Objective    The C-terminal domain of rodent Muc3 is proteolytically cleaved. This study is to explore the relationship between N-linked oligosaccharides in SEA module and the proteolytic cleavage within C-terminal domain of rodent Muc3.     Methods    Truncated rodent Muc3 C-terminal domains with complete SEA module (p20SEA) were produced by site-directed mutagenesis to insert a stop code in the required place. Proteins were detected by pulse/chase and immunoprecipitation method

or SDS/PAGE and Western blot. Inhibition of glycosylation of the expressed protein was performed by using tunicamycin.     Results    Muc3 C-terminal domain was posttranslationally cleaved to produce a V5-tagged 30 000 extracellular glycopeptide and a Myc-tagged 49 000 membrane-associated glycopeptide. Treatment with tunicamycin to transfected COS-1 cells led to the abundant production of 60 000 uncleaved and whole-length Muc3 C-terminal domain

the 30 000 N-terminal fragment shifted to 22 000 and 49 000 C-terminal fragment shifted to 41 000 after deglycosylation. The truncated Muc3 C-terminal domain containing complete SEA module but without the following residues led to production of 36 000 uncleaved and whole-length protein

and 30 000 cleaved product shifted to 22 000 after deglycosylation.     Conclusion    Proteolytic cleavage in both complete rodent C-terminal domain and complete SEA module without the following residues were partially inhibited by tunicamycin.