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.
Highlights
From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, W12 0NN, UK; §Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215; ¶Department of Chemistry, UCIBIONOVA University of Lisbon, 1099085, Portugal, ʈDivision of Infectious Diseases, Cincinnati Children’s Hospital Medical Center and **University of Cincinnati College of Medicine, Cincinnati, Ohio 45229
Among the mucins were those derived from human ovarian cystadenomas, meconia, and porcine stomach mucin (PSM) and bovine submaxillary mucin (BSM)
There was binding by both VP8* proteins to preparations of all five of the meconiumderived, eleven of the cystadenoma-derived mucins as well as to the PSM and BSM (Fig. 2A and 2B and supplemental Fig. S2)
Summary
O-glycome Beam Search Arrays technology coupled with mass spectrometry has undergone sequential developments (17, 18), and has been the basis of the first microarray system for sequence-defined glycans (19). This is a state-of-the-art platform with the glycan probes robotically arrayed in a liposomal formulation at low femtomoles per spot (20, 21). As an exemplar study-case, applying the approach to a ligand-bearing mucin, we identify O-glycan ligands for the cell-adhesion proteins, VP8*, of two rotaviruses P[19] and P[10]. These are evolutionarily closely related but of distinct genotypes.
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