Hans L. Vos

Cell membrane-associated mucins and their adhesion-modulating property.

A class of highly sialylated glycoproteins with very large mucin-like domains that protrude high above the plasma membrane have been shown to strongly reduce cellular adhesion. In normal epithelial cells, where the expression is restricted to the luminal side of the cell, these molecules may prevent inadvertent closing of the lumen as a result of weak, non-specific protein-protein interactions between opposite luminal membranes. In malignant tumors cellular polarization is often lost, which can lead to the entire cell surface being covered by these molecules. The resulting strongly reduced adhesion and immune recognition properties may play an important role during invasion and metastasis.

Is episialin/MUC1 involved in breast cancer progression?

Episialin, also designated MUC1, CA 15-3 antigen and PEM, is an established serum marker for breast cancer. Its function and possible involvement in tumor progression has not yet been completely established. The molecule is an extended rod-like molecule protruding high above the cell surface. It is often highly overexpressed in breast cancer relative to normal breast epithelium cells. Overexpression of episialin on cells in vitro reduces cell-cell and cell-extracellular matrix adhesion, because the rod-like molecule masks the adhesion receptors. Episialin also exerts its anti-adhesion effect in vivo. In certain human tumors, where episialin was present at the basal side of the cell, abnormal contacts between the plasma membrane and the stroma were observed. As a consequence of its anti-adhesion properties, episialin overexpression reduces the sensitivity of the cells for cytotoxic lymphocytes. This might be one of the reasons why episialin transfected cells are more potent to form experimental metastases after i.v. injection into nude mice.

The mouse episialin (Muc1) gene and its promoter: Rapid evolution of the repetitive domain in the protein

We have cloned the Muc1 gene of the mouse, encoding the murine equivalent of human episialin (also known as EMA or PEM), a mucin-like glycoprotein that is overexpressed in carcinoma cells. The extracellular domain of the mouse protein, that mainly consists of tandem repeats, contains 16 repeats of variable length and sequence, whereas the human protein usually contains between 30 and 90 nearly identical repeats. The murine repeats contain more potential O-glycan side chains and this may result in a more extended conformation of the murine protein. The transmembrane and cytoplasmic domains of the protein show about 90% conservation. The promoter region shows many conserved regions that could function as transcription factor binding sites.

Characterization of the human extracellular matrix protein 1 gene on chromosome 1q21

Ecm1, the mouse gene encoding extracellular matrix protein 1, is highly expressed in bone and cartilage as well as in osteogenic, preosteoblastic and chondroblastic cell lines. Ecm1 was recently localized to a chromosomal region in mouse syntenic to human chromosome 1q21, establishing this gene as a prime candidate gene for pycnodysostosis, a rare, autosomal recessive sclerosing skeletal dysplasia. Shortly thereafter, it was determined that cathepsin K is the pycnodysostosis gene. We now report the radiation hybrid mapping of human ECM1 to 1q21, and the gene structure and coding sequence of human ECM1.

Biosynthesis and shedding of epiglycanin: a mucin-type glycoprotein of the mouse TA3Ha mammary carcinoma cell

Epiglycanin is a mucin-type glycoprotein present at the surface of TA3Ha mouse mammary tumour cells. It is a long rod-like glycoprotein with a molecular mass of 500 kDa. Its function has not yet been established but its overexpression can affect cell-cell and cell-matrix adhesion. To understand better the biological function of epiglycanin, we have studied the biochemical structure and biosynthesis of epiglycanin in TA3Ha cells. Pulse-chase labelling experiments with [(3)H]threonine revealed an early precursor with a molecular mass of approx. 300 kDa containing approx. 5-10 kDa of N-linked glycans. The precursor was gradually converted into a high-molecular-mass mature form, owing mainly, if not entirely, to O-glycosylation. The mature molecule consists of two major glycoforms that differ in sialylation. Unlike secreted mucins, epiglycanin did not form cysteine-bound multimers, providing further evidence that epiglycanin belongs to the class of membrane-associated mucins. The mature form, but not the precursor form, is shed from the cell surface. The half-life of epiglycanin on the cell surface was found to be approx. 60 h. These results provide the first detailed analysis of the biochemical structure and biosynthesis of epiglycanin.

Cell surface associated mucins: Structure and effects on cell adhesion

Cell surface associated mucins are a class of transmembrane glycoproteins that protrude high above the cell surface and can mask other cell surface molecules, thereby interfering with the function of the latter molecules. In particular, adhesion molecules are affected, which has important consequences for cellular processes that are dependent on adhesion such as cytolysis by immune cells and probably also invasion and metastasis. Episialin is such a cell-associated mucin which is highly overexpressed on certain carcinomas and it can serve as a prototypic cell associated mucin. Here, we discuss its structure, its effect on cellular adhesionin vitro andin vivo and the consequences for LAK cell and CTL mediated cytolysis. In addition, we discuss the biological impact of other related molecules.

Membrane-associated mucins

Publisher Summary This chapter discusses membrane-associated mucins. Several mucin-like proteins have been described which contain an extended domain rich in O-linked glycans that has all the characteristics of a genuine mucin. However, these proteins are monomeric transmembrane molecules and are, therefore, classified as membrane-associated mucins. Their O-glycosylated domains may be relatively small, such as in the LDL receptor, in which case the mucin-like domain is mainly intended to lift the receptor domain above the glycocalyx, but it may also encompass most of the extracellular part of a glycoprotein. This is the case for episialin/MUC1, one of the best characterized mucin-like proteins. The chapter also discusses the properties of CD43, epiglycanin, and ascites sialoglycoprotein (ASGP). Episialin is encoded by the MUCl gene and is also known as the MUCl protein, polymorphic epithelial mucin (PEM), epithelial membrane antigen (EMA), the CA 15.3 antigen, and by various other names. The large mucin-like domain of episialin contains a large array of almost identical 20 amino acid repeats. ASGP-1 (ascites sialoglycoprotein) has many properties in common with episialin with respect to biosynthesis and processing. It is made from a precursor that is later split into a mucin-like domain (ASGP-1) and a cell-anchoring domain (ASGP-2).

39 Cell surface bound mucins and tumor progression

We have investigated the role of episialin, a surface bound mucin also known as EMA, PEM, CA 15-3 etc. encoded by the MUCI gene, in tumor progression. The molecule has an extended rod-like structure protruding more than 200xa0nm above the plasma membrane. The expression of the molecule is usually more than ten times that in normal epithelia as determined by in situ hybridization and can be extremely high on metastatic cells present in pleural effusions. Episialin overexpression strongly reduces cell-cell and cell-matrix adhesion. The anti-adhesion properties of the molecule are due to the extreme length of the molecule since genetically modified molecules with a reduced length did not exhibit the anti-adhesion effect. E-cadherin/episialin double transfectants showed that episialin can prevent E-cadherin mediated cell-cell adhesion. Decreased E-cadherin mediated cell-cell interactions are known to promote invasion. Episialin overexpression is expected to have the same effect. Indeed, episialin overexpression promoted invasion in matrigel. Episialin overexpression at the cell-stroma boundary in primary breast cancers caused large “clefts” between the stroma and tumor cells. These results suggest that episialin has the same anti-adhesion properties in vivo . Episialin also interfered with immune recognition. Melanoma transfectants expressing high levels of episialin were less susceptible to lysis by LAK cells and allogeneically stimulated T-lymphocytes. The same transfectants had a significantly higher propensity to form lung metastases after i. v. injection in nude mice than episialin negative revertants of the same clones. Episialin may protect the tumor cells against NK cells and/or episialin expressing cells are more likely to metastasize as a result of the decreased cell-cell interactions. Our results strongly suggest that episialin overexpression is an important factor in tumor progression.