Glycobiology Advance Access published online on June 26, 2009
Glycobiology, doi:10.1093/glycob/cwp095
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Role of N-glycosylation of the SEA module of rodent Muc3 in posttranslational processing of its carboxy-terminal domain
Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing 400038, P. R. of China
¶ Corresponding Author: Rongquan Wang MD Ph.D Prof, Department of Gastroenterology, Southwestern Hospital, Third Military Medical University, Chongqing 400038, P. R. of China, Telephone: 023-65478216 (H), 023-68754124, Email: rongquanw{at}hotmail.com
Received on April 10, 2009; accepted on June 22, 2009
A prominent feature of the rodent Muc3 SEA module is the precursor cleavage event that segregates the O-glycosylated N-terminal fragment and transmembrane domain into the noncovalently attached heterodimer. There are seven potential N-glycosylation sites that occur in a cluster in the SEA module of Muc3. However, it is unknown if these sites are modified or what the function of these N-glycans may be in the SEA module. Our data show that the proteolytic cleavage of the rodent Muc3 SEA module was partially prevented by treatment with tunicamycin, an inhibitor of N-glycosylation. Each single mutant of the seven N-glycosylation sites (N1A, N2A, N3A, N4A, N5A, N6A and N7A) and multiple mutants, including double (N34A) and triple (N345A) mutants, and mutants with four (N3457A), five (N34567A), six (N134567A and N234567A), seven (N1234567A) mutations, confirmed that all seven of these potential sites are N-glycosylated simultaneously. The proteolytic cleavage of the SEA module was not affected when it lacked only one, two or three N-glycans, but was partially inhibited when lacking four, five and six N-glycans. In all, 1.99%, 48.34%, 85.03% and 72.98% of the products from N3457A, N34567A, N134567A and N234567A transfectants, respectively, remained uncleaved. The proteolytic cleavage was completely prevented in the N1234567A transfectant, which eliminated all seven N-glycans in the SEA module. The interaction of the heterodimer was independent of the N-glycans within the rodent Muc3 SEA module. Thus, the N-glycosylation pattern constituted a control point for the modulation of the proteolytic cleavage of the SEA module.
Key words: N-glycan / proteolytic cleavage / mutation
* : This work was supported by the State Foundation for Natural Sciences of the People's Republic of China (No. 30300121, 30470401) and the Natural Science Foundation Project of CQ CSTC 2008BA5034