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.     

Lectin Receptor Kinases in Plants

Referee: Dr. Philip Becraft, Zoology and Genetics/Agronomy Depts., 2116 Molecular Building, lowa State University, Ames, IA 50011 Forty-two lectin receptor kinase (lecRK)-related sequences and nine related soluble legume lectin sequences were identified in the Arabidopsis thaliana genome. The genes are scattered as a single or gathered copies at different loci throughout the five chromosomes, and four predicted lecRK probably correspond to pseudogenes. Both structural alignments and molecular modeling revealed striking similarities between the lectinlike domain of lecRK, and related A. thaliana soluble lectins and legume lectins. The hydrophobic cavity is extremely conserved, whereas most of the residues forming the monosaccharide-binding site and the bivalent cation-binding site of legume lectins are poorly conserved. LecRK should be unable to bind the simple sugars usually recognized by genuine legume lectins. Molecular modeling of the kinase domain suggests that, except for two apparently inactive receptors, all other lecRK contain a putative functional Ser/Thr kinase catalytic domain. Both the juxtamembrane and C-terminal domains, which are considered important regions for regulating the kinase activity, exhibit a few specific stretches of amino acid residues. Some phylogenetic relationships are inferred from the phylogenetic trees built up from the different lecRK domain sequences. LecRK cluster in three distinct classes (A,B,C), one of them (B) being more closely related to soluble lectins of A. thaliana and legume lectins.     

Polymorphism of lectin genes in Lathyrus 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.     

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 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 Trijilium repens, T. pratense, and T. tri-chocephalum 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.     

Étude des réactions croisées entre les lectines de légumineuses: In systématique et phylogénétique

Seed extracts from 56 legume species were examined for immunological relatedness using 11 different lectin antisera. The immunological cross-reactions observed indicate that only lectins from species belonging to the tribe Vicieae share common antigenic determinants and are evolutionary closely related. These results suggest that the immunochemical study of the Vicieae lectins may provide a powerful tool for determining the systematic and phylogenetic relationships within the tribe.     

The bark of Robinia pseudoacacia contains a complex mixture of lectins.Characterization of the proteins and the cDNA clones.

Two lectins were isolated from the inner bark of Robinia pseudoacacia (black locust). The first (and major) lectin (called RPbAI) is composed of five isolectins that originate from the association of 31.5- and 29-kD polypeptides into tetramers. In contrast, the second (minor) lectin (called RPbAII) is a hometetramer composed of 26-kD subunits. The cDNA clones encoding the polypeptides of RPbAI and RPbAII were isolated and their sequences determined. Apparently all three polypeptides are translated from mRNAs of approximately 1.2 kb. Alignment of the deduced amino acid sequences of the different clones indicates that the 31.5- and 29-kD RPbAI polypeptides show approximately 80% sequence identity and are homologous to the previously reported legume seed lectins, whereas the 26-kD RPbAII polypeptide shows only 33% sequence identity to the previously described legume lectins. Modeling the 31.5-kD subunit of RPbAI predicts that its three-dimensional structure is strongly related to the three-dimensional models that have been determined thus far for a few legume lectins. Southern blot analysis of genomic DNA isolated from Robinia has revealed that the Robinia bark lectins are the result of the expression of a small family of lectin genes.     

Detection of lectin-related sequences in the genome of Sesbania rostrata

The extensive homologies between the amino-acid sequences of lectins extracted from the seeds of Leguminous plants have been confirmed by using cDNAs corresponding to the lectin genes from Pea, French bean, and Soybean. These cDNAs, used as probes, were hybridized with restriction fragments of genomic DNA from Pea. Good cross-hybridizations were observed. This strategy was extended to another plant: Sesbania rostrata, a tropical legume for which the presence of lectins has not been reported yet. Several genomic fragments hybridize with the lectin cDNA probes, indicating the existence of lectin-related sequences.     

Archeal lectins: An identification through a genomic search

Forty‐six lectin domains which have homologues among well established eukaryotic and bacterial lectins of known three‐dimensional structure, have been identified through a search of 165 archeal genomes using a multipronged approach involving domain recognition, sequence search and analysis of binding sites. Twenty‐one of them have the 7‐bladed β‐propeller lectin fold while 16 have the β‐trefoil fold and 7 the legume lectin fold. The remainder assumes the C‐type lectin, the β‐prism I and the tachylectin folds. Acceptable models of almost all of them could be generated using the appropriate lectins of known three‐dimensional structure as templates, with binding sites at one or more expected locations. The work represents the first comprehensive bioinformatic study of archeal lectins. The presence of lectins with the same fold in all domains of life indicates their ancient origin well before the divergence of the three branches. Further work is necessary to identify archeal lectins which have no homologues among eukaryotic and bacterial species. Proteins 2016; 84:21–30. © 2015 Wiley Periodicals, Inc.     

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.     

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.     

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.     

Host-symbiont interactions.I. The lectins of legumes interact with the O-antigen-containing lipopolysaccharides of their symbiont Rhizobia

A specific interaction between the O-antigen-containing lipopolysaccharides of $\text{Rhizobia}$ and the lectins of their legume hosts has been demonstrated. The lectins have been purified from the seeds of four legumes and the lectins covalently attached to Agarose. The lipopolysaccharides were isolated from the four $\text{Rhizobial}$ symbionts of the legumes. These four lipopolysaccharides were passed through the four lectin columns. In each case, the lipopolysaccharide from a $\text{Rhizobium}$ interacts with the lectin column of its symbiont but not with the other lectin columns.     

Isolation and partial characterisation of galactose-specific lectins from African yam beans, Sphenostyles stenocarpa Harms.

A new galactose-specific lectin was isolated from African yam bean (Sphenostyles stenocarpa Harms) by affinity chromatography on galactose-Sepharose 4B. SDS-PAGE analysis resulted in four polypeptide bands of approximately 27, 29, 32 and 34 kDa, respectively. Based on the analysis of carbohydrate content and native PAGE, it is likely that the Sphenostyles lectin is a tetrameric glycoprotein with M(r) of approximately 122 kDa. N-terminal protein sequencing of purified lectins from four different Sphenostyles accessions shows that the four polypeptides have largely identical amino acid sequences. The sequences contain the conserved consensus sequence F-F-LILG characteristic of legume lectins, as well as Phaseolus vulgaris proteins in the arcelin-alpha-amylase inhibitor gene family. The lectin agglutinates both rabbit and human erythrocytes, but with a preference for blood types A and O. Using Western blotting, the lectin was shown to accumulate rapidly during seed development, but levels dropped slightly as seeds attained maturity. This is the first time a lectin has been purified from the genus Sphenostyles. The new lectin was assigned the abbreviation LECp.SphSte.se.Hga1.     

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.     

Unexpectedly diverse Mesorhizobium strains and Rhizobium leguminosarum nodulate native legume genera of New Zealand, while introduced legume weeds are nodulated by Bradyrhizobium species

The New Zealand native legume flora are represented by four genera, Sophora, Carmichaelia, Clianthus, and Montigena. The adventive flora of New Zealand contains several legume species introduced in the 19th century and now established as serious invasive weeds. Until now, nothing has been reported on the identification of the associated rhizobia of native or introduced legumes in New Zealand. The success of the introduced species may be due, at least in part, to the nature of their rhizobial symbioses. This study set out to address this issue by identifying rhizobial strains isolated from species of the four native legume genera and from the introduced weeds: Acacia spp. (wattles), Cytisus scoparius (broom), and Ulex europaeus (gorse). The identities of the isolates and their relationship to known rhizobia were established by comparative analysis of 16S ribosomal DNA, atpD, glnII, and recA gene sequences. Maximum-likelihood analysis of the resultant data partitioned the bacteria into three genera. Most isolates from native legumes aligned with the genus Mesorhizobium, either as members of named species or as putative novel species. The widespread distribution of strains from individual native legume genera across Mesorhizobium spp. contrasts with previous reports implying that bacterial species are specific to limited numbers of legume genera. In addition, four isolates were identified as Rhizobium leguminosarum. In contrast, all sequences from isolates from introduced weeds aligned with Bradyrhizobium species but formed clusters distinct from existing named species. These results show that native legume genera and these introduced legume genera do not have the same rhizobial populations.     

Sequence studies of peanut agglutinin

Sequence studies have been performed on affinity purified peanut agglutinin, a galactose binding lectin. 161 residues have been compared to homologous residues in soybean agglutinin and favin. Extensive similarities have been uncovered, confirming the conservation of lectin sequences among all legume lectins. Evidence is presented for the existence of internal duplications and/or isolectins.     

Cloning and characterization of novel ficolins from the solitary ascidian, Halocynthia roretzi

Ficolins are animal lectins with collagen-like and fibrinogen-like domains. They are involved in the first line of host defense against pathogens. Human ficolin/P35 as well as mannose-binding lectin (MBL) activates the complement lectin pathway in association with MBL-associated serine proteases. To elucidate the origin and evolution of ficolins, we separated approximately 40 kDa (p40) and approximately 50 kDa (p50) N-acetylglucosamine-binding lectins from hemolymph plasma of the solitary ascidian. Binding assays revealed that p40 recognizes N-acetyl groups in association with a pyranose ring and that p50 recognizes N-acetylglucosamine alone. Based on the amino acid sequences of the proteins, we isolated two clones each of p40 and p50 from the ascidian hepatopancreas cDNA and determined the entire coding sequences of these clones. Because all of the clones contained both collagen-like and fibrinogen-like domains, we concluded that these were homologs of the mammalian ficolin family and designated ascidian ficolins (AsFCNs). The fibrinogen-like domain of the AsFCNs shows 45.4-52.4% amino acid sequence identity with the mammalian ficolin family. A phylogenetic tree of the fibrinogen-like sequences shows that all the fibrinogen-like domains may have evolved from a common ancestor that branched off an authentic fibrinogen. These results suggest that AsFCNs play an important role with respect to ascidian hemolymph lectin activity and the correlation of different functions with binding specificity.     

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.