N-linked oligosaccharides play a role in disulphide-dependent dimerization of intestinal mucin Muc2.
Within the C-terminal domain of many secretory mucins is a 'cystine knot' (CK), which is needed for dimer formation in the endoplasmic reticulum. Previous studies indicate that in addition to an unpaired cysteine, the three intramolecular cystine bonds of the knot are important for stability of the dimers formed by rat intestinal mucin Muc2. The present study was undertaken to determine whether the two N-glycans N9 and N10, located near the first and second cysteines of the knot, also play a role in dimer formation. The C-terminal domain of rat Muc2 (RMC), a truncated RMC mutant containing the CK, and mutants lacking N9 and N10 sites, were expressed in COS-1 cells and the products monitored by radioactive [(35)S]Met/Cys metabolic pulse-chase and immunoprecipitation. Mutation of N9, but not N10, caused increased synthesis of dimers over a 2-h chase period. The N9 mutant remained associated with calreticulin for a prolonged period. About 34-38% of the total labelled products of RMC and its mutants was secreted into the media by 2 h, but the proportion in dimer form was dramatically reduced for the N9 mutant, suggesting lower dimer stability relative to RMC or its N10 mutant. We conclude that under normal conditions the presence of the N9 glycan functions to maintain a folding rate for mucin monomers that is sufficiently slow to allow structural maturation and stability of Muc2 dimers. To our knowledge this report is the first demonstration that a specific N-glycan plays a definitive role in mucin dimer formation.
Division of Structural Biology & Biochemistry, Research Institute, The Hospital for Sick Children and the University of Toronto, 555 University Avenue, Toronto, ON M5G 1X8, Canada.
Sherilyn L
SL
Bell
Gongqiao
G
Xu
Ismat A
IA
Khatri
Rongquan
R
Wang
Sameera
S
Rahman
Janet F
JF
Forstner
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D017931:DNA Primers; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007422:Intestines / Q000737:chemistry*; D055262:Mucin-2; D009077:Mucins / Q000737:chemistry*; D009844:Oligosaccharides / Q000737:chemistry*; D051381:Rats
D017931:DNA Primers; D004220:Disulfides; C524628:Muc2 protein, rat; D055262:Mucin-2; D009077:Mucins; D009844:Oligosaccharides