MUC1 and MUC5B Can Form a Protective Mucin Scaffold

Both secreted mucins (MUC5B and MUC7) and membrane-bound mucins (MUC1 and MUC4) are found in the oral cavity. Secreted mucins facilitate speech and swallowing, aid in microbial clearance and participate in heterotypic complexes with other salivary proteins. Membrane bound mucins form a protective layer on epithelial surfaces. Objective: The aim of this study was to determine whether interactions between salivary membrane bound and secreted mucins occur, and to map the sites of these interactions. Methods: The N-terminal region of MUC1 (residue 1-107) was expressed as a GST fusion protein in E. coli (GST-M1) and affinity purified. MUC5B (MG1) was isolated from SMSL. Interactions between GST-M1 and MUC5B were evaluated in pull-down assays where MUC5B was incubated with GST-M1 and protein complexes were collected on GST Sepharose. Bound proteins were eluted, electrophoresed, blotted and the blots probed with anti-MG1 antibodies. Interactions were also examined by Far-Western blotting where blots containing GST-M1 fusion protein were incubated with MUC5B and probed with the anti-MG1 antibody. Interacting domains were then identified using the yeast two hybrid system with pairwise transformations containing a M1 bait vector and one of a series of MUC5B target vectors expressing different C-terminal domains. Results: Pull-down assays showed that MUC5B formed a complex with the N-terminal domain of MUC1 and this interaction was confirmed in Far Western blot assays. Yeast two hybrid mapping showed that this interaction probably occurs through multiple MUC5B domains including the C-terminal CK domain. Conclusion: Interactions between the membrane-bound mucin, MUC1 and the secreted mucin MUC5B could form a protective scaffold on the surface of the oral epithelium. The properties of the scaffold could be modulated by the stoichiometry of the mucin components and the nature of other bound salivary proteins. (Supported in part by NIH grants DE 14080, DE 11691 and DE 07652).