Development and Distribution of a Lectin from the Stems and Leaves of Dolichos biflorus

The stems and leaves of the Dolichos biflorus plant contain a lectin that cross-reacts with antiserum against the seed lectin. This cross-reactive material (CRM) was followed during early seedling growth, stem elongation, and seed development using a specific radioimmunoassay.

No CRM was detected in developing seeds, but very low levels were found in dormant and imbibed seeds. As germination proceeds, the CRM accumulates at the apex of both etiolated and green seedlings in the epicotyl and leaves. Lower amounts of CRM are found in the cotyledons and hypocotyl, but no CRM was detected in the roots.

The amount of CRM in the first and second stem internodes increases during elongation and gradually declines after the completion of elongation. Approximately 80% of the CRM in the stems of 19-day-old Dolichos biflorus plants is associated with the elongating tissues. These results are discussed with respect to the possible roles of lectins in plants.

     

Production of a Lectin in Tissue Cultures of Dolichos biflorus

Callus cultures have been produced from the epicotyl and leaves, hypocotyl, and roots of germinating Dolichos biflorus seeds. These cultures were initiated on media containing 2,4-dichlorophenoxyacetic acid and kinetin, transferred to media with increased amounts of these hormones, and then maintained on hormone-free media. Extracts of these cultures were examined by radioimmunoassays specific for the lectin from the seeds of this plant and for a lectin that is present only in the stems and leaves of the intact plant. Although the seed lectin was not detected in any cultures, the stem and leaf lectin was produced in those cultures grown on the hormone free media. Lectin isolated from these cultures had subunits identical in electrophoretic mobilities to the subunits from the lectin isolated from intact stems and leaves. Levels of this lectin decreased when the cells were transferred back to media containing hormones and increased again upon transfer to the hormone-free media. The absence of exogenous hormones and the production of lectin were also correlated with the rapid growth and greening of the cells. Immunofluorescence and immunocytochemical studies on sections of cultured cells indicated that the stem and leaf lectin is associated with the cytoplasm as well as the cell wall as has been found in previous studies on the subcellular localization of this lectin in the intact plant.     

Subcellular Distribution of Ependymins in Goldfish Brain Measured by Radioimmunoassay

Goldfish CNS was fractionated by differential and density gradient centrifugation. The fractions obtained were characterized by marker enzymes typical of various subcellular organelles. They were further analyzed by radioimmunoassay for their contents of ependymins, two CNS glycoproteins known to participate in biochemical reactions after learning events. Ependymins were shown to be major constituents of the soluble cytoplasm (5.6% of the total protein content). The nuclear fraction was virtually devoid of ependymins (0.6% of protein). Small amounts were observed in the crude synaptosomal and microsomal fractions (1.0 and 3.5%, respectively). The highest steady-state concentration of ependymins, however, was measured in the brain extracellular fluid (15.6% of the protein), including the CSF. The specificity of the distribution was examined by intracerebroventricular injection of 125I-labeled ependymins as exogenous marker substances. No indication of an artificial redistribution of the radiolabel during homogenization and fractionation was obtained. The exogenous analogues of ependymins were, however, incorporated in vivo into organelles recovered in the nuclear and crude synaptosomal fractions. Our results suggest that ependymins may interact with synaptic membranes from the extracellular fluid, although so far no evidence for a specific receptor-type binding site could be obtained in vitro.     

Subcellular Localizations of Two Dolichos biflorus Lectins

The subcellular localizations of the Dolichos biflorus seed lectin and the structurally related lectin (cross-reactive material [CRM]) from the stems and leaves of this plant were determined by immunofluorescence, immunocytochemistry, and cell fractionation procedures. Subcellular fractionation of the cotyledons using a nonaqueous procedure to minimize disruption of the protein bodies showed that the majority of the seed lectin was associated with the protein body fraction and some lectin was also present in the starch granules. Immunofluorescence and immunocytochemistry at the light microscopic level showed that the seed lectin was mainly localized at the peripheries of these organelles. Lectin was also found in the cytoplasm of the cells, although the amount appeared to be dependent upon the degree of protein body disruption.

Immunofluorescence and immunocytochemistry studies of the stem and leaf lectin (CRM) indicated that a significant portion of this lectin may be associated with the cell walls, although lectin was also seen in the cytoplasm of plasmolyzed cells. Extraction and cell fractionation studies showed that a large portion of the CRM is readily solubilized and most of the remainder is pelleted at 1000g. The CRM can be extracted from these pellets by treatment with cellulase and pectinase; other reagents such as NaCl, detergents, and EDTA could also release significant amounts of CRM. These studies suggest that the CRM is noncovalently bound to the cell walls. A comparison of the distribution of exogenously supplied [¹²⁵I]CRM with the endogenous CRM during extraction and cell fractionation indicates that soluble CRM is not adsorbed to the 1000g pellet during fractionation.

The different subcellular distributions of these two structurally related lectins suggest that different tissues of the same plant may utilize lectins for different functions.

     

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.     

An isoflavone diglycoside from the seeds of Dolichos biflorus

-5-Hydroxy-7,3′,4′-trimethoxy-8-methylisoflavone 5-neohesperidoside has been identified from the seeds of Dolichos biflorus.     

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.     

Dolichins A and B,two pterocarpans from bacteria-treated leaves of Dolichos biflorus

Two minor isoflavonoids isolated from bacteria-inoculated leaves of Dolichos biflorus have been identified as the 6aR; 11aR; 2′S and 6aR; 11aR; 2′S isomers of 3,9-dihydroxy-10-2′-hydroxy-3′-methyl-3′-butenyl pterocarpan.     

Organ-related differences in binding of Dolichos biflorus agglutinin to vascular endothelium

We screened cryostat sections of tissues from 10 inbred strains of mice with a panel of conjugated lectins, in a search for polymorphisms which could be used as markers in mouse chimeras. In DDK and RIII mice, but not the other strains tested, there are binding sites on the membrane of vascular endothelial cells for Dolichos biflorus agglutinin (DBA), which is specific for terminal nonreducing N-acetyl galactosamine residues. The expression of DBA binding sites on RIII and DDK endothelium is not, however, uniform: there are consistent differences between blood vessels in different organs. These observations provide evidence of an organ-related heterogeneity in vascular endothelium which is open to biochemical analysis, and which raises the possibility that endothelial cells carry a “tissue address.”     

Circular dichroism and conformational transition of Dolichos biflorus and Robinia pseudoacacia lectins.

The conformation of the lectins from Dolichos biflorus and Robinia pseudoacacia was studied by means of circular dichroism (CD). It was found that N-acetyl-D-galactosamine induced significant changes in the near-ultraviolet CD spectrum of Dolichos lectin but was ineffective with the lectin from Robinia. Tyrosine and tryptophan chromophores were chiefly involved in this saccharide-lectin interaction. The far-ultraviolet CD spectra indicated that both lectins have a significant content of the pleated sheet conformation, but not much, if any, alpha-helix. The predominant conformation in these lectins is the aperiodic bend structure which is stabilized chiefly by hydrophobic interactions. This was ascertained by the effect of sodium dodecylsulfate on these proteins.     

Properties of the human erythrocyte membrane receptors for peanut and Dolichos biflorus lectins.

Neuraminidase treatment of blood type A and B human erythrocytes, which is required for the agglutination of these cells by peanut (Arachis hypogaea) lectin, increased the number of receptor sites for the lectin from about 5 × 10⁴ to 1.8 × 10⁶ sites/ cell for both blood types. The same treatment also increased the agglutinability of type A cells by the blood group A-specific Dolichos biflorus lectin, but the number of receptor sites for this lectin (~6 × 10⁵ sites/cell) did not change. D. biflorus lectin binding and agglutination of blood type B cells were negligible both before and after neuraminidase treatment. To isolate the peanut agglutinin receptor from the membrane of these cells, washed type A erythrocytes were incubated with neuraminidase and galactose oxidase and then treated with NaB³H₄, thus labeling the galactose residues on the membrane. For measuring peanut agglutinin receptor activity, a radioaffinity assay was developed based on the displacement of [¹⁴C]asialofetuin from peanut agglutinin by receptor and precipitation of the complex in the presence of polyethyleneglycol. Membranes were isolated by hypotonic lysis and were solubilized in 0.5% Empigen BB, a zwitterionic detergent, which was found to be superior to Triton X-100 for this purpose. The cell extract, after centrifugation, was subjected to affinity chromatography on peanut agglutinin-polyacrylhydrazido-Sepharose. Elution with lactose afforded a peak of radioactivity (32% yield) containing 70% of the applied receptor activity. The eluting sugar and the receptor were separated by chromatography on Bio-Gel P-2 with subsequent dialysis against 80% acetone to remove the detergent. The bulk of the isolated receptor radioactivity (91%) precipitated with peanut agglutinin. The amino acid composition, the glucosamine and galactosamine content and the electrophoretic mobility, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the peanut receptor were similar to those of asialoglycophorin. In addition, the peanut receptor coprecipitated with asialoglycophorin and with isolated erythrocyte T antigen on Ouchterlony double-diffusion plates against peanut agglutinin and the Ricinus communis lectin, but not with D. biflorus lectin, suggesting that the receptor for the latter lectin is distinct from the peanut agglutinin receptor.     

Formation of Bowman-Birk inhibitors during the germination of horsegram (Dolichos biflorus)

Three Bowman-Birk type inhibitors (HGGI-I, II and III), which appear in the cotyledons of 120 h germinated horsegram (Dolichos biflorus) seeds have been purified to homogeneity by size-exclusion chromatography and ion-exchange chromatography. HGGI-I, HGGI-II and HGGI-III differ from each other and from the dormant seed inhibitors in amino acid composition, molecular size and charge. The amino-terminal sequence analyses indicate that these inhibitors are derived from the isoinhibitors of the dormant seed by a limited proteolysis and not by de novo synthesis. These inhibitors differ from each other at their amino-terminus. HGGI-II identical to HGGI-I except for the loss of a single amino-terminal aspartyl residue, where as HGGI-III shows the loss of a pentapeptide. All the three inhibitors are potent competitive inhibitors of trypsin and chymotrypsin. The dissociation constants (K(i)s) for trypsin inhibition indicate that amino-terminal tail of the inhibitors play a role in trypsin binding probably through electrostatic interaction.     

Application of Dolichos biflorus in immunoassay detection of kidney collecting duct biomarkers

Currently there are no biomarkers for detecting collecting duct damage in man. Antibodies to several collecting duct-specific antigens exist but sandwich assays have been difficult to establish due to the need for two different antibodies to the same protein. We hypothesized that a collecting duct-specific lectin could be used in combination with a collecting duct-specific antibody to negate the need for two different antibodies. The collecting duct specificity of selected antibodies (NiCa II 13C2, Pap XI 3C7, HuPaP VII 2B11 and aquaporin 2), was verified by immunohistochemistry. Aquaporin 2 and Pap XI 3C7 were used successfully in setting up assays with the lectin Dolichos biflorus, using the Meso Scale Discovery (MSD) platform. Antigen expression was highest in the papillae of rat and human kidney (corresponding to the greatest density of collecting ducts) and was also present in normal urine. We propose that further qualification and validation would lead to an assay for detecting collecting duct damage in man.     

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 and characterization of subunits from the predominant form of Dolichos biflorus lectin.

The subunits of the two molecular forms (A and B) of the Dolichos biflorus lectin were isolated by ion-exchange chromatography on DEAE-cellulose in 8.0 M urea. Subunits IA and IIA which comprise the predominant molecular form A of the lectin were found to have molecular weights of 27,700 and 27,300, respectively, as determined by sedimentation equilibrium studies in 8.0 M urea. These subunits have similar amino acid compositions and each have alanine at their amino-terminal ends. Comparison of the IA and IIA subunits by immunodiffusion against antisera to the seed extract as well as to subunits IA and IIA showed no antigenic differences between the two subunits. Carboxyl terminal analyses of subunits IA and IIA with carboxypeptidase A produced an essentially simultaneous release of both leucine and valine residues from subunit IA; no detectable amino acids were released from subunit IIA under identical conditions. The data suggest that the molecular form A of the lectin (molecular weight 113,000, Carter and Etzler, 1975) consists of four subunits with a possible stoichiometry of IA2IIA2. Other possible arrangements of the subunits are discussed.     

On the frequency of isothermal germination in seeds of Dolichos biflorus L.

The relative frequencies of seed germination show positivelyskewed distributions along the time of isothermal incubation.Unimodal patterns prevailing between 19.3? and 31.4?C changeinto progressively polymodal distributions both below 19.3?and above 31.4?C. None of the distributions fit the adjustedgaussians, except at 8.0? and at 40.6?C. (Received September 29, 1977; )     

Nature of the tryptic/chymotryptic inhibitor from horsegram (Dolichos biflorus).

A protease inhibitor specific to trypsin and chymotrypsin was purified from horsegram (Dolichos biflorus) with the inhibition index 0.24 micrograms/micrograms for trypsin and 0.36 micrograms/micrograms for chymotrypsin. In SDS-PAGE, the inhibitor protein was seen as a single band with apparent molecular mass Mr = 15,500. However, on fast protein liquid chromatography (FPLC) or non-denaturating PAGE, the inhibitor resolved into four components revealing the existence of isoinhibitors. Data on amino acid analysis indicate that the isoinhibitors are closely related. The major amino acids in the inhibitor are half cystine (18.9 mole %), aspartic acid (12.7 mole %) and serine (14.3 mole %). The inhibitor was partially stable to 0.1% sodium dodecyl sulphate, 8M urea or 6M guanidine hydrochloride. The inhibitory activity was lost on reduction or carboxamidomethylation or acetylation. Modification of the arginine groups or CNBr cleavage of the protein did not result in significant loss of either tryptic or chymotryptic inhibitory activities. The isoinhibitors separated by FPLC reacted with polyclonal antibody raised in rabbits and had pI values ranging from 4.8-5.1. The horsegram inhibitor thus resembles other Bowman-Birk protease inhibitors.     

Purification and properties of a subtilisin inhibitor and an associated trypsin inhibitor from Dolichos biflorus

A subtilisin inhibitor and an associated trypsin inhibitor from Dolichos biflorus were purified to homogeneity by conventional methods such as chromatography on DEAE-cellulose, gel filtration on Sephadex G-75, PAGE and affinity chromatography. The final preparations were homogeneous on PAGE. Their pI were 7.66 and 7.70, respectively. The dissociation constant of the complex of the inhibitor with subtilisin was 2.69.10(-10) M. Both the inhibitors were stable to heat, TCA and ethanol. The molecular weights of the subtilisin inhibitor and the associated trypsin inhibitor by gel filtration were 7500 and 8200, respectively.