Contribution Of Conservative Cleavage Motif To The Proteolytic Cleavage Within Carboxyl Terminal Domain Of Rodent Muc3

Object:To demonstrate how the conservative cleavage motif,LS₁KGS₂IV₁V₂,contributes the proteolytic cleavage through the mutagenesis experiment of each residue in the LS₁KGS₂I V₁V₂ motif of rodent Muc3 mucin.Methods:Constructs p201/a,p20s₁/a,p20k/a,p20i/a,p20v₁/a,p20v₂/a were produced by Site-directed mutagenesis of p20.Mutations were confimed by DNA sequencing.Each construct was transfected into COS-7 cell by cation lipoid mediating methed.The cell lysates were detected by SDS/PAGE and Western blotting using anti-V5 and anti-Myc antibodies.The membranes were scanned and semiquantified for intensities of bands,the percentages of the uncleaved carboxyl domain of Muc3 was determined according to the following method:Ratio of the uncleaved Muc3=Intensity of the uncleaved carboxyl domain of Muc3/ (Intensity of the uncleaved carboxyl domain of Muc3+Intensity of V5 tagged N-terminal fragment)×100%.Results:Mutants of the conservative cleavage motif,LS₁KGS₂IV₁V₂,were constructed successfully according to DNA sequencing and alignment with clustalx1.83.The results of Western blotting demonstrate that S₂ mutation to alanine prevents cleavage completely, 79%,39%,22%,17%and 14%products from p20g/a(G mutated to A),p20i/a(I mutated to A),p201/a(L mutated to A),p20v₂/a(V₂ mutated to A) and p20v₁/a(V₁ mutated to A) were remaining to be uncleaved,p20k/a(K mutated to A) and p20s₁/a(S₁ mutated to A)(6%and 3%) exerted almost same result as the wild type of p20 transfectant(4%).The Mt of the cleaved N-terminal fragment of p20s₁/a transfectant was 26 kDa,but the N-terminal fragments from p20,p20g/a,p201/a,p20k/a,p20i/a,p20v₁/a and p20v₂/a were 30 kDa. There was 4 kDa difference in molecular weight between p20s₁/a and the other mutants and the wild type.Conclusion:Our studies indicate that S₂,G,I,L,V₁,V₂ within the conservative cleavage motif, LS₁KGS₂IV₁V₂,are important for the cleavage to happen and contribute the structural formation and conformational stress of the small loop betweenβ2 andβ3 strands.But K and S₁ residues within LS₁KGS₂IV₁V₂ motif have no effect to the cleavage of rodent Muc3. S₁ residue might be O-glycosylated and abolishment of O-glycosylation on the S₁ residue d??not affect the cleavage of LS₁KGS₂IV₁V₂ motif.