sequences of other proteins reveals that the H. roretzi lectin has a structure consisting of a twice-repeated sequence, a fibrinogen-related sequence and a C-type lectin-homologous sequence. The above amino acid sequence was verified by cDNA cloning of this lectin. Three cDNA clones that have single ORFs encoding the lectin precursor were isolated from an H. roretzi hepatopancreas cDNA library. The deduced amino acid sequences in the three cDNA clones contain the same sequence of the mature lectin molecule and the same putative signal sequence. In addition, it was demonstrated that this lectin can enhance phagocytosis by H. roretzi hemocytes. Thus, the plasma lectin is constructed into an oligomer structure via intermolecular disulfide bonds and plays a role in the biological defense of H. roretzi as a defense molecule.

Amino Acid Sequence↗
PubMed2000

Lectin binding characteristics of mouse placental cells.

The lectin binding characteristics of mouse placental cells were examined. Wax embedded tissue sections of placentae from d 14 pregnant mice were stained with 26 lectins, with a wide range of sugar specificities. Cell cultures prepared from d 14 mouse placentae and cultured for 24 h were stained with 7 of the lectins to determine if they could be used as markers for the different trophoblast cells in culture. In tissue sections all placental cell populations bound lectin but no lectin bound specifically to any single trophoblast population. All the lectins which bound to layer 1 cytotrophoblast lining the maternal blood spaces of the labyrinthine placenta also bound to the fetal endothelium of the labyrinthine placenta. Binding of lectin appeared strongest on the adluminal membrane of these cell populations suggesting a role for the carbohydrate moieties in nutrient transfer. Few lectins bound to junctional zone trophoblast. Overall, the binding of lectin to cultured cells did not correlate exactly with lectin binding to the cell populations in tissue sections. The value of lectins as markers for placental cells in culture was therefore found to be limited. Our findings indicate that carbohydrate expression by at least some placental cells may vary in culture from that expressed by the cells in vivo with obvious concerns for the validity of functional in vitro studies.

Animals↗
PubMed1995

Identification of the galactose-adherence lectin epitopes of Entamoeba histolytica that stimulate tumor necrosis factor-alpha production by macrophages.

The 170-kDa subunit of the galactose-adherence lectin (Gal-lectin) of Entamoeba histolytica mediates adherence to human colonic mucins and intestinal epithelium as a prerequisite to amebic invasion. The Gal-lectin is an immunodominant molecule and a protective antigen in the gerbil model of amebiasis. Tumor necrosis factor alpha (TNF-alpha) produced by activated macrophages enhances nitric oxide-dependent cytotoxicity in host defense against E. histolytica. The purpose of this study was to identify the Gal-lectin epitopes which stimulate TNF-alpha production by macrophages. Murine bone marrow-derived macrophages (BMMs) exposed to Gal-lectin (100-500 ng/ml) stimulated stable expression of TNF-alpha mRNA (8-fold increase) and TNF-alpha production similar to that of lipopolysaccharide-stimulated cells (100 ng/ml). Polyclonal anti-lectin serum specifically inhibited TNF-alpha mRNA induction in response to the Gal-lectin but not to lipopolysaccharide. Anti-lectin monoclonal antibodies 8C12, H85 and 1G7, which recognize nonoverlapping epitopes of the cysteine-rich region of the 170-kDa heavy subunit, inhibited both amebic adherence to mammalian cells and Gal-lectin-stimulated TNF-alpha mRNA expression by BMMs,but monoclonal antibody 7F4 did neither. As these inhibitory antibodies map to amino acids 596-1082 of the 170-kDa Gal-lectin, our results have identified the functional region that mediates amebic adherence and TNF-alpha mRNA induction in BMMMs; thus, this region of the Gal-lectin is a subunit vaccine candidate.

Animals↗
PubMed2003

Purification, characterization, molecular cloning, and expression of novel members of jacalin-related lectins from rhizomes of the true fern Phlebodium aureum (L) J. Smith (Polypodiaceae).

A lectin was purified from rhizomes of the fern Phlebodium aureum by affinity chromatography on mannose-Sepharose. The lectin, designated P. aureum lectin (PAL), is composed of two identical subunits of approximately 15 kDa associated by noncovalent bonds. From a cDNA library and synthetic oligonucleotide probes based on a partial amino acid sequence, 5'- and 3'-rapid amplification of cDNA ends allowed the generation of two similar full-length cDNAs, termed PALa and PALb, each of which had an open reading frame of 438 bp encoding 146 amino acid residues. The two proteins share 88% sequence identity and showed structural similarity to jacalin-related lectins. PALa contained peptide sequences exactly matching those found in the isolated lectin. PALa and PALb were expressed in Escherichia coli using pET-22b(+) vector and purified by one-step affinity chromatography. Native and recombinant forms of PAL agglutinated rabbit erythrocytes and precipitated with yeast mannan, dextran, and the high mannose-containing glycoprotein invertase. The detailed carbohydrate-binding properties of the native and recombinant lectins were elucidated by agglutination inhibition assay, and native lectin was also studied by isothermal titration calorimetry. Based on the results of these assays, we conclude that this primitive vascular plant, like many higher plants, contains significant quantities of a mannose/glucose-binding protein in its storage tissue, whose binding specificity differs in detail from either legume mannose/glucose-binding lectins or monocot mannose-specific lectins. The identification of a jacalin-related lectin in a true fern reveals for the first time the widespread distribution and molecular evolution of this lectin family in the plant kingdom.

Amino Acid Sequence↗
PubMed1985

Lectin binding affinities of induced pancreatic lesions in the hamster model.

We examined the binding pattern of nine lectins to N-nitrosobis(2-oxopropyl)amine (BOP)-induced pancreatic lesions in Syrian hamsters. These lectins were Arachis hypogaea (PNA), Dolichos biflorus (DBA), Griffonia simplicifolia I (GS-I), Helix aspersa (HAA), Helix pomatia (HPA), Sophora japonica (SJA), Ricinus communis I (RCA-I), Triticum vulgaris (WGA) and Ulex europaeus I (UEA-I). All of the lectins reacted in untreated control hamsters to varying intensities with cytoplasmic components of acinar cells. GS-I, HPA, RCA-I and UEA-I bound to the basolateral surface and PNA, HAA and HPA to the luminal surface of these cells. All but GS-I, RCA-I and UEA-I stained the cytoplasm of islet cells diffusely. In untreated control hamsters, some ductal cells bound PNA, HAA and RCA-I, whereas these cells reacted negatively to the remaining six lectins. Ductular cells did not bind any of the nine lectins. Hyperplastic ductal cells in untreated hamsters were reactive with all nine lectins; however the intensity of the reactivity, cellular localization and extent differed for each lectin. In carcinogen-treated hamsters, the binding pattern of the lectins to acinar and islet cells did not differ significantly from that in untreated hamsters, whereas cells of induced ductal and ductular lesions bound each of the lectins in different patterns and intensities. The reaction of UEA-I to induced lesions was most consistent, specific and strong, thereby indicating the presence of L-fucose in glycoproteins produced by altered cells. Although the binding affinity of the lectins to induced hyperplastic lesions differed in both a quantitative and qualitative fashion, all dysplastic and malignant lesions were reactive to each lectin. This result indicates a heterogeneity in the carbohydrate structure of the glycoproteins produced by pancreatic cells during carcinogenicity.

Animals↗
PubMed1997

Cloning and expression of a Xenopus laevis oocyte lectin and characterization of its mRNA levels during early development.

cDNA clones encoding a soluble, calcium-dependent, melibiose-binding lectin from Xenopus laevis oocytes have been isolated, characterized, and expressed in bacteria. This lectin has been shown by others to be localized in oocyte cortical granules where it ultimately is released and participates in the formation of the fertilization envelope. A lectin with similar specificity has been purified by others from blastula and immunolocalized to specific locations in developing embryos, which suggests it may also function after fertilization in regulating cell adhesion and migration. We have used melibiose affinity chromatography to isolate the oocyte lectin (monomer molecular masses of about 45 and 43 kDa) and shown that after exhaustive treatment with N-glycanase, only one major protein band at 35 kDa was observed, suggesting that a single polypeptide with variable N-linked glycosylation is expressed in the oocyte. After obtaining internal peptide sequences, a PCR-based cloning approach allowed the isolation of full length cDNAs from an ovary lambda gt11 library encoding a protein of 313 amino acids with three potential N-linked oligosaccharide sites. Although this lectin, termed XL35, requires calcium ions for oligosaccharide binding, its sequence does not contain the sequence motif defined for "C-type" lectins. A 6-Histagged from of the lectin was expressed in E. coli and purified on a Ni(2+)-NTA column from bacterial extracts. The recombinant lectin was active using an agglutination assay, and this activity was inhibitable by EDTA and melibiose, properties exhibited by the native lectin. Southern blot analysis revealed a single hybridizing band, arguing against the existence of a multigene family. Northern blot analysis demonstrated that the lectin mRNA is expressed in oocytes and remains at relatively high levels through late gastrulae, continuing until tadpole stages. The persistence of the lectin mRNA, coupled with results from earlier studies, strongly suggests that XL35 is zygotically expressed and functions during morphogenesis.

Amino Acid Sequence↗
PubMed1987

Further characterization and structural studies on human placenta lectin.

The properties of a previously purified beta-galactoside-binding lectin of human placenta were studied in detail. Isoelectric focusing gave multiple bands around pH 4.9, although the lectin preparation was homogenous in SDS-polyacrylamide gel electrophoresis. High-performance gel chromatography suggested that the lectin exists mainly as the monomer and that a small fraction forms a dimer. From all the criteria examined, human placenta lectin resembles one of the chick lectins obtained from embryonic skin or adult intestine (subunit molecular weight: 14,000). The lectin was inactivated by thiol-modifying reagents, p-chloromercuribenzoic acid and N-ethylmaleimide. Reduced and carboxymethylated lectin contained five carboxymethylated cysteines per subunit, and five free thiol groups were titrated by using 5,5'-dithio-bis(2-nitrobenzoic acid). Preliminary sequence analysis showed the presence of a region highly homologous to the corresponding region of the chick lectin (13 identical residues out of 18 from number 70 to 87 of the chick lectin), suggesting a close evolutionary relation between these lectins and the importance of this conserved region in the function of the lectins.

Amino Acid Sequence↗
PubMed1988

Purification and characterization of a lectin-like molecule specific for galactose/N-acetyl-galactosamine from tumoricidal macrophages.

A lectin-like molecule (macrophage lectin) was purified from murine peritoneal exudate macrophages which had been induced with an antitumor streptococcal preparation, OK-432. The purified macrophage lectin from both 3H-labeled and unlabeled macrophages after rechromatography on a beta-D-galactose-Bio-Gel P-100 column gave a broad single band corresponding to 45-60 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The broadness of this band was due to high N-glycosylation of the lectin, because the lectin gave a compact band corresponding to 35 kDa on SDS-PAGE after deglycosylation. The lectin required Ca2+ for binding and showed an optimum pH of around 6. The sugar specificity of the lectin was examined by means of an inhibition assay using simple sugars and neoglycoproteins. The lectin was found to be specific for D-galactose/N-acetyl-D-galactosamine, and not inhibited with D-mannose or N-acetyl-D-glucosamine at all. The lectin was detected on the surface of OK-432-elicited and thioglycolate-elicited macrophages, but it was not detected on resident macrophages. Moreover, the binding of tumor cells to macrophages was inhibited by the addition of the purified lectin to the binding mixture. These results suggest that this lectin is expressed on the surface of activated macrophages, and that it participates in the interaction between tumoricidal macrophages and tumor cells.

Animals↗
PubMed1994

Kinetic measurement of the interaction between an oligosaccharide and lectins by a biosensor based on surface plasmon resonance.

Kinetic measurements of the interaction between an oligosaccharide and various lectins were performed using a biosensor based on surface plasmon resonance (SPR). A glycopeptide, prepared from asialofetuin and having a nearly homogeneous N-linked sugar chain, was immobilized on the surface of a sensor chip via the amino groups of its peptide moiety. The interactions of this bound glycopeptide with six lectins [Sambucus sieboldiana lectin, Maackia amurensis lectin, Aleuria aurantia lectin, Ricinus communis agglutinin-120 (RCA120), Datura stramonium lectin (DSA) and Phaseolus vulgaris leukoagglutinating lectin] were monitored in real-time with the change in the SPR response. Of these lectins, only RCA120 and DSA showed an increase in the SPR response, indicating that these two lectins bound specifically to the immobilized glycopeptide. The other lectins did not show any significant changes in the SPR response. These results are in good agreement with the binding specificity previously demonstrated with affinity chromatography. The association-rate constant (kass) and the dissociation-rate constant (kdiss) for the glycopeptide-RCA120 interaction were 3.4 x 10(5) M-1 s-1 and 2.1 x 10(-3) s-1, respectively. The kass and kdiss determined for DSA were 5.7 x 10(5) M-1 s-1 and 1.3 x 10(-3) s-1, respectively. Furthermore, the relative binding molar ratio to the glycopeptide was three times higher for RCA120 than for DSA, suggesting that this sugar chain possesses three binding sites for RCA120 and one for DSA. These parameters are expected to provide useful information for defining the interaction between oligosaccharides and lectins.

Asialoglycoproteins↗