Production of pea lectin in Escherichia coli

In order to explore the molecular basis for the glycopeptide specificity of legume lectins, we have developed an experimental system in which specific amino acid alterations can be introduced into the carbohydrate binding site of pea lectin. This system is based on the production of pea lectin in Escherichia coli. The plasmid coding for the lectin was constructed from two lectin cDNA sequences isolated from Pisum sativum seeds (Higgins, T. J. V., Chandler, P. M., Zurawski, G., Button, S. C., and Spencer, D. (1983) J. Biol. Chem. 258, 9544-9549) and an expression vector based on the gene for the outer membrane lipoprotein of E. coli (Nakamura, K., and Inouye, M. (1982) EMBO J. 1, 771-775). The lectin is produced as a single polypeptide chain and forms insoluble aggregates in E. coli cells (2-5 mg/liter). Functional lectin is recovered by solubilization of the aggregates in guanidinium hydrochloride, renaturation in the presence of MnCl2 and CaCl2, and affinity purification on Sephadex. This procedure yields a homogeneous 28,000-dalton protein. Comparison of the recombinant lectin with natural pea lectin in an inhibition of hemagglutination assay demonstrated that there is no detectable difference in the carbohydrate binding properties of the two lectins.     

Sugar-quantum dot conjugates for a selective and sensitive detection of lectins

One-pot synthesized neoglycoconjugates with a reactive thiol group are introduced here for functionalization with carbohydrates for solubilization and stabilization of CdSe-ZnS quantum dots in aqueous solution. Three different sizes of quantum dots (QDs) with lactose, melibiose, and maltotriose on their surface have been utilized, for the first time, for lectin detection through agglutination assay. The sugar-QDs thus synthesized were characterized by transmission election microscopy (TEM), fluorescence, and absorption spectroscopy. Agglutination of sugar-QDs by three different lectins occurred through specific multivalent carbohydrate-lectin interactions and was studied extensively by monitoring the scattered light at 600 nm. This assay was very selective, which has been demonstrated by a more selective binding of soybean agglutinin (SBA) with melibiose-QD, as compared to lactose-QD, and specific deagglutination caused by alpha-d-galactose, while alpha-d-mannose did not show any effect. The detection sensitivity of the maltotriose-QD was tested with Concanavalin A (ConA), and as little as 100 nM of the lectin was detected using light scattering. The detection sensitivity of this protocol has been enhanced considerably by the fluorescence properties of QDs. This agglutination process seems to occur through formation of smaller soluble aggregates, which further associate to form larger aggregates.     

New GlcNAc/GalNAc-specific lectin from the ascidian Didemnum ternatanum

Previously we isolated GlcNAc-specific lectin (DTL) from the ascidian Didemnum ternatanum by affinity chromatography on cross-linked ovalbumin. Here we report the purification and characterization of new D-GlcNAc/D-GalNAc-specific lectin DTL-A from the same ascidian. This lectin was isolated from non-bound cross-linked ovalbumin fraction and further was purified by gel filtration on Sepharose CL-4B, affinity chromatography on GlcNAc-agarose and gel filtration on Superdex 200. SDS-polyacrylamide gel electrophoresis and gel filtration of purified lectin on Sepharose CL-4B indicates that it exists as large aggregates in the native state. Investigations of the carbohydrate specificity of DTL-A by enzyme-linked lectin assay suggest the multi-specificity of this lectin. DTL-A binds BSM, asialo-BSM as well as heparin and dextran sulfate. The binding of DTL-A to BSM was inhibited by monosaccharides D-GlcNAc and D-GalNAc, their alpha- but not beta-anomers. Among polysaccharides and glycoconjugates, DTL-A binding to BSM was effectively inhibited by BSM, asialo-BSM, pronase-treated BSM and synthetic alpha-D-GalNAc-PAA. Fetuin and asialofetuin showed a much lower inhibitory potency, heparin and dextran sulfate were noninhibitory. On the other hand, DTL-A binding to heparin was effectively inhibited by dextran sulfate, fucoidan, whereas BSM showed insignificantly inhibitory effect. DTL-A binding to heparin was not inhibited by D-GlcNAc and D-GalNAc.     

Release of beta-D-galactoside-binding lectins into the cavities of aggregates of chick extraembryonic endoderm cells

Dissociated cells from the extraembryonic endoderm of gastrulating chick embryos form aggregates when cultured in rotating flasks. The large cellular aggregates are initially solid but subsequently cavitate to form hollow, thin-walled vesicles. These cells also contain an endogenous beta-D-galactoside-binding lectin. Previous work has shown that high extracellular concentrations of this lectin are associated with decreased cell-cell adhesion [Milos, N. and S.E. Zalik: Differentiation 21, 175-182 (1982)]. We have removed the fluid contents from aggregates cultured for 24 and 48 h and tested them for the presence of lectin activity. The results demonstrate that lectin activity is detectable in a higher number of aggregates cultured for 24 as opposed to 48 h (75% vs. 28%, respectively). The lectin activity per aggregate is also higher in aggregates cultured for 24 h (180 vs. 67 hemagglutinating units, respectively, for 24- and 48-h aggregates). Thus, at the time when cells are moving apart from one another during aggregate cavitation, detectable lectin activity is released into the vesicular contents of the aggregate.     

Food Quality in Lusaka National Park: Tracking Mortality in Black Lechwe Antelopes

Translocation is one of the fundamental tools in wildlife management but only if appropriately undertaken. In 2009, 30 black lechwe antelopes were suddenly translocated from the State House Habitat (STH) into the newly established Lusaka National Park (LNP). However, within 4 months of being translocated to LNP, 28 black lechwes (93%) died. A pathological report produced by veterinarians following a postmortem examination suggested no disease incidence affected the antelopes. The food quality of LNP was tested and compared to that in the STH and the antelopes’ native habitat of the Bangweulu wetlands (BGW) to establish if variations in food quality were responsible for the antelopes’ mortality. The findings suggest that the food quality in LNP was greatly inferior to that in STH, which could explain the observed high mortality of the antelopes in LNP. Further, the quality of food in LNP did not widely differ from that in the BGW, suggesting that the antelopes might not have survived had they been translocated to their native habitat, as they had already adapted to feeding on highly nutritious supplementary feed at the STH.     

LNP-CpG ODN-adjuvanted varicella-zoster virus glycoprotein E induced comparable levels of immunity with ShingrixTM in VZV-primed mice

Latent varicella-zoster virus (VZV) may be reactivated to cause herpes zoster, which affects one in three people during their lifetime. The currently available subunit vaccine Shingrix^TM is superior to the attenuated vaccine Zostavax^® in terms of both safety and efficacy, but the supply of its key adjuvant component QS21 is limited. With ionizable lipid nanoparticles (LNPs) that were recently approved by the FDA for COVID-19 mRNA vaccines as carriers, and oligodeoxynucleotides containing CpG motifs (CpG ODNs) approved by the FDA for a subunit hepatitis B vaccine as immunostimulators, we developed a LNP vaccine encapsulating VZV-glycoprotein E (gE) and CpG ODN, and compared its immunogenicity with Shingrix^TM in C57BL/6J mice. The results showed that the LNP vaccine induced comparable levels of gE-specific IgG antibodies to Shingrix^TM as determined by enzyme-linked immunosorbent assay (ELISA). Most importantly, the LNP vaccine induced comparable levels of cell-mediated immunity (CMI) that plays decisive roles in the efficacy of zoster vaccines to Shingrix^TM in a VZV-primed mouse model that was adopted for preclinical studies of Shingrix^TM. Number of IL-2 and IFN-γ secreting splenocytes and proportion of T helper 1 (Th1) cytokine-expressing CD4⁺ T cells in LNP-CpG-adjuvanted VZV-gE vaccinated mice were similar to that of Shingrix^TM boosted mice. All of the components in this LNP vaccine can be artificially and economically synthesized in large quantities, indicating the potential of LNP-CpG-adjuvanted VZV-gE as a more cost-effective zoster vaccine.     

Production of limit size nanoliposomal systems with potential utility as ultra-small drug delivery agents

Previous studies from this group have shown that limit size lipid-based systems – defined as the smallest achievable aggregates compatible with the packing properties of their molecular constituents – can be efficiently produced using rapid microfluidic mixing technique. In this work, it is shown that similar procedures can be employed for the production of homogeneously sized unilamellar vesicular systems of 30–40?nm size range. These vesicles can be remotely loaded with the protonable drug doxorubicin and exhibit adequate drug retention properties in vitro and in vivo. In particular, it is demonstrated that whereas sub-40?nm lipid nanoparticle (LNP) systems consisting entirely of long-chain saturated phosphatidylcholines cannot be produced, the presence of such lipids may have a beneficial effect on the retention properties of limit size systems consisting of mixed lipid components. Specifically, a 33-nm diameter doxorubicin-loaded LNP system composed of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1,2-dipalmitoyl phosphatidylcholine (DPPC), cholesterol, and PEGylated lipid (DSPE-PEG2000) demonstrated adequate, stable drug retention in the circulation, with a half-life for drug release of ～12?h. These results indicate that microfluidic mixing is the technique of choice for the production of bilayer LNP systems with sizes less than 50?nm that could lead to development of a novel class of ultra-small drug delivery vehicles.     

Antibody-modified lipid nanoparticles for selective delivery of siRNA to tumors expressing membrane-anchored form of HB-EGF

An Fab’ antibody against heparin-binding epidermal growth factor-like growth factor (HB-EGF) was applied to achieve advanced tumor-targeted delivery of siRNA. Lipid nanoparticles (LNP) encapsulating siRNA (LNP-siRNA) were prepared, pegylated, and surface modified with Fab’ fragments of anti-HB-EGF antibody (αHB-EGF LNP-siRNA). αHB-EGF LNP-siRNA showed high-binding affinity to recombinant human HB-EGF in a Biacore assay. In addition, αHB-EGF LNP-siRNA selectively associated with cells expressing HB-EGF in vitro. Confocal microscopic images showed that siRNA formulated in αHB-EGF LNP-siRNA was efficiently internalized into MDA-MB-231 human breast cancer cells, on which HB-EGF is highly expressed. In addition, siRNA encapsulated in αHB-EGF LNP induced obvious suppression of both target mRNA and protein levels in MDA-MB-231 cells. These results indicate that αHB-EGF LNP have excellent potential to deliver siRNA to target cancer cells, resulting in effective gene silencing.     

Concanavalin A increases spontaneous beat rate of embryonic chick heart cell aggregates

The plant lectin concanavalin A (Con A), at concentrations of 5–200 μg/ml, induced a twofold to fivefold increase in spontaneous beat rate of cultured aggregates of ventricular cells from seven-day chick embryos. This response was time, dose, and temperature dependent and was accompanied by a decrease in transmembrane potential. It could be blocked or reversed by α-methyl-D-mannoside but was not reversed by dilution alone. Binding of the lectin occurred in the cold, but a temperature-dependent process was also necessary to produce the response. Divalent (succinyl) Con A did not cause a beat rate increase. Whole heart aggregates responded similarly but less intensely than ventricular aggregates. Atrial aggregates, and whole heart aggregates treated with 5 μg/ml of Con A, produced a biphasic chronotropic response, first decreasing then increasing their beat rates. These results suggest that saccharide-bearing macromolecules on the heart cell surface play a role in regulating spontaneous beat rate.     

Azolla fern lectins that specifically recognize endosymbiotic cyanobacteria

Lectins were extracted from whole fern grindings ofAzolla pinnata (AP) andAzolla filiculoides (AF) by precipitation with ammonium sulfate to 20% of saturation. At high pH both lectins dissociate into inactive subunits (5000 mol wt) which reassociate into active aggregates (>500,000 mol wt) following concentration by ammonium sulfate precipitation or freezing and thawing. Although amino sugars inhibited hemagglutinating activity of both AP and AF lectins,d-fructose was inhibitory only to the AP lectin hemagglutinating activity, andd-galactose was slightly inhibitory to the AP lectin but not to the AF lectin. Both lectins exhibited specificity for freshly extracted cyanobionts from homologous fern species: AP lectin agglutinated cyanobiont filaments from AP, but not from AF; AF lectin agglutinated cyanobiont filaments from AF, but not AP. Neither lectin reacted with cultured cyanobionts from either fern species. Hemagglutinating titers were likewise reduced by adsorption of these lectins to homologous cyanobiont cells. This report provides strong suggestive evidence for specificity in this N-fixing symbiosis between aquatic fern and cyanobacterium.     

mRNA lipid nanoparticle phase transition

Crucial for mRNA-based vaccines are the composition, structure, and properties of lipid nanoparticles (LNPs) as their delivery vehicle. Using all-atom molecular dynamics simulations as a computational microscope, we provide an atomistic view of the structure of the Comirnaty vaccine LNP, its molecular organization, physicochemical properties, and insight in its pH-driven phase transition enabling mRNA release at atomistic resolution. At physiological pH, our simulations suggest an oil-like LNP core that is composed of the aminolipid ALC-0315 and cholesterol (ratio 72:28). It is surrounded by a lipid monolayer formed by distearoylphosphatidylcholine, ALC-0315, PEGylated lipids, and cholesterol at a ratio of 22:9:6:63. Protonated aminolipids enveloping mRNA formed inverted micellar structures that provide a shielding and likely protection from environmental factors. In contrast, at low pH, the Comirnaty lipid composition instead spontaneously formed lipid bilayers that display a high degree of elasticity. These pH-dependent lipid phases suggest that a change in pH of the environment upon LNP transfer to the endosome likely acts as trigger for cargo release from the LNP core by turning aminolipids inside out, thereby destabilizing both the LNP shell and the endosomal membrane.     

Heparin-binding lectin from human placenta: purification and partial molecular characterization and its relationship to basic fibroblast growth factors

The heparin-binding lectin from human placenta is isolated on the basis of its tendency to form large aggregates by gel filtration and on the basis of its affinity for heparin by affinity chromatography. The purified lectin dissociates into up to four distinct polypeptides with molecular weight values of 14,400, 15,000, 16,200, and 16,700 and a single isoelectric point of 9.0. Molecular heterogeneity is not due to different degrees of glycosylation, as evidenced by gel electrophoretic analysis after extensive treatment with various endoglycosidases. Despite its similarities of affinity to heparin, molecular size, and isoelectric point to the basic fibroblast growth factor (bFGF), the comparatively high yield of the lectin (approximately 1.5 mg/100 g of placenta), the occurrence of proteolytic fragmentation in the presence of heparin, and the lack of homology to the amino-terminal sequence of the lectin argue against any notable relationship to bFGF. Most importantly, the lack of mitogenic activity in a commonly used bioassay with quiescent 3T3 fibroblasts rules out any FGF-like activity on cell proliferation. The heparin-binding lectin is thus clearly distinguishable from heparin-binding growth factors. By employing biotinylated heparin as labeled ligand to visualize and quantify heparin binding, hapten inhibition in a solid-phase assay reveals that except for heparin no other vertebrate glycosaminoglycan but the sulfated fucan fucoidan can effectively reduce the Ca2+-independent ligand binding. Proteolytic fragmentation by chymotrypsin in two independent assays demonstrates that a fragment of Mr 7800 still retains ability to bind heparin. The interaction of this lectin with naturally occurring heparin-like molecules may physiologically be involved in modulatory regulation of heparin-mediated processes.     

Examination of Le and lele Genotypes of Glycine max (L.) Merr. for Membrane-Bound and Buffer-Soluble Soybean Lectin

Membrane fractions from seedlings of four soybean [Glycine max (L.) Merr.] lines were examined by radioimmunoassay and hemagglutination assay for the 120,000 dalton soybean lectin. Two of the lines (Sooty and T-102) are genotypically lele and lack buffer-soluble soybean lectin; the remaining two lines (Beeson and Harosoy 63) are Le and produce seeds that contain the lectin (Su et al. 1980 Biochim. Biophys. Acta 629: 292-304). Both Triton X-100 (0.5% v/v) and nonidet P-40 (0.05% v/v) solubilized soybean lectin from membrane fractions of Le cotyledons. Triton X-100 interfered substantially with the assay of protein and hemagglutinating activity and was unacceptable for use in quantitative measurements. The nonidet P-40-solubilized soybean lectin from Le cotyledons was consistently present both in washed 13,000g and 82,500g membrane fractions, but it accounted for less than 1.5% of the total (buffer-soluble plus membrane-bound) soybean lectin. The membrane lectin was purified by the affinity chromatography procedure devised for soluble soybean lectin, and it was immunologically indistinguishable from authentic soybean lectin. Membrane fractions from Le cotyledons contained insignificant amounts of radioisotope-labeled soybean lectin that had been added during homogenization, and purified membrane fractions did not bind the lectin in the presence of the hapten, d-galactose. These controls make it unlikely that the membrane soybean lectin was of cytoplasmic origin. Soybean lectin and other hemagglutinins were not present in buffer-soluble or membrane fractions from lele cotyledons or from roots and hypocotyls of any of the lines.     

In vitro study of the comparative behaviour of a human acute lymphoblastic leukemia cell line and a reference normal cell line towards the effects of a mitogenic lectin

An in vitro study of the behaviour of a human acute lymphoblastoid leukemia cell line (REH) towards the action of a mitogenic lectin of Robinia pseudoacacia was carried out. The results were compared with those a reference cell line (LHN13) established from normal human lymphocytes. In both cell lines, the lectin induces agglutination (measured by counting the number of aggregates as well as the number of cells in each aggregate) and decrease of growth (measured by counting the number of cells and the incorporation of tritiated thymidine into TCA-precipitable material per 10(6) cells). The agglutination and the decrease of growth are produced at the doses of 0.5 and 1 microgram/ml of culture medium and after 4 h of exposure of cells to the lectin, respectively. These effects increase progressively with higher doses of lectin and continues throughout the culture. However, the REH line is less sensitive than the LHN13 line to the effects of lectin. Both agglutination and growth decrease of REH as well as LHN13 cell lines by the lectin are reversible; this is confirmed by the fact that the monospecific anti-Robinia lectin serum suppresses these effects.     

Purification and characterization of a Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc-specific lectin from the fruiting body of the polypore mushroom Polyporus squamosus

A lectin has been purified from the carpophores of the mushroom Polyporus squamosus by a combination of affinity chromatography on beta-D-galactosyl-Synsorb and ion-exchange chromatography on DEAE-Sephacel. Gel filtration chromatography, SDS-polyacrylamide gel electrophoresis, and N-terminal amino acid sequencing indicated that the native lectin, designated P. squamosus agglutinin, is composed of two identical 28-kDa subunits associated by noncovalent bonds. P. squamosus agglutinin agglutinated human A, B, and O and rabbit red blood cells but precipitated only with human alpha(2)-macroglobulin, of many glycoproteins and polysaccharides tested. The detailed carbohydrate binding properties of the purified lectin were elucidated using three different approaches, i.e. precipitation inhibition assay (in solution binding assay), fluorescence quenching studies, and glycolipid binding by lectin staining on high-performance thin layer chromatography (solid-phase binding assay). Based on the results obtained by these assays, we conclude that although the P. squamosus lectin binds beta-D-galactosides, it has an extended carbohydrate-combining site that exhibits highest specificity and affinity toward nonreducing terminal Neu5Acalpha2, 6Galbeta1,4Glc/GlcNAc (6'-sialylated type II chain) of N-glycans (2000-fold stronger than toward galactose). The strict specificity of the lectin for alpha2,6-linked sialic acid renders this lectin a valuable tool for glycobiological studies in biomedical and cancer research.     

Distribution of cell surface saccharides on pancreatic cells

We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a “universal” affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.     

An enzyme-linked lectin binding assay for quantitative determination of lectin receptors

An enzyme-linked lectin binding assay (ELBA) has been developed for the detection of soluble lectin binding substances (receptors) and the determination of their relative affinity for the lectin. The assay is based on competitive binding to enzyme-labeled lectin of a known lectin receptor, bound to a solid phase, and unknown sample receptors. In this paper the assay is exemplified with the mannose/glucose-specific pea lectin, with the glycoprotein ovalbumin as its receptor, and with horseradish peroxidase (EC 1.11.1.7) as the enzyme used for labeling. Also a method was developed for the preparation of peroxidase-labeled lectin. Labeling was started by mixing equimolar amounts of lectin and periodate-oxidized enzyme at pH 4.5 at a final concentration of 10(-4)M, after which conjugation was started by raising the pH to 9.5. This resulted in complete conjugation, after which the product could be diluted 50-500 times for application in ELBA. For the ELBA ovalbumin was adsorbed onto polystyrene microtiter plates. Sample receptors, added together with the enzyme-labeled lectin, inhibited binding of the latter to ovalbumin. Bound enzyme activity was colorimetrically determined after addition of o-phenylenediamine. Relative lectin affinity (KL) was expressed as (formula; see text) in which [X]50% is the concentration of sample receptor necessary to inhibit 50% of the binding of a certain amount of lectin, and [M]50% is the concentration of D-mannose necessary to inhibit 50% binding of the same amount of lectin. With this technique lectin affinity of both monovalent and polyvalent lectin binding substances can be estimated: low KL values mean high lectin affinity.     

Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery

Lipid nanoparticles (LNP) modified with cell-penetrating peptides (CPP) were prepared for the delivery of small interfering RNA (siRNA) into cells. Lipid derivatives of CPP derived from protamine were newly synthesized and used to prepare CPP-decorated LNP (CPP-LNP). Encapsulation of siRNA into CPP-LNP improved the stability of the siRNA in serum. Fluorescence-labeled siRNA formulated in CPP-LNP was efficiently internalized into B16F10 murine melanoma cells in a time-dependent manner, although that in LNP without CPP was hardly internalized into these cells. In cells transfected with siRNA in CPP-LNP, most of the siRNA was distributed in the cytoplasm of these cells and did not localize in the lysosomes. Analysis of the endocytotic pathway indicated that CPP-LNP were mainly internalized via macropinocytosis and heparan sulfate-mediated endocytosis. CPP-LNP encapsulating siRNA effectively induced RNA interference-mediated silencing of reporter genes in B16F10 cells expressing luciferase and in HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. These data suggest that modification of LNP with the protamine-derived CPP was effective to facilitate internalization of siRNA in the cytoplasm and thereby to enhance gene silencing.     

Role of Lectins in Plant-Microorganism Interactions: II. Distribution of Soybean Lectin in Tissues of Glycine max (L.) Merr

Three different assay procedures have been used to quantitate the levels of soybean (Glycine max [L.] Merr.) lectin in various tissues of soybean plants. The assays used were a standard hemagglutination assay, a radioimmunoassay, and an isotope dilution assay. Most of the lectin in seeds was found in the cotyledons, but lectin was also detected in the embryo axis and the seed coat. Soybean lectin was present in all of the tissues of young seedlings, but decreased as the plants matured and was not detectable in plants older than 2 to 3 weeks. Soybean lectin isolated from seeds of several soybean varieties were identical when compared by several methods.