The lectin-binding protein from the pea (Pisum sativum); properties and interactions

The lectin-binding protein (lectin binder) from the garden pea (Pisum sativum) was studied. It is a glycoprotein composed of four subunits of about 50 000 Da. Its amino-acid composition and molecular mass differ from those of lectin and of storage proteins. The interaction between lectin and lectin binder is demonstrated and quantified by several different methods and is shown to be specifically sugar-dependent. A biological function of lectin binders and lectins is discussed.     

Interactions between lectins and other components of leguminous protein bodies

Previous work from this laboratory had shown that Leguminosa seed extracts contain lectin-bound proteins. In the present paper, the isolation of protein bodies from the seeds of 7 Leguminosa species (Canavalia ensiformis, Lens culinaris, Pisum sativum, Glycine max, Sophora japonica, Wisteria floribunda and Phaseolus vulgaris) is described. Protein bodies were characterized microscopically and by their constituents, storage proteins, lectins and some glycosidases. From the protein bodies, lectin-bound proteins were isolated and were shown to be identical with those from whole seed extracts. This indicates a common localization of lectin-bound proteins and of lectins. Lectin-bound proteins belong to the storage proteins and to the proteins with glycosidase activity. The common localization of these proteins and interactions between them suggest a biological role of seed lectins: during maturation they may act as a packaging aid for storage proteins and enzymes into developing protein bodies. Lectins thus may contribute to an ordered construction and degradation of protein bodies.     

Concanavalin A binds to a mannose-containing ligand in the cell wall of some lichen phycobionts.

Concanavalin A, the lectin from Canavalia ensiformis, develops arginase activity depending on Mn(2+). The cation cannot be substituted by Ca(2+) which, in addition, inhibits Mn(2+)-supported activity. Fluorescein-labeled Concanavalin A is able to bind to the cell wall of algal cells recently isolated from Evernia prunastri and Xanthoria parietina thalli. This binding involves a ligand, probably a glycoprotein containing mannose, which can be isolated by affinity chromatography. Analysis by SDS-PAGE reveals that the ligand is a dimeric protein composed by two monomers of 54 and 48 kDa. This ligand shows to be different from the receptor for natural lichen lectins, previously identified as a polygalactosylated urease.     

Further characterization of monoclonal antibody 6,F-8 reacting with Lathyrus, Lens and Pisum lectins

The murine monoclonal antibody (MoAB) 6,F-8 made against the glucose/mannose-specific Lathyrus odoratus mitogen has previously been shown to react with Lens culinaris and Pisum sativum lectins, but not with the lectin from Vicia faba [(1988) Biol. Chem. Hoppe-Seyler 369, 365-370]. The reactivity against seven other completely sequenced Lathyrus lectins has now been tested after separation of the subunits by SDS-polyacrylamide gel electrophoresis and electroblotting to nitrocellulose filters. Two of these lectins reacted with the antibody. Comparison of the amino acid sequences of the examined lectins and the predicted hydrophilic, flexible and accessible regions of Pisum sativum suggest that valine-147 is involved in antibody binding.     

Binding of Lectins to Culture and Vector Forms of Trypanosoma rangeli Tejera, 1920 (Protozoa, Kinetoplastida) and to Structures of the Vector Gut

Culture forms of Trypanosoma rangeli could be agglutinated with Canavalia ensiformis (Con A) lectin and, less effectively with Pisum sativum agglutinin (PEA), at a concentration of 200 μg/ml. Ricinus communis agglutinin I (RCA I) agglutinated trypanosomes only if they were not previously washed with physiological Ringer's solution. Three other lectins did not react with the same parasite forms. Direct or indirect lectin-gold labeling techniques were applied to LR-White embedded thin sections of T. rangeli culture forms and to forms in the gut, hemolymph, and salivary glands of Rhodnius prolixus. Under these conditions, Con A was the only lectin out of 9 that bound to the surface of trypanosomes from culture and from the bug hemolymph. Con A did not react with any midgut or salivary gland forms. The preservation of the biological activity of the lectin-gold complexes that did not bind to the parasite surface was confirmed by reactions with structures of the invertebrate host.     

Quantification of lectin receptors on B, T, T-gamma and T-mu lymphocytes. I. Concanavalin A, Pisum sativum, Lens culinaris and wheat-germ agglutinin

The immune system is formed of different lymphocyte subpopulations, each one having a defined role to defend the organism. Their plasma membranes present differences in the glycoproteinic or/and glycolipidic composition, as detected with labelled 125I-lectins. B lymphocytes have a greater number of receptors for the Pisum sativum, Lens culinaris and WGA lectins than T lymphocytes. On the other hand, T lymphocytes bind a greater number of Concanavalin A molecules than B lymphocytes. WGA lectin appeared to be more specific for T mu subpopulation, while Con A and Pisum sativum lectins were bound preferentially to T gamma lymphocytes while no significant differences were observed between both subpopulations for Lens culinaris lectin. From the affinity of each lectin to each lymphocyte population it could be deduced that the receptor structure, conformation and arrangement on the membrane was optimal in B lymphocytes for Con A and WGA binding, and T lymphocytes for Lens culinaris and Pisum sativum binding.     

Binding of lectins to culture and vector forms of Trypanosoma rangeli Tejera, 1920 (Protozoa, Kinetoplastida) and to structures of the vector gut

Culture forms of Trypanosoma rangeli could be agglutinated with Canavalia ensiformis (Con A) lectin and, less effectively with Pisum sativum agglutinin (PEA), at a concentration of 200 micrograms/ml. Ricinus communis agglutinin I (RCA I) agglutinated trypanosomes only if they were not previously washed with physiological Ringer's solution. Three other lectins did not react with the same parasite forms. Direct or indirect lectin-gold labeling techniques were applied to LR-White embedded thin sections of T. rangeli culture forms and to forms in the gut, hemolymph, and salivary glands of Rhodnius prolixus. Under these conditions, Con A was the only lectin out of 9 that bound to the surface of trypanosomes from culture and from the bug hemolymph. Con A did not react with any midgut or salivary gland forms. The preservation of the biological activity of the lectin-gold complexes that did not bind to the parasite surface was confirmed by reactions with structures of the invertebrate host.     

Comparison of the amino acid sequences of the lectins from seeds of Dioclea lehmanni and Canavalia maritima.

The amino acid sequences of the major lectins from the seeds of Dioclea lehmanni and Canavalia maritima were determined by DABITC/PITC microsequence analysis of peptides derived from the proteins by enzymatic digestions with trypsin, chymotrypsin and the protease from S. aureus V8. These sequences were found to be very similar to those of the lectins from Dioclea grandiflora and Canavalia ensiformis (Con A). The D. lehmanni lectin was unusual amongst legume lectins in that it contained a single Cys.     

Immunochemical analysis of lectin acceptors in the structure of the fertility alpha 2-microglobulin

Fertility alpha 2-microglobulin reacts with 3 out of 8 lectins, which possess affinity to monosaccharides (glucose and mannose) and acetylamino sugar. The affinity is most marked to concanavalin A and is considerably weaker to Pisum sativum and Vicia faba lectins, whereas the protein gives no reaction with other lectins.     

The binding of lectins to components of plasma membranes from porcine submaxillary lymph node lymphocytes

By sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis the plasma membranes from porcine lymphocytes contain at least 30--35 glycopolypeptides and one or more glycolipids to which one or more of 12 purified lectins bind. The specificities of binding generally followed the same pattern as those of the reaction of the lectin with intact pig lymphocytes. Some lectins (e.g., the isolectin pair, Agaricus bisporus lectins A and B and a group consisting of the Lens culinaris A and B isolectins and the closely related Pisum sativum lectins) bind to almost identical populations of plasma membrane components and compete with each other for all their binding sites. Others (e.g., Concanavalin A and the Lens culinaris-Pisum sativum group and a group consisting of phytohemagglutinin-L, Ricinus communis lectin-60 and Ricinus communis lectin-120 bind in a cross reactive manner to some common binding moieties but, in addition, to certain nonshared ones. Still others (e.g., soybean agglutinin, peanut agglutinin and wheat germ agglutinin) do not share any common binding moieties with the other lectins. The amount of lectin binding and the number of membrane components to which a lectin binds is directly related to the Ka of binding of the lectin to the intact lymphocyte. Those with high Ka (Cocanavalin A Lens culinaris lectins, Pisum sativum lectins, phytohemagglutinin-L), bind to 20-30 different components giving very complex binding patterns while those with lower Ka (Agaricus bisporus lectins, wheat germ agglutinin, peanut agglutinin, and soybean agglutinin) bind to 8--13 components with easily distinguishable patterns. Soybean agglutinin binds almost exclusively to a glycolipid fraction while for the others one or more glycopolypeptides served as the major lectin-binding molecule. The Ricinus lectins, two lymphocyte toxins, bind to essentially every plasma membrane component to which the mitogen phytohemagglutinin-L binds, in fact competing for most of those plasma membrane moieties which bind phytohemagglutinin-L.     

Studies of storage proteins of higher plants: I. Concanavalin a from three species of the genus canavalia

Concanavalin A, the lectin of the Jack bean, Canavalia ensiformis, was extracted and compared with homologous proteins from Canavalia gladiata and Canavalia maritima. All proteins were bound to Sephadex G-100 and eluted from the gel with buffered glucose solution. Quantitative recoveries indicated that large quantities (23 to 28% of dry seed protein) of these lectins are synthesized by all three species. Antibody preparations made against C. ensiformis lectin failed to discriminate among the three proteins; the pattern of the precipitin bands indicated identical antigenic determinants in the Ouchterlony double-diffusion assay. Native and sodium dodecyl sulfate polyacryl-amide gel electrophoresis also failed to distinguish differences in the proteins. The storage protein active in carbohydrate binding is composed, in each case, of identical subunits. However, the amino acid composition of the subunit chains from the three sources is not identical. In particular, the lectins from C. ensiformis and C. gladiata contain two methionine residues per protein subunit, while only one methionine residue is found in the C. martima lectin. Cyanogen bromide cleavage of the purified subunit from C. maritima yieded two fragments with molecular weights estimated at 20,400 and 4,600, respectively. Amino acid analysis of the separated fragments indicated that the methionine residue at position 130 in C. ensiformis is absent in the lectin from C. maritima.     

Interactions between Rhizobia and Lectins of Lentil, Pea, Broad Bean, and Jackbean

A quantitative method was developed to measure the binding of fluorescent-labeled lentil (Lens esculenta Moench), pea (Pisum sativum L.), broad bean (Vicia faba L.), and jackbean (Canavalia ensiformis L., DC.) lectins to various Rhizobium strains. Lentil lectin bound to three of the five Rhizobium leguminosarum strains tested. The number of lentil lectin molecules bound per R. leguminosarum 128C53 cell was 2.1 × 10⁴. Lentil lectin also bound to R. japonicum 61A133. Pea and broad bean lectins bound to only two of the five strains of R. leguminosarum, whereas concanavalin A (jackbean lectin) bound to all strains of R. leguminosarum, R. phaseoli, R. japonicum, and R. sp. tested. Since these four lectins have similar sugarbinding properties but different physical properties, the variation in bindings of these lectins to various Rhizobium strains indicates that binding of lectin to Rhizobium is determined not only by the sugar specificity of the lectin but also by its physical characteristics.     

Reactivity with legume lectins of three monoclonal antibodies made against the Lathyrus odoratus lectin

Three murine IgG1 monoclonal antibodies (MoAb) were produced against the glucose/mannose-specific two-chain mitogen from seeds of Lathyrus odoratus (sweet pea) belonging to the Vicieae tribe of the Leguminosae family. Their antigenic specificities were tested against subunits of two-chain and one-chain legume lectins separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose filters. Different binding to native and detergent-treated lectins in dot-blots indicated that two of the antibodies bound to conformational epitopes which by immunoblotting were shown to be expressed on the heavy subunits from all the five tested two-chain lectins and on the six one-chain lectins, including PHA (Phaseolus vulgaris) and Con A (Canavalia ensiformis). However, ELISA cross-inhibition studies showed that these two antibodies bound to different epitopes on the lectin molecules. The third MoAb reacted with a continuous epitope not revealed by classical taxonomy since only three of five heavy subunits of the two-chain lectins stained with the antibody. Comparison of the known amino-acid sequences of the chains indicates eight positions as possible binding sites for this antibody.     

Lectin histochemistry of feline sphingomyelinosis.

The brain from a Siamese cat with sphingomyelinosis was examined with lectin histochemistry. Swollen neurons were stained with Canavalia ensiformis agglutinin (Con A). Some of them were also stained with Ricinus communis agglutinin-I (RCA-I) and Ulex europaeus agglutinin-I (UEA-I). A small number of axonal spheroids and glia cells were positive for Con A, RCA-I, UEA-I and wheat germ agglutinin. Control tissues were weakly stained with Con A, but not with any of the other lectins. These results indicate that affected neurons contain mannose and glucose residues in addition to sphingomyelin. This study points to the possibility that the characteristics of lectin histochemical study might be helpful for the diagnosis of sphingomyelinosis.     

Lectin-enzyme assay as a method of estimation of immunoglobulins' glycosylation

The mechanism of interaction of lectins with IgG molecules by the method of the lectin-enzyme assay has been described that allows to register a degree of human serum IgG molecules' glycosylation (mannosylation in case of lectin of Pisum sativum) in norm and at pathology. To detect an authentic difference in a glycosylation degree between control and pathological IgG, the wells of an ELISA plate were coated with an antibody in concentration of 1 microg/ml. Introducing alpha-D-mannose between the stages of incubation of immunoglobulin and lectin showed, that alpha-D-mannose inhibits the affinity of lectins for IgG. The preliminary incubation of lectin with IgG molecules stabilizes the activity of horseradish peroxidase, which labeled the lectins. Lectin-enzyme assay, in which Fab and Fc fragments of IgG were used, showed that lectin of Pisum sativum possesses a higher affinity for Fab regions. These findings and the glycosylation analysis of paraproteins and Bence-Jones proteins of multiple myeloma patients help to understand the details of interaction of immunoglobulins and lectins.     

Seed lectin from pisum arvense: isolation, biochemical characterization and amino acid sequence

A glucose/mannose lectin was purified by affinity chromatography from Pisum arvense seeds (PAL) and the 50 kDa molecular mass in solution determined by size exclusion chromatography. SDS-PAGE and electrospray ionization mass spectrometry showed two distinct polypeptide chains: alpha (Mr. 5591 Da) and beta (19986 Da). The lectin was extensively characterized in terms of its biochemical and biological aspects. The amino acid sequence was established by Edman degradation of overlapping peptides. PAL in solution behaves as a dimer and has its monomeric structure formed by two distinct polypeptide chains named alpha (Mr. 5591 Da) and beta (19986 Da) by Electrospray ionization (ESI) mass spectrometry. PAL possesses identical amino acid sequences to that of pea seed lectin but undoubtedly does not exhibit sequence heterogeneity. It is discussed that P. arvense should be considered as a synonym of P. sativum. Furthermore, like pea lectin, PAL discriminates biantennary fucosylated glycan, determined by surface plasmon resonance.     

Purification and characterization of a mitogenic lectin and a lectin-binding protein from Vicia sativa

From the seeds of Vicia sativa, a novel mitogenic lectin was isolated. Purification was carried out by affinity chromatography on Sephadex G-100. The tetrameric lectin is a glycoprotein with a molecular weight of Mr 40 000; it consists of two large beta-subunits (Mr 14 000) and two small alpha-subunits (Mr 6000). The N-terminal sequence of both subunits and their amino acid compositions were determined. The lectin agglutinates human erythrocytes, preferring group B, and erythrocytes from rabbits and horses; no agglutination takes place with sheep erythrocytes. Agglutination is inhibited by mono-, di- and tri-saccharides with the configuration of glucose at the free 4-hydroxyl group. The lectin stimulates mitosis in lymphocytes of mice. From the seeds of the same plant, a protein was isolated which binds to the lectin described above. The lectin binder consists of subunits with a molecular weight of 53 500.     

Comparative analysis of membrane glycoproteins of normal and chronic lymphatic leukemia B lymphocytes by lectins

Eight plant lectins were used to investigate membrane alterations in lymphocytes from patients with chronic lymphocytic leukemia (CLL). By rosetting with lectins attached to latex particles, the cell percentages with the abundance of each lectin receptor were compared in B normal and leukemic lymphocytes. Comparing these data with the number of lectin molecules bound to each cell and the affinity, which are values calculated with 125I-labeled lectins, it was possible to deduce differences in the composition of glycoproteins in B normal and B-CLL lymphocytes membrane. Compared to B normal, B-CLL lymphocytes had fewer receptors for WGA and more for Lens culinaris, SBA and Tetragonolobus purpureus lectins. Receptors for Concanavalin A, Pisum sativum, PHA and Tetragonolobus purpureus showed a higher affinity with B normal lymphocytes, while the other lectins assayed showed more affinity with B-CLL lymphocytes. So, it is possible to establish a comparative analysis about the plasma membrane glycoproteins in the B normal and CLL lymphocytes by lectin binding studies.     

Carbohydrate binding properties of banana (Musa acuminata) lectin I. Novel recognition of internal alpha1,3-linked glucosyl residues.

Examination of lectins of banana (Musa acuminata) and the closely related plantain (Musa spp.) by the techniques of quantitative precipitation, hapten inhibition of precipitation, and isothermal titration calorimetry showed that they are mannose/glucose binding proteins with a preference for the alpha-anomeric form of these sugars. Both generate precipitin curves with branched chain alpha-mannans (yeast mannans) and alpha-glucans (glycogens, dextrans, and starches), but not with linear alpha-glucans containing only alpha1,4- and alpha1,6-glucosidic bonds (isolichenan and pullulan). The novel observation was made that banana and plantain lectins recognize internal alpha1,3-linked glucosyl residues, which occur in the linear polysaccharides elsinan and nigeran. Concanavalin A and lectins from pea and lentil, also mannose/glucose binding lectins, did not precipitate with any of these linear alpha-glucans. This is, the authors believe, the first report of the recognition of internal alpha1,3-glucosidic bonds by a plant lectin. It is possible that these lectins are present in the pulp of their respective fruit, complexed with starch.     

Isolation of lectin and albumin from Pisum sativum var. macrocarpon ser. cv. sugar snap

A mannose- and glucose-binding lectin bearing considerable sequence similarity to other legume lectins was isolated using a simple procedure, from legumes of the sugar snap Pisum sativum var. macrocarpon. The lectin was unadsorbed on Affi-gel blue gel and Q-Sepharose in 10 mM Tris-HCl buffer (pH 7.2) and adsorbed on SP-Toyopearl in 50 mM NaOAc buffer (pH 5). An albumin could also be purified at the same time. It was unadsorbed on Affi-gel Blue gel, adsorbed on Q-Sepharose and unadsorbed on SP-Toyopearl under the aforementioned chromatographic conditions. The lectin was almost identical in N-terminal sequences of its alpha- and beta-subunit to lectin from P. sativum L. var. Feltham First except for the 19th N-terminal residue of the beta-subunit. The lectin was devoid of antifungal activity. Out of the 15 N-terminal amino acids examined in pea albumin, three were different between the two varieties of P. sativum.