Binding of activated macrophages to tumor cells through a macrophage lectin and its role in macrophage tumoricidal activity

A 45-60 kDa Gal/GalNAc-specific macrophage lectin was found to participate in the interaction between tumor cells and tumoricidal macrophages activated by an antitumor streptococcal preparation, OK-432, and in the tumoricidal activity of the activated macrophages. The binding between OK-432-elicited activated macrophages and murine mastocytoma P-815 cells was inhibited on preincubation of the macrophages with a neoglycoprotein (Gal-BSA) or a complex-type glycopeptide (unit B) which was a specific inhibitor of the macrophage lectin. This binding of the macrophages to P-815 cells was also inhibited on the addition of anti-macrophage lectin antiserum. Contrary to the case of OK-432-elicited macrophages, the binding of thioglycolate-elicited (responsive) macrophages to P-815 cells was inhibited only a little by Gal-BSA and unit B, and not inhibited by the antiserum. Furthermore, the tumoricidal activity of the activated macrophages was inhibited by the addition of the anti-macrophage lectin antiserum. These results suggest that the binding of activated macrophages to tumor cells through the Gal/GalNAc-specific macrophage lectin is an important part of the tumor cell killing mechanism.     

Molecular cloning of the lectin and a lectin-related protein from common Solomon's seal (Polygonatum multiflorum)

The most prominent protein ofPolygonatum multiflorum (common Solomon's seal) rhizomes has been identified as a mannose-binding lectin. Analysis of the purified lectin demonstrated that it is a tetramer of four identical subunits of 14 kDa. Molecular cloning further revealed that the lectin from this typical Liliaceae species belongs to the superfamily of monocot mannose-binding proteins. Screening of cDNA libraries constructed with RNA isolated from buds, leaves and flowers ofP. multiflorum also yielded cDNA clones encoding a protein, which contains two tandemly arranged domains with an obvious sequence homology to the mannose-binding lectins. Molecular modelling of thePolygonatum lectin and lectin-related protein indicated that the three-dimensional structure of both proteins strongly resembles that of the snowdrop lectin. In addition, this approach suggested that the presumed carbohydrate-binding sites of the lectin can accommodate a mannose residue whereas most of the carbohydratebinding sites of the lectin-related protein cannot.Abbreviations GNA Galanthus nivalis agglutinin - HCA hydrophobic cluster analysis - LECPMA cDNA clone encoding PMA - PM30 30 kDa protein isolated fromPolygonatum multiforum - PMA Polygonatum multiflorum agglutinin - PMLRP Polygonatum multiflorum lectin-related protein     

Formation of homogeneous carbohydrate-lectin cross-linked precipitates from mixtures of D-galactose/N-acetyl-D-galactosamine-specific lectins and multiantennary galactosyl carbohydrates.

Quantitative precipitation studies have shown that the Man/Glc-specific lectin concanavalin A (ConA) forms homogeneous (homopolymeric) cross-linked precipitates with individual asparagine-linked oligomannose and bisected hybrid-type glycopeptides in the presence of binary mixtures of the carbohydrates [Bhattacharyya, L., Khan, M. I. & Brewer, C. F. (1988) Biochemistry 27, 8762-8767]. The results indicate that the ConA-glycopeptide precipitates are highly organized cross-linked lattices that are unique for each carbohydrate. Using similar techniques, the present study shows that the Gal-specific lectins from Erythrina indica and Ricinus communis (agglutinin I) form homogeneous cross-linked complexes with individual carbohydrates in binary mixtures of triantennary and tetraantennary complex-type oligosaccharides with terminal Gal residues. Conversely, binary mixtures of Gal/GalNAc-specific lectins from E. indica, Erythrina cristagalli, Erythrina flabelliformis, R. communis, soybean (Glycine max), and Wistaria floribunda (tetramer) in the presence of a naturally occurring or synthetic branched-chain oligosaccharide with terminal GalNAc or Gal residues provide evidence for the formation of separate cross-linked lattices between each lectin and the carbohydrate. The present results therefore demonstrate the formation of homogeneous lectin-carbohydrate cross-linked lattices in (a) a mixture of branched-chain complex-type oligosaccharides in the presence of a specific Gal/GalNAc-binding lectin, and (b) a mixture of lectins with similar physicochemical and carbohydrate binding properties in the presence of an oligosaccharide. These findings show that lectin-carbohydrate cross-linking interactions provide a high degree of specificity which may be relevant to their biological functions as receptors.     

Cryptosporidium p30, a galactose/N-acetylgalactosamine-specific lectin, mediates infection in vitro

Cryptosporidium sp. cause human and animal diarrheal disease worldwide. The molecular mechanisms underlying Cryptosporidium attachment to, and invasion of, host cells are poorly understood. Previously, we described a surface-associated Gal/GalNAc-specific lectin activity in sporozoites of Cryptosporidium parvum. Here we describe p30, a 30-kDa Gal/GalNAc-specific lectin isolated from C. parvum and Cryptosporidium hominis sporozoites by Gal-affinity chromatography. p30 is encoded by a single copy gene containing a 906-bp open reading frame, the deduced amino acid sequence of which predicts a 302-amino acid, 31.8-kDa protein with a 22-amino acid N-terminal signal sequence. The p30 gene is expressed at 24-72 h after infection of intestinal epithelial cells. Antisera to recombinant p30 expressed in Escherichia coli react with an approximately 30-kDa protein in C. parvum and C. hominis. p30 is localized to the apical region of sporozoites and is predominantly intracellular in both sporozoites and intracellular stages of the parasite. p30 associates with gp900 and gp40, Gal/GalNAc-containing mucin-like glycoproteins that are also implicated in mediating infection. Native and recombinant p30 bind to Caco-2A cells in a dose-dependent, saturable, and Gal-inhibitable manner. Recombinant p30 inhibits C. parvum attachment to and infection of Caco-2A cells, whereas antisera to the recombinant protein also inhibit infection. Taken together, these findings suggest that p30 mediates C. parvum infection in vitro and raise the possibility that this protein may serve as a target for intervention.     

Current status of ribosome inactivating proteins

Ribosome inactivating proteins (RIPs) are a group of naturally occurring plant proteins with a RNA-N-glycosidases activity which depurinate rRNA at a specific universally conserved position (i.e. cleavage of N-glycosidic bond of a specific adenine of 28S rRNA). These proteins are found in different parts of plants, in concentrations ranging from a few micrograms to several hundred mg per 100 g of plant tissues. RIPs exist in two forms, type 1 having a single polypeptide chain with a molecular mass of approximately 30 kDa possessing N-glycosidase activity; and type 2 with two or four polypeptide chains having a molecular mass of approximately 60 kDa and approximately 120 kDa respectively showing lectin activity along with N-glycosidase moiety. Such biomolecules causing cytotoxicity are being exploited for designing immunotoxins/hormonotoxins using heterobifunctional conjugates. These carrier conjugates with the RIPs can influence cellular trafficking and inhibition of protein synthesis. We are witnessing a novel protein from plants that can be utilised for various therapeutical treatments ranging from cancers, AIDS and other viral diseases of present times.     

Elderberry bark lectins evolved to recognize Neu5Ac alpha2,6Gal/GalNAc sequence from a Gal/GalNAc binding lectin through the substitution of amino-acid residues critical for the binding to sialic acid

Bark lectins from the elderberry plants belonging to the genus Sambucus specifically bind to Neu5Acalpha2,6Gal/GalNAc sequence and have long been used for the analysis of sialoglycoconjugates that play important roles in many biological phenomena. However, molecular basis of such a unique carbohydrate binding specificity has not been understood. To answer these questions, we tried to identify the amino-acid residues in the Japanese elderberry bark lectin, Sambucus sieboldiana agglutinin that enabled the lectin to recognize sialic acid by using in silico docking simulation and site-directed mutagenesis. These studies showed that three amino-acid residues, S(197), A(233) and Q(234), in the C-terminal subdomain of SSA-B chain are critical for the binding to the sialic acid in Neu5Acalpha2,6Gal/GalNAc sequence. Replacement of one of these residues to the one in the corresponding position of ricin B-chain completely abolished the binding to a sialoglycoprotein, fetuin. Conserved presence of these amino acid residues in the corresponding sequences of two other elderberry lectins with similar binding specificity further supported the conclusion. These findings indicated that the replacement of the corresponding amino-acid residues in a putative Gal/GalNAc-specific ancestral lectin to these amino-acid residues generated the unique Neu5Acalpha2,6Gal/GalNAc-specific elderberry lectins in the course of molecular evolution.     

LOCALIZATION AND IDENTIFICATION OF GALACTOSE/N-ACETYLGALACTOSAMINE AND SIALIC ACID-CONTAINING PROTEINS IN CHINESE HAMSTER METAPHASE CHROMOSOMES

Four lectins were used to recognize galactose/N-acetyl-galactosamine (Gal/GalNAc) and sialic acid residues in proteins of Chinese hamster metaphase chromosomes. In situ binding pattern of a fluorescein isothiocyanate-labelled (Gal/GalNAc)-specific lectin Sophora japonica agglutinin (SJA) showed that chromosomal SJA-binding proteins are primarily localized to the helically coiled substructure of chromatids. Numerous SJA-binding proteins were identified in Western blots of chromosomal proteins, their molecular weights ranging from 26 to 200kDa. Another Gal/GalNAc-specific lectin, peanut agglutinin (PNA), with a slightly different sugar binding specificity, did not bind to Chinese hamster metaphase chromosomes, and in Western blots only two chromosomal protein bands were faintly stained. The in situ labelling patterns of two sialic acid-specific lectins, Maackia amurensis (MAA) and Sambucus nigra (SNA) agglutinins, both showed that the helically coiled substructure of chromatids is also enriched in sialylated proteins. In Western blot analysis 11 MAA-binding protein bands with molecular weights ranging from 54 to 215kDa were identified, while SNA only bound to one protein band of 67kDa. MAA and SNA are specific for α (2|ad3)- and α (2|ad6)-linked sialic acid residues, respectively. Thus, it is likely that α (2|ad3)-linked sialic acid residues are more common in chromosomal proteins than α(2|ad6)-linked sialic acid residues. These data suggest that Gal/GalNAc and sialic acid-containing glycoproteins exist in metaphase chromosomes and that these proteins may have a role in the formation of higher order metaphase chromosome structures.     

Nematotoxicity of Marasmius oreades agglutinin (MOA) depends on glycolipid binding and cysteine protease activity

Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a Galα1,3Gal/GalNAc-specific lectin from the fairy ring mushroom that consists of an N-terminal ricin B-type lectin domain and a C-terminal dimerization domain. The latter domain shows structural similarity to catalytically active proteins, suggesting that, in addition to its carbohydrate-binding activity, MOA has an enzymatic function. Here, we demonstrate toxicity of MOA toward the model nematode Caenorhabditis elegans. This toxicity depends on binding of MOA to glycosphingolipids of the worm via its lectin domain. We show further that MOA has cysteine protease activity and demonstrate a critical role of this catalytic function in MOA-mediated nematotoxicity. The proteolytic activity of MOA was dependent on high Ca(2+) concentrations and favored by slightly alkaline pH, suggesting that these conditions trigger activation of the toxin at the target location. Our results suggest that MOA is a fungal toxin with intriguing similarities to bacterial binary toxins and has a protective function against fungivorous soil nematodes.     

Sequence comparison and phylogenetic analysis by the Maximum Likelihood method of ribosome-inactivating proteins from angiosperms

Ribosome-inactivating proteins (RIPs) from angiosperms are rRNA N-glycosidases that have been proposed as defence proteins against virus and fungi. They have been classified as type 1 RIPs, consisting of single-chain proteins, and type 2 RIPs, consisting of an A chain with RIP properties covalently linked to a B chain with lectin properties. In this work we have carried out a broad search of RIP sequence data banks from angiosperms in order to study their main structural characteristics and phylogenetic evolution. The comparison of the sequences revealed the presence, outside of the active site, of a novel structure that might be involved in the internal protein dynamics linked to enzyme catalysis. Also the B-chains presented another conserved structure that might function either supporting the beta-trefoil structure or in the communication between both sugar-binding sites. A systematic phylogenetic analysis of RIP sequences revealed that the most primitive type 1 RIPs were similar to that of the actual monocots (Poaceae and Asparagaceae). The primitive RIPs evolved to the dicot type 1 related RIPs (like those from Caryophyllales, Lamiales and Euphorbiales). The gene of a type 1 RIP related with the actual Euphorbiaceae type 1 RIPs fused with a double beta trefoil lectin gene similar to the actual Cucurbitaceae lectins to generate the type 2 RIPs and finally this gene underwent deletions rendering either type 1 RIPs (like those from Cucurbitaceae, Rosaceae and Iridaceae) or lectins without A chain (like those from Adoxaceae).     

Changes in the level of lectins in the mantle of the mussel Mytilus trossulus in response to anthropogenic contaminants

The lectin activity in an extract from the mantle of the mussel Mytilus trossulus was tested for the first time. Using the method of the hemagglutination inhibition assay, it was shown that lectins were Gal/GalNAc-specific and best agglutinated with rabbit erythrocytes. The influence of foreign compounds on the lectin level in the M. trossulus mantle was examined. Upon cadmium acetate exposure, the level of lectin activity exhibited phasic alterations and depended on the contaminant concentration or the time of exposure. During exposure of mussels to a synthetic detergent or diesel fuel, changes in lectin contents were dependent on the time of exposure.     

Domain-specific distribution of carbohydrates in human fibronectins and the transformation-dependent translocation of branched type 2 chain defined by monoclonal antibody C6

Fibronectins purified from human plasma (termed pFN), spent culture media of human fibroblasts WI38 (termed cFN), and SV40 virus-transformed WI38/VA13 cells (termed tFN) and their cleavage fragments were compared with respect to their binding activities to lectins and anti-carbohydrate antibodies reacting with chemically well-defined structures. The following findings were of particular interest. About 25-35% of cFN and tFN carried a binary sialosyl type 2 chain (NeuAc alpha 2----3/or 6Gal beta 1----4GlcNAc) linked beta 1----3/beta 1----6 to the galactose residue and defined by monoclonal antibody C6. This structure was not detected in pFN. In cFN, the C6-defined structure was localized within the gelatin-binding domain, whereas in tFN the same structure was absent from this domain but was located at the NH2-terminal region of the central domain. Other carbohydrate determinants, defined by Ricinus communis lectin and concanavalin A before and after sialidase treatment, showed essentially identical domain distribution patterns among cFN, tFN, and pFN and were all located at the gelatin-binding domain (44 kDa), its precursor (60 kDa), and the Cell/Hep-2 domain (155/145 kDa). Although both cFN and tFN were reactive with lentil lectin, pFN was not. Fibronectin from transformed cells (tFN) showed much greater reactivity than cFN and pFN with wheat germ lectin before sialidase treatment and showed enhanced reactivity with R. communis lectin and peanut lectin after sialidase treatment, indicating that tFN is more highly sialylated than cFN and pFN. All fibronectins examined were strongly reactive with monoclonal antibody AH8-28, which binds to Gal beta 1----3GalNAc residues, and this reactivity was localized to both the NH2-terminal half and COOH-terminal half of the S-cyanylation-cleaved fibronectin molecule.     

Isolation and characterization of a second lectin (SNA-II) present in elderberry (Sambucus nigra L.) bark

A second lectin (SNA-II) has been isolated from elderberry (Sambucus nigra L.) bark by affinity chromatography on immobilized asialo-glycophorin. This lectin is a blood group nonspecific glycoprotein containing 7.8% carbohydrate and which is rich in asparagine/aspartic acid, glutamine/glutamic acid, glycine, valine, and leucine. Gel filtration on Superose 12 gave a single symmetrical peak corresponding to Mr, 51,000; SDS-acrylamide electrophoresis gave a single polypeptide, Mr, 30,000. Hence SNA-II appears to be a homodimer. The lectin is a Gal/GalNAc-specific lectin which is precipitated by glycoproteins containing GalNAc-terminated oligosaccharide chains (e.g., asialo-ovine submaxillary and hog gastric mucins), and by glycoproteins and polysaccharides having multiple terminal nonreducing D-galactosyl groups as occur in asialoglycophorin, asialo-laminin and Type 14 pneumococcal polysaccharide. The carbohydrate binding specificity of SNA-II was studied by sugar hapten inhibition of the asialo-glycophorin precipitation reaction. The lectin's binding site appears to be most complementary to Gal-NAc linked alpha to the C-2, C-3, or C-6 hydroxyl group of galactose. These disaccharide units are approximately 100 times more potent than melibiose, 60 times more potent than N-acetyllactosamine, and 30 times more potent than lactose. Interestingly, the blood group A-active trisaccharide containing an L-fucosyl group linked alpha 1-2 to galactose was 10-fold poorer as an inhibitor than the parent oligosaccharide (GalNAc alpha 1-3Gal), suggesting steric hindrance to binding by the alpha-L-fucosyl group; this explains the failure of the lectin to exhibit blood group A specificity.     

Purification and properties of lung lectin. Rat lung and human lung beta-galactoside-binding proteins.

Lung is one of the organs of the rat with a particular abundance of haemagglutinating activity that is inhibited by beta-galactosides. This lectin activity can be attributed to a single protein that has been purified from rat lung; a similar protein has been purified from human lung. The molecular weights and subunit structures were estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the human lung lectin appeared to be composed to two identical subunits, mol.wt. 14500, whereas rat lung lectin was observed as both a dimer and a tetramer of one subunit type, mol.wt. 13500. Both lectins bind to disaccharides or oligosaccharides with terminal beta-linked galactose residues. The carbohydrate moiety may be free [lactose or D-galactopyranosyl-beta-(1 leads to 4)-thiogalactopyranoside], protein-bound (asialofetuin) or lipid-bound (cerebrosides). The molecular properties of the beta-galactoside-binding proteins of rat lung and human lung are closely similar to those of embryonic chick muscle lectin [Nowak, Kobiler, Roel & Barondes (1977) Proc. Natl. Acad. Sci. U.S.A. 73, 1383--1387] and calf heart lectin [De Waard, Hickman & Kornfeld (1976) J. Biol. Chem. 251, 7581--7587].     

Purification and carbohydrate-binding specificities of a blood type B binding lectin from hemolymph of a crab (Charybdis japonica)

A blood type B binding lectin (CJA-B) was isolated from the hemolymph of the crab Charybdis japonica by affinity chromatography on Sephadex G-200. The molecular mass of the native lectin was determined to be 300 kDa by gradient polyacrylamide gel electrophoresis under nondenaturing conditions. On SDS-polyacrylamide gel electrophoresis, the lectin gave a single protein band with molecular masses of 19 and 38 kDa in the presence and absence of 2-mercaptoethanol, respectively. CJA-B contained mannose, N-acetylglucosamine, xylose, and fucose in the molar ratio of 3.0:1.6:1.2:1.1. The protein required calcium ions for hemagglutinating activity and showed specificities for alpha-galactosyl and alpha-glucosyl residues. Studies on hemagglutination inhibition by Synsorbs, which are synthetic oligosaccharides coupled chemically to crystalline silica, showed that the lectin mainly interacts with Gal alpha 1-3Gal.     

Antisense inhibition of expression of the light subunit (35 kDa) of the Gal/GalNac lectin complex inhibits Entamoeba histolytica virulence

One of the under-represented genes identified by cDNA representational difference analysis (RDA) between avirulent Entamoeba histolytica strain Rahman and virulent strain HM-1:IMSS was the amoebic light (35 kDa) subunit of the Gal/GalNac lectin complex. This lectin complex, which mediates the adhesion of the parasite to the target cell, also contains a heavy (170 kDa) subunit, which has the carbohydrate-binding domain. Stable transfectants of the virulent strain in which the expression of the 35 kDa subunit was inhibited by antisense RNA were not significantly affected in their adhesion activity to mammalian or bacterial cells but were strongly inhibited in their cytopathic activity, cytotoxic activity and in their ability to induce the formation of liver lesions in hamsters. These findings suggest that the 35 kDa subunit may have a specific function in the pathogenic pathway and provides a new insight into the role of this component of the Gal/GalNac lectin complex in amoebic virulence.     

Effect of alpha-sarcin and ribosome-inactivating proteins on the interaction of elongation factors with ribosomes.

alpha-Sarcin from Aspergillus giganteus and the ribosome-inactivating proteins (RIPs) from higher plants inactivate the 60 S ribosomal subunit. The former is an RNAase, whereas RIPs are N-glycosidases. The site of cleavage of RNA and that of N-glycosidic depurinization are at one nucleotide distance in 28 S rRNA [Endo & Tsurugi (1987) J. Biol. Chem. 262, 8128-8130]. The effect of alpha-sarcin and that of RIPs on the interaction of elongation factors with Artemia salina (brine shrimp) ribosomes have been investigated. alpha-Sarcin inhibits both the EF1 (elongation factor 1)-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of EF2 (elongation factor 2) to ribosomes, whereas two of the RIPs tested, ricin from Ricinus communis (castor bean) and volkensin from Adenia volkensii (kilyambiti), inhibit only the latter reaction. EF2 protects ribosomes from inactivation by both alpha-sarcin and ricin. The EF1-binding site is affected only by alpha-sarcin. The sensitivity of this site to alpha-sarcin is increased by pretreatment of ribosomes with ricin. A. salina ribosomes were highly resistant to the third RIP tested, namely gelonin from Gelonium multiflorum. All four proteins tested have, however, a comparable activity on the rabbit reticulocyte-lysate system.     

The differences in structural specificity for recognition and binding between asialoglycoprotein receptors of liver and macrophages

The Gal/GalNAc-specific lectin on the surface of rat peritoneal macrophages (macrophage asialoglycoprotein binding protein, M-ASGP-BP), which consists of a single polypeptide chain of 42 kDa, can form a homooligomeric receptor exhibiting high affinity for asialoorosomucoid (ASOR) [Ozaki K., Ii M., Itoh N., Kawasaki T. (1992)J Biol Chem 267: 9229–35]. In this study, the binding affinity of M-ASGP-BP was studied by using a series of synthetic or natural glycosides as inhibitors of¹²⁵I-ASOR binding to recombinant M-ASGP-BP expressed on COS-1 cells (rM-ASGP-BP), and the results were compared with those of human hepatic lectin (HHL) on Hep G2 cells. Clustering of multiple Gal (or GalNAc) residues increased the binding affinity to M-ASGP-BP as well as to HHL. In contrast to HHL and other mammalian hepatic lectins, rM-ASGP-BP bound Gal residues tighter than GalNAc residues. A galactose-terminated triantennary N-glycoside, having oneN-acetyl-lactosamine unit on the 6 branch and twoN-acetyl-lactosamine units on the 3 branch of the trimannosyl core structure, showed affinity enhancement of 10⁵ over a monovalent ligand for HHL, while the same glycopeptide showed enhancement of about 2000-fold for rM-ASGP-BP. These results suggest that spatial arrangements of sugar combining sites and subunit organization of macrophage and hepatic lectins are different.     

The Hexapeptide inhibitor of Galbeta 1,3GalNAc-specific alpha 2,3-sialyltransferase as a generic inhibitor of sialyltransferases

The mammalian Galbeta1,3GalNAc-specific alpha2,3-sialyltransferase (ST3Gal I) was expressed as a secreted glycoprotein in High Five (Trichoplusia ni) cells. Using this recombinant ST3Gal I, we screened the synthetic hexapeptide combinatorial library to explore a sialyltransferase inhibitor. We found that the hexapeptide, NH(2)-GNWWWW, exhibited the most strong inhibition of ST3Gal I among five different hexapeptides that were finally selected. The kinetic analysis of ST3Gal I inhibition demonstrated that this hexapeptide could act as a competitive inhibitor (K(i) = 1.1 microm) on CMP-NeuAc binding to the enzyme. Moreover, the hexapeptide was shown to strongly inhibit both N-glycan-specific alpha2,3- and alpha2,6-sialyltranferase in vitro, suggesting that this peptide may inhibit the broad range of sialyltransferases regardless of their linkage specificity. The inhibitory activity in vivo was investigated by RCA-I lectin blot analyses and by metabolic d-[6-(3)H]GlcNH(2) radiolabeling analyses of N- and O-linked oligosaccharides in Chines hamster ovary cells. Our results demonstrate that the hexapeptide can act as a generic inhibitor of the N- and O-glycan-specific sialyltransferases in mammalian cells, which results in the significantly reduced NeuAc expression on cellular glycoproteins in vivo.     

Isolation and characterization of ribosome-inactivating proteins from Cucurbitaceae

Due to their RNA-N-glycosidase activity, ribosome-inactivating proteins (RIPs) are attractive candidates as antitumor and antiviral agents in biomedical and agricultural research. We have isolated and characterized two such proteins, foetidissimin II and texanin, from two Cucurbitaceae species. Foetidissimin II, obtained from the roots of Cucurbita foetidissima, was identified as a type-2 RIP, with a molecular weight of 61 kDa, as estimated by gel electrophoresis. It is composed of two chains, a 29-kDa chain A, and a 32-kDa chain B. Texanin, isolated from the fruits of Cucurbita texana, is a type-I RIP, with a single chain of molecular weight 29.7 kDa, as estimated by MALDI-TOF-MS. Both proteins exhibit RNA-N-glycosidase activity, with aniline playing a critical role in rRNA cleavage. The IC50 value of foetidissimin II, determined by cell-free protein-synthesis inhibition, was 0.251 muM. In an in vitro cytotoxicity assay, foetidissimin II exhibited IC50 values of ca. 70 nM to both adenocarcinoma and erythroleukemia cells. Texanin exhibited a weaker anticancer activity against erythroleukemia cells, with an IC50 value of 95 microM, but no activity against adenocarcinoma cells. The N-terminal sequences of both proteins were compared with those of reported RIPs.     

Molecular cloning of the bark and seed lectins from the Japanese pagoda tree (Sophora japonica)

cDNA clones encoding the bark and seed lectins from Sophora japonica were isolated and their sequences analyzed. Screening of a cDNA library constructed from polyA RNA isolated from the bark resulted in the isolation of three different lectin cDNA clones. The first clone encodes the GalNAc-specific bark lectin which was originally described by Hankins et al. whereas the other clones encode the two isoforms of the mannose/glucose-specific lectin reported by Ueno et al.. Molecular cloning of the seed lectin genes revealed that Sophora seeds contain only a GalNAc-specific lectin which is highly homologous to though not identical with the GalNAc-specific lectin from the bark. All lectin polypeptides are translated from mRNAs of ca. 1.3 kb encoding a precursor carrying a signal peptide. In the case of the mannose/glucose-specific bark lectins this precursor is post-translationally processed in two smaller peptides. Alignment of the deduced amino acid sequences of the different clones revealed striking sequence similarities between the mannose/glucose-binding and the GalNAc-specific lectins. Furthermore, there was a high degree of sequence homology with other legume lectins which allowed molecular modelling of the Sophora lectins using the coordinates of the Pisum sativum, Lathyrus ochrus and Erythrina corallodendron lectins.