Automated motif discovery from glycan array data.

Assessing interactions of a glycan-binding protein (GBP) or lectin with glycans on a microarray generates large datasets, making it difficult to identify a glycan structural motif or determinant associated with the highest apparent binding strength of the GBP. We have developed a computational method, termed GlycanMotifMiner, that uses the relative binding of a GBP with glycans within a glycan microarray to automatically reveal the glycan structural motifs recognized by a GBP. We implemented the software with a web-based graphical interface for users to explore and visualize the discovered motifs. The utility of GlycanMotifMiner was determined using five plant lectins, SNA, HPA, PNA, Con A, and UEA-I. Data from the analyses of the lectins at different protein concentrations were processed to rank the glycans based on their relative binding strengths. The motifs, defined as glycan substructures that exist in a large number of the bound glycans and few non-bound glycans, were then discovered by our algorithm and displayed in a web-based graphical user interface ( http://glycanmotifminer.emory.edu ). The information is used in defining the glycan-binding specificity of GBPs. The results were compared to the known glycan specificities of these lectins generated by manual methods. A more complex analysis was also carried out using glycan microarray data obtained for a recombinant form of human galectin-8. Results for all of these lectins show that GlycanMotifMiner identified the major motifs known in the literature along with some unexpected novel binding motifs.

[1]  R. Cummings,et al.  Affinity of galectin-8 and its carbohydrate recognition domains for ligands in solution and at the cell surface. , 2007, Glycobiology.

[2]  R. Cummings,et al.  [18] Lectin affinity chromatography of glycopeptides , 1987 .

[3]  G. Uhlenbruck,et al.  On the specificity of lectins with a broad agglutination spectrum , 1969, Blut.

[4]  David F. Smith,et al.  Dimeric Galectin-8 Induces Phosphatidylserine Exposure in Leukocytes through Polylactosamine Recognition by the C-terminal Domain* , 2008, Journal of Biological Chemistry.

[5]  J. Turnbull,et al.  Saccharide microarrays for high-throughput interrogation of glycan-protein binding interactions. , 2009, Methods in molecular biology.

[6]  G. Bird.  Anti‐T in Peanuts , 1964, Vox sanguinis.

[7]  J. Turnbull,et al.  Fabrication of carbohydrate microarrays on gold surfaces: direct attachment of nonderivatized oligosaccharides to hydrazide-modified self-assembled monolayers. , 2006, Analytical chemistry.

[8]  David F. Smith,et al.  Novel fluorescent glycan microarray strategy reveals ligands for galectins. , 2009, Chemistry & biology.

[9]  I. Goldstein,et al.  Immunochemical studies on the interaction between synthetic glycoconjugates and alpha-L-fucosyl binding lectins. , 1986, Biochemistry.

[10]  T. Osawa,et al.  Purification and characterization of an anti-H(O) phytohemagglutinin of Ulex europeus. , 1969, Biochimica et biophysica acta.

[11]  Yun Chi,et al.  Frequent Subtree Mining - An Overview , 2004, Fundam. Informaticae.

[12]  James C Paulson,et al.  Glycan microarrays for decoding the glycome. , 2011, Annual review of biochemistry.

[13]  Richard D. Cummings,et al.  The repertoire of glycan determinants in the human glycome. , 2009, Molecular bioSystems.

[14]  I. Goldstein,et al.  Studies on the combining sites of concanavalin A. , 1975, Advances in experimental medicine and biology.

[15]  G. Uhlenbruck,et al.  An Agglutinin from Helix Pomatia, which Reacts with Terminal N‐Acetyl‐D‐Galactosamine , 1966, Vox sanguinis.

[16]  M. Fukuda,et al.  Immunohistochemical Demonstration of α1,4-N-acetylglucosaminyltransferase that Forms GlcNAcα1,4Galβ Residues in Human Gastrointestinal Mucosa , 2001 .

[17]  A. Misra,et al.  Expression cloning of a human α1,4-N-acetylglucosaminyltransferase that forms GlcNAcα1→4Galβ→R, a glycan specifically expressed in the gastric gland mucous cell-type mucin , 1999 .

[18]  J. Hirabayashi,et al.  Glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of glycan-binding proteins. , 2008, Glycobiology.

[19]  A. M. Wu,et al.  Coding and classification of d-galactose, N-acetyl-d-galactosamine, and β-d-Galp-[1→3(4)]-β-d-GlcpNAc, specificities of applied lectins , 1991 .

[20]  Richard D Cummings,et al.  Use of glycan microarrays to explore specificity of glycan-binding proteins. , 2010, Methods in enzymology.

[21]  S. Ito,et al.  Expression cloning of a human alpha1, 4-N-acetylglucosaminyltransferase that forms GlcNAcalpha1-->4Galbeta-->R, a glycan specifically expressed in the gastric gland mucous cell-type mucin. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[22]  B. Haab,et al.  The fine specificity of mannose-binding and galactose-binding lectins revealed using outlier motif analysis of glycan array data. , 2012, Glycobiology.

[23]  I. Goldstein,et al.  PROTEIN-CARBOHYDRATE INTERACTION. II. INHIBITION STUDIES ON THE INTERACTION OF CONCANAVALIN A WITH POLYSACCHARIDES. , 1965, Biochemistry.

[24]  Minoru Kanehisa,et al.  Mining significant tree patterns in carbohydrate sugar chains , 2008, ECCB.

[25]  S. Baldus,et al.  Characterization of the binding specificity ofAnguilla anguilla agglutinin (AAA) in comparison toUlex europaeus agglutinin I (UEA-I) , 1996, Glycoconjugate Journal.

[26]  Ten Feizi,et al.  Oligosaccharide microarrays to decipher the glyco code , 2004, Nature Reviews Molecular Cell Biology.

[27]  David F. Smith,et al.  A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses* , 2011, The Journal of Biological Chemistry.

[28]  G. Strecker,et al.  Specificity of Twelve Lectins Towards Oligosaccharides and Glycopeptides Related to N‐Glycosylproteins , 2005 .

[29]  I. Goldstein,et al.  The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac(alpha 2-6)Gal/GalNAc sequence. , 1987, The Journal of biological chemistry.

[30]  Anne Imberty,et al.  Biochemical and Structural Analysis of Helix pomatia Agglutinin , 2006, Journal of Biological Chemistry.

[31]  J. Mikkelsen,et al.  Sugar‐coated microarrays: A novel slide surface for the high‐throughput analysis of glycans , 2002, Proteomics.

[32]  Edward Suh,et al.  A motif-based analysis of glycan array data to determine the specificities of glycan-binding proteins. , 2010, Glycobiology.

[33]  R. Cummings,et al.  Use of lectins in analysis of glycoconjugates. , 1994, Methods in enzymology.

[34]  Richard D. Cummings,et al.  Quantifiable fluorescent glycan microarrays , 2007, Glycoconjugate Journal.

[35]  G. Springer,et al.  Common precursors of human blood group MN specificities. , 1974, Biochemical and biophysical research communications.

[36]  Chi-Huey Wong,et al.  Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[37]  E. Kabat,et al.  Further immunochemical studies on the combining sites of Lotus tetragonolobus and Ulex europaeus I and II lectins. , 1982, Carbohydrate research.

[38]  R. Cummings,et al.  Lectin affinity chromatography of glycopeptides. , 1987, Methods in enzymology.

[39]  M. Fukuda,et al.  Immunohistochemical demonstration of alpha1,4-N-acetylglucosaminyltransferase that forms GlcNAcalpha1,4Galbeta residues in human gastrointestinal mucosa. , 2001, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[40]  R. Lotan,et al.  The purification, composition, and specificity of the anti-T lectin from peanut (Arachis hypogaea). , 1975, The Journal of biological chemistry.

[41]  Ten Feizi,et al.  Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions , 2002, Nature Biotechnology.

[42]  J U Baenziger,et al.  Structural determinants of concanavalin A specificity for oligosaccharides. , 1979, The Journal of biological chemistry.

[43]  David F. Smith,et al.  Innate immune lectins kill bacteria expressing blood group antigen , 2010, Nature Medicine.

[44]  E. Kabat,et al.  Studies on specificity and binding properties of the blood group A reactive hemagglutinin from Helix pomatia. , 1971, Biochemistry.