discovered that these adhesins show highly distinct binding preferences for O-glycan patterns displayed on distinct mucins, providing a new level of complexity and diversity to interactions with the mucin glycome. We showed that the mucin display platform is ideal for the discovery and exploration of mucin-degrading enzymes, and we discovered a small mucin-binding module X409 on the mucinase StcE6). Probing the X409 module with the cell-based mucin TR display revealed that this module bound to select human mucin TRs and for example not to MUC1, and interestingly the binding to these mucin TRs was not dependent on particular O-glycan structures. The X409 mucin-binding module provides a novel concept for selective binding to mucins without requirement of particular O-glycan structures, and the class of X409 mucin-binding modules may be useful for targeting the mucosal surfaces. Display of the Human Mucinome with Defined O-Glycans by Gene Engineered Cells – Potential for mucin therapeuticsYoshiki Narimatsumucins, and the secreted reporters were expressed stably. We reasoned that most of the features of human mucin TRs could be displayed in shorter segments of 150–200 amino acids and used a GFP-tagged expression design containing representative O-glycodomains of most human secreted and membrane-bound mucins as well as a large number of O-glycoproteins mucin-like O-glycodomain5,6) and used to produce a library of the cell membrane and secreted mucin TR reporters with defined O-glycan structure.The mucin display platform was developed and validated by a variety of means including using immune/lectin studies and structural analysis of cells and reporter molecules produced in these6). Secreted mucin TR reporters were stably expressed in cells and isolated by Ni-chromatography and characterized. We discovered that mucin TR reporters with around 200 amino acids could be produced as rather homogeneous molecules with near full O-glycan occupancies and distinct custom-designed O-glycan structures, which enabled us to characterize these reporters (at least for the simplest glycoforms) by intact mass spectrometry (MS)6-8). Display of these reporters as cell membrane proteins provides the first cell-based display of the human mucins for interrogation of biological interactions and enabled us to probe and dissect the binding specificities of microbial adhesins, influenza virus6) as well as Siglecs5). We Figure 2. Potencial application study and academic community use of Mucin display platformProspectiveThe cell-based mucin display platform presented here offers a unique resource with wide applications and opportunities for the discovery and dissection of molecular properties of natural human mucins and other glycoproteins with mucin domains. The informational cues harbored in mucin TRs with their distinct patterns and structures of O-glycans can now be addressed with well-defined molecules in a variety of assay formats. Mucin TR 114
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