Intracellular Trafficking and Glycobiology of TbPDI2, a Stage-Specific Protein Disulfide Isomerase in Trypanosoma brucei

Trypanosoma brucei protein disulfide isomerase 2 (TbPDI2) is a bloodstream stage-specific lumenal endoplasmic reticulum (ER) glycoprotein. ER localization is dependent on the TbPDI2 C-terminal tetrapeptide (KQDL) and is mediated by TbERD2, an orthologue of the yeast ER retrieval receptor. Consistent with this function, TbERD2 localizes prominently to ER exit sites, and RNA interference (RNAi) knockdown results in specific secretion of a surrogate ER retention reporter, BiPN:KQDL. TbPDI2 is highly N-glycosylated and is reactive with tomato lectin, suggesting the presence of poly-N-acetyllactosamine modifications, which are common on lyso/endosomal proteins in trypanosomes but are inconsistent with ER localization. However, TbPDI2 is reactive with tomato lectin immediately following biosynthesis—far too rapidly for transport to the Golgi compartment, the site of poly-N-acetyllactosamine addition. TbPDI2 also fails to react with Erythrina cristagalli lectin, confirming the absence of terminal N-acetyllactosamine units. We propose that tomato lectin binds the Manβ1-4GlcNAcβ1-4GlcNAc trisaccharide core of paucimannose glycans on both newly synthesized and mature TbPDI2. Consistent with this proposal, α-mannosidase treatment renders oligomannose N-glycans on the T. brucei cathepsin L orthologue TbCatL reactive with tomato lectin. These findings resolve contradictory evidence on the location and glycobiology of TbPDI2 and provide a cautionary note on the use of tomato lectin as a poly-N-acetyllactosamine-specific reagent.     

Tomato lectin resists digestion in the mammalian alimentary canal and binds to intestinal villi without deleterious effects

Experiments were designed to investigate whether orally consumed tomato lectin could resist the digestive process and function as a lectin within the alimentary canal. Rats fed on a tomato lectin-rich diet passed faeces containing serologically detectable tomato lectin, and the lectin could be shown by immunoperoxidase staining bound to intestinal villi. Moreover, radioactivity was mainly recovered from the alimentary canal 3h after 125I-labelled tomato lectin administration with only traces in the circulation or internal organs. Radioactivity absorbed into the human circulation after consumption of 125I-labelled tomato lectin was also less than that expected for a digestable protein.     

Molecular Structure and Properties of Lectin from Tomato Fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro₄ motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.     

Molecular structure and properties of lectin from tomato fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro(4) motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.     

Analysis of sugar chain-binding specificity of tomato lectin using lectin blot: recognition of high mannose-type N-glycans produced by plants and yeast

The sugar chain-binding specificity of tomato lectin (LEA) against glycoproteins was investigated qualitatively using lectin blot analysis. Glycoproteins containing tri- and tetra-antennary complex-type N-glycans were stained with LEA. Unexpectedly, glycoproteins containing high mannose-type N-glycans and a horseradish peroxidase were stained with LEA. LEA blot analysis of the glycoproteins accompanied by treatment with exoglycosidase revealed that the binding site of LEA for the complex-type N-glycans was the N-acetyllactosaminyl side chains, whereas the proximal chitobiose core appeared to be the binding site of LEA for high mannose-type N-glycans. Despite these results, the glycoproteins did not inhibit the hemagglutinating activity of LEA. Among the chitin-binding lectins compared, potato tuber lectin showed specificity similar to LEA on lectin blot analysis, while Datura stramonium lectin and wheat germ agglutinin (WGA) did not interact with glycoproteins containing high mannose-type N-glycans, except that RNase B was stained by WGA. Based on these observations, LEA blot analysis was applied to sugar chain analysis of tomato glycoproteins. The most abundant LEA-reactive glycoprotein was purified from the exocarp of ripe tomato fruits, and was identified as the tomato anionic peroxidase1 (TAP1). These results suggest that LEA interacts with glycoproteins produced by tomatoes, which participate in biological activities in tomato plants.     

番茄凝集素的分离纯化及某些性质的研究

番茄成熟果实经抽提上鸡卵类粘蛋白(OM)-Sepharose 4B亲和层析柱分离纯化制得番茄凝集素。制品在SDS—聚丙烯酰胺凝胶电泳上呈一条带,表观分子量为205,000。Sephadex G200凝胶过滤行为呈单一吸收峰,分子量为180,000。等电聚焦测得等电点为7.6和9.4。它能使人和多种动物红细胞,以及某些培养细胞凝集,但效价不同。N—乙酰葡萄糖胺寡聚糖是其专一性结合糖。     

Control of Root-knot Nematodes on Tomato by Lectins

Significant control of tomato root knot was achieved by applications of the lectins Concanavalin A (Con A) and Limax flavus agglutinin in greenhouse, growth chamber, and microplot trials. Four consecutive weekly applications at lower concentrations of Con A yielded better control than single applications at a higher total concentration. The present state of knowledge on binding of Con A to soil nematodes and the in vitro effect of this lectin in chemotactic behavior are discussed. The mode of action of Con A on root-knot control is unknown.     

Implication of Pectic Components in Cell Surface Interactions between Tomato Root Cells and Fusarium oxysporum f. sp. radicis-lycopersici: A Cytochemical Study by Means of a Lectin with Polygalacturonic Acid-Binding Specificity

Aplysia gonad lectin, a polygalacturonic acid-binding lectin isolated from the sea mollusc Aplysia depilans, was complexed to colloidal gold and used for localizing polygalacturonic-acid-containing molecules in tomato root tissues infected with Fusarium oxysporum f. sp. radicis-lycopersici (FORL). Colonization of host tissues by FORL was associated with striking wall modifications including disruption and even loss of middle lamellae. According to the labeling pattern observed in host wall areas adjacent to fungal penetration channels, it is likely that FORL pectolytic enzymes act through localized wall degradation. The release of polygalacturonic acid-rich wall fragments and the accumulation of polygalacturonic acid-containing molecules in some altered phloem cells were frequently observed and considered to be specific host reactions to fungal attack. The heavy deposition of such molecules at strategic sites such as wall oppositions and intercellular spaces provides support to their implication in the plant defense system. The possible interrelation between polygalacturonic acid-containing molecules and other polymers such as lignin and phenolic compounds remains to be investigated further. The role of these molecules in host-pathogen interactions is discussed in relation to plant defense.     

Characterization and sequence of tomato 2S seed albumin: a storage protein with sequence similarities to the fruit lectin

We found a 2S storage albumin from the seed of tomato ( Lycopersicon esculentum L. cv. Cherry) that cross-reacted with antiserum to the fruit lectin, and named it Lec2SA. According to its size and basicity, Lec2SA was classified into four isoforms. These isoforms have an M(r) of approximately 12,000, and are composed of a small subunit (M(r) 4,000) and a large subunit (M(r) 8,000) linked by disulfide bonds. The complete amino acid sequence of Lec2SA was determined. The small subunit was composed of 32 amino acids, whereas the large subunit contained 70 amino acids with a pyroglutamine as the N-terminal residue. The sequence of Lec2SA was similar to that of 2S albumins from different plants, such as Brazil nut and castor beans. Furthermore, a sequence similarity was found between the large subunit of Lec2SA and the peptide sequence from tomato lectin. Although these similarities were found, Lec2SA did not show hemagglutinating activity or sugar-chain-binding activity, indicating that Lec2SA lacks the carbohydrate-binding domain. These results suggest that tomato lectin is a chimeric lectin sharing the seed storage protein-like domain that is incorporated into the gene encoding tomato lectin through gene fusion.     

Purification of acetyllactosamine-specific tomato lectin by erythroglycan-sepharose affinity chromatography

Tomato lectin is specific for oligomers of poly-N-acetyllactosamine containing 3 repeating Gal(beta 1-4)GlcNAc (beta 1-3)-disaccharides. As such it is highly useful for purifying oligosaccharides or glycopeptides with poly-N-acetyllactosamine character. We have found the lectin very useful as an affinity reagent for isolating glycoproteins or glycoprotein domains having poly-N-acetyllactosamine glycosylation. Conventional preparation of tomato lectin by ovomucoid-Sepharose affinity chromatography was found to be unsatisfactory due to instability of column and bleeding of ovomucoid into eluents requiring the necessity for additional purification steps following affinity chromatography. We prepared a column of human erythrocyte band 3 carbohydrate glycopeptide (erythroglycan) attached to Sepharose as an affinity matrix. The purification of tomato lectin to homogeneity in one step on this column matrix is described in this report.     

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis (which is the branching off of major vessels) and vasculogenesis (de novo vessel formation), perfusion mapping techniques such as resin casts, in vivo ultrasound imaging, and micro-dissection have been limited in demonstrating the intimate relationships between these two processes and developing renal structures within the embryo. Here, we describe the procedure of in utero intra-cardiac ultrasound-guided FITC-labeled tomato lectin microinjections on mouse embryos to gauge the ontogeny of renal perfusion. Tomato lectin (TL) was perfused throughout the embryo and kidneys harvested. Tissues were co-stained for various kidney structures including: nephron progenitors, nephron structures, ureteric epithelium, and vasculature. Starting at E13.5 large caliber vessels were perfused, however peripheral vessels remained unperfused. By E15.5 and E17.5, small peripheral vessels as well as glomeruli started to become perfused. This experimental technique is critical for studying the role of vasculature and blood flow during embryonic development.     

Purification and separation of tomato isolectins by chromatofocusing

Tomato lectin can be rapidly separated from a crude extract of tomato fruit proteins by chromatofocusing. The lectin is recovered from a PBE94 column in two peaks, each with a specific activity comparable to that of lectin purified by affinity chromatography on ovomucoid-Sepharose. Both isolectins consist of a single polypeptide chain (Mr 68,000) and have similar properties.     

Partial purification and characterization of 2, 4-diacetylphloroglucinol producing Pseudomonas fluorescens VSMKU3054 against bacterial wilt disease of tomato

We find out the antimicrobial potential of partially purified 2,4-diacetylphloroglucinol (DAPG) against Ralstonia solanacearum and fungal plant pathogens isolated from tomato rhizobacterium Pseudomonas fluorescens VSMKU3054. The present study is mainly focused on the control of wilt disease of tomato by our isolate VSMKU3054 and DAPG. The cell free culture filtrate of P. fluorescens VSMKU3054 was significantly arrested the growth of R. solanacearum and fungal pathogens such as Rhizoctonia solani, Sclerotium rolfsii, Macrophomina phaseolina and Fusarium oxysporum compared to control. The existence of DAPG from the crude metabolites of P. fluorescens VSMKU3054 was confirmed on TLC with Rf value 0.34, which is coincide with that of authentic phloroglucinol. The partially purified DAPG exhibited much higher activity against R. solanacearum at 30 µg/ml than the fungal plant pathogens compared to control. The antimicrobial partially purified compound was identified as DAPG by UV, FT-IR and GC–MS analysis. The percentage of live cells of R. solanacearum when supplemented with DAPG at 30 µg/ml, significantly controlled the living nature of R. solanacearum up to 68% compared to tetracycline and universal control observed under high content screening analysis. The selected isolate P. fluorescens VSMKU3054 and DAPG significantly controlled wilt disease of tomato up to 59.5% and 42.12% on 3rd and 7th days compared to positive and negative control by detached leaf assay. Further, in silico analysis revealed that high interaction of DAPG encoding protease with lectin which is associated with R. solanacearum. Based on our findings, we confirmed that P. fluorescens VSMKU3054 and DAPG could be used a potential bio inoculants for the management of bacterial wilt disease of tomato.     

Sucrose-binding lectin in regenerating cockroach (Periplaneta americana) legs: Its purification from adult hemolymph

Previously, we purified Periplaneta lectin from the hemolymph of adult Periplaneta americana (American cockroach) (Kubo and Natori Eur. J. Biochem. 168, 75–82, 1987). Immunoblotting analysis using antibody against Periplaneta lectin showed that the cockroach hemolymph contains another lectin that cross reacted immunologically with Periplaneta lectin. We have purified this new lectin (regenectin) to homogeneity. Affinity purified antibody against regenectin cross reacted with Periplaneta lectin. Thus, Periplaneta lectin and this new lectin were found to be different lectins sharing common antigenicity. After leg amputation, this new lectin was found to appear transiently at a specific stage of regeneration as revealed by immunoblotting, suggesting that it plays a role in the regeneration process.     

Artificial colonization of non-symbiotic plants roots with the use of lectins

Rhizobia have the ability to increase growth of non-legume plants due to the production of phytohormones and protection of plant from diseases and pathogens. However, the practical use of these beneficial bacteria sometimes fails because of their inability to effectively colonize rhizoplane and rhizosphere of inoculated plants. We chose the legume lectins as a factor that allows plants to form associative symbiosis with rhizobia. To test the fact that transgenic tobacco, tomato and rape roots with pea lectin gene may affect specific interaction with rhizobia, transgenic roots have been artificially inoculated by fluorescently-labeled pea rhizobia R. leguminosarum and east galega rhizobia Rhizobium galega. Microscopic and microbiological tests have shown that the number of adhered R. leguminosarum onto tobacco, rape and tomato roots which transformed with pea lectin gene is higher in comparison with the control, but no such effect through inoculation of these plants with R. galegae has been found. This confirms the interaction of R. leguminosarum with pea lectin at the surface of transformed roots. Undoubtedly, the improvement of recognition and attachment processes by using lectins can lead to the achievement of a stable associative relationship between non-symbiotic plants and rhizobia.     

Properties of Lectins in the Root and Seed of Lotononis bainesii

A lectin was purified from the root of Lotononis bainesii Baker by affinity chromatography on Sepharose-blood group substance A + H. The molecular weight of the lectin was estimated by gel filtration to be 118,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the lectin was a tetramer composed of two slightly different subunits with respective molecular weights of 32,000 and 35,000. The lectin had a hexose content of 12% (w/w) and contained the sugars fucose, glucosamine, mannose, and xylose. Root lectin hemagglutination was preferentially inhibited by disaccharides with terminal nonreducing galactose residues. Antigens capable of cross-reaction with root lectin antibody were not detected in the seed of L. bainesii.

A lectin from the seed of L. bainesii was partially purified by adsorption to pronase-treated rabbit erythrocytes. The lectin preparation had a molecular weight of approximately 200,000. Galactose and galactono-1,4-lactone inhibited seed lectin hemagglutination but lactose was ineffective. There was no evidence that the root of L. bainesii contained material antigenically related to the seed lectin.

     

Hormonal Regulation of the Lectin Biosynthesis in Callus Culture of the Phaseolus vulgaris Plant

Callus cultures established from Phaseolus vulgaris seedlings were used to investigate hormonal influence on lectin biosynthesis. The plant tissue cultures were initiated using defined levels of both a cytokinin (kinetin) and an auxin (2,4-dichlorophenoxyacetic acid) and were then transferred to media containing different amounts of these hormones. The lectin content of each callus culture was determined using an enzyme immunoassay specific for the seed lectin of the P. vulgaris plant. The lectin biosynthesis was directly affected by the levels of auxin and cytokinin in the culture media and no lectin was detected in hormone-free medium. This enabled us to compose culture media yielding a maximal or minimal lectin content of the callus cultures, illustrating the ability to induce an enhancement or suppression of the in vitro lectin biosynthesis. The lectin level of callus tissue during the growth cycle of a culture was, furthermore, related to the cellular growth rate which might indicate an involvement of the lectin in cellular events during rapid cell division.     

Purification and characterization of an α-d-galactosyl-binding lectin from Artocarpus lakoocha seeds

A lectin from Artocarpus lakoocha seeds has been purified by affinity chromatography on a melibiose-agarose column. The homogeneity of the purified lectin was tested by several criteria, viz., poly(acrylamide)-gel electrophoresis, ultracentrifugal analysis, and gel filtration. The molecular weight of the lectin was estimated to be ∼70,000 as determined by Sephadex gel filtration. SDS-poly-(acrylamide)-gel electrophoresis gave a single component of molecular weight 18,000, suggesting that the lectin is a tetramer composed of four apparently identical subunits. The lectin agglutinated human erythrocytes, regardless of blood group. Artocarpus lakoocha lectin is a glycoprotein, and contains 11.7% of carbohydrates, in which d-xylose (6%) is the main sugar, with smaller proportions of d-galactose, d-glucose, d-mannose, N-acetyl-d-glucosamine, and N-acetyl-d-mannosamine. Amino acid analysis of the lectin revealed a high content of acidic and hydroxylic amino acids, a relatively low proportion of basic amino acids, and a trace of cysteine and methionine. In hapten-inhibition assays with simple sugars, glycosides of α-d-galactopyranose and N-acetyl-d-galactosamine were potent inhibitors of the purified lectin.     

Biosynthesis of the Snowdrop (Galanthus nivalis) Lectin in Ripening Ovaries

The biosynthesis and processing of the Galanthus nivalis agglutinin were studied in vivo in ripening snowdrop ovaries. Using labeling and pulse chase labeling experiments it could be demonstrated that the snowdrop lectin is synthesized as a precursor of relative molecular weight (M_r) 15,000 which is posttranslationally converted into the authentic lectin polypeptide of M_r 13,000 with a half-life of about 6 hours. Gel filtration of an extract of [³H]leucine labeled ovaries on Sepharose 4B showed that a significant portion of the newly synthesized lectin is associated with the particulate fraction. When the organellar fraction was fractionated on isopycnic sucrose gradients this lectin banded in the same density region as the endoplasmic reticulum (ER) marker enzyme NADH cytochrome c reductase. Both radioactivity in lectin and in enzyme activity shifted towards a higher density in the presence of 2 millimolar Mg-acetate indicating that the labeled lectin was associated with the rough ER. Labeled lectin could be chased from the ER with a half-life of 4 hours and then accumulated in the soluble fraction. Whereas the ER-associated lectin contains exclusively polypeptides of M_r 15,000 the soluble fraction contains both precursor molecules and mature lectin polypeptides. The snowdrop lectin in the ER is fully capable of binding immobilized mannose. It is associated into tetramers with an appropriate molecular weight of 60,000. These results indicate that newly synthesized snowdrop lectin is transiently associated with the ER before transport and processing.