Induction of cytoplasmic mannose-binding jacalin-related lectins is a common phenomenon in cereals treated with jasmonate methyl ester

Treatment of whole plants with jasmonic acid methyl ester induces lectin activity in leaves of Oryza sativa, Hordeum vulgare, Triticum vulgare, Secale cereale and Zea mays. Purification and characterization of the lectins revealed that they all have a very similar molecular structure and carbohydrate-binding properties. Further analysis of the cDNA clones encoding the lectins revealed that they all belong to the family of cytoplasmic mannose-specific jacalin-related lectins.     

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.     

Fruit-specific lectins from banana and plantain

 One of the predominant proteins in the pulp of ripe bananas (Musa acuminata L.) and plantains (Musa spp.) has been identified as a lectin. The banana and plantain agglutinins (called BanLec and PlanLec, respectively) were purified in reasonable quantities using a novel isolation procedure, which prevented adsorption of the lectins onto insoluble endogenous polysaccharides. Both BanLec and PlanLec are dimeric proteins composed of two identical subunits of 15 kDa. They readily agglutinate rabbit erythrocytes and exhibit specificity towards mannose. Molecular cloning revealed that BanLec has sequence similarity to previously described lectins of the family of jacalin-related lectins, and according to molecular modelling studies has the same overall fold and three-dimensional structure. The identification of BanLec and PlanLec demonstrates the occurrence of jacalin-related lectins in monocot species, suggesting that these lectins are more widespread among higher plants than is actually believed. The banana and plantain lectins are also the first documented examples of jacalin-related lectins, which are abundantly present in the pulp of mature fruits but are apparently absent from other tissues. However, after treatment of intact plants with methyl jasmonate, BanLec is also clearly induced in leaves. The banana lectin is a powerful murine T-cell mitogen. The relevance of the mitogenicity of the banana lectin is discussed in terms of both the physiological role of the lectin and the impact on food safety. Received: 1 December 1999 / Accepted: 31 January 2000     

Isolation and characterization of a jacalin-related mannose-binding lectin from salt-stressed rice (Oryza sativa) plants

A novel plant lectin was isolated from salt-stressed rice (Oryzasativa L.) plants and partially characterized. The lectin occurs as a natural mixture of two closely related isoforms consisting of two identical non-covalently linked subunits of 15 kDa. Both isoforms are best inhibited by mannose and exhibit potent mitogenic activity towards T-lymphocytes. Biochemical analyses and sequence comparisons further revealed that the rice lectins belong to the subgroup of mannose-binding jacalin-related lectins. In addition, it could be demonstrated that the lectins described here correspond to the protein products of previously described salt-stress-induced genes. Our results not only identify the rice lectin as a stress protein but also highlight the possible importance of protein-carbohydrate interactions in stress responses in plants. Received: 27 July 1999 / Accepted: 11 November 1999     

A lectin and a lectin-related protein are the two most prominent proteins in the bark of yellow wood (Cladrastis lutea).

Using a combination of cDNA cloning and protein purification it is demonstrated that bark of yellow wood (Cladrastis lutea) contains two mannose/glucose binding lectins and a lectin-related protein which is devoid of agglutination activity. One of the lectins (CLAI) is the most prominent bark protein. It is built up of four 32 kDa monomers which are post-translationally cleaved into a 15 kDa and a 17 kDa polypeptide. The second lectin (CLAII) is a minor protein, which strongly resembles CLAI except that its monomers are not cleaved into smaller polypeptides. Molecular cloning of the Cladrastis lectin family revealed also the occurrence of a lectin-related protein (CLLRP) which is the second most prominent bark protein. Although CLLRP shows sequence homology to the true lectins, it is devoid of carbohydrate binding activity. Molecular modelling of the three Cladrastis proteins has shown that their three-dimensional structure is strongly related to the three-dimensional models of other legume lectins and, in addition, revealed that the presumed carbohydrate binding site of CLLRP is disrupted by an insertion of three extra amino acids. Since it is demonstrated for the first time that a lectin and a noncarbohydrate binding lectin-related protein are the two most prominent proteins in the bark of a tree, the biological meaning of their simultaneous occurrence is discussed.     

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     

The amino-acid sequence of the glucose/mannose-specific lectin isolated from Parkia platycephala seeds reveals three tandemly arranged jacalin-related domains.

A mannose/glucose-specific lectin was isolated from seeds of Parkia platycephala, the most primitive subfamily of Leguminosae plants. The molecular mass of the purified lectin determined by mass spectrometry was 47 946 +/- 6 Da (by electrospray ionization) and 47 951 +/- 9 Da (by matrix-assisted laser-desoption ionization). The apparent molecular mass of the lectin in solutions of pH in the range 4.5-8.5 determined by analytical ultracentrifugation equilibrium sedimentation was 94 +/- 3 kDa, showing that the protein behaved as a non-pH-dependent dimer. The amino-acid sequence of the Parkia lectin was determined by Edman degradation of overlapping peptides. This is the first report of the primary structure of a Mimosoideae lectin. The protein contained a blocked N-terminus and a single, nonglycosylated polypeptide chain composed of three tandemly arranged homologous domains. Each of these domains shares sequence similarity with jacalin-related lectin monomers from Asteraceae, Convolvulaceae, Moraceae, Musaceae, Gramineae, and Fagaceae plant families. Based on this homology, we predict that each Parkia lectin repeat may display a beta prism fold similar to that observed in the crystal structure of the lectin from Helianthus tuberosus. The P. platycephala lectin also shows sequence similarity with stress- and pathogen-upregulated defence genes of a number of different plants, suggesting a common ancestry for jacalin-related lectins and inducible defence proteins.     

Expression analysis of the nucleocytoplasmic lectin 'Orysata' from rice in Pichia pastoris

The Oryza sativa lectin, abbreviated Orysata, is a mannose-specific, jacalin-related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin-related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N-glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high-mannose structures, Man9-11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high-mannose as well as with more complex N-glycan structures. Orysata has potent anti-human immunodeficiency virus and anti-respiratory syncytial virus activity in cell culture compared with other jacalin-related lectins.     

Cloning and characterization of mannose binding jacalin related lectin encoding gene from Indian Cycas ( Cycas annaikalensis Singh and Radha)

Mannose specific jacalin-related lectins or agglutinins (mJRLs) constitute an important superfamily of proteins known to play vital roles in various biological processes. In the present study, a cDNA having 876 bp open reading frame (ORF) coding for mJRL of 291 amino acids residues was cloned from pinna of Cycas annaikalensis which is endemic to Western Ghats, India and designated as C. annaikalensis pinna lectin (CAPL). Expression of the coding sequence under the control of a T7 promoter in E. coli produced 31 kDa protein. The purified recombinant protein had shown agglutination with erythrocytes of rabbit blood. The deduced amino acid sequence of CAPL showed two sugar binding sites (also determined to be jacalin-like lectin domains) and 95% similarity with C. revoluta leaf lectin (CRLL) protein. Further, a monomeric protein of CAPL consisting of mannose binding residues and jacalin motifs that are having 35–90% similarities with mJRLs which have already been reported. A phylogenetic tree exhibited the grouping of CAPL into a subclade different from that of the CRLL. Also, a model of cycas leaf lectin was built by homology modeling using 1ZGRA (Parkia platycephala seed lectin) as a template for the construction of three-dimensional structures. Structural modeling and docking studies were completed using Discovery studio version 2.1. This study, first of its kind, reports mJRLs from the Indian gymnosperm.     

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.     

Amino acid sequence of beta-galactoside-binding bovine heart lectin. Member of a novel class of vertebrate proteins

A variety of animal tissues contain beta-galactoside-binding lectins with molecular masses in the range 13-17 kDa. There is evidence that these lectins may constitute a new protein family although their function in vivo is not yet clear. In this work the major part of the amino acid sequence of the 13 kDa lectin from bovine heart muscle has been determined. Comparison of this sequence with the cDNA-deduced sequence published for the chick embryo skin lectin showed 58% homology. Comparison of the bovine lectin sequence with partial sequences from two cDNA clones from a human hepatoma library and partial amino acid sequences of human lung lectin showed 70, 40 and 85% homology, respectively. The sequences of these vertebrate lectins are thus clearly related, supporting earlier results of immunological cross-reactivity within this group of proteins. Computer searching of protein sequence databases did not detect significant homologies between the bovine lectin sequence and other known proteins.     

Identification, cloning and tissue localization of a rainbow trout (Oncorhynchus mykiss) intelectin-like protein that binds bacteria and chitin

Intelectins are a recently identified group of animal lectins involved in innate immune surveillance. This paper describes the primary structure, expression and immunohistochemical localization of a rainbow trout plasma intelectin (RTInt). RTInt exhibited calcium-dependent binding to N-acetylglucosamine (GlcNAc) and mannose conjugated Toyopearl Amino 650 M matrices. When GlcNAc eluates from chromatography matrices were analyzed by reducing 1D PAGE and Western blots, the lectin appeared as approximately 37 kDa and approximately 72 kDa bands. Similar analysis of plasma revealed a single 72 kDa band under reducing conditions. MALDI-TOF MS demonstrated five, approximately 37 kDa isoforms (pI 5.3-6.1) separated by 2D-PAGE. A 975 bp cDNA sequence obtained by RT-PCR from liver and spleen tissue encoded a 325 amino acid secretory protein with homology to human and murine intelectins, which bind bacterial components and are induced during parasitic infections. Gene expression and immunohistochemistry detected RTInt in gill, spleen, hepatic sinusoid, renal interstitium, intestine, skin, swim bladder and within leukocytes. Direct binding assays demonstrated the ability of RTInt to bind relevant bacterial and chitinous targets. These findings suggest that RTInt plays a role in innate immune defense against bacterial and chitinous microbial organisms.     

Isolation,characterization and molecular cloning of the mannose-binding lectins from leaves and roots of garlic (Allium sativum L.)

Two novel lectins were isolated from roots and leaves of garlic. Characterization of the purified proteins indicated that the leaf lectin ASAL is a dimer of two identical subunits of 12 kDa, which closely resembles the leaf lectins from onion, leek and shallot with respect to its molecular structure and agglutination activity. In contrast, the root lectin ASARI, which is a dimer of subunits of 15 kDa, strongly differs from the leaf lectin with respect to its agglutination activity. cDNA cloning of the leaf and root lectins revealed that the deduced amino acid sequences of ASAL and ASARI are virtually identical. Since both lectins have identical N-terminal sequences the larger Mr of the ASARI subunits implies that the root lectin has an extra sequence at its C-terminus. These results not only demonstrate that virtually identical precursor polypeptides are differently processed at their C-terminus in roots and leaves but also indicate that differential processing yields mature lectins with strongly different biological activities. Further screening of the cDNA library for garlic roots also yielded a cDNA clone encoding a protein composed of two tandemly arrayed lectin domains. Since the presumed two-domain root lectin has not been isolated yet, its possible relationship to the previously described two-domain bulb lectin could not be studied at the protein level.     

Cloning and characterization of the lectin cDNA clones from onion,shallot and leek

Characterization of the lectins from onion (Allium cepa), shallot (A. ascalonicum) and leek (A. porrum) has shown that these lectins differ from previously isolated Alliaceae lectins not only in their molecular structure but also in their ability to inhibit retrovirus infection of target cells.cDNA libraries constructed from poly(A)-rich RNA isolated from young shoots of onion, shallot and leek were screened for lectin cDNA clones using colony hybridization. Sequence analysis of the lectin cDNA clones from these three species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level.Apparently the onion, shallot and leek lectins are translated from mRNAs of ca. 800 nucleotides. The primary translation products are preproproteins (ca. 19 kDa) which are converted into the mature lectin polypeptides (12.5–13 kDa) after post-translational modifications.Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.     

Two distinct jacalin-related lectins with a different specificity and subcellular location are major vegetative storage proteins in the bark of the black mulberry tree

Using a combination of protein isolation/characterization and molecular cloning, we have demonstrated that the bark of the black mulberry tree (Morus nigra) accumulates large quantities of a galactose-specific (MornigaG) and a mannose (Man)-specific (MornigaM) jacalin-related lectin. MornigaG resembles jacalin with respect to its molecular structure, specificity, and co- and posttranslational processing indicating that it follows the secretory pathway and eventually accumulates in the vacuolar compartment. In contrast, MornigaM represents a novel type of highly active Man-specific jacalin-related lectin that is synthesized without signal peptide or other vacuolar targeting sequences, and accordingly, accumulates in the cytoplasm. The isolation and cloning, and immunocytochemical localization of MornigaG and MornigaM not only demonstrates that jacalin-related lectins act as vegetative storage proteins in bark, but also allows a detailed comparison of a vacuolar galactose-specific and a cytoplasmic Man-specific jacalin-related lectin from a single species. Moreover, the identification of MornigaM provides the first evidence, to our knowledge, that bark cells accumulate large quantities of a cytoplasmic storage protein. In addition, due to its high activity, abundance, and ease of preparation, MornigaM is of great potential value for practical applications as a tool and bioactive protein in biological and biomedical research.     

A Rainbow Trout Lectin with Multimeric Structure

A novel lectin has been identified in rainbow trout serum and plasma. The lectin binds to Sepharose (an agarose polymer) in a calcium-dependent manner. Glucose, N-acetyl-glucosamine, mannose, N-acetyl-mannosamine, l-fucose, maltose and α-methyl-mannoside are good inhibitors of this binding, whereas glucosamine and d-fucose inhibits to a lesser degree and mannosamine and galactose do not inhibit the binding to Sepharose. When analysed by SDS-PAGE under non-reducing conditions, the lectin appears as a characteristic ladder of bands with approximately 16 kDa between consecutive bands. Upon reduction, the lectin appears as a 16-kDa band. On size-exclusion chromatography of trout serum and plasma, the protein emerges over a broad range corresponding to sizes from about 2000 kDa to less than 200 kDa. The NH₂-terminal sequence (AAENRNQXPPG) shows no significant homology with known proteins. Because of the characteristic appearance in non-reducing SDS-PAGE and the lectin activity, we propose to name the protein “ladderlectin.”     

Genomic Cloning and Characterization of a Novel Lectin Gene from <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis>

A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5′ flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3′ flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5′ flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3′-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49–54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms.     

The lectin from leaves of Japanese cycad, Cycas revoluta Thunb. (gymnosperm) is a member of the jacalin-related family.

A novel lectin was isolated from leaves of the Japanese cycad, Cycas revoluta Thunb. (gymnosperm), and its characteristics including amino acid composition, molecular mass, carbohydrate binding specificity and partial amino acid sequences were examined. The inhibition analysis of hemagglutinating activity with various sugars showed that the lectin has a carbohydrate-binding specificity similar to those of mannose recognizing, jacalin-related lectins. Partial amino acid sequences of the lysylendopeptic peptides shows that the lectin might have a repeating structure and belong to the jacalin-related lectin family.     

Human surfactant protein D: SP-D contains a C-type lectin carbohydrate recognition domain

Lung surfactant protein D (SP-D) shows calcium-dependent binding to specific saccharides, and is similar in domain structure to certain members of the calcium-dependent (C-type) lectin family. Using a degenerate oligomeric probe corresponding to a conserved peptide sequence derived from the amino-terminus of the putative carbohydrate binding domain of rat and bovine SP-D, we screened a human lung cDNA library and isolated a 1.4-kb cDNA for the human protein. The relationship of the cDNA to SP-D was established by several techniques including amino-terminal microsequencing of SP-D-derived peptides, and immunoprecipitation of translation products of transcribed mRNA with monospecific antibodies to SP-D. In addition, antibodies to a synthetic peptide derived from a predicted unique epitope within the carbohydrate recognition domain of SP-D specifically reacted with SP-D. DNA sequencing demonstrated a noncollagenous carboxy-terminal domain that is highly homologous with the carboxy-terminal globular domain of previously described C-type lectins. This domain contains all of the so-called "invariant residues," including four conserved cysteine residues, and shows high homology with the mannose-binding subfamily of C-type lectins. Sequencing also demonstrated an amino-terminal collagenous domain that contains an uninterrupted sequence of 59 Gly-X-Y triplets and that also contains the only identified consensus for asparagine-linked oligosaccharides. The studies demonstrate that SP-D is a member of the C-type lectin family, and confirm predicted structural similarities to conglutinin, SP-D, and the serum mannose binding proteins.     

Characterization and cloning of GNA-like lectin from the mushroom Marasmius oreades

A new mannose-recognizing lectin (MOL) was purified on an asialofetuin-column from fruiting bodies of Marasmius oreades grown in Japan. The lectin (MOA) from the fruiting bodies of the same fungi is well known to be a ribosome-inactivating type lectin that recognizes blood-group B sugar. However, in our preliminary investigation, MOA was not found in Japanese fruiting bodies of M. oreades, and instead, MOL was isolated. Gel filtration showed MOL is a homodimer noncovalently associated with two subunits of 13 kDa. The N-terminal sequence of MOL was blocked. The sequence of MOL was determined by cloning from cDNA and by protein sequencing of enzyme-digested peptides. The sequence shows mannose-binding motifs of bulb-type mannose-binding lectins from plants, and similarity to the sequences. Analyses of sugar-binding specificity by hemagglutination inhibition revealed the preference of MOL toward mannose and thyroglobulin, but asialofetuin was the strongest inhibitor of glycoproteins tested. Furthermore, glycan-array analysis showed that the specificity pattern of MOL was different from those of typical mannose-specific lectins. MOL preferred complex-type N-glycans rather than high-mannose N-glycans.