O-Glycome Beam Search Arrays for Carbohydrate Ligand Discovery

Abstract

O-glycosylation is a post-translational modification of proteins crucial to molecular mechanisms in health and disease. O-glycans are typically highly heterogeneous. The involvement of specific O-glycan sequences in many bio-recognition systems is yet to be determined because of a lack of efficient methodologies. We describe here a targeted microarray approach: O-glycome beam search that is both robust and efficient for O-glycan ligand-discovery. Substantial simplification of the complex O-glycome profile and facile chromatographic resolution is achieved by arraying O-glycans as branches, monitoring by mass spectrometry, focusing on promising fractions, and on-array immuno-sequencing. This is orders of magnitude more sensitive than traditional methods. We have applied beam search approach to porcine stomach mucin and identified extremely minor components previously undetected within the O-glycome of this mucin that are ligands for the adhesive proteins of two rotaviruses. The approach is applicable to O-glycome recognition studies in a wide range of biological settings to give insights into glycan recognition structures in natural microenvironments. O-glycosylation is a post-translational modification of proteins crucial to molecular mechanisms in health and disease. O-glycans are typically highly heterogeneous. The involvement of specific O-glycan sequences in many bio-recognition systems is yet to be determined because of a lack of efficient methodologies. We describe here a targeted microarray approach: O-glycome beam search that is both robust and efficient for O-glycan ligand-discovery. Substantial simplification of the complex O-glycome profile and facile chromatographic resolution is achieved by arraying O-glycans as branches, monitoring by mass spectrometry, focusing on promising fractions, and on-array immuno-sequencing. This is orders of magnitude more sensitive than traditional methods. We have applied beam search approach to porcine stomach mucin and identified extremely minor components previously undetected within the O-glycome of this mucin that are ligands for the adhesive proteins of two rotaviruses. The approach is applicable to O-glycome recognition studies in a wide range of biological settings to give insights into glycan recognition structures in natural microenvironments. Mucins are glycoproteins crucial in molecular mechanisms in health and disease (1.Andrianifahanana M. Moniaux N. Batra S.K. Regulation of mucin expression: Mechanistic aspects and implications for cancer and inflammatory diseases.BBA-Rev. Cancer. 2006; 1765: 189-222PubMed Google Scholar, 2.Tran D.T. Ten Hagen K.G. Mucin-type O-Glycosylation during Development.J. Biol. Chem. 2013; 288: 6921-6929Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 3.Arike L. Hansson G.C. The Densely O-Glycosylated MUC2 Mucin Protects the Intestine and Provides Food for the Commensal Bacteria.J. Mol. Biol. 2016; 428: 3221-3229Crossref PubMed Scopus (92) Google Scholar). They occur in secretions and in membrane-associated forms on epithelia, endothelia and leukocytes being associated with tissue- and cell-specific activities such as cell-attachment of commensal and pathogenic microbes, targeting of leukocytes to endothelial cells and modulation of immune responses (4.van Kooyk Y. Rabinovich G.A. Protein-glycan interactions in the control of innate and adaptive immune responses.Nat. Immunol. 2008; 9: 593-601Crossref PubMed Scopus (561) Google Scholar). The glycans on mucins are O-glycosidically linked to serine or threonine, and range from a single to 20 or more monosaccharide residues (5.Robbe C. Capon C. Coddeville B. Michalski J.C. Structural diversity and specific distribution of O-glycans in normal human mucins along the intestinal tract.Biochem. J. 2004; 384: 307-316Crossref PubMed Scopus (248) Google Scholar). The O-glycans are known to express species- and tissue-specific antigens that change during cell differentiation and oncogenesi