Role Of N-glycosylation Of The SEA Module Of Rodent Muc3 In Posttranslational Processing Of Its Carboxy-terminal Domain

Objective: To demonstrate how seven N-glycans residing SEA module influence its proteolytic cleavage within LSKGSIVV motif, association between N- and C-terminal cleaved fragments, and cell surface targeting of rat Muc3 carboxyl terminus.Methods: All constructs, including the single mutants and multiple mutants, which were produced by Site-directed mutagenesis to abolish the N-glycosation sites based on p20, a eukaryotic expression vector containing the cDNA of rat Muc3 carboxy-terminal domain. Mutations were confimed by DNA sequencing. Mutants were transfected into COS-7 cells by cation lipoid mediating method. The cell lysates were identified by Western blotting using anti-V5 or anti-Myc antibodies. Positive bands were detected with the NBT/BCIP substrate system. FITC labeled antibodies were used to determine the cell surface targeting of rat Muc3 carboxyl terminus.Results:1. All constructs which abolished the N-glycosation sites were produced by Site-directed mutagenesis of p20. Mutations were confimed by DNA sequencing.2. The molecular weights of the N-terminal fragments from the p20 transfectant (wild type) and the N4A, N5A, N6A and N7A mutants were 26-30 kDa. The products were primarily 30 kDa. The smear below 30 kDa represents incompletely glycosylated product. In contrast, the molecular weights of the N-terminal fragments from the N1A, N2A or N3A mutants were primarily 26 kDa with some smeared products below 26 kDa that may also be due to incompletely glycosylated product. The cleaved C-terminal fragments from N1A, N2A, N3A or p20 transfectants were mainly 49 kDa, but the molecular weights of the C-terminal cleaved fragments from N4A, N5A, N6A and N7A were different from those of N1A, N2A, N3A and wild type. Strong bands ranging from 45-46 kDa in N4A, N5A, N6A and N7A mutants were observed.3. The N-terminal fragment from wild type transfectants was 30 kDa, the N-terminal fragments from N1A, N2A and N3A were around 26 kDa and the N-terminal fragments from N34A, N345A, N3457 and N34567A were similar in size to the single mutant, N3A. The molecular weight of the N-terminal fragments from both of the mutants with six mutations decreased to 23 kDa. It was consistent with their speculated molecular weights of the N-terminal fragments from all kinds of mutants. Interestingly, when all seven N-glycosylation sites were abolished, the N-terminal cleaved fragment disappeared.4. 100% of the product from the N1234567A transfectant was uncleaved. N134567A and N234567A produced 85.03% and 72.98% uncleaved products, respectively. In contrast, 48.34% of the product from the N34567A mutant was uncleaved, 1.99% of the product from the N3457A mutant was uncleaved and the products from the N34A and N345A mutants were the same as wild type.5. No immunoreactive product was found in the spent media from the different mutants or wild type using the anti-V5 antibody.6. Immunofluorescence experiments using an FITC labeled antibodies showed that the distribution of fluorescence of all transfectants was similar except N2A,N234567A and N1234567A which have no fluorescence on plasma membrane not only in the fixed transfected cells but non-fixed transfected cells.Conclusion: Our studies indicated that all seven N-glycosylation sites in the SEA module of rMuc3 were occupied simultaneously, association between the two cleaved fragments did not involve the N-linked polysaccharides in the SEA module of the rodent Muc3 carboxyl terminus. Only the second N-linked polysaccharide in the SEA module of the rodent Muc3 carboxyl terminus (N2) is essential for cell membrane targeting. This finding has therapeutic implications for the treatment of mucous obstruction in cystic fibrosis patients, in which a soluble rodent Muc3 was one of the constituents in a mouse model.