Purification,Characterization, and Antiproliferative Activity of a Single-Chain Lectin from Vicia palaestina (Fabaceae) Seeds

Vicia palaestina Boiss. is an annual herb that grows in dry areas of eastern Mediterranean countries. It belongs to section Cracca subgenus Vicilla, which is characterized by having a high content in the non-protein amino acid canavanine. The seeds from some of these vetches are also rich in lectins. The purification and characterization of a single-chain lectin from the seeds of V. palaestina is described here. This lectin was the most abundant protein in albumin extracts. It has affinity for the glycoconjugate N-acetylgalactosamine and inhibits proliferation of the cancerous Caco-2 and THP-1 cell lines. In addition to their high nutritional value, the seeds from V. palaestina represent a source of lectins with health promoting and pharmacological potential because of their antiproliferative activity.     

S-Nitrosylation of mannose binding lectin regulates its functional activities and the formation of autoantibody in rheumatoid arthritis

The possibility of post-translational modifications of mannose binding lectin (MBL) leading to functional impairment of the MBL pathway and the presence of anti-MBL autoantibodies were reported earlier in rheumatoid arthritis (RA). MBL was observed to be S-nitrosylated (S-nitrosated) in vitro. HepG2 cells were stimulated with 10% synovial fluid from RA patients to produce increased levels of MBL and nitric oxide. Under these experimental conditions MBL was observed to be S-nitrosated using biotin switch assay. The plasma of RA patients was also found to contain higher levels of S-nitrosylated MBL (SNO-MBL) in comparison to the healthy controls. Functional activities of SNO-MBL were compared with normal MBL. Mannan binding and C4 deposition ability of MBL was found to decrease after S-nitrosylation. It was also observed that S-nitrosylation of MBL leads to a decrease in the bacterial phagocytosis and apoptotic cell binding as measured by fluorescence microscopy and FACS analysis. These results indicate that the carbohydrate binding ability of MBL was affected by S-nitrosylation (S-nitrosation). High levels of anti-MBL autoantibodies were detected against SNO-MBL in plasma of RA patients in comparison to normal MBL suggesting a role of SNO-MBL in generation of autoantibodies in RA patients.     

A fungal lectin and its apparent receptors on a nematode surface

Abstract A lectin was isolated from the nematode-trapping fungus Arthrobotrys oligospora . This carbohydrate-binding protein was developmentally regulated and was found only on trap-bearing mycelia. The lectin receptor on the surface of the nematode Panagrellus redivivus has, furthermore, been investigated using homogenates from whole nematodes or nematode cuticle. The ability of macromolecules from nematodes, fractionated according to M _r and lectin affinities, to inhibit the capture of nematodes by A. oligospora was tested using an inhibition assay, based on a simple dialysis membrane technique. One major lectin-binding glycoprotein, with apparent M _r of 65 000, was isolated from the nematode cuticle.     

Phosphorylation of Streptomycin by an Intracellular Enzyme of Streptomyces griseus

A zinc-binding protein was purified to homogeneity for the first time from the gonad parts of scallops, Patinopecten yessoensis, kept in filtered seawater to which no heavy metals were added. Based upon the elution profiles in two chromatographic systems, spectrophotometric analysis, and amino acid composition of the purified preparation, the protein met the criteria for classification as a metallothionein; i.e., low molecular weight (about 9000), paucity of both aromatic amino acid residues and absorbance at 280 nm, and abundance of both cysteinyl residues (> 25%) and absorbance at 215 and 254 nm. Furthermore, the results of chromatographies on a Sephacryl S-300 column and electrophoresis with or without SDS suggested that the protein molecules would be in several polymeric forms in vivo. The antiserum prepared with the purified protein as the antigen was shown to have immunocross-reactivity to neither an extract of the surf clam, Pseudocardium sybillae, nor the whelk, Neptunea arthritica, indicating the heterogeneity of the proteins in marine shellfishes. These results suggested that the Zn-binding protein purified in this study was characteristic of scallops and involved in zinc storage of this organism.     

Effects of Food Lectins on the Transport System of Human Intestinal Caco-2 Cell Monolayers

The effects of 16 lectins isolated from foodstuff on the transport system across human intestinal Caco-2 cell monolayers were investigated by using four fluorescent markers: lucifer yellow (LY) for the paracellular pathway, fluorescein (FL) for the monocarboxylic acid transporter-mediated pathway, rhodamine 123 for the P-glycoprotein-mediated efflux pathway, and calcein for the multidrug resistance associated protein-related efflux pathway. The transepithelial electrical resistance (TER) values for the monolayers were also measured. WGA from wheat germ, ABA from white mushroom, AOL from Aspergillus oryzae, and CSL3 from chum salmon eggs (each at 100 µg/mL) decreased the TER value by 20–40% which resulted in increased LY transport. These lectins, as well as such other lectins as SBA from soybean, RBA from rice bran, and Con A from jack bean, affected other transport pathways too. These results indicate that the lectins modulated the transepithelial transport system in different ways, probably because of their specific binding characteristics toward Caco-2 cell monolayers.     

Engineering a versatile tandem repeat-type α2-6sialic acid-binding lectin

Previously, we developed an α2-6-sialic acid (Sia)-specific lectin (SRC) starting from an R-type galactose-specific lectin C-terminal domain. However, it showed relatively low affinity because of its monovalency. Here, we engineered a tandem repeat construct (SRC2) showing substantial affinity for α2,6-sialylated N-glycans (in the order of 10⁻⁶ M in K_d), almost comparable to a natural α2-6Sia-specific lectin from Sambucus sieboldiana (SSA). Notably, its binding to branched N-glycans was found to be more selective than SSA. Nevertheless, SRC2 showed no apparent hemagglutinating activity, while it exerted strong erythrocyte-binding activity. This unique feature will help flow cytometry analysis, where usual lectins including SSA agglutinate cells. Some other biochemical properties investigated for SRC2, e.g., high productivity in bacteria and easy release of captured glycoproteins with lactose have demonstrated versatility of this mutant protein as a powerful tool for sialoglycomics.     

Crystalline Features of Streptococcal (l→3)-α-D-Glucans of Human Saliva

Crystalline polymorphs of the backbone (l→3)-α-D-glucans of two streptococcal α-glucans were studied by X-ray diffraction measurements in comparison with that of a fungal (l→3)-α-D-glucan. The glucan produced by S. salivarius changed its polymorph from the hydrated form at 100% relative humidity to the dehydrated form under vacuum, that produced by cariogenic S. mutans took the dehydrated form only, and the fungal glucan always showed the hydrated form. The difference of polymorphic behavior was ascribed to the molecular weight of the glucan since the fungal glucan showed the highest viscosity, the saliverius glucan, middle, and the mutans glucan, the lowest.     

A lectin method for investigating the glycosylation of nanogram amounts of purified glycoprotein

To unravel the complexities of the glycosylation of a protein is a substantial task, which requires considerable effort and resources. However, in many situations this is unnecessary, because only a limited amount of information is required. A new lectin-binding assay is described which is rapid, cheap and versatile. A purified glycoprotein is absorbed on to the plastic surface of a microtitre plate. After removing unbound protein by washing, uncoated sites on the plate are blocked and digoxigenin or biotin-labelled lectin is added. The degree of lectin binding is measured using either an anti-DIG antibody or streptavidin conjugated enzyme, which is subsequently used to develop a colour reaction. Using this method it is possible to screen multiple specimens with high sensitivity and excellent precision. In addition, very small amounts of lectin are used, background absorbances are low, and the procedure does not require a high degree of technical skill. Because very small amounts of glycoprotein are needed, a glycoprotein can often be rapidly purified by batch affinity chromatography. The method has been successfully applied to several purified proteins using the lectins, Con A, LCA, LTA, MAA, and SNA, and the information obtained agrees with that produced by more sophisticated approaches, eg Dionex Carbohydrate Analyser. Using a panel of lectins, a carbohydrate structural profile is quickly built-up, and subtle differences in glycosylation identified. This method should be particularly useful for screening glycosylation in multiple clinical specimens; in specimens where very small amounts of material are available, such as membrane molecules; and in the screening of recombinant proteins produced commercially. This revised version was published online in November 2006 with corrections to the Cover Date.