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      A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine

      * , * , * , * , * , * , * , * , * , , * , , § , 1
      Biochemical Journal
      Portland Press Ltd.
      Agrocybe aegerita, anti-hepatoma activity, fungal lectin, glycan-binding property, N-acetylglucosamine, AAL-2, Agrocybe aegerita lectin 2, BLL, Boletopsis leucomelas lectin, GlcNAc, N-acetylglucosamine, GSL-II, Griffonia simplicifolia lectin-II, HPA, Helix pomatia agglutinin, IPTG, isopropyl β-D-thiogalactopyranoside, ITC, isothermal titration calorimetry, LacNAc, N-acetyl-lactosamine, MAL, Maackia amurensis lectin, MALDI, matrix-assisted laser-desorption ionization, MS/MS, tandem MS, nAAL-2, native AAL-2, ORF, open reading frame, PI, propidium iodide, PVL, Psathyrella velutina lectin, rAAL-2, recombinant AAL-2, RFU, relative fluorescence units, SLL, Solanum lycopersicum lectin, TBS, Tris-buffered saline, TOF, time-of-flight, WGA, wheatgerm agglutinin

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          A novel lectin was isolated from the mushroom Agrocybe aegerita (designated AAL-2) by affinity chromatography with GlcNAc ( N-acetylglucosamine)-coupled Sepharose 6B after ammonium sulfate precipitation. The AAL-2 coding sequence (1224 bp) was identified by performing a homologous search of the five tryptic peptides identified by MS against the translated transcriptome of A. aegerita. The molecular mass of AAL-2 was calculated to be 43.175 kDa from MS, which was consistent with the data calculated from the amino acid sequence. To analyse the carbohydrate-binding properties of AAL-2, a glycan array composed of 465 glycan candidates was employed, and the result showed that AAL-2 bound with high selectivity to terminal non-reducing GlcNAc residues, and further analysis revealed that AAL-2 bound to terminal non-reducing GlcNAc residues with higher affinity than previously well-known GlcNAc-binding lectins such as WGA (wheatgerm agglutinin) and GSL-II ( Griffonia simplicifolia lectin-II). ITC (isothermal titration calorimetry) showed further that GlcNAc bound to AAL-2 in a sequential manner with moderate affinity. In the present study, we also evaluated the anti-tumour activity of AAL-2. The results showed that AAL-2 could bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro. Furthermore, AAL-2 exerted an anti-hepatoma effect via inhibition of tumour growth and prolongation of survival time of tumour-bearing mice in vivo.

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          Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling.

          Glycans have important roles in living organisms with their structural diversity. Thus, glycomics, especially aspects involving the assignment of functional glycans in a high-throughput manner, has been an emerging field in the postproteomics era. To date, however, there has been no versatile method for glycan profiling. Here we describe a new microarray procedure based on an evanescent-field fluorescence-detection principle, which allows sensitive, real-time observation of multiple lectin-carbohydrate interactions under equilibrium conditions. The method allows quantitative detection of even weak lectin-carbohydrate interactions (dissociation constant, K(d) > 10(-6) M) as fluorescent signals for 39 immobilized lectins. We derived fully specific signal patterns for various Cy3-labeled glycoproteins, glycopeptides and tetramethylrhodamine (TMR)-labeled oligosaccharides. The obtained results were consistent with the previous reports of glycoprotein and lectin specificities. We investigated the latter aspects in detail by frontal affinity chromatography, another profiling method. Thus, the developed lectin microarray should contribute to creation of a new paradigm for glycomics.
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            Innate immune lectins kill bacteria expressing blood group antigen

            The expression of ABO(H) blood group antigens causes deletion of cells that generate self anti-blood group antibodies, but this deletion limits adaptive immunity toward pathogens bearing cognate blood group antigens. To explore potential defense mechanisms against these pathogens, given such limitations in adaptive immunity, we screened for innate proteins that could recognize human blood group antigens. Here we report that two innate immune lectins, galectins-4 and -8, which are expressed in the intestinal tract, recognize and kill human blood group antigen-expressing E. coli, while failing to alter viability of other E. coli strains or other gram-negative or gram-positive organisms both in vitro and in vivo. Killing by both galectins-4 and -8 resides within their C-terminal domains, occurs rapidly and independently of complement, and is accompanied by disruption of membrane integrity. These results demonstrate that innate defense lectins can provide immunity against pathogens that display blood group self-antigens on their surface.
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              O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry.

              O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification. We report methods for direct enrichment and identification of in vivo O-GlcNAc-modified peptides through lectin weak affinity chromatography (LWAC) and mass spectrometry. The effectiveness of this strategy on complex peptide mixtures was demonstrated through enrichment of 145 unique O-GlcNAc-modified peptides from a postsynaptic density preparation. 65 of these O-GlcNAc-modified peptides were sequenced and belonged to proteins with diverse functions in synaptic transmission. Beta-elimination/Michael addition, MS(3) on O-GlcNAc neutral loss ions, and electron capture dissociation were shown to facilitate analysis of O-GlcNAc-modified peptides/sites from lectin weak affinity chromatography enriched postsynaptic density samples. Bassoon and Piccolo, proteins critical to synapse assembly and vesicle docking, were extensively modified by O-GlcNAc. In some cases, O-GlcNAc was mapped to peptides previously identified as phosphorylated, indicating potential interplay between these modifications. Shared substrate amino acid context was apparent in subsets of O-GlcNAc-modified peptides, including "PVST" and a novel "TTA" motif (two hydroxyl-containing amino acids adjacent to an alanine). The results suggest specific roles for O-GlcNAc modification in synaptic transmission, establish a basis for site-specific regulatory studies, and provide methods that will facilitate O-GlcNAc proteome analysis across a wide variety of cells and tissues.

                Author and article information

                Biochem J
                Biochem. J
                Biochemical Journal
                Portland Press Ltd.
                23 January 2012
                27 March 2012
                15 April 2012
                : 443
                : Pt 2
                : 369-378
                *College of Life Sciences, Wuhan University, Wuhan 430072, People's Republic of China
                †Department of Clinical Immunology, Guangdong Medical College, Dongguan 523808, People's Republic of China
                ‡Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, Wuhan 430071, People's Republic of China
                §State Key Laboratory of Virology, Wuhan University, Wuhan 430072, People's Republic of China
                Author notes

                The nucleotide sequence data for Agrocybe aegerita lectin 2 will appear in the GenBank®, EMBL, DDBJ and GSDB Nucleotide Sequence Databases under accession number JN001164.

                The glycan array data of AAL-2 were deposited in the Consortium for Functional Glycomics database with the labels primscreen_3312–primscreen_3315.

                1To whom correspondence should be addressed (email sunhui@ 123456whu.edu.cn ).
                © 2012 The Author(s) The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                : 22 November 2011
                : 28 December 2011
                : 23 January 2012
                Page count
                Figures: 7, Tables: 1, Equations: 2, References: 49, Pages: 10
                Research Article

                pvl, psathyrella velutina lectin,mal, maackia amurensis lectin,iptg, isopropyl β-d-thiogalactopyranoside,orf, open reading frame,ms/ms, tandem ms,itc, isothermal titration calorimetry,raal-2, recombinant aal-2,wga, wheatgerm agglutinin,lacnac, n-acetyl-lactosamine,rfu, relative fluorescence units,bll, boletopsis leucomelas lectin,sll, solanum lycopersicum lectin,naal-2, native aal-2,anti-hepatoma activity,maldi, matrix-assisted laser-desorption ionization,agrocybe aegerita,glcnac, n-acetylglucosamine,fungal lectin,pi, propidium iodide,gsl-ii, griffonia simplicifolia lectin-ii,aal-2, agrocybe aegerita lectin 2,glycan-binding property,n-acetylglucosamine,hpa, helix pomatia agglutinin,tof, time-of-flight,tbs, tris-buffered saline


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