SEA (sea-urchin sperm protein, enterokinase and agrin)-module cleavage, association of fragments and membrane targeting of rat intestinal mucin Muc3.
In a previous study we showed, by transient expression studies in COS-1 cells, that the C-terminal domain of rat intestinal membrane mucin Muc3 was cleaved between glycine and serine within a GSIVV (one-letter) amino acid sequence during its residence in the endoplasmic reticulum. The extracellular domain fragment remained linked to the membrane-associated fragment by non-covalent interactions. The present study demonstrates that cleavage depends not only on the presence of the G/SIVV site (where G/S is the glycine downward arrow serine cleavage site), but also on more distant C-terminal sequences in the SEA (sea-urchin sperm protein, enterokinase and agrin) module. Inhibition of N-glycosylation by tunicamycin treatment of transfected cells did not prevent re-association of fragments, although cleavage was partially impaired, as some of the non-glycosylated, non-cleaved products were seen to accumulate in cells. Membrane targeting of the Muc3 domain and its cleavage products occurred in transfected cells and was not impaired in mutants in which the cleavage site was mutated. Targeting was also not impaired for products devoid of N-linked oligosaccharides. Our studies thus indicate that (a) cleavage within the SEA module of rat Muc3 requires participation of peptide sequences located C-terminal of and distant from the cleavage site, (b) re-association of the fragments requires the SEA module, but is independent of N-linked oligosaccharides, and (c) membrane targeting of the mucin is independent of the SEA-module-cleavage reaction.
Khatri
Ismat A
IA
Research Institute, Department of Structural Biology and Biochemistry, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8.
Wang
Rongquan
R
Forstner
Janet F
JF
D018171:Agrin / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004765:Enteropeptidase / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism; D008297:Male; D055263:Mucin-3; D009077:Mucins / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D051381:Rats; D012617:Sea Urchins / Q000378:metabolism*; D013094:Spermatozoa / Q000378:metabolism
D018171:Agrin; C524651:Muc3 protein, rat; D055263:Mucin-3; D009077:Mucins; D009844:Oligosaccharides; D004765:Enteropeptidase