Hydrocarbon-Binding Peptides from Legume Lectins in Relation to Different Host Specificity upon Legume–Rhizobium Symbiosis

The nucleotide sequences of 280–360-bp domains of lectin genes from 20 legume species belonging to 17 genera have been determined. A computer analysis of the sequences has been performed with the LASERGENE package. Based on this analysis, we constructed the phylogenetic tree of the lectins, which reflects their phylogenetic and evolutionary relationships, and predicted the amino-acid sequences of the corresponding protein domains. Features of the structure of the hydrocarbon-binding lectin domains were elucidated in some species of legume genera from the temperate climatic zone. The domains were highly variable and contained the consensus sequence AspTrePheXxxAsxXxxXxxTrpAspProXxxXxxIns/DelArgHis bearing the bulk of amino acid replacements, insertions, and deletions. An association between legume groups (including species from different genera and tribes) symbiotic with the same rhizobium species and the similarity between the hydrocarbon-binding domains of lectins from these plants was found.     

Characterization of the Arabidopsis lecRK-a genes: members of a superfamily encoding putative receptors with an extracellular domain homologous to legume lectins

An Arabidopsis cDNA clone that defines a new class of plant serine/threonine receptor kinases was found to be a member of a family of four clustered genes (lecRK-a1–a4) which have been cloned, sequenced and mapped on chromosome 3. This family belongs to a large superfamily encoding putative receptors with an extracellular domain homologous to legume lectins and appears to be conserved at least among dicots. In the Columbia ecotype only the lecRK-a1 and perhaps the lecRK-a3 gene is functional, since lecRK-a2 is disrupted by a Ty-copia retroelement and lecRK-a4 contains a frameshift mutation. Structural analysis of the lecRK-a1 and lecRK-a3 deduced amino-acid sequences suggests that the lectin domain is unlikely to be involved in binding monosaccharides but could interact with complex glycans and/or with hydrophobic ligands. Immunodetection of lecRK gene products in plasma membranes purified by free-flow electrophoresis showed that the lecRK-a proteins are probably highly glycosylated integral plasma membrane components.     

Polymorphism of lectin genes in <Emphasis Type="Italic">Lathyrus</Emphasis> plants

The carbohydrate-binding sequences of the lectin genes from spring vetchling Lathyrus vernus (L.) Bernh., marsh vetchling L. palustris (L.), and Gmelin’s vetchling L. gmelinii (Fitsch) (Fabaceae) were determined. Computer-aided analysis revealed substantial differences between nucleotide and predicted amino acid sequences of the lectin gene regions examined in each of the three vetchling species tested. In the phylogenetic trees based on sequence similarity of carbohydrate-biding regions of legume lectins, the sequences examined formed a compact cluster with the lectin genes of the plants belonging to the tribe Fabeae. In each plant, L. vernus, L. palustris, and L. gmelinii, three different lectin-encoding genes were detected. Most of the substitutions were identified within the gene sequence responsible for coding the carbohydrate-binding protein regions. This finding may explain different affinity of these lectins to different carbohydrates, and as a consequence, can affect the plant host specificity upon development of symbiosis with rhizobium bacteria.     

The Arabidopsis LecRK‐VI.2 associates with the pattern‐recognition receptor FLS2 and primes Nicotiana benthamiana pattern‐triggered immunity

Pattern‐triggered immunity (PTI) is broad spectrum and manipulation of PTI is believed to represent an attractive way to engineer plants with broad‐spectrum disease resistance. PTI is activated upon perception of microbe‐associated molecular patterns (MAMPs) by pattern‐recognition receptors (PRRs). We have recently demonstrated that the L‐type lectin receptor kinase‐VI.2 (LecRK‐VI.2) positively regulates Arabidopsis thaliana PTI. Here we show through in vitro pull‐down, bimolecular fluorescence complementation and co‐immunoprecipitation analyses that LecRK‐VI.2 associates with the PRR FLS2. We also demonstrated that LecRK‐VI.2 from the cruciferous plant Arabidopsis remains functional after interfamily transfer to the Solanaceous plant Nicotiana benthamiana. Wild tobacco plants ectopically expressing LecRK‐VI.2 were indeed more resistant to virulent hemi‐biotrophic and necrotrophic bacteria, but not to the fungal pathogen Botrytis cinerea suggesting that, as with Arabidopsis, the LecRK‐VI.2 protective effect in N. benthamiana is bacteria specific. Ectopic expression of LecRK‐VI.2 in N. benthamiana primed PTI‐mediated reactive oxygen species production, mitogen‐activated protein kinase (MAPK) activity, callose deposition and gene expression upon treatment with the MAMP flagellin. Our findings identified LecRK‐VI.2 as a member of the FLS2 receptor complex and suggest that heterologous expression of components of PRR complexes can be used as tools to engineer plant disease resistance to bacteria.     

Purification and cDNA cloning of a lectin and a lectin-like protein from Apios americana Medikus tubers

An Apios americana lectin (AAL) and a lectin-like protein (AALP) were purified from tubers by chromatography on Butyl-Cellulofine, ovomucoid-Cellulofine, and DEAE-Cellulofine columns. AAL showed strong hemagglutinating activity toward chicken and goose erythrocytes, but AALP showed no such activity toward any of the erythrocytes tested. The hemagglutinating activity of AAL was not inhibited by mono- or disaccharides, but was inhibited by glycoproteins, such as asialofetuin and ovomucoid, suggesting that AAL is an oligosaccharide-specific lectin. The cDNAs of AAL and AALP consist of 1,093 and 1,104 nucleotides and encode proteins of 302 and 274 amino acid residues, respectively. Both amino acid sequences showed high similarity to known legume lectins, and those of their amino acids involved in carbohydrate and metal binding were conserved.     

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.     

Mutational analysis of the sugar-binding site of pea lectin

Comparison of x-ray crystal structures of several legume lectins, co-crystallized with sugar molecules, showed a strong conservation of amino acid residues directly involved in ligand binding. For pea (Pisum sativum) lectin (PSL), these conserved amino acids can be classified into three groups: (I) D81 and N125, present in all legume lectins studied so far; (II) G99 and G216, conserved in almost all legume lectins; and (III) A217 and E218, which are only found in Vicieae lectins and are possibly determinants of sugar-binding specificity. Each of these amino acids in PSL was changed by site-directed mutagenesis, resulting in PSL molecules with single substitutions: for group I D81A, D81N, N125A; for group II G99R, G216L; and for group III A217L, E218Q, respectively. PSL double mutant Y124R; A126S was included as a control. The modified PSL molecules appeared not to be affected in their ability to form dimeric proteins, whereas the sugar-binding activity of each of the PSL mutants, with the exception of the control mutant (as shown by haemagglutination assays), was completely eliminated. These results confirm the model of the sugar-binding site of Vicieae lectins as deduced from X-ray analysis.     

Purification,Chemical, and Immunochemical Properties of a New Lectin from Mimosoideae (Parkia Discolor)

ABSTRACT

A glucose/mannose-binding lectin was isolated from seeds of Parkia discolor (Mimosoideae) using affinity chromatography on Sephadex G-100 gel. The protein presented a unique component in SDS-PAGE corresponding to a molecular mass of 58,000 Da, which is very similar to that of a closely related lectin from Parkia platycephala. Among the simple sugars tested, mannose was the best inhibitor, but biantennary glycans, containing the trimannoside core, present in N-glycoproteins, also seem to be powerful inhibitors of the haemagglutinating activity induced by the purified lectin. The protein was characterised by high content of glycine and proline and absence of cysteine. Rabbit antibodies, anti-P. platycephala seed lectin, recognised the P.discolor lectin. However, no cross-reaction was observed when a set of other legume lectins from sub-family Papilionoideae and others from families Moraceae and Euphorbiaceae were assayed with the Parkia lectins. This suggests that Parkia lectins comprise a new group of legume lectins exhibiting distinct characteristics.     

Leaves of the Lamiaceae species Glechoma hederacea (ground ivy) contain a lectin that is structurally and evolutionary related to the legume lectins

A novel lectin has been isolated and cloned from leaves of Glechoma hederacea (ground ivy), a typical representative of the plant family Lamiaceae. Biochemical analyses indicated that the G. hederacea agglutinin (Gleheda) is a tetrameric protein consisting of four subunits pairwise linked through an interchain disulphide bridge and exhibits a preferential specificity towards N-acetylgalactosamine. Cloning of the corresponding gene and molecular modeling of the deduced sequence demonstrated that Gleheda shares high sequence similarity with the legume lectins and exhibits the same overall fold and three-dimensional structure as the classical legume lectins. The identification of a soluble and active legume lectin ortholog in G. hederacea not only indicates that the yet unclassified Lamiaceae lectins belong to the same lectin family as the legume lectins, but also sheds a new light on the specificity, physiological role and evolution of the classical legume lectins.     

Hydrocarbon-binding peptides from bean plant lectins in connection with various host specific macrosymbionts during development of symbiosis with rhizobium bacteria

The nucleotide sequences of 280-360-bp domains of lectin genes from 20 legume species belonging to 17 genera have been determined. A computer analysis of the sequences has been performed with the LASERGENE package. Based on this analysis, we constructed the phylogenetic tree of the lectins, which reflects their phylogenetic and evolutionary relationships, and predicted the amino-acid sequences of the corresponding protein domains. Features of the structure of the hydrocarbon-binding lectin domains were elucidated in some species of legume genera from the temperate climatic zone. The domains were highly variable and contained the consensus sequence AspTrePheXxxAsxXxxXxxTrpAspProXxxXxxIns/DelArgHis bearing the bulk of amino acid replacements, insertions, and deletions. An association between legume groups (including species from different genera and tribes) symbiotic with the same rhizobium species and the similarity between the hydrocarbon-binding domains of lectins from these plants was found.     

A Nod factor-binding lectin is a member of a distinct class of apyrases that may be unique to the legumes

Recent studies from our laboratory have found that a root lectin from the legume Dolichos biflorus is present on the root surface, binds rhizobial Nod factor and has apyrase activity. To assess the broader significance of this lectin/nucleotide phosphohydrolase (Db-LNP), we have cloned a second related cDNA (Db-apyrase-2) from D. biflorus, as well as related cDNAs from the legumes Lotus japonicus and Medicago sativa, and from Arabidopsis thaliana, a non-legume. The deduced amino acid sequences of these apyrases were aligned with one another and with the sequences of other apyrases from plants, animals, yeast and protozoa. Phylogenetic analysis shows that Db-LNP has closely related orthologs only in other legumes, while Db-apyrase-2 is more closely related to apyrase sequences from non-leguminous plants. We also show that the orthologs of Db-LNP from M. sativa and Pisum sativum have carbohydrate binding activity. The results suggest that legume LNPs may represent a special class of apyrases that arose by gene duplication and subsequent specialization. Received: 11 March 1999 / Accepted: 14 June 1999     

Structures of sugar-binding peptides of <Emphasis Type="Italic">Galega orientalis</Emphasis> and <Emphasis Type="Italic">G. officinalis</Emphasis> lectins determine the choice of partner in rhizobium—Legume symbiosis

RAPD and RFEL analyses revealed appreciable genetic heterogeneity of Rhizobium galegae bv. officinalis and R. galegae bv. orientalis, which are nitrogen-fixing symbiosis partners of Galega officinalis and G. orientalis, respectively, and do not form a single cross-inoculation group. Comparison of nucleotide and amino acid sequences for their lectins revealed relatively high general homology, testifying again to their close phylogenetic relationships. Yet the lectin region of the carbohydrate-binding peptide (CBP) proved to differ considerably, being TYCNPGWDPRDR in G. orientalis and TFYNEEWDLVIKDEH in G. officinalis. Conserved positions in the CBP were observed for amino acid residues involved in binding Ca²⁺ and Mn²⁺ and stabilizing the spatial structure of the carbohydrate-binding pocket. These findings confirm the role in Rhizobium— legume symbiosis for lectins and especially for their carbohydrate-binding domains.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 103–111.Original Russian Text Copyright © 2005 by Baimiev, Gubaidullin, Chemeris, Vakhitov.     

The carbohydrate-binding sequences in lectins of the clovers Trifolium repens, T. pratense, and T. trichocephalum

The carbohydrate-binding sequences (CBS) in the lectin genes of Trifolium repens, T. pratense, and T. trichocephalum were sequenced. The gene regions encoding lectin CBS of T. pratense and T. repens displayed a considerable similarity; however, the CBS of these species differed essentially. Moreover, T. repens formed a compact cluster with Melilotus albus and M. officinalis in the phylogenetic trees constructed according to the nucleotide sequences and the corresponding CBS of legume lectins. T. trichocephalum does not fall into the group of the tribe Trifolieae members according to both the amino acid sequence of lectin carbohydrate-binding region and the nucleotide sequence of lectin gene.     

Structural and functional conservation profiles of novel cathepsin L-like proteins identified in the Drosophila melanogaster genome

Cathepsin L is a cysteine protease which degrades connective tissue proteins including collagen, elastin, and fibronectin. In this study, five well-characterized cathepsin L proteins from different arthropods were used as query sequences for the Drosophila genome database. The search yielded 10 cathepsin L-like sequences, of which eight putatively represent novel cathepsin L-like proteins. To understand the phylogenetic relationship among these cathepsin L-like proteins, a phylogenetic tree was constructed based on their sequences. In addition, models of the tertiary structures of cathepsin L were constructed using homology modeling methods and subjected to molecular dynamics simulations to obtain reasonable structure to understand its dynamical behavior. Our findings demonstrate that all of the potential Drosophila cathepsin L-like proteins contain at least one cathepsin propeptide inhibitor domain. Multiple sequence alignment and homology models clearly highlight the conservation of active site residues, disulfide bonds, and amino acid residues critical for inhibitor binding. Furthermore, comparative modeling indicates that the sequence/structure/function profiles and active site architectures are conserved.     

Direct cryopreservation of winter-acclimated buds of Dracocephalum austriacum (Lamiaceae) from field material

Mistletoes are semiparasite plants containing pharmaceutical proteins with applications in cancer treatment. Previous research has demonstrated that somaclonal variation can lead to the biosynthesis of novel proteins from mistletoe callus cultures. The protein content of Viscum album subsp. abietis tissues and biotechnologically propagated calluses, was analyzed to identify proteins with putative anticancer properties. In addition, evolutionary relations among linked species to Viscum were studied. Calluses were propagated from stem explants. The protein extracts mass spectra were processed with Proteome Discoverer and a search was performed using as reference the Uniprot V. album reviewed database. A phylogenetic tree was reconstructed using the LG amino acid substitution model by homologous sequences for Beta galactoside-specific lectin 2. The homology modeling of the Beta-galactoside-specific lectin 2 was carried out using Modeller software. Considerable differences were observed by comparing the protein content of the calluses and the maternal tissues. Four mistletoe lectins, six viscotoxins and the chitin binding lectin-cbML were identified within the species tissues. An in silico phylogenetic and structural study provides insights to the role of these lectins and the mechanism of semiparasite survival and evolution, towards a novel anticancer and immune system modulation pipeline. Callogenesis exhibited protein biosynthesis alterations and novel protein isoforms expression. Phyllogenetic analysis revealed evolutionary relations primarily within the Viscum genus and other species containing 2-ribosome inactivating proteins. The homology modeling of the mistletoe lectin 2 revealed possible structure related anticancer properties. In conclusion, mistletoe calluses were shown to possess a unique protein biosynthetic profile compared to donor plant tissues.

     

Differential accumulation of the transcripts of 22 novel protein kinase genes in Arabidopsis thaliana

22 novel members of the Arabidopsis thaliana protein kinase family (AKs) were identified by using degenerate oligonucleotide primers directed to highly conserved amino acid sequences of the protein kinase (PK) catalytic domain. Of these 22 genes, 16 turned out to carry intron sequences. Homologies of AK sequences were detected to S-locus receptor protein kinases (SRKs) from Brassica spp., to SRK-like PKs from maize and A. thaliana and to several other receptor PKs from A. thaliana. Sequence similarity was also detected to Ca²⁺-dependent PKs (CDPKs) from rape and soybean, to SNF1 and to CDC2 homologues. The genomic organization and the accumulation of the mRNAs from these 22 AK genes were investigated.     

Isolectins I-A and I-B of Griffonia (Bandeiraea) simplicifolia. Crystal structure of metal-free GS I-B(4) and molecular basis for metal binding and monosaccharide specificity.

Seeds from the African legume shrub Griffonia simplicifolia contain several lectins. Among them the tetrameric lectin GS I-B(4) has strict specificity for terminal alpha Gal residues, whereas the closely related lectin GS I-A(4) can also bind to alpha GalNAc. These two lectins are commonly used as markers in histology or for research in xenotransplantation. To elucidate the basis for the fine difference in specificity, the amino acid sequences of both lectins have been determined and show 89% identity. The crystal structure of GS I-B(4), determined at 2.5-A resolution, reveals a new quaternary structure that has never been observed in other legume lectins. An unexpected loss of both Ca(2+) and Mn(2+) ions, which are necessary for carbohydrate binding in legume lectins, may be related to a particular amino acid sequence Pro-Glu-Pro in the metal binding loop. Comparison with demetallized concanavalin A reveals a different process for the loss of metal ions and for the subsequent loss of carbohydrate binding activity. The GS I-A x alpha GalNAc and GS I-B x alpha Gal complexes were constructed using homology modeling and docking approaches. The unusual presence of an aromatic amino acid at position 47 (Tyr in I-A and Trp in I-B) explains the strong preference for alpha-anomeric sugars in both isolectins. Alteration at one amino acid position, Ala(106) in I-A versus Glu(106) in I-B, is the basis for the observed specificities toward alpha GalNAc and alpha Gal.