Structure-function studies of tumor-associated MUC1: Primary sequence of MUC1 involved in its post-translational processing and function

Studies described in this dissertation were designed to elucidate the relationship between the structure and function of tumor-associated MUC1. Towards this goal I have performed site-directed mutagenesis and domain deletions in MUC1 to determine their contribution to the post-translational processing and function of MUC1. MUC1 is overexpressed and aberrantly glycosylated by a variety of tumor cells. Studies presented focus on investigating the ability of MUC1 to mediate or block binding to adhesion molecules including E-, P- and L-selectin and members of the siglec (sialic acid-binding immunoglobulin superfamily lectins) class of adhesion molecules (sialoadhesin, myelin associated glycoprotein (MAG), CD22 and CD33). The tandem repeat domain of MUC1 was shown to specifically inhibit E-selectin binding to its tumor-associated ligand. In addition, sialylation of MUC1 is shown to mediate binding to siglec family members sialoadhesin and MAG. These results demonstrate for the first time that MUC1 can simultaneously mediate and block binding to adhesion molecules. Investigation of the molecular mechanisms involved in the described anti-adhesion and adhesion functions of MUC1 are also presented in this thesis.; Proteolytic cleavage of the full length protein results in two fragments that remain non-covalently associated. Studies presented were designed to elucidate the mechanism mediating the noncovalent heterodimerization of MUC1 subunits. We hypothesized that one or more extracellular Tyr residues are sulfated and that tyrosine sulfation of MUC1 plays a role in the heterodimerization of MUC1 subunits. These hypotheses were disproved. However, conservative mutation of the five Tyr residues to phenylalanines (Phe) demonstrated that the hydrogen bonding properties of Tyr residues are involved in the association of the two MUC1 polypeptides. This is the first report defining elements of the primary sequence of MUC1 that are involved in association of MUC1 subunits. In addition, I have demonstrated that the heterodimerization of MUC1 subunits plays a role in the trafficking of MUC1 subunits to the cell surface and in the glycosylation of MUC1.