Fishing for lectins from diverse sequence libraries by yeast surface display - an exploratory study

The establishment of a robust technology platform for the expression cloning of carbohydrate-binding proteins remains a key challenge in glycomics. Here we explore the utility of using yeast surface display (YSD) technology in the interaction-based lectin cloning from complete cDNA libraries. This should pave the way for more detailed studies of protein-carbohydrate interactions. To evaluate the performance of this system, lectins representing three different subfamilies (galectins, siglecs, and C-type lectins) were successfully displayed on the surface of Saccharomyces cerevisiae and Pichia pastoris as a-agglutinin and/or alpha-agglutinin fusions. The predicted carbohydrate-binding activity could be detected for three out of five lectins tested (galectin-1, galectin-3, and siaoadhesin). For galectin-4 and E-selectin, no specific carbohydrate-binding activity could be detected. We also demonstrate that proteins with carbohydrate affinity can be specifically isolated from complex metazoan cDNA libraries through multiple rounds of FACS sorting, employing multivalent, fluorescent-labeled polyacrylamide-based glycoconjugates.     

Galectin-loaded cells as a platform for the profiling of lectin specificity by fluorescent neoglycoconjugates: a case study on galectins-1 and -3 and the impact of assay setting

The involvement of galectins as pleiotropic regulators of cell adhesion and growth in disease progression explains the interest to define their ligand-binding properties. Toward this end, it is desirable to approach in vivo conditions to attain medical relevance. In order to simulate physiological conditions with cell surface glycans as recognition sites and galectins as mediators of intercellular contacts we developed an assay using galectin-loaded Raji cells. The extent of surface binding of fluorescent neoglycoconjugates depended on the lectin presence and the type of lectin, the nature of the probes' carbohydrate headgroup and the density of unsubstituted beta-galactosides on the cell surface. Using the most frequently studied galectins-1 and -3, application of this assay led to rather equal binding levels for linear and branched oligomers of N-acetyllactosamine. A clear preference of galectin-3 for alpha1-3-linked galactosylated lactosamine was noted. In parallel, a panel of 24 neoglycoconjugates was tested as inhibitors of galectin binding from solution to N-glycans of surface-immobilized asialofetuin. These two assays differ in presentation of the galectin and ligand, facilitating identification of assay-dependent properties. Under the condition of the cell assay, selectivity among oligosaccharides for the lectins was higher, and extraordinary affinity of galectin-1 to 3'-O-sulfated probes in a solid-phase assay was lost in the cell assay. Having introduced and validated a cell assay, the comprehensive profiling of ligand binding to cell-surface-presented galectins is made possible.     

外源毒性物质及其在植物抗虫品种培育中的应用概况

对昆虫具有毒性的外源毒性物质广泛存在于微生物、植物和一些动物中。概述了Bt晶体蛋白、蛋白酶抑制剂、植物凝集素、淀粉酶抑制剂、几丁质酶等几种外源毒性物质的特征、杀虫机理及其基因在植物抗虫品种培育方面的应用概况,并对今后转外源毒性基因抗虫植物的主要研究方向作了探讨。     

香蕉凝集素基因的克隆及在成熟果实中的特异性表达

采用RT-PCR的方法克隆香蕉凝集素(Bankc)基因,对其全序列进行了测定并与已发表的BanLec基因序列进行了比较。采用定量PCR的方法对该基因在果实成熟过程中和香蕉不同组织中的表达进行了研究,结果表明：BanLec基因的表达同时具有发育特异性和组织特异性,即仅在果实中表达,而且其表达量随果实成熟度的变化而变化。     

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].     

Low-dose intragastric administration of Phaseolus vulgaris agglutinin (PHA) does not induce immunoglobulin E (IgE) production in Sprague-Dawley rats

Native Phaseolus vulgaris agglutinin (PHA) poses a potential health threat, when ingested with improperly cooked red kidney beans. Since PHA triggers human basophilic granulocytes in culture to rapidly release considerable amounts of interleukin-(IL-)4 and IL-13, key cytokines for inducing immunoglobulin E (IgE) production, the question was addressed whether this lectin can evoke in vivo IgE production. IgE-low-responder (Sprague-Dawley) rats received PHA (6 mg/rat/day) intragastrically by gavage over a period of 10 days. Up to day 35, there was no IgE induction regardless of whether the animals were boostered subcutaneously with PHA or not, indicating that PHA cannot be regarded as a general IgE inducer in rats.     

Lectin histochemistry of normal human gastric mucosa

Information about the saccharides expressed in gastric mucosa is mostly limited to the glycan content of gastric mucins and there are only a few studies of the glycoprofiling of the constituent cells and their components. Knowledge of the glycan expression of normal gastric mucosa is necessary for the interpretation of the significance of changes of expression in disease. A lectin histochemical study of normal human gastric (body) mucosa was performed using 27 lectins chosen to probe for a wide range of oligosaccharide sequences within several categories of glycoprotein glycans. There were marked differences in staining reactions in the various microanatomical structures of the mucosa, particularly between pits and glands with the former more closely resembling the surface epithelium. A notable feature was the degree of difference in the staining between a substantial sub-population of cells within the neck region and the epithelium of both the pits and glands. These neck cells resembled the pit cells with some lectins, glandular cells with some others and neither with some other lectins. Overall, the differences between the pit, gland and neck epithelia were diverse and numerous, and could not be explained by altered activity of a small set of glycosyltransferases. Widespread alterations of glycans must have occurred (affecting terminal and internal parts of their structures) and the very different glycotypes of the pit, neck and gland epithelia are, therefore, suggestive of the existence of three cell lineages within normal gastric epithelium. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Influence of plant development and environment on transgene expression in potato and consequences for insect resistance

Clonal replicates of different transformed potato plants expressing transgene constructs containing the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter, and sequences encoding the plant defensive proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA), and bean chitinase (BCH) were propagated in tissue culture. Plants were grown to maturity, at first under controlled environmental conditions, and later in the glasshouse. For a given transgene product, protein accumulation was found to vary between the different lines of clonal replicates (where each line was derived from a single primary transformant plant), as expected. However, variability was also found to exist within each line of clonal replicates, comparable to the variation of mean expression levels observed between the different clonal lines. Levels of GNA, accumulated in different parts of a transgenic potato plant, also showed variation but to a lesser extent than plant–plant variation in expression. With the majority of the clonal lines investigated, accumulation of the transgene product was found to increase as the potato plant developed, with maximum levels found in mature plants. The variation in accumulation of GNA among transgenic plants within a line of clonal replicates was exploited to demonstrate that the enhanced resistance towards larvae of the tomato moth, Lacanobia oleracea L., caused by expression of this protein in potato, was directly correlated with the level of GNA present in the plants, and that conditions under which the plants were grown affect the levels of GNA expression and subsequent levels of insect resistance.     

Isolation of Lectins by Affinity Chromatography with Porcine Plasma Proteins Immobilized on Agarose

To develop a convenient method to isolate lectins, we prepared an affinity gel by coupling plasma proteins with agarose beads under conditions where the pH did not exceed 7.5. The validity of the use of this affinity gel in combination with elution using a hapten saccharide was confirmed by isolation of concanavalin A from Jack bean meal. Successful application of the method was demonstrated by isolation of two novel vegetable lectins from udo (Aralia cordate) and wasabi (Wasabia japonica). The method would be useful to isolate new lectins from various sources including plant and animal tissues.