Mucin-type glycans are conjugated glycans linked through N-acetylgalactosamine at the reducing end to ser or thr in the peptide. They are found primarily in secreted mucins and membrane-bound glycoproteins, including some mucins. One major function of membrane-associated mucin-type glycans is to guide the migration of circulating leukocytes to site of injury and lymphoid tissues through interaction of sialyl Lewis x- containing glycans located at the non-reducing termini with selectins. The major function of mucin-type glycans in secreted mucins is to protect mucus-secetroy tissues by retention of water, and trapping and clearance of airborne and injected pathogens. Functions of both types of glycans are controlled to a large extent by ?6GlcNAc branch structures;for membrane-associated mucin-type glycans, it is core 2 and for mucin-type glycans found in secreted mucins, it is core 2, core 4, and blood group I antigen. Core 2 can be synthesized by core 2 N-acetylglucosaminyl transferase (C2GnT)-L, -M and -T isozymes, core 4 by C2GnT- M only, and I antigen by C2GnT-M and IGnT. Therefore, alteration of the expression of these branching enzymes can have a significant impact on the production of biologically important glycotopes and thus the functions of mucins. It is not clear why membrane-bound mucins contain only core 2 while secreted mucins contain all three branch structures. Literature information suggests that non-tandem repeat peptide sequence unique to each mucin and the microenvironment in the Golgi stacks where the branching enzymes reside are the key determinants. The goal of this application is to test the hypothesis that synthesis of mucin glycan branch structures in secreted and membrane-bound mucins is controlled by different sub-Golgi localization of C2GnT-M and C2GnT-L.
The specific aims of this application are to demonstrate that: (1) overexpression of C2GnT-M would not affect the levels of core 2 structure in MUC1 and overexpression of C2GnT-L would not affect the levels of core 2 structure in MUC5AC, (2) targeting C2GnT-M and core 3 synthase to the sub-Golgi locations of C2GnT-L and core 1 synthase, respectively would generate MUC1 containing core 4, and (3) C2GnT-L and C2GnT-M are localized at separate sub- Golgi compartments by immunogold electron microscopy. H292-MUC1 cells which express C2GnT-L, C2GnT-M, MUC1, and MUC5AC will be used as the cell model. Mucin glycans released from MUC1 and MUC5AC will be analyzed by HPLC and Maldi-Tof-Ms. C2GnT-M to be targeted to C2GnT-L location will be generated by replacing its N-terminal region with that of C2GnT-L. Core 3 synthase to be targeted to core 1 synthase location will be similarly prepared. Proof of this hypothesis would advance our understanding of how synthesis of different mucin glycan branch structures is controlled, which can help develop strategies to design therapeutics to treat patients with lung diseases associated with altered mucin glycosylation.
The work proposed could advance our fundamental understanding of the key factors that control the synthesis of mucin carbohydrates, the main determinants of mucin functions. The knowledge gained could help design strategy to develop therapeutic agents to treat patients with mucus hypersecretory lung diseases and cancer.
