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Synthesis and secretion of Muc4/sialomucin complex: implication of intracellular proteolysis.

ABSTRACT: Muc4/sialomucin complex (SMC) is a heterodimeric glycoprotein complex implicated in epithelial protection and overexpressed in some tumours. It is encoded by a single gene, and the two subunits are produced by proteolytic cleavage at a time before substantial O-glycosylation, near the time of transit from the endoplasmic reticulum to the Golgi. Although Muc4/SMC is translated as a membrane protein, it is produced as a soluble form in many epithelia. Transfection experiments using Cos-7, HBL-100 human epithelial, MCF-7 human breast tumour and HC11 mouse mammary cell lines showed that soluble rat Muc4/SMC is produced by a proteolytic cleavage mechanism and released by secretion from multiple cell lines, including both human and mouse normal epithelial cells and tumour cells. Similar transfection experiments demonstrated the same mechanism for the human analogue MUC4. Gel electrophoresis analyses of deglycosylated membrane and soluble transmembrane subunits and of the membrane-associated cleavage fragment indicated a released cleavage product of 25 kDa, resulting from cleavage between two epidermal growth factor-like domains. Further evidence for this site was obtained from deletion mutants removing this region of the protein, which blocked secretion. Finally, pulse-chase analyses of Muc4/SMC biosynthesis indicated no kinetic difference between the timing of the cleavage to release the soluble form and that to produce the two subunits, indicating that the soluble form is created early in transit to the cell surface. These studies provide the first clear evidence that membrane mucins can be released from cells by an intracellular proteolytic mechanism that leads to secretion of the soluble form of the mucin.

SUBMITTER: Komatsu M

PROVIDER: S-EPMC1222983 | biostudies-other | 2002 Nov

REPOSITORIES: biostudies-other

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Project description:Sialomucin complex (SMC/Muc4) is a heterodimeric glycoprotein complex consisting of a mucin subunit ascites sialoglycoprotein-1 (ASGP-1) and a transmembrane subunit (ASGP-2), which is aberrantly expressed on the surfaces of a variety of tumour cells. SMC is transcribed from a single gene, translated into a large polypeptide precursor, and further processed to yield the mature ASGP-1/ASGP-2 complex. SMC has complex spatial and temporal expression patterns in the normal rat, suggesting that it has complex regulatory mechanisms. A crude exon/intron map of the 5' regions of the SMC/Muc4 gene generated from clones isolated from a normal rat liver genomic DNA library reveals that this gene has a small first exon comprising the 5' untranslated region and signal peptide, followed by a large intron. The second exon appears to be large, comprising the 5' unique region and a large part (probably all) of the tandem repeat domain. This structure is strikingly similar to that reported for the human MUC4 gene. Using PCR-based DNA walking, 2.4 kb of the 5'-flanking region of the SMC/Muc4 gene was cloned and characterized. Promoter-pattern searches yielded multiple motifs commonly found in tissue-specific promoters. Reporter constructs generated from this 2.4 kb fragment demonstrate promoter activity in primary rat mammary epithelial cells (MEC), the human colon tumour cell line HCT-116, and the human lung carcinoma cell line NCI-H292, but not in COS-7 cells, suggesting epithelial cell specificity. Deletion constructs of this sequence transfected into rat MEC or HCT-116 cells demonstrate greatly varying levels of activity, suggesting that there are positive and negative, as well as tissue-specific, regulatory elements in this sequence. Taken together, these data suggest that the rat SMC/Muc4 promoter has been identified, that it is tissue- (epithelial cell-) specific, and that there are both positive and negative, as well as tissue-specific, regulatory elements in the sequence.

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Synthesis and secretion of Muc4/sialomucin complex: implication of intracellular proteolysis.

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