A New Look into Protein C Inhibitor Posttranslational Modifications and their Functions

Sammanfattning: The influences of posttranslational modifications on the functions of the versatile serpin protein C inhibitor (PCI) were studied. PCI is a serine protease inhibitor that is expressed in many tissues and secreted to various fluids in human, including blood plasma, seminal plasma, and urine. PCI in blood can act both as an anticoagulant and a procoagulant and is believed to play a role in pathogen defence. PCI in reproductive tissues is believed to regulate human reproduction at several steps, including the fertilization process. Due to the broad protease specificity and the contradictory activities, the physiological role of PCI is elusive. In this work the inhibitor was purified from blood and seminal plasma by immunoaffinity chromatography. Blood-derived PCI was found to be highly heterogeneous, due to variations in posttranslational modifications. The occupancy and structures of N- and O-glycans attached to blood plasma PCI and N-glycans of seminal plasma PCI were determined by mass spectrometry. An O-glycosylation site at Thr 20 was identified in PCI derived from blood. N-glycan structures of PCI isolated from blood and seminal plasma differed markedly, demonstrating that they are expressed in a tissue-specific manner. Proteolytic processing also appeared to be tissue-specific, since N-terminally cleaved PCI was found in PCI isolated both from blood and seminal plasma, but the length of the lacking segment differed. The effects of the N-linked glycans and the N-terminus of PCI on protease inhibition were determined using enzymatic measurements with chromogenic substrates. The N-glycans and the N-terminus had different effects on the inhibition of thrombin, factor Xa and prostate specific antigen, demonstrating that posttranslational modifications of PCI affect its functional specificity. These findings enhance the understanding of the regulation of the various functions of PCI and may potentially be used for the production of specialized PCI variants for medical purposes.