The binding characteristics and utilization of Aleuria aurantia,Lens culinaris and few other lectins in the elucidation of fucosyltransferase activities resembling cloned FT VI and apparently unique to colon cancer cells

Human colon carcinoma cell fucosyltransferase (FT) in contrast to the FTs of several human cancer cell lines, utilized GlcNAcbeta1,4GlcNAcbeta-O-Bn as an acceptor, the product being resistant to alpha1,6-L-Fucosidase and its formation being completely inhibited by LacNAc Type 2 acceptors. Further, this enzyme was twofold active towards the asialo agalacto glycopeptide as compared to the parent asialoglycopeptide. Only 60% of the GlcNAc moieties were released from [14C]fucosylated asialo agalacto triantennary glycopeptide by jack bean beta-N-acetylhexosaminidase. These alpha1,3-L-fucosylating activities on multiterminal GlcNAc residues and chitobiose were further examined by characterizing the products arising from fetuin triantennary and bovine IgG diantennary glycopeptides and their exoglycosidase-modified derivatives using lectin affinity chromatography. Utilization of [14C]fucosylated glycopeptides with cloned FTs indicated that Lens culinaris lectin and Aleuria aurantia lectin (AAL) required, respectively, the diantennary backbone and the chitobiose core alpha1,6-fucosyl residue for binding. The outer core alpha1,3- but not the alpha-1,2-fucosyl residues decreased the binding affinity of AAL. The AAL-binding fraction from [14C]fucosylated asialo fetuin, using colon carcinoma cell extract, contained 60% Endo F/PNGaseF resistant chains. Similarly AAL-binding species from [14C]fucosylated TFA-treated bovine IgG using colon carcinoma cell extract showed significant resistance to endo F/PNGaseF. However, no such resistance was found with the corresponding AAL non- and weak-binding species. Thus colon carcinoma cells have the capacity to fucosylate the chitobiose core in glycoproteins, and this alpha1,3-L-fucosylation is apparently responsible for the AAL binding of glycoproteins. A cloned FT VI was found to be very similar to this enzyme in acceptor substrate specificities. The colon cancer cell FT thus exhibits four catalytic roles, i.e., alpha1,3-L-fucosylation of: (a) Galbeta1,4GlcNAcbeta-; (b) multiterminal GlcNAc units in complex type chain; (c) the inner core chitobiose of glycopeptides and glycoproteins; and (d) the nonreducing terminal chiotobiose unit.     

Multiple Layers of Complexity in O-Glycosylation Illustrated With the Urinary Glycoproteome

While N-glycopeptides are relatively easy to characterize, O-glycosylation analysis is more complex. In this article, we illustrate the multiple layers of O-glycopeptide characterization that make this task so challenging. We believe our carefully curated dataset represents perhaps the largest intact human glycopeptide mixture derived from individuals, not from cell lines. The samples were collected from healthy individuals, patients with superficial or advanced bladder cancer (three of each group), and a single bladder inflammation patient. The data were scrutinized manually and interpreted using three different search engines: Byonic, Protein Prospector, and O-Pair, and the tool MS-Filter. Despite all the recent advances, reliable automatic O-glycopeptide assignment has not been solved yet. Our data reveal such diversity of site-specific O-glycosylation that has not been presented before. In addition to the potential biological implications, this dataset should be a valuable resource for software developers in the same way as some of our previously released data has been used in the development of O-Pair and O-Glycoproteome Analyzer. Based on the manual evaluation of the performance of the existing tools with our data, we lined up a series of recommendations that if implemented could significantly improve the reliability of glycopeptide assignments.     

Wheat Germ Agglutinin Inhibits the Effects of Nerve Growth Factor on the Phosphorylation of Proteins in PC12h Cells

Treatment of PC12h cells in tissue culture with nerve growth factor (NGF) led to an increased incorporation of [32P]orthophosphoric acid into specific proteins. The increased phosphorylation of 60,000-dalton and 20,000-dalton proteins in the 0.2% Triton X-100 detergent-soluble fraction, of 35,000-dalton protein in the 0.2% Triton X-100 detergent-insoluble fraction, and of slow migrating protein (SMP) in the nonhistone nuclear fraction was observed upon NGF treatment. On the other hand, wheat germ agglutinin (WGA) treatment of PC12h cells induced a slightly decreased phosphorylation of these NGF-responsive proteins. Incubation of cell-free extracts from PC12h cells with [gamma-32P]ATP led to the phosphorylation of a 100,000-dalton protein. In extracts from cells treated with NGF, the labeling of the 100,000-dalton protein was substantially and selectively reduced. In contrast, treatment of PC12h cells with WGA led to an increased phosphorylation of the 100,000-dalton protein in cell-free extracts. Thus, NGF and WGA showed opposite effects on the phosphorylation of specific proteins in both intact cells and cell-free extracts. In addition, it was also observed in both systems that pre- and posttreatment of PC12h cells with WGA abolished the effects of NGF on the phosphorylation and produced a phosphorylation pattern similar to that from PC12h cells treated only with WGA. In parent PC12 cells, it has been reported that the treatment of cells with WGA inhibits NGF binding to its receptors and converts the rapidly dissociating receptors to slowly dissociating receptors. Thus, WGA in conjunction with NGF, results in the practical disappearance of rapidly dissociating receptors on cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental Changes in the Molecular Weights of Polypeptides in the Human CNS That Carry the HNK-1 Epitope and Bind Phaseolus vulgaris Lectins

The binding patterns of electrophoresed polypeptides from homogenates of human frontal lobe, cerebellum, and spinal cord obtained at various stages of development were determined for several lectins with specificities for a wide range of oligosaccharides. A discrete developmental change in the molecular-weight pattern was seen only among polypeptides binding the two Phaseolus vulgaris agglutinins, E-phytohemagglutinin (E-PHA) and L-PHA. With increasing maturity, the apparent molecular weights of the major polypeptides binding these two lectins progressively decreased. Furthermore, at all stages of development, E-PHA and L-PHA bound to the same polypeptides as the monoclonal antibody HNK-1, which recognizes a carbohydrate epitope on polypeptides that may play roles in cell adhesion. Based on the carbohydrate specificities of the two PHAs, we conclude that it is likely that the HNK-1 epitope resides on a triantennary N-linked oligosaccharide bisected by N-acetylglucosamine.     

利用小RNA深度测序技术鉴定半夏病毒病病原

RNA沉默是真核生物体内由病毒来源的干扰小RNA（virus derived small interfering RNA, vsiRNA）沉默复合物介导目标RNA特异降解的一种保守机制,通过对vsiRNA分析可进行植物病毒病原鉴定。本文利用小RNA深度测序技术对感病半夏叶片进行鉴定,结果发现,表现典型花叶症状的半夏叶片受到大豆花叶病毒（Soybean mosaic virus, SMV）、黄瓜花叶病毒（Cucumber mosaic virus, CMV）、芋花叶病毒（Dasheen mosaic virus, DsMV）、魔芋花叶病毒（Konjac mosaic virus, KoMV）、烟草花叶病毒（Tobacco mosaic virus, TMV）等多种病毒的复合侵染。为明确SMV山西半夏分离物（SMV-SXBX）的进化关系,进行SMV-SXBX全基因组克隆与分析,获得SMV-SXBX全长为9 735 nt,编码一个由3 105个氨基酸组成的多聚蛋白质。通过核苷酸与氨基酸序列比对发现,SMV-SXBX与半夏分离物P同源性最高,分别为91.1%和94.1%,且系统发育分析表明,SMV-SXBX与半夏SMV分离物P聚为一簇。同时,也对vsiRNA进行了系统分析,研究结果有望为半夏SMV的有效防治提供一定科学依据。     

Gold-conjugated Reagents for the Labeling of Carbohydrate-Recognition Domains and Glycoconjugates on Nematode Surfaces

Various fluorescent conjugated lectins have been used for the detection of glycoconjugates on nematode surfaces under light microscopy. Several problems have been experienced with these reagents including penetration of the cuticle by fluorescent lectins, non-glycoconjugate specificity, strong nematode autofluorescence at the emission wavelength of the fluorescent dye, and prevention of persistent visualization due to rapid quenching of the fluorescent components. Gold-conjugated reagents combined with silver enhancement alleviated these difficulties when working with three phytonematode species (Heterodera avenae, H. latipons, and Meloidogyne javanica) and two entomopathogenic species (Steinernema carpocapsae and S. glaseri) under light-microscopy visualization of binding by fluorescent lectins and neoglycoproteins. Moreover, gold-conjugated reagents resulted in stable bindings that enabled long-term observations.     

Resistance ofUrtica dioica to mycorrhizal colonization: a possible involvement ofUrtica dioica agglutinin

Roots of stinging nettle (Urtica dioica L.) were sampled at different sites around Basel (Switzerland) and examined under the microscope. They were completely devoid of mycorrhizal structures. Similarly, stinging nettle plants grown in the greenhouse in the presence of the arbuscular mycorrhizal fungusGlomus mosseae did not show any signs of mycorrhiza formation. Spread ofG. mosseae through the rhizosphere of stinging nettle plants was inhibited, and application of extracts of stinging nettle roots and rhizomes to hyphal tips ofG. mosseae reduced hyphal growth.Urtica dioica agglutinin, an antifungal protein present in the rhizomes of stinging nettle, inhibited hyphal growth in a similar way as the crude root extract. The possibility thatUrtica dioica agglutinin is at least partially responsible for the inability of stinging nettle to form the arbuscular mycorrhizal symbiosis withG. mosseae is discussed.     

Antifeedant effects of plant lectins and an enzyme on the adult stage of the rice brown planthopper,Nilaparvata lugens

The feeding activity of adult rice brown planthopper,Nilaparvata lugens (Stal) on selected proteins incorporated into artificial diets was examined, based on quantitative analysis of honeydew excretion, in order to determine the mechanism of action of antimetabolic proteins towards homopteran pests. The lectinsGalanthus nivalis agglutinin (GNA), wheat germ agglutinin (WGA) and the enzyme soybean lipoxygenase (LPO), previously demonstrated to be toxic to this insect, reduced honeydew excretion levels of adultNilaparvata lugens over a 24 h period when incorporated into artificial diet at 0.1% {w:v}, indicating that these proteins acted as antifeedants. Of the proteins tested GNA was the most effective antifeedant, reducing honeydew droplet production by 96%, although after 24 h there was some recovery in the honeydew excretion levels and thus the insects appeared to tolerate the presence of the antifeedant with time. The lectinPisum sativum agglutinin (PSA), previously demonstrated to be non-toxic, showed no antifeedant properties.     

Lectin binding distinguishes between neuroendocrine and neuronal derivatives of the sympathoadrenal neural crest

Lectin cytochemistry was used to identify surface epitopes selectively expressed by chromaffin cell chemoreceptors (glomus cells) in the rat carotid body. Unexpectedly, these studies revealed that binding sites for peanut agglutinin (PNA; Arachis hypogea) were highly expressed by all neuroendocrine derivatives of the sympathoadrenal neural crest, including glomus cells, small, intensely fluorescent cells, and adrenal chromaffin cells in situ. In contrast, principal sympathetic neurons did not express PNA receptors. PNA binding was inhibited by 2% galactose. To determine whether expression of PNA receptors was selectively induced by neuroendocrine differentiation of sympathoadrenal precursors, we compared PNA labeling of embryonic sympathoblasts in the presence of either nerve growth factor (NGF) or the synthetic glucocorticoid dexamethasone (DEX). Dex-treated cells, which expressed several neuroendocrine traits, bound PNA, whereas NGF-treated neuronal derivatives did not. In addition, to examine whether expression of existing PNA receptors was down-regulated by neuronal differentiation of chromaffin cells, we compared labeling of PC12 cells, which normally bind PNA, in the presence and absence of NGF. Although PC12 cells acquired characteristic neuronal morphologies in the presence of NGF, they did not lose PNA labeling, even after 8 days of NGF treatment. These findings indicate that neuronal and neuroendocrine derivatives of the sympathoadrenal lineage can be distinguished by differential expression of carbohydrate epitopes and suggest that PNA receptors are induced by neuroendocrine differentiation. © 1995 John Wiley & Sons, Inc.     

Lectin-induced alterations on the proliferation of three human prostatic cancer cell lines

Summary While lectins are known to influence the cell growth of several types of normal and neoplastic tissues, their roles in the case of prostatic cancer cells remain relatively unexplored. In the present work, we report thein vitro influence of five lectins, namely peanut (PNA), wheat germ (WGA), Concanavalin A (Con A),Griffonia simplicifolia (GSA-IA₄), andPhaseolus vulgaris (PHA-L) agglutinins, on the cell proliferation of one androgen-sensitive (LNCaP) and two androgen-insensitive (PC-3 and DU 145) human prostatic cancer cell lines cultured in either 10% or 1% fetal bovine serum (FBS)-supplemented media. The cell proliferation was assessed by means of the colorimetric 3-(4,5-dimethythiazol-2-yle)2,5-diphenyltetrazolium bromide. (MTT) assay. Four lectin concentrations were tested (i.e., 0.1, 1, 10, and 100 μg/ml) at five experimental states (i.e., 2, 3, 5, 7, and 9 d following the addition of each lectin to the culture media). Our results demonstrated that the five lectins under study had a globally significant dose-dependent toxic effect on prostatic cancer cell proliferation. Nevertheless, low doses of GSA-IA₄ and PHA-L significantly (P<0.05 toP<0.001) increased the cell proliferation of confluent PC-3 cells. Increasing the FBS from 1% to 10% in the culture media significantly antagonized lectin-induced toxicity in the three prostatic cell lines. In conclusion, the present data strongly suggest that some lectins might influence the proliferation of prostatic carcinoma cells. In addition, because lectins are present in our diet, and are able to pass into the systemic circulation and thus reach the prostate, the present results suggest that some lectins might exert an influence on prostate cancer growth under clinical conditions.