The role of the SEA (sea urchin sperm protein, enterokinase and agrin) module in cleavage of membrane-tethered mucins.
The membrane-tethered mucins are cell surface-associated dimeric or multimeric molecules with extracellular, transmembrane and cytoplasmic portions, that arise from cleavage of the primary polypeptide chain. Following the first cleavage, which may be cotranslational, the subunits remain closely associated through undefined noncovalent interactions. These mucins all share a common structural motif, the SEA module that is found in many other membrane-associated proteins that are released from the cell surface and has been implicated in both the cleavage events and association of the subunits. Here we examine the SEA modules of three membrane-tethered mucins, MUC1, MUC3 and MUC12, which have significant sequence homology within the SEA domain. We previously identified the primary cleavage site within the MUC1 SEA domain as FRPG/SVVV a sequence that is highly conserved in MUC3 and MUC12. We now show by site-directed mutagenesis that the F, G and S residues are important for the efficiency of the cleavage reaction but not indispensable and that amino acids outside this motif are probably important. These data are consistent with a new model of the MUC1 SEA domain that is based on the solution structure of the MUC16 SEA module, derived by NMR spectroscopy. Further, we demonstrate that cleavage of human MUC3 and MUC12 occurs within the SEA domain. However, the SEA domains of MUC1, MUC3 and MUC12 are not interchangeable, suggesting that either these modules alone are insufficient to mediate efficient cleavage or that the 3D structure of the hybrid molecules does not adequately re-create an accessible cleavage site.
Palmai-Pallag
Timea
T
Paediatric Molecular Genetics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.
Khodabukus
Naila
N
Kinarsky
Leo
L
Leir
Shih-Hsing
SH
Sherman
Simon
S
Hollingsworth
Michael A
MA
Harris
Ann
A
D018171:Agrin / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003110:Colonic Neoplasms / Q000378:metabolism; D017124:Conserved Sequence; D004765:Enteropeptidase / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008969:Molecular Sequence Data; D009077:Mucins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012617:Sea Urchins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013094:Spermatozoa / Q000378:metabolism
D018171:Agrin; D009077:Mucins; D004765:Enteropeptidase