cDNA cloning and in vitro synthesis of the Dolichos biflorus seed lectin

The Dolichos biflorus seed lectin contains two structurally related subunits. A cDNA library was constructed using RNA isolated from D. biflorus seeds actively synthesizing the seed lectin. The library was expressed in Escherichia coli using a lambda Charon 16 vector, and lectin-specific antiserum was used to isolate a seed lectin cDNA. Hybridization of the D. biflorus seed lectin cDNA to RNA isolated from seeds actively producing both lectin subunits identifies a single-size RNA of 1100 bases. An oligodeoxyribonucleotide probe, constructed from an amino acid sequence common to both lectin subunits, detects the same size RNA. Translation of seed mRNA in vitro and immunoprecipitation of translation products using a lectin-specific antiserum yields a single polypeptide of slightly higher molecular mass than the largest seed lectin subunit. This seed lectin precursor is indistinguishable from a polypeptide synthesized from mRNA hybrid selected by the seed lectin cDNA. These data support the existence of a single polypeptide precursor for both subunit types of the D. biflorus seed lectin and suggest that differences between the subunit types arise by posttranslational processing.     

Primary structure of the Dolichos biflorus seed lectin

The Dolichos biflorus seed lectin is a tetramer composed of equal amounts of two subunit types. The subunit types are structurally very similar, yet only the larger subunit exhibits the ability to bind carbohydrate. A cDNA clone representing the entire coding region of the D. biflorus lectin mRNA has been sequenced. This cDNA represents 1075 nucleotides of seed lectin mRNA encoding a polypeptide of Mr = 29,674. Analysis of the deduced sequence indicates that the NH2 termini and COOH termini of both lectin subunits are present within the mRNA coding region. This information supports previous data indicating that both subunits of the lectin are encoded by a single mRNA and that the difference between the subunit types apparently arises by the proteolytic removal of a 10-amino acid sequence from the COOH terminus of the larger subunit. Comparison of the D. biflorus seed lectin sequence to the sequence of other leguminous seed lectins indicates regions of extensive homology. The residues of concanavalin A involved in metal binding are highly conserved in the D. biflorus lectin, but those involved in saccharide binding show a much lower degree of conservation. Prediction of the secondary conformation of the D. biflorus polypeptide suggests that structures involved in the formation of quaternary structure in concanavalin A are also conserved.     

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.     

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.     

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.     

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.     

The Lectins of Sophora japonica: I. Purification Properties and N-Terminal Amino Acid Sequences of Two Lectins from Leaves

Two lectins, Leaf Lectin I and Leaf Lectin II (LLI and LLII) were purified from the leaves of Sophora japonica. Like the Sophora seed lectin, LLI and LLII are tetrameric glycoproteins containing a single subunit with respect to size. The subunits of LLI (32 kilodaltons) and LLII (34 kilodaltons) are slightly larger than those of the seed lectin (29.5 kilodaltons). The three Sophora lectins display indistinguishable specificities, amino acid compositions, specific hemagglutinin activities, and extinction coefficients. Although very closely related to the seed lectin, the leaf and seed lectins are not immunologically identical and they differ in subunit molecular weights, carbohydrate content, and in the pH sensitivity of their hemagglutinin activities. N-terminal amino acid sequence analysis shows that although they are homologous proteins, the three Sophora lectins are products of distinct genes.     

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.     

The closely related homomeric and heterodimeric mannose-binding lectins from garlic are encoded by one-domain and two-domain lectin genes, respectively.

Lectin cDNA clones for two different lectins from garlic (Allium sativum L.) bulbs, ASAI and ASAII (ASA, Allium sativum agglutinin), were isolated and characterized. The first lectin, ASAI, is a heterodimer composed of two different subunits of 11.5 kDa and 12.5 kDa. It is translated from an mRNA of 1400 nucleotides encoding a polypeptide of 306 amino acids with two very similar domains. N-terminal sequencing of the two polypeptides of the mature lectin confirmed that both subunits are derived from the same precursor and that each corresponds to one of the two domains in the sequence. In contrast to ASAI, the second garlic lectin, ASAII, is a homodimer of two identical 12-kDa subunits. It is translated from an mRNA of approximately 800 nucleotides encoding a polypeptide of 154 amino acids. Interestingly, the coding region of the ASAII cDNA clones is almost identical to that of the second domain of the ASAI cDNA clones.     

Isolation and characterization of a lectin gene from seeds of chickpea (Cicer arietinum L.).

A cDNA library was constructed in lambda TriplEx2 vector using poly (A(+)) RNA from immature seeds of Cicer arietinum. The lectin gene was isolated from seeds of chickpea through library screening and RACE-PCR. The full-length cDNA of Chichpea seed lectin(CpGL)is 972 bp and contains a 807 bp open reading frame encoding a 268 amino acid protein. Analysis shows that CpSL gene has strong homology with other legume lectin genes. Phylogenetic analysis showed the existence of two main clusters and clearly indicated that CpSL belonged to mannose-specific family of lectins. RT-PCR revealed that CAA gene expressed constitutively in various plant tissues including flower, leaf, root and stem. When chickpea lectin mRNA level was checked in developing seeds, it was higher in 10 DAF seeds and decreased throughout seed development.     

Structural differences between two lectins from Cytisus scoparius, both specific for D-galactose and N-acetyl-D-galactosamine.

Three lectin fractions were obtained from seeds of the leguminous plant Cytisus scoparius (Scotch broom) by means of affinity chromatography on a N-acetyl-D-galactosamine medium. The first fraction, termed CSIa, was equally well inhibited in haemagglutination experiments by D-galactose and by N-acetyl-D-galactosamine and consisted of a group of isolectins formed from closely related polypeptide chains of approx. Mr 30000. The second fraction, CSIb, was closely related to CSIa in specificity, c.d. and other properties. The third fraction contained a homogeneous lectin, CSII, formed from subunits again of approx. Mr 30000. CSII was 100 times more readily inhibited by N-acetyl-D-galactosamine than by D-galactose. Despite the similarity in specificity, comparative studies of their amino acid composition, c.d. and N-terminal amino acid sequence showed that the CSIa and CSII lectins diverged considerably in structure. The lectin from Cytisus sessilifolius, specific for chitobiose, was also examined and resembled CSIa in composition and c.d. properties.     

The seed lectins of black locust (robinia pseudoacacia) are encoded by two genes which differ from the bark lectin genes

Two lectins were isolated from Robinia pseudoacacia (black locust) seeds using affinity chromatography on fetuin-agarose, and ion exchange chromatography on a Neobar CS column. The first lectin, R. pseudoacacia seed agglutinin I, referred to as RPsAI, is a homotetramer of four 34 kDa subunits whereas the second lectin, referred to as RPsAII, is composed of four 29 kDa polypeptides. cDNA clones encoding the polypeptides of RPsAI and RPsAII were isolated and their sequences were determined. Both polypeptides are translated from mRNAs of ca. 1.2 kb encoding a precursor carrying a signal peptide. Alignment of the deduced amino acid sequences of the different clones indicates that the 34 and 29 kDa seed lectin polypeptides show 95% sequence identity. In spite of this striking homology, the 29 kDa polypeptide has only one putative glycosylation site whereas the 34 kDa subunit has four of these sites. Carbohydrate analysis revealed that the 34 kDa possesses three carbohydrate chains whereas the 29 kDa polypeptide is only partially glycosylated at one site. A comparison of the deduced amino acid sequences of the two seed and three bark lectin polypeptides demonstrated unambiguously that they are encoded by different genes. This implies that five different genes are involved in the control of the expression of the lectins in black locust.Abbreviations LECRPAs cDNA clone encoding Robinia pseudoacacia seed lectin - LoLI Lathyrus ochrus isolectin I - PsA Pisum sativum agglutinin - RPbAI Robinia pseudoacacia bark agglutinin I - RPbAII Robinia pseudoacacia bark agglutinin II - RPsAI Robinia pseudoacacia seed agglutinin I - RPsAII Robinia pseudoacacia seed agglutinin II     

Isolation and characterization of a cDNA clone encoding the lima bean lectin

The complete amino acid sequence of the lima bean (Phaseolus lunatus) lectin was deduced from the nucleotide sequence of a cDNA clone. The lectin appears to be synthesized as a prepeptide consisting of a signal sequence of 21 residues and a mature protein of 241 amino acids. Comparison of the lima bean lectin sequence to the sequences of other leguminous seed lectins indicates regions of extensive homology. Northern blot analysis showed absence of lectin mRNA in the leaves, roots, or stems of 16-day-old lima bean plants.     

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.     

Isolation and characterization of a lectin from the roots of Dolichos biflorus

A lectin has been isolated from the roots of 7-day-old Dolichos biflorus plants and has been compared with the D. biflorus seed lectin. The root lectin differs from the seed lectin in molecular weight, subunit stoichiometry, amino acid composition, amino terminal amino acid sequence, and isoelectric focusing pattern. However, the root lectin has in common with the seed lectin a specificity for N-acetyl-D-galactosamine, and upon denaturation the root lectin will react weakly with antiserum made to denatured seed lectin. Distribution studies of this lectin in germinating seedlings show that the highest levels of lectin are found in 1-day-old roots. Upon dissection and analysis of 7-day-old roots, the highest levels of the lectin are in the uppermost segment. In addition, isoforms of this lectin also exist in the stems and leaves of the plant.     

A C-type lectin like-domain (CTLD)-containing protein (PtLP) from the swimming crab Portunus trituberculatus

C-type lectins play important roles in the non-self innate immune system of invertebrates. In this study, we isolated the full-length cDNA of the C-type lectin like-domain (CTLD)-containing protein, designated PtLP, from the hepatopancreas of the swimming crab Portunus trituberculatus, one of the most common edible crabs of East Asia. The PtLP cDNA consists of 923bp and encodes a polypeptide of 164 amino acids containing a well-conserved C-type lectin like-domain (CTLD). The deduced amino acid sequence of PtLP shows 29-36% amino acid sequence identity to other crustacean C-type lectin sequences. A phylogenetic analysis revealed that PtLP is in a large cluster together with black tiger shrimp PmAV, a gene involved in virus resistance of shrimp, and all of the C-type lectins from the various shrimps. Quantitative RT-PCR analysis showed that the PtLP mRNA was expressed highly in hepatopancreas and moderately in gills, hemocytes, and ovary of normal swimming crabs.     

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.     

cDNA cloning and expression of Bauhinia purpurea lectin.

Bauhinia purpurea lectin (BPA) was purified from seeds of B. purpurea alba. The purified lectin was digested with an endoproteinase, Asp-N, or trypsin and then the amino acid sequences of the resultant fragments were analyzed. Furthermore, a cDNA library for BPA was constructed using RNA isolated from germinated Bauhinia purpurea seeds. By gene cloning, the nucleotide sequence of BPA cDNA and its deduced amino acid sequence were analyzed. The cloned BPA cDNA comprised 1,152 nucleotides and the open reading frame of the cDNA encodes a polypeptide of 290 amino acids including a signal peptide composed of 28 amino acids. BPA expressed in Escherichia coli showed a relative molecular mass of 29 kDa on sodium dodecyl sulfate-polyacrylamide gel. On comparison of its sequence with those of other leguminous seed lectins, BPA showed high homology to the others.     

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.