The MUC1 SEA Module Is a Self-cleaving Domain

Abstract

MUC1, a glycoprotein overexpressed by a variety of human adenocarcinomas, is a type I transmembrane protein (MUC1/TM) that soon after its synthesis undergoes proteolytic cleavage in its extracellular domain. This cleavage generates two subunits, ␣ and ␤, that specifically recognize each other and bind together in a strong noncovalent interaction. Proteolysis occurs within the SEA module, a 120-amino acid domain that is highly conserved in a number of heavily glycosylated mucin-like proteins. Post-translational cleavage of the SEA module occurs at a site similar to that in MUC1 in the glycoproteins IgHepta and MUC3. However, as in the case of other proteins containing the cleaved SEA module, the mechanism of MUC1 proteolysis has not been elucidated. Alternative splicing generates two transmembrane MUC1 isoforms, designated MUC1/Y and MUC1/X. We demonstrated here that MUC1/X, whose extracellular domain is comprised solely of the SEA module in addition to 30 MUC1 N-terminal amino acids, undergoes proteolytic cleavage at the same site as the MUC1/TM protein. In contrast, the MUC1/Y isoform, composed of an N-terminally truncated SEA module, is not cleaved. Cysteine or threonine mutations of the MUC1/X serine residue (Ser-63) immediately C-terminal to the cleavage site generated cleaved proteins, whereas mutation of the Ser-63 residue of MUC1/X to any other of 17 amino acids did not result in cleavage. In vitro incubation of highly purified precursor MUC1/X protein resulted in self-cleavage. Furthermore, addition of hydroxylamine, a strong nucleophile, markedly enhanced cleavage. Both these features are signature characteristics of self-cleaving proteins, and we concluded that MUC1 undergoes autoproteolysis mediated by an N 3 O-acyl rearrangement at the cleavage site followed by hydrolytic resolution of the unstable ester and concomitant cleavage. It is likely that all cleaved SEA module-containing proteins follow a similar route.The MUC1 gene is highly expressed in a number of human epithelial malignancies, including breast, prostate, and colon carcinomas, as well as on the malignant plasma cells of multiple myeloma (1-9). As a well characterized tumor-associated protein, it has generated considerable interest as a tumor marker for disease prognosis (10 -14) as well as a target for tumor cell killing (15-18). Although alternative splicing can generate multiple MUC1 protein forms (19 -23), the most intensively studied MUC1 protein is a type I transmembrane protein comprised of a heavily glycosylated extracellular domain containing a tandem-repeat array, a transmembrane domain, and a cytoplasmic domain (Fig. 1, MUC1/TM) (24 -26). MUC1/TM is proteolytically cleaved soon after its synthesis, generating two subunits, ␣ and ␤, that specifically recognize each other and bind together by a strong noncovalent interaction (27).Cleavage occurs within the SEA module (28 -30), a highly conserved protein module so-called from its initial identification in a sperm protein, in enterokinase, and in agrin (31), that is found in a numb...

MUC16 is produced in tracheal surface epithelium and submucosal glands and is present in secretions from normal human airway and cultured bronchial epithelial cells

“…MUC16 has a SEA module in the C-terminal domain (O'Brien et al, 2001) and in MUC1, MUC3, MUC13, this module contains a conserved sequence (GSVVV) where each of these mucins may be cleaved (Palmai-Pallag et al, 2005) -with MUC1 undergoing an autocatalytic cleavage (Levitin et al, 2005). In secreted MUC16 the most C-terminal peptide identified with mass spectroscopy was a unique peptide found at the N-terminal end of this SEA domain.…”

MUC1 oncoprotein is targeted to mitochondria by heregulin-induced activation of c-Src and the molecular chaperone HSP90

“…MUC1 forms a heterodimer following synthesis as a single polypeptide and cleavage of the precursor into two subunits in the endoplasmic reticulum (Ligtenberg et al, 1992) that may be mediated by an autocatalytic process (Levitan et al, 2005). The >250 kDa MUC1 subunit contains variable numbers of 20 amino-acid tandem repeats that are imperfect with highly conserved variations and are modified by O-linked glycans (Gendler et al, 1988;Siddiqui et al, 1988).…”

Evolution of the human MUC1 oncoprotein

“…The membrane-bound mucins typically have a sea urchin sperm protein, enterokinase and agrin (SEA) domain that resides between the glycosylated ectodomain and the transmembrane domain. Autoproteolysis of the MUC1 SEA domain results in the formation of an N-terminal ectodomain and a C-terminal transmembrane subunit that in turn form a stable non-covalent heterodimer (2)(3)(4).…”