Changes in S-type lectin localization in neuroblastoma cells (N1E115) upon differentiation

The distribution of a 14.4 kDa S-type lectin was examined in murine neuroblastoma cells, either undifferentiated or after differentiation induced by dibutyryl-cyclic adenosine monophosphate. In undifferentiated cells the immunoreactivity was detected extracellularly, associated with the plasma membrane and in bulges released into the extracellular milieu. Important modifications of the lectin localization were associated with the differentiation process that induced an increased cytosolic expression and a decreased externalization. Possible functions for the lectin expressed intracellularly in the differentiated cells are also considered.     

Protection of catheter surfaces from adhesion of Pseudomonas aeruginosa by a combination of silver ions and lectins

A catheter surface was modified by coating a cellulose acetate polymer. Adhesion of Pseudomonas aeruginosa ATCC 27853 to the surface was investigated by exposing bacterial cultures to three treatments: polymer impregnated with silver ions (Ag⁺), polymer surfaces coated with lectins and a combination of Ag⁺ and a lectin coating. The effective concentration of Ag⁺ providing protection against bacterial biofilm development was 100g/ml and higher. Lectins alone at 10% also showed inhibition of bacterial attachment. However, the best result was achieved against bacterial adhesion and growth on surfaces using a combination of 100 g Ag⁺/ml and a lectin coating as a surface treatment. This surface treatment was also effective against both fresh culture and a two-week-old culture containing P. aeruginosa producing exopolymers. Our results suggest that Ag⁺impregnation combined with a lectin coating warrants further investigation as a potential means of protecting catheters.     

Glycomics-driven discoveries in schistosome research

Schistosome glycans and glycoconjugates play a prominent role in the parasite's biology, in particular in the interaction with the human host. A large amount of structural data regarding glycosylation of different schistosome life stages and glycoconjugate subsets has been collected in the last decade, but many significant gaps in our knowledge of the schistosomal glycome remain. Here we will present a concise review of the already available data guided by a selection of recently generated stage-specific glycan profiles, and discuss implications and prospects of glycomics studies of schistosomes.     

从绿藻礁膜（Monostroma nitidum Wittr）分离半乳糖专一的凝集素

礁膜（Monostroma nitidum Wittr）经 25%～80%硫酸铵分级、DEAE-纤维素52离子交换层析和Sephadex G-200凝胶过滤层析,得到纯化礁膜凝集素（Monostroma nitidum lectin,MNL）,在SDS-PAGE上显示单一蛋白染色带. 用Sephadex G-200层析测得其分子质量为66.6 kD, 用SDS-PAGE测得其分子质量为66.2 kD．该凝集素可以凝集人A、B、AB、O型红细胞,且凝集活性相同. 在对人（A、B、AB、O）、兔、鲤、鲫、鼠、羊、鸡、狗的红细胞凝集作用中,兔凝集作用最强．该凝集素在pH 4.00～10.53范围内均有活性,但在pH 5.20～9.40范围内活性最大．经100 ℃热处理30 min后,该凝集素对兔红细胞血凝活性保留25%,活性最大的温度范围为25～55 ℃．MNL被EDTA抑制,最小抑制浓度为3.13 mmol/L,但对 Ca^２＋和Mg^２＋不敏感．该凝集素凝集兔红细胞的作用不被D -果糖、D-甘露糖、D-葡萄糖、蔗糖、麦芽糖、γ-球蛋白、牛甲状腺球蛋白所抑制,但被D- 半乳糖和乳糖抑制,最小抑制浓度分别为5 mmol/L和2.5 mmol/L．     

A predominantly hydrophobic recognition of H-antigenic sugars by winged bean acidic lectin: a thermodynamic study

The thermodynamics of binding of winged bean (Psophocarpus tetragonolobus) acidic agglutinin to the H-antigenic oligosaccharide (Fucalpha1-2Galbeta1-4GlcNAc-oMe) and its deoxy and methoxy congeners were determined by isothermal titration calorimetry. We report a relatively hydrophobically driven binding of winged bean acidic agglutinin to the congeners of the above sugar. This conclusion is arrived, from the binding parameters of the fucosyl congeners, the nature of the enthalpy-entropy compensation plots and the temperature dependence of binding enthalpies of some of the congeners. Thus, the binding site of winged bean acidic agglutinin must be quite extended to accommodate the trisaccharide, with non-polar loci that recognize the fucosyl moiety of the H-antigenic determinant.     

Isolation of mannose-binding C-type lectin from Heliothis virescens pupae

A mannose-binding C-type lectin (MBL) was isolated by affinity chromatography from Heliothis virescens immune pupal hemolymph. The immune pupal hemolymph was obtained after bacterial injection of live Enterobacter cloacae bacteria. MBL in mammals acts as an opsonin for phagocytosis and activates the lectin complement pathway of the innate immune response, which leads to killing of gram-negative bacteria and enveloped viruses. The affinity-purified and reduced pupal MBL showed a single band of 36 kDa by SDS-PAGE (12% gel). A dot-immunoblot ELISA (using guinea pig anti-MBL IgG as primary antibody) demonstrated specificity of the antibody for the affinity-purified pupal MBL. The immune pupal hemolymph contained 21 microg of MBL per ml of hemolymph. The amino acid composition of the purified pupal MBL was determined with high amounts of arginine and histidine detected. The presence of MBL in insect pupae has not before been reported and could be important in pupal innate immunity to bacterial infection.     

Topology of the 10 subunits within the Decamer of KLH, the hemocyanin of the marine gastropod Megathura crenulata

Immunoelectron microscopy has been performed using negatively stained immune complexes of keyhole limpet hemocyanin isoform 1 (KLH1) decamers and a functional unit-specific monoclonal antibody anti-KLH1-c1. The antibody links hemocyanin molecules at both the collar and the collarless edge of the decamer, indicating a peripheral localization of functional units c. In isoform 2 (KLH2) the positions of functional units c have been identified with the peanut agglutinin (PNA), which has previously been shown to exclusively bind to KLH2-c. Ferritin linked to PNA was used to visualize labeled molecules electron microscopically. The pattern of labeling also indicates a peripheral localization of the c functional units. The data presented in this paper support only one of two possible models for the subunit orientation within the hemocyanin decamer.     

二龄草鱼脾脏、肝脏组织高表达甘露糖结合凝集素mRNA

Innate immunity is expected to be very important in fish. Mannose-bingding lectin (MBL) participates in the innate immune system as an activator of the complement system and as an opsonin after binding to certain carbohydrate structures on microorganisms. In this experiment, total mRNA was isolated from spleen, liver, gills, thymus, head kidney and kidney of adult and immature grass carp Ctenopharygodon idllus. The cDNA of MBL was obtained by RT-PCR using total mRNA from the spleen of carp as template. Such cDNA was labled with ^32p and used as probe for Northern analysis, and autoradiographic signals were quantified by densitometry analysis. The results showed that MBL was high expressed in the spleen and liver and low in gills, thymus, head kidney and kidney of adult grass carp, and MBL was much lower expressed in spleen and liver of immature grass carp than those of adult grass carp. The results might partially explain why immature grass carp are vulnerable to grass carp hemorrhage virus (GCHV) whereas adult grass carp are not.This suggested that MBL mav be an imoortant anti-GCHV factor [Acta Zoologica Sinica 50 (1): 137 - 140. 2004].     

Expression of frutalin, an alpha-D-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris

Frutalin is an α-d-galactose-binding lectin expressed in breadfruit seeds. Its isolation from plant is time-consuming and results in a heterogeneous mixture of different lectin isoforms. In order to improve and facilitate the availability of the breadfruit lectin, we cloned an optimised codifying frutalin mature sequence into the pPICZαA expression vector. This expression vector, designed for protein expression in the methylotrophic yeast Pichia pastoris, contains the Saccharomyces α-factor preprosequence to direct recombinant proteins into the secretory pathway. Soluble recombinant frutalin was detected in the culture supernatants and recognised by native frutalin antibody. Approximately 18–20 mg of recombinant lectin per litre medium was obtained from a typical small scale methanol-induced culture purified by size-exclusion chromatography. SDS–PAGE and Edman degradation analysis revealed that frutalin was expressed as a single chain protein since the four amino-acid linker peptide “T-S-S-N”, which connects α and β chains, was not cleaved. In addition, incomplete processing of the signal sequence resulted in recombinant frutalin with one Glu-Ala N-terminal repeat derived from the α-factor prosequence. Endoglycosidase treatment and SDS–PAGE analysis revealed that the recombinant frutalin was partly N-glycosylated. Further characterisation of the recombinant lectin revealed that it specifically binds to the monosaccharide Me-α-galactose presenting, nevertheless, lesser affinity than the native frutalin. Recombinant frutalin eluted from a size-exclusion chromatography column with a molecular mass of about 62–64 kDa, suggesting a tetrameric structure, however it did not agglutinate rabbit erythrocytes as native frutalin does. This work shows that the galactose-binding jacalin-related lectins four amino-acid linker peptide “T-S-S-N” does not undergo any proteolytic cleavage in the yeast P. pastoris and also that linker cleavage might not be essential for lectin sugar specificity.