ABSTRACT The enzyme gamma-glutamyl transpeptidase (GGT) is over-expressed by liver tumors and is elevated in the serum of patients with these tumors. It is also elevated in the serum of patients with liver cirrhosis, cholestatic liver disease, alcoholism and other non-malignant liver diseases. However, multiple, large cohort studies over the past thirty years have shown that patients with hepatocellular carcinoma (HCC) have tumor-specific isoforms of GGT in their serum that can be resolved on native polyacrylamide gels. GGT is a type II transmembrane protein. The dominant liver-tumor specific form is of GGT in the serum is hydrophilic, lacking its transmembrane domain. It is not known how the liver-tumor-specific hydrophilic form of GGT is cleaved from its transmembrane domain. Preliminary data indicate that there are liver-tumor specific post-translational modifications of GGT. The mechanism by which GGT is released from the cell and enters the serum may also differ between tumors and non-malignant disease and, thus, contribute to the unique migration of the liver tumor-specific GGT on native polyacrylamide gels forms. This proposal has three specific aims. The first is to characterize the glycans and other post-translational modifications on GGT from normal human liver, kidney and pancreas to identify tissue-specific patterns of post-translational modifications. The second is to determine the cleavage site and the protease in liver tumors that cleaves the hydrophilic, liver-tumor specific form of GGT from its transmembrane domain. We will also determine whether this cleavage site is unique to GGT released from liver-tumor cells. The third specific aim is to identify post-translational modifications on GGT that are specific to liver tumors. These data will define the molecular mechanisms that give rise to the liver tumor specific forms of GGT. Changes in pathways that regulate post-translational modifications of GGT are likely to affect the post-translational modifications of many other proteins. The consistency with which the liver tumor- specific isoforms of GGT are observed in the serum of patients with HCC indicates that GGT may be a reporter of changes in critical pathways that provide the tumor cell with a selective growth advantage during tumor formation or progression. Identifying these pathways and other proteins that are similarly modified will provide novel targets for diagnosis and therapy.