Contribution of the conservative cleavage motif to posttranslational processing of the carboxyl terminal domain of rodent Muc3. In previous works, we showed by transient expression studies in COS-1 cells that the C-terminal domain of rat intestinal membrane mucin (rMuc3) that was cloned in the pSecTag2 plasmid (named as p20) is posttranslationally cleaved twice. One location is between the glycine and the serine within a LS1KGS2IV1V2 motif, and the other is in the 49 kDa membrane-tethered fragment at an undefined site. The sea-urchin sperm protein, enterokinase and agrin module of rMuc3 is responsible for the cleavage and association of the cleaved fragments. The present study demonstrates how the conservative cleavage motif LS1KGS2IV1V2 contributes to posttranslational processing through mutagenesis of each residue in the LS(1)KGS2IV1V2 motif. Mutation of S2 to alanine (p20s2/a) completely prevented cleavage. While p20k/a (in this construct the K is replaced by A) and p20s1/a (in this construct the S1 is replaced by A) (6 and 3%) showed almost the same result as the wild-type p20 transfectant (4%), 79, 39, 22, 17, and 14% of the products from p20g/a (in this construct the G is replaced by A), p20i/a (in this construct the I is replaced by A), p20l/a (in this construct the L is replaced by A), p20v2/a (in this construct the V2 is replaced by A), and p20v1/a (in this construct the V1 is replaced by A) remained uncleaved. The cleaved N-terminal fragment of the p20s1/a transfectant was 26 kDa, but the N-terminal fragments from p20, p20g/a, p20l/a, p20k/a, p20i/a, p20v1/a, and p20v2/a were 30 kDa. The S1 residue was possibly O-glycosylated, which was supported by deglycosylation with O-cocktail (a mixture of glycosidases). The N-terminal fragment of p20s1/a transfected cells was present at high levels in the spent media. Thus, the S2, G, I, L, V2, and V1 residues within the conserved cleavage motif, LS1KGS2IV1V2, are important for cleavage and contribute to the structural formation and conformational stress of the small loop between the beta2 and the beta3 strands. The S1 residue is possibly O-glycosylated, and mutation of S1 residue to alanine does not affect the cleavage of the LS1KGS2IV1V2 motif, but it is important for the dissociation and further release of the cleaved N-terminal fragment from the cell surface.