The effects of Phaseolus vulgaris erythro- and leucoagglutinating isolectins (PHA-E and PHA-L) delivered via artificial diet and transgenic plants on the growth and development of tomato moth (Lacanobia oleracea) larvae; lectin binding to gut glycoproteins in vitro and in vivo

Red kidney bean, Phaseolus vulgaris, contains a lectin phytohemagglutinin (PHA) with toxicity towards higher animals. PHA exists in the isoforms PHA-E and PHA-L, which agglutinate erythrocytes and lymphocytes, respectively. Lacanobia oleracea larvae were reared from hatch on artificial diets containing PHA-E or PHA-L at 2% (w/w) dietary protein, and on transgenic Arabidopsis plants expressing either lectin at 0.4-0.6% of total soluble proteins. In artificial diet bioassays neither lectin affected larval survival, development, growth nor consumption. In transgenic plant bioassays both PHA-E and PHA-L promoted larval growth and development. This effect was greatest for PHA-E. Mean larval biomass of insects fed on plants expressing PHA-E was significantly greater (up to two-fold) than controls during the final two instars and the insects developed at a significantly greater rate so that after 26 days 83% of PHA-E exposed insects were in the final instar compared to 44% for control insects. PHA-E and PHA-L were detected by Western blotting in haemolymph, sampled from insects fed diets or plant material containing the lectins. However, despite the demonstrated potential for both isolectins to bind to gut glycopolypeptides in vitro neither was found to accumulate in vivo in the guts of exposed insects. Since lectin binding to gut polypeptides is thought to be necessary for insecticidal activity the failure of PHA-E and PHA-L to bind in vivo may account for their lack of toxicity to L. oleracea.     

Differences in expression between two seed lectin alleles obtained from normal and lectin-deficient beans are maintained in transgenic tobacco

Using Agrobacterium-mediated transformation, two genes for phytohemagglutinin-L (PHA-L), the lectin seed protein of the common bean Phaseolus vulgaris, were stably integrated into the tobacco genome. The two alleles for PHA-L, dlec2 and pdlec2, were obtained from a normal cultivar (Greensleeves) and a lectin-deficient cultivar (Pinto) respectively. In the bean embryos, the expression of dlec2 is 30 times greater than the expression of pdlec2. In the dlec2-transformed tobacco, PHA-L accumulated specifically in the seeds at the same stages as the tobacco seed storage proteins and was degraded after germination. PHA-L was found in the embryo, and at a 5–7 times lower concentration in the endosperm tissue of the mature tobacco seeds. No PHA could be detected in other parts of the plants. We conclude that the signals for temporal and spatial regulation of the dlec2 gene are present in the DNA fragment used for transformation. Transformation with the second PHA-L allele pdlec2 from the cultivar Pinto caused the accumulation of about 50 times less PHA-L in tobacco seeds when compared to dlec2. We conclude from analyzing the 5' sequences of dlec2 and Pdlec2 that the low expression phenotype of the Pdlec2 allele could be due to the absence or mutation of a cis-acting element carried by the dlec2 fragment.     

Correct glycosylation,Golgi-processing,and targeting to protein bodies of the vacuolar protein phytohemagglutinin in transgenic tobacco

We used a heterologous system (transgenic Nicotiana tabacum L.) to investigate the processing, assembly and targeting of phytohemagglutinin (PHA), the lectin of the common bean, Phaseolus vulgaris L. In the bean, this glycoprotein accumulates in the protein bodies of the storage parenchyma cells in the cotyledons, and each polypeptide has a high-mannose glycan attached to Asn12 and a complex glycan on Asn60. The gene for PHA-L, dlec2, with 1200 basepairs (bp) 5 upstream and 1600 bp 3 downstream from the coding sequence was introduced into tobacco using Agrobacterium-mediated transformation (T. Voelker et al., 1987, EMBO J. 6, 3571–3577). Examination of thin sections of tobacco seeds by immunocytochemistry with antibodies against PHA showed that PHA-L accumulated in the amorphous matrix of the protein bodies in the embryo and endosperm. This localization was confirmed using a non-aqueous method to isolate the protein bodies from mature tobacco seeds. The biochemical analysis of tobacco PHA indicated that the signal peptide had been correctly removed, and that the polypeptides formed 6.4 S oligomers; tobacco PHA had a high-mannose glycan at Asn12 and a complex glycan at Asn60. The presence of the complex glycan shows that transport to the protein bodies was mediated by the Golgi complex. At seed maturity, a substantial portion of the PHA-L remained associated with the endoplasmic reticulum and the Golgi complex, as indicated by fractionation experiments using aqueous media and the presence of two high-mannose glycans on some of the polypeptides. Taken together, these data show that insertion of the nascent PHA into the endoplasmic reticulum, signal peptide processing, glycosylation, assembly into oligomers, glycan modification in the Golgi, and targeting of the protein occur faithfully in this heterologous system, although transport may not be as efficient as in bean cotyledons.Abbreviations Asn asparagine - Endo H endoglycosidase H - HPLC high-performance liquid chromatography - IgG immunoglobulin G - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PHA phytohemagglutinin - SDS sodium dodecylsulfate - TFMS trifluoromethanesulfonic acid     

Synthesis of Lectin-Like Protein in Developing Cotyledons of Normal and Phytohemagglutinin-Deficient Phaseolus vulgaris

The genome of the common bean Phaseolus vulgaris contains a small gene family that encodes lectin and lectin-like proteins (phytohemagglutinin, arcelin, and others). One of these phytohemagglutinin-like genes was cloned by L. M. Hoffman et al. ([1982] Nucleic Acids Res 10: 7819-7828), but its product in bean cells has never been identified. We identified the product of this gene, referred to as lectin-like protein (LLP), as an abundant polypeptide synthesized on the endoplasmic reticulum (ER) of developing bean cotyledons. The gene product was first identified in extracts of Xenopus oocytes injected with either cotyledonary bean RNA or LLP-mRNA obtained by hybrid-selection with an LLP cDNA clone. A tryptic map of this protein was identical with a tryptic map of a polypeptide with the same SDS-PAGE mobility detectable in the ER of bean cotyledons pulse-labeled with either [³H]glucosamine or [³H]amino acids, both in a normal and in a phytohemagglutinin-deficient cultivar (cultivars Greensleeves and Pinto UI 111). Greensleeves LLP has M_r 40,000 and most probably has four asparagine-linked glycans. Pinto UI 111 LLP has M_r 38,500. Unlike phytohemagglutinin which is a tetramer, LLP appears to be a monomer by gel filtration analysis. Incorporation of [³H]amino acids indicates that synthesis of LLP accounts for about 3% of the proteins synthesized on the ER, a level similar to that of phytohemagglutinin.     

Gene Expression and Synthesis of Phytohemagglutinin in the Embryonic Axes of Developing Phaseolus vulgaris Seeds

Phytohemagglutinin (PHA), the major seed lectin of the common bean (Phaseolus vulgaris), is found largely in the cotyledons, but is also present in the embryonic axis. At mid-maturation, the percentage of total protein synthesis which is directed towards making PHA is 5 to 10 times greater in the cotyledons than in the axes. This lower rate of synthesis in the axes is correlated with a lower abundance of mRNA for PHA, as determined by dot blot hybridization using a cDNA clone for PHA. Manen and Pusztai (Planta 1982 155: 328-334) have claimed on the basis of immunocytochemical evidence that, in the axis, PHA is found in the cytosol although it is present in protein bodies in the cotyledons. In the cotyledons, PHA is synthesized on rough endoplasmic reticulum, and its transport to the protein bodies via the Golgi complex is associated with specific posttranslational processing steps (Vitale and Chrispeels, J Cell Biol 1984 In press). A cytosolic localization of axis PHA would be an indication of a different site of synthesis and transport pathway. The results presented here indicate that the site of synthesis of PHA and the posttranslational modifications of PHA are the same in the axes as in the cotyledons. Since in the cotyledons these modifications take place in the endoplasmic reticulum, the Golgi, and the protein bodies, it appears that the transport pathway and the site of accumulation of PHA in the axes is similar to that in the cotyledons. On the basis of our evidence, we suggest that the subcellular localization of PHA in the axes should be reexamined.     

Secretion of phytohemagglutinin by monkey COS cells

The entire coding region of a gene, which encodes a polypeptide of phytohemagglutinin (PHA-L), obtained from a library of genomic DNA of the common bean Phaseolus vulgaris cv. Greensleeves, was introduced into the SV40 expression vector pJC119. Monkey COS1 cells were transfected with the recombinant clone and the synthesis, glycosylation, and transport of PHA-L studied and compared with the normal processes in bean cotyledons. In the bean, phytohemagglutinin is synthesized on the rough endoplasmic reticulum and transported via the Golgi complex to protein bodies, vacuole-like organelles. Phytohemagglutinin was synthesized and glycosylated at the ER and processed in the Golgi apparatus of the transfected COS1 cells. After passing the Golgi apparatus, PHA-L was slowly secreted into the culture medium (half-time of 3-6 h), a result indicating that the signals for targeting proteins beyond the Golgi apparatus in plant cells are different from those in animal cells. PHA, which is stored in protein bodies in the plant cells, is secreted by animal cells. Tunicamycin inhibited both glycosylation and secretion of PHA by the COS1 cells, a finding indicating an essential role of the oligosaccharides for transport of PHA in these cells in contrast to the situation found in bean cotyledons. PHA, secreted into the culture medium, was partially sensitive to endo H, a result indicating the presence of one high-mannose and one complex oligosaccharide chain, a situation identical to that in beans.     

Characterisation of N-glycans bound to IGFBP-3 in sera from healthy adults

Human IGFBP-3 contains three potential N-linked glycosylation sites. Published data concerning the type and saccharide composition of the N-glycans is scarce. The aim of this study was to characterise N-glycans covalently attached to IGFBP-3 from sera of healthy adults (men and women). In order to do that a panel of eight lectins covering broad saccharide specificity was used: agarose-immobilised SNA (Sambucus nigra agglutinin), Con A (lectin from Canavalia ensiformis), RCA I (Ricinus communis agglutinin I), PHA-E (Phaseolus vulgaris erythroagglutinin), PHA-L (P. vulgaris leukoagglutinin), succinylated WGA (wheat germ agglutinin), ECL (Erythrina cristagalli lectin) and UEA (Ulex europaeus agglutinin). IGFBP-3 interacted with SNA, Con A, RCA I, PHA-E and, to a much lesser extent, with PHA-L. These results indicate that human IGFBP-3 bears mostly biantennary complex type N-glycans with a very high content of α-2,6-linked Sia at their termini. Hybrid type and high-mannose type N-glycans are present, as well as a bisecting GlcNAc residue, which may be core fucosylated. N-glycosylation of IGFBP-3 follows the N-glycosylation pattern of major serum proteins. This study represents a ground for the future research of glycosylation pattern of IGFBP-3 from the circulation of men and women diagnosed with different illnesses.     

Mutation of Asn128 to Asp of Phaseolus vulgaris leucoagglutinin (PHA-L) eliminates carbohydrate-binding and biological activity

Phytohaemagglutinin (PHA) is the major lectin present in theseeds of the common bean, Phaseolus vulgaris, and PHA-L is theleucocyte-agglutinating form of this lectin. This tetramericglycoprotein accumulates in the vacuoles of storage parenchymacells. Based on amino acid sequence comparisons of legume lectinsand the three-dimensional structure of lectin-carbohydrate complexes,Asn¹²⁸ can be identified as a likely candidate for site-directedmutagenesis to create a mutant PHA-L that does not bind carbohydrate.PHA-L N¹²⁸     

Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA) expressed in Pichia pastoris correct N-terminal processing and secretion of heterologous proteins expressed using the PHA-E signal peptide.

Phytohemagglutinin (Phaseolus vulgaris agglutinin; PHA; E- and L-forms) and snowdrop lectin (Galanthus nivalis agglutinin; GNA) were expressed in Pichia pastoris using native signal peptides, or the Saccharomyces alpha-factor preprosequence, to direct proteins into the secretory pathway. PHA and GNA were present as soluble, functional proteins in culture supernatants when expressed from constructs containing the alpha-factor preprosequence. The recombinant lectins, purified by affinity chromatography, agglutinated rabbit erythrocytes at concentrations similar to the respective native lectins. However, incomplete processing of the signal sequence resulted in PHA-E, PHA-L and GNA with heterogenous N-termini, with the majority of the protein containing N-terminal extensions derived from the alpha-factor prosequence. Polypeptides in which most of the alpha-factor prosequence was present were also glycosylated. Inclusion of Glu-Ala repeats at the C-terminal end of the alpha-factor preprosequence led to efficient processing N-terminal to the Glu-Ala sequence, but inefficient removal of the repeats themselves, resulting in polypeptides with heterogenous N-termini still containing N-terminal extensions. In contrast, PHA expressed with the native signal peptide was secreted, correctly processed, and also fully functional. No expression of GNA from a construct containing the native GNA signal peptide was observed. The PHA-E signal peptide directed correct processing and secretion of both GNA and green fluorescent protein (GFP) when used in expression constructs, and is suggested to have general utility for synthesis of correctly processed proteins in Pichia.     

The plant vacuolar protein, phytohemagglutinin, is transported to the vacuole of transgenic yeast

Phytohemagglutinin (PHA), the major seed lectin of the common bean, Phaseolus vulgaris, accumulates in the parenchyma cells of the cotyledons. It has been previously shown that PHA is cotranslationally inserted into the endoplasmic reticulum with cleavage of the NH2-terminal signal peptide. Two N-linked oligosaccharide side chains are added, one of which is modified to a complex type in the Golgi apparatus. PHA is then deposited in membrane-bound protein storage vacuoles which are biochemically and functionally equivalent to the vacuoles of yeast cells and the lysosomes of animal cells. We wished to determine whether yeast cells would recognize the vacuolar sorting determinant of PHA and target the protein to the yeast vacuole. We have expressed the gene for leukoagglutinating PHA (PHA-L) in yeast under control of the yeast acid phosphatase (PHO5) promoter. Under control of this promoter, PHA-L accumulates to 0.1% of the total yeast protein. PHA-L produced in yeast is glycosylated as expected for a yeast vacuolar glycoprotein. Cell fractionation studies show that PHA-L is efficiently transported to the yeast vacuole. This is the first demonstration that vacuolar targeting information is recognized between two highly divergent species. A small proportion of yeast PHA-L is secreted which may be due to inefficient recognition of the vacuolar sorting signal because of the presence of an uncleaved signal peptide on a subset of the PHA-L polypeptides. This system can now be used to identify the vacuolar sorting determinant of a plant vacuolar protein.     

Subcellular effects and localization of binding sites of phytohemagglutinin in the potato leafhopper, Empoasca fabae (Insecta: Homoptera: Cicadellidae)

To identify the means by which phytohemagglutinin (PHA) exerts its toxicity on the potato leafhopper, four different methods (thick and semi-thin sectioning combined with immunofluorescent staining, in vitro receptor autoradiography, and immunoelectron microscopy) were used to elucidate the PHA target tissue, binding site, and its effects on this tissue. Sixteen 1- or 2-day-old female potato leafhoppers were fed for 36 h on each of three treatments: a control, diet or a diet containing either the PHA-E subunit or the PHA-L subunit. The PHA-E subunit, but not PHA-L, had previously been shown to be lethal. The insects were then prepared for both light and confocal microscopy. Analysis of images showed that PHA bound only to the surface of midgut epithelial cells of the potato leafhopper. PHA-E caused severe disruption, disorganization, and elongation of the brush border microvilli, and swelling of the epithelial cells into the lumen of the gut, leading to complete closure of the lumen. Furthermore, PHA-E stimulated the division of midgut epithelial cell nuclei, leading to two nuclei in each cell. Nuclei later elongated and degraded. In contrast, PHA-L had little effect on the epithelial cells of the midgut. It did not strongly bind to the surface of epithelial cells and caused much less disruption of brush-border microvilli, less disorganization of the cells and less elongation of nuclei. Strong binding of PHA occurred solely on the cell membrane of the brush border microvilli of epithelial cells. In contrast, the controls (i.e., midgut tissue, blocking agent, PHA, and antibodies) showed that midgut tissue was not autofluorescent and showed no fluorescent binding signal. Analysis of both bright- and dark-field images obtained by autoradiography and immunoelectron microscopy confirmed these findings.     

A short domain of the plant vacuolar protein phytohemagglutinin targets invertase to the yeast vacuole.

Phytohemagglutinin (PHA), the seed lectin of the common bean, accumulates in protein storage vacuoles of storage parenchyma cells in cotyledons. When expressed in yeast, PHA is efficiently targeted to the yeast vacuole [Tague and Chrispeels (1987). J. Cell Biol. 105, 1971-1979]. To identify vacuolar sorting information in PHA, a series of 3' deletions of the PHA gene were fused in-frame to a truncated yeast invertase gene. An amino-terminal portion of PHA composed of a 20-residue signal sequence and 43 residues of the mature protein efficiently targeted invertase to the yeast vacuole. Internal deletions in a short PHA-invertase fusion showed that targeting information exists between residues 14 and 23 of mature PHA. Based on examinations of three-dimensional structures of related lectins, only a portion of these residues would be available on the surface of PHA for interaction with a putative receptor. Amino acid replacements at these positions in a PHA-invertase hybrid caused secretion of the invertase. The results indicate the presence of a vacuolar targeting domain in PHA that is centered at position 19 of the mature protein. This sequence of PHA also shows sequence identity to a vacuolar sorting domain characterized in yeast carboxypeptidase Y. Single amino acid alterations in a short PHA-invertase hybrid protein that caused the highest levels of secretion introduced a glycosylation site at position 21 of PHA. This observation suggests that glycan addition may interfere with recognition of a sorting determinant. These same amino acid changes did not dramatically increase secretion in a long PHA-invertase fusion or in PHA itself. Thus, a second domain of PHA may function in concert with the first one to bring about correct targeting of PHA.     

Isolation and characterization of two tightly linked catalase genes from castor bean that are differentially regulated

Two catalase genes,cat1 andcat2, have been isolated from the castor bean genome. They were located in the same direction on a chromosome at a distance of 2.4 kb,cat1 being on the downstream side ofcat2. The two genes contained introns at the same positions except that one of the 7 introns incat1 is missing incat2 and the corresponding introns differed in size and sequence between the two genes. The translated regions of the two genes had the same number of nucleotides and exhibited 81.3% nucleotide sequence identity. In addition to introns, the nucleotide sequences of the 5-and 3-flanking regions are highly divergent between the two genes. In etiolated seedlings,cat1 mRNA was present abundantly in endosperms and cotyledons and only in a small amount in roots. Thecat1 mRNA could not be detected in hypocotyls. By contrast,cat2 mRNA is most abundant in hypocotyls and roots, while endosperms and cotyledons contained only low levels ofcat2 mRNA. Although neithercat1 norcat2 mRNA could be detected in dry seeds, both mRNAs showed temporal accumulation in the endosperm in response to germination. These results suggest that expression of two tightly linked catalase genes of castor bean,cat1 andcat2, are differentially regulated during development.     

Isolation and Partial Characterization of a Seed Lectin from Tepary Bean that Delays Bruchid Beetle Development

Four isolectin forms of a seed lectin from mature seed of tepary bean (Phaseolus acutifolius) were isolated using solubility fractionation, affinity chromatography, and high performance liquid chromatography. The subunits are polypeptides with an apparent molecular mass of 30,000 daltons. The 30 kilodalton subunits are produced starting approximately 13 days after flowering and subsequently comprise a major fraction of the proteins found in the mature seed. The amino terminus of each isolectin fraction was determined to be highly homologous with that of the subunits of common bean (Phaseolus vulgaris L.) phytohemagglutinin (PHA). The tepary isolectin cross-reacts with both erythroagglutinating and leucoagglutinating subunits of PHA antibodies, although differential cross-reactivity was noted. A seed protein fraction enriched in tepary bean lectin was found to be toxic to bean bruchid beetles (Acanthoscelides obtectus), when incorporated into their diets at incremental concentrations from (1-5% w/w) above that of PHA concentrations in mature seeds of the susceptible common bean variety “Red Kidney.”     

Posttranslational processing of proteins in vacuoles and protein bodies is inhibited by monensin

A number of proteins that accumulate in vacuoles and protein bodies undergo posttranslational processing at these accumulation sites. These processing steps include proteolytic cleavage (e.g. pea lectin, soybean glycinin, and rice lectin) and the removal of some sugar residues from oligosaccharide side-chains (e.g. bean phytohemagglutinin). Treatment of immature rice embryos with the sodium ionophore monensin slows down the proteolytic processing of the rice lectin precursor (M_r 23,000) to mature rice lectin (M_r 10,000 and 8,000). Treatment of developing bean cotyledons with monensin slows down the removal of peripheral N-acetylglucosamine residues from the oligosaccharide side-chains of phytohemagglutinin. The results are consistent with the interpretation that these processing steps, which occur in vacuoles or protein bodies, are carried out by enzymes with an acidic pH optimum, and that monensin slows down processing by alkalinization of the vacuoles or protein bodies.     

Cassettes for seed-specific expression tested in transformed embryogenic cultures of soybean

Soybean (Glycine max [L.] Merrill) lectin is a seed protein that accumulates in protein bodies of cotyledons during seed development. We have constructed two expression cassettes containing the 5′ and 3′ regions of the soybean lectin gene connected by aNot I restriction site. One vector also contains the 32 amino acid signal sequence. Using polymerase chain reaction (PCR), the coding region of the β-glucuronidase (uidA) gene was inserted into theNot I site of each vector. We tested the function of the expression cassettes in transformed embryogenic cultures of soybean. Development-specific GUS expression was observed in developing somatic embryos transformed with the chimeric lectin promoter-GUS constructs as determined by histochemical assays. Our data indicate that these cassettes could be used to drive expression of foreign genes to modify embryo-specific traits of soybean as protein quality or quantity in the seed.     

Dynamic of lectin activity during germination of bean seeds (Phaseolus vulgaris L.)

PHA quantity and activity dynamics during early germination of bean seed were investigated. Electrophoretic characteristics, subunits composition and carbohydrate-binding specificity of lectin extracted from white kidney bean cv. Bilozerna were studied. It was shown that investigated lectin consisted of 2 subunits E and L with molecular weight 34 and 36 kDa, respectively, analogously to purified PHA ("Serva", Germany), and specifically bound N-acetyl-D-galactosamin and galactose. During germination both quantity and activity of PHA were dramatically decreasing in embryonic axes and in cotyledons, possibly, as a result of the lectin release from seeds to the environment. It is very likely that one of the defence mechanisms of germinating seeds is related with the releasing of lectins that are able to bind components of the bacterial cell wall and to inhibit their growth.