Weak protein-protein interactions in lectins: the crystal structure of a vegetative lectin from the legume Dolichos biflorus.

The legume lectins are widely used as a model system for studying protein-carbohydrate and protein-protein interactions. They exhibit a fascinating quaternary structure variation, which becomes important when they interact with multivalent glycoconjugates, for instance those on cell surfaces. Recently, it has become clear that certain lectins form weakly associated oligomers. This phenomenon may play a role in the regulation of receptor crosslinking and subsequent signal transduction. The crystal structure of DB58, a dimeric lectin from the legume Dolichos biflorus reveals a separate dimer of a previously unobserved type, in addition to a tetramer consisting of two such dimers. This tetramer resembles that formed by DBL, the seed lectin from the same plant. A single amino acid substitution in DB58 affects the conformation and flexibility of a loop in the canonical dimer interface. This disrupts the formation of a stable DBL-like tetramer in solution, but does not prohibit its formation in suitable conditions, which greatly increases the possibilities for the cross-linking of multivalent ligands. The non-canonical DB58 dimer has a buried symmetrical alpha helix, which can be present in the crystal in either of two antiparallel orientations. Two existing structures and datasets for lectins with similar quaternary structures were reconsidered. A central alpha helix could be observed in the soybean lectin, but not in the leucoagglutinating lectin from Phaseolus vulgaris. The relative position and orientation of the carbohydrate-binding sites in the DB58 dimer may affect its ability to crosslink mulitivalent ligands, compared to the other legume lectin dimers.

Localization of a Nod factor-binding protein in legume roots and factors influencing its distribution and expression.

The roots of the legume Dolichos biflorus contain a lectin/nucleotide phosphohydrolase (Db-LNP) that binds to the Nod factor signals produced by rhizobia that nodulate this plant. In this study we show that Db-LNP is differentially distributed along the surface of the root axis in a pattern that correlates with the zone of nodulation of the root. Db-LNP is present on the surface of young and emerging root hairs and redistributes to the tips of the root hairs in response to treatment of the roots with a rhizobial symbiont or with a carbohydrate ligand. This redistribution does not occur in response to a non-symbiotic rhizobial strain or a root pathogen. Db-LNP is also present in the root pericycle where its level decreases upon initiation of nodule formation. Maximum levels of Db-LNP are found in 2-d-old roots, and the expression of this root protein is increased when the plants are grown in the absence of NO(3)(-) and NH(4)(+). These results support the possibility that Db-LNP is involved in the initiation of the Rhizobium legume symbiosis.

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 hifloris 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. hiflorus, 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.

A nod factor binding lectin with apyrase activity from legume roots.

A lectin isolated from the roots of the legume, Dolichos biflorus, binds to Nod factors produced by rhizobial strains that nodulate this plant and has a deduced amino acid sequence with no significant homology to any lectin reported to date. This lectin also is an enzyme that catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside di- and triphosphates; the enzyme activity is increased in the presence of carbohydrate ligands. This lectin-nucleotide phosphohydrolase (LNP) has a substrate specificity characteristic of the apyrase category of phosphohydrolases, and its sequence contains four motifs characteristic of this category of enzymes. LNP is present on the surface of the root hairs, and treatment of roots with antiserum to LNP inhibits their ability to undergo root hair deformation and to form nodules on exposure to rhizobia. These properties suggest that this protein may play a role in the rhizobium-legume symbiosis and/or in a related carbohydrate recognition event endogenous to the plant.

Carbohydrate binding, quaternary structure and a novel hydrophobic binding site in two legume lectin oligomers from Dolichos biflorus.

The seed lectin (DBL) from the leguminous plant Dolichos biflorus has a unique specificity among the members of the legume lectin family because of its high preference for GalNAc over Gal. In addition, precipitation of blood group A+H substance by DBL is slightly better inhibited by a blood group A trisaccharide (GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal) containing pentasaccharide, and about 40 times better by the Forssman disaccharide (GalNAc(alpha1-3)GalNAc) than by GalNAc. We report the crystal structures of the DBL-blood group A trisaccharide complex and the DBL-Forssman disaccharide complex.A comparison with the binding sites of Gal-binding legume lectins indicates that the low affinity of DBL for Gal is due to the substitution of a conserved aromatic residue by an aliphatic residue (Leu127). Binding studies with a Leu127Phe mutant corroborate these conclusions. DBL has a higher affinity for GalNAc because the N-acetyl group compensates for the loss of aromatic stacking in DBL by making a hydrogen bond with the backbone amide group of Gly103 and a hydrophobic contact with the side-chains of Trp132 and Tyr104. Some legume lectins possess a hydrophobic binding site that binds adenine and adenine-derived plant hormones, i.e. cytokinins. The exact function of this binding site is unknown, but adenine/cytokinin-binding legume lectins might be involved in storage of plant hormones or plant growth regulation. The structures of DBL in complex with adenine and of the dimeric stem and leaf lectin (DB58) from the same plant provide the first structural data on these binding sites. Both oligomers possess an unusual architecture, featuring an alpha-helix sandwiched between two monomers. In both oligomers, this alpha-helix is directly involved in the formation of the hydrophobic binding site. DB58 adopts a novel quaternary structure, related to the quaternary structure of the DBL heterotetramer, and brings the number of know legume lectin dimer types to four.

Crystallization of two related lectins from the legume plant Dolichos biflorus.

The seed lectin DBL and the related stem and leaves lectin DB58 of the tropical legume Dolichos biflorus were crystallized, as well as complexes of DBL with adenine and with GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal. The different crystal forms of DBL diffract to about 2.8 A, while DB58 crystals diffract to 3.3 A.

Oligosaccharide signaling of plant cells.

A variety of oligosaccharide signals have been identified that function in the regulation of plant development, defense, and other interactions of plants with the environment. Some of these oligosaccharides are produced by various pathogens or symbionts, whereas others are synthesized by the plant itself. This mini-review summarizes our present state of information on these oligosaccharide signals and provides an overview of approaches being used to identify receptors for these signals and gain an understanding of the mechanism(s) by which these signals activate downstream events. Possible biotechnological applications of future work in this field are also considered.

Transferred nuclear Overhauser enhancement (NOE) and rotating-frame NOE experiments reflect the size of the bound segment of the Forssman pentasaccharide in the binding site of Dolichos biflorus lectin.

A complex between the Forssman pentasaccharide alpha-D-GalNAc-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Gal-(1-->4)-beta-D- Gal-(1-->4)-D-Glc and the seed lectin from Dolichos biflorus was studied using transfer-NOESY and transfer rotating frame NOE spectroscopy (ROESY) experiments. The evolution of transferred NOEs and ROEs as a function of the pentasaccharide/lectin ratio was different for the non-reducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNac compared to the rest of the molecule, which reflects distinct relaxation properties and effects of exchange broadening of the corresponding ligand resonances. Significantly, several intermolecular transferred NOEs were observed between protons of the nonreducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and aliphatic as well as aromatic amino acid side chain protons in the binding pocket of the lectin. It is concluded that the non-reducing disaccharide fragment is buried in the lectin-binding pocket, whereas the reducing trisaccharide portion alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-D-Glc has no immediate contacts with the protein. The experimental transfer NOE data were qualitatively compared to theoretical proton-proton distances from a model that was based on a previous homology modeling study of a complex between the disaccharide fragment alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and D. biflorus lectin. It was found that all intermolecular transferred NOEs matched short interatomic distances between ligand protons and aliphatic or aromatic amino acid side chain protons predicted by the theoretical model.

Conformational analysis of blood group A trisaccharide in solution and in the binding site of Dolichos biflorus lectin using transient and transferred nuclear Overhauser enhancement (NOE) and rotating-frame NOE experiments.

The present study is concerned with the elucidation of the conformation of the blood group A trisaccharide (alpha-D-GalNAc(1-3)[alpha-L-Fuc(1-->2)] beta-D-Gal-O-R) in the combining site of Dolichos biflorus seed lectin by use of 400-MHz and 600-MHz NMR spectroscopy. D. biflorus lectin displays a unique specificity for GalNAc residues. It occurs in solution as a tetrameric assembly having a molecular mass of 110 kDa, with two carbohydrate-binding sites per molecule. First, NOE build-up curves were obtained for the free blood group A trisaccharide from one-dimensional transient NOE experiments. Simulated NOE build-up curves were constructed from an ensemble of low-energy conformers derived from previous investigations. The comparison of theoretical and experimental data indicates that an equilibrium between two families of low-energy conformers most likely reflects the solution behavior of the trisaccharide in solution. Two-dimensional transferred NOE and rotating-frame enhancements (ROE) were subsequently measured for the trisaccharide complexed with the D. biflorus seed lectin. In addition to the NOEs observed for the free trisaccharide, the transferred NOESY spectrum showed several new NOEs that were identified as spin diffusion using a rotating-frame NOESY (ROESY) experiment. Experimental interglycosidic transferred nuclear Overhauser effect (TRNOE) build-up curves were compared to theoretical curves calculated for both low-energy conformers located in the D. biflorus lectin-binding site. Calculations of theoretical TRNOE were performed using a combination of the full relaxation matrix and the protein-ligand exchange matrix. Comparison between experimental and simulated TRNOE volumes leads to the conclusion that one conformation of blood group A trisaccharide is selected upon binding by D. biflorus lectin.

A comparison of the carbohydrate binding properties of two Dolichos biflorus lectins.

The carbohydrate binding properties of the Dolichos biflorus seed lectin and DB58, a vegetative tissue lectin from this plant, were compared using two types of solid phase assays. Both lectins bind to hog blood group A + H substance covalently coupled to Sepharose 4B and this binding can be inhibited with free blood group A + H substance. However, the binding of the seed lectin is inhibited by D-GalNAc whereas DB58 binding was not inhibited by any monosaccharide tested, thus suggesting that its carbohydrate combining site may be more extensive than that of the seed lectin. The activities of these two lectins also differ from one another in ability to recognize blood group A + H substance adsorbed on to plastic and in the effects of salt and urea on their carbohydrate binding activities. Neither lectin showed glycosidase activity with p-nitrophenyl alpha-D-GalNAc or p-nitrophenyl beta-D-GalNAc.

Molecular modelling of the Dolichos biflorus seed lectin and its specific interactions with carbohydrates: alpha-D-N-acetyl-galactosamine, Forssman disaccharide and blood group A trisaccharide.

The three-dimensional structure of Dolichos biflorus seed lectin has been constructed using five legume lectins for which high resolution crystal structures were available. The validity of the resulting model has been thoroughly investigated. Final structure optimization was conducted for the lectin complexed with alpha GalNAc, providing thereby the first three-dimensional structure of lectin/GalNAc complex. The role of the N-acetyl group was clearly evidenced by the occurrence of a strong hydrogen bond between the protein and the carbonyl oxygen of the carbohydrate and by hydrophobic interaction between the methyl group and aromatic amino acids. Since the lectin specificity is maximum for the Forssman disaccharide alpha GalNAc(1-3) beta GalNAc-O-Me and the blood group A trisaccharide alpha GalNAc(1-3)[alpha Fuc(1-2)] beta Gal-O-Me, the complexes with these oligosaccharides have been also modelled.

Expression and partial characterization of Dolichos biflorus seed lectin in Escherichia coli.

The seed lectin from the legume, Dolichos biflorus, was expressed in Escherichia coli using the pET expression vector. Replacement of the 22-amino acid signal sequence of this lectin with a methionine increased the level of lectin expression greater than 100-fold. Approximately 20% of the expressed seed lectin was soluble; the remainder was solubilized in 8 M urea and renatured by rapid dilution. No difference in physicochemical properties or activity was detected between the soluble and renatured forms. NH2-terminal amino acid analysis and immunoblots, using antibodies that recognize the COOH-terminus of only the nontruncated subunit of the native heteroligomer, established that the expressed lectin has a primary structure equivalent to subunit I of the native seed lectin. The expressed seed lectin is active as evidenced by its ability to bind to blood group A + H substance-Sepharose and to be specifically eluted from this column with N-acetylgalactosamine. However, a comparison of the activity of the expressed lectin with the native seed lectin using a sensitive ELISA showed that the expressed lectin has a slightly lower affinity for blood group A + H substance than the native seed lectin. The expressed lectin also had a lower M(r) than the seed lectin as determined by molecular exclusion chromatography.

Isolation and characterization of subunits of DB58, a lectin from the stems and leaves of Dolichos biflorus.

The DB58 lectin of the stems and leaves of Dolichos biflorus is a heterodimer composed of two closely related subunits, alpha and beta. These subunits were dissociated from one another in urea and isolated by high-performance anion-exchange chromatography. Steric exclusion chromatography of the isolated subunits in 6 M guanidine hydrochloride showed molecular weights of 30,900 and 29,800 for the alpha and beta subunits, respectively. The subunits have very similar amino acid compositions and are glycosylated at each of their two N-glycosylation consensus sites. Each of the subunits had weak carbohydrate binding activity. Reverse-phase chromatography of tryptic digests of the subunits showed identical peptide maps with the exception of peaks identified as COOH-terminal peptides. Analyses of these peptides, COOH-terminal amino acid analyses, and the small differences in amino acid composition between the 2 subunits establish that the beta subunit differs from the alpha subunit by the absence of 11 or 12 amino acids from its COOH terminus. This structural difference, combined with information from previous biosynthetic studies, establishes that the beta subunit is derived from the alpha subunit by posttranslational proteolytic modification at the COOH terminus. The heterogeneity in the extent of truncation suggests that this conversion occurs by sequential removal of amino acids rather than by endoproteolytic cleavage. The possible physiological significance of this modification is discussed.

Incorrect targeting of plant vacuolar lectins in yeast.

Two soluble vacuolar lectins, seed lectin and DB58, from the legume, Dolichos biflorus, were expressed in Saccharomyces cerevisiae using both low and high copy number plasmids under the control of the GAL1 promoter. When expressed at low levels, these lectins were each secreted by the yeast at all stages of growth. Expression of the lectins at high levels resulted in the retention of most of the lectins in the cell. Cell fractionation studies showed that this retained lectin was not associated with the yeast vacuoles. The differential COOH-terminal processing of these lectins, that results in the production of heteroligomers in plants, did not occur in yeast. Site-directed mutagenesis was employed to produce a construct encoding the shorter subunit of the seed lectin. Expression of this truncated subunit in yeast produced the same results as found with the larger subunit, thus indicating that this modification does not provide the vacuolar targeting signal. The inability of these two vacuolar proteins from different plant tissues to be transported to the yeast vacuole suggests that plants and yeast utilize different signals for targeting soluble vacuolar proteins.

Biosynthesis of DB58 lectin in cell suspension cultures of Dolichos biflorus.

The stem and leaf lectin of the legume, Dolichos biflorus, was found to be expressed in cell suspension cultures derived from calli from this plant. The lectin is present at levels equivalent to the amount of lectin in the plant and its expression is correlated with the exponential growth phase of the cells. In vitro translation of mRNA isolated from these cultures, followed by immunoprecipitation with antibodies to the lectin, yields a single polypeptide precursor for this lectin. In vivo pulse chase labeling of the DB58 lectin yields a single glycosylated precursor that ultimately gives rise to the mature alpha and beta subunits of this heterodimer. Chemical deglycosylation of the labeled precursors and products shows that the alpha and beta subunits do not differ simply by their extent of glycosylation. Antibodies generated against a synthetic peptide representing the deduced COOH-terminus of the nascent protein react only with the alpha subunit. These data support a mechanism of lectin subunit generation involving differential carboxyl terminal modification of a single polypeptide precursor.

Photoaffinity labeling of the adenine binding sites of two Dolichos biflorus lectins.

Two differentially expressed lectins from the legume Dolichos biflorus, the seed lectin and a stem and leaf lectin (DB58), were photoaffinity-labeled at their adenine binding sites using the probe [2-3H]8-azidoadenine. Both heteromeric subunits I and II of the seed lectin and alpha and beta of DB58 were specifically labeled. This result, combined with the adenine binding site stoichiometries of two identical sites/seed lectin tetramer or one site/DB58 dimer, indicates that the adenine binding site resides at a heterologous subunit interface. Three radiolabeled peaks from seed lectin and one from DB58 were isolated from chymotryptic digests of the labeled lectins by reverse phase chromatography at pH 7.0. From these four peaks, six unique peptide sequences were determined. When aligned with the concanavalin A sequence, four of these peptides map to three loops in the metal binding domain of concanavalin A. The remaining two sequences represent carboxyl-terminal peptides unique to the D. biflorus lectins which may extend to the putative binding site from adjacent, heterologous subunits. It thus appears that the adenine binding sites of these D. biflorus lectins are within the metal binding domain and adjacent to the carbohydrate binding site.

Directional coupling of synthetic peptides to poly-L-lysine and applications to the ELISA.

Synthetic peptides are increasingly being used as antigens to generate highly specific antisera. Screening the antipeptide sera by enzyme-linked immunosorbent assay (ELISA) can suffer from carrier crossreactivity or lack sensitivity due to poor adsorption or presentation of the peptides. In this work we describe a procedure utilizing a heterobifunctional crosslinker to effect the directional coupling of synthetic peptides to poly-L-lysine preadsorbed to microtiter plates. Plates prepared by this method conferred precision, sensitivity, and specificity to an ELISA for antipeptide antisera. In a competitive ELISA format this method permitted detection of specific peptides to 3.7 x 10(-10) M and provided an assay sensitive to protein structure in solution.

Characterization of the adenine binding sites of two Dolichos biflorus lectins.

The seed lectin and a stem and leaf lectin (DB58) from Dolichos biflorus have high-affinity hydrophobic sites that bind to adenine. The present study employs a centrifugal filtration assay to characterize these sites. The seed lectin contains two identical sites with Ka's of 7.31 x 10(5) L/mol whereas DB58 has a single site with a Ka of 1.07 x 10(6) L/mol. The relative affinities of these sites for a host of adenine analogs and derivatives were determined by competitive displacement assays. The most effective competitors for adenine were the cytokinins, a class of plant hormone, for which the lectins had apparent Ka's of 1.96 x 10(5)-4.90 x 10(4) L/mol. Direct binding of the cytokinin 6-(benzylamino)purine (BAP) to both lectins showed positive cooperativity for only the seed lectin, indicating the interaction of this ligand with more than one class of hydrophobic binding site. Fluorescence enhancement assays demonstrate cooperativity between hydrophobic sites of the seed lectin and also suggest that BAP binds to more than one class of site.

Two lectin genes differentially expressed in Dolichos biflorus differ primarily by a 116-base pair sequence in their 5' flanking regions.

Previous studies in our laboratory have shown that the Dolichos biflorus plant contains two similar lectins, a seed lectin and a stem and leaf lectin called DB58, that are present at different stages in the plant's life cycle. We have now established that each of these lectins is encoded by a separate gene by isolating these lectin genes from a library of D. biflorus nuclear DNA. Restriction mapping and nucleotide sequencing analyses show that the seed lectin and DB58 genes are located in the same transcriptional orientation within 3-kilobase pairs of one another. The lectin genes contain no introns and show greater than 90% nucleotide sequence identity in their protein coding and untranslated regions. This sequence similarity extends to both the 5' and 3' flanking regions of the genes; the major exception is that a 116-base pair segment located at position -215 to -100 from the transcription start site of the seed lectin gene is missing in the 5' flanking region of the DB58 gene. The possible significance of this segment with respect to the differential expression of these genes is discussed.

In Vivo Biosynthetic Studies of the Dolichos biflorus Seed Lectin.

The in vivo biosynthesis of the Dolichos biflorus seed lectin was studied by pulse-chase labeling experiments using [(35)S] methionine and [(14)C]glucosamine. These studies demonstrate that each of the two mature lectin subunit types are derived by the processing of separate glycosylated precursors. The appearance of the precursor to subunit I before the precursor to subunit II supports the possibility raised by previous studies that both subunit types of this lectin may originate from a single gene product.