Interaction of protein C inhibitor with retinoids and fatty acids: Biochemical characteristics and biological relevance

Serpins (Serine protease inhibitors) are a family of related glycoproteins, which not only includes inhibitors of proteolytic enzymes, but also non-inhibitory members with other biological functions, such as the hormone precursor angiotensinogen or the binding proteins for hydrophobic hormones corticosteroid-binding globulin and thyroxin-binding globulin. My research group has been working on protein C inhibitor (PCI), a secreted inhibitory member of the serpin family for many years. PCI is synthesized by many human tissues and is present in many body fluids. PCI seems to be a nonspecific protease inhibitor, which inhibits not only activated protein C, but also many other intra-and extravascular proteases. We have recently generated PCI-deficient mice by targeted disruption of the PCI gene. All mice were normal at birth; they developed normally and appeared healthy. However, male homozygous PCI- deficient mice were infertile because of abnormal spermatogenesis. The fact that there were no other abnormalities was not surprising, since our studies also revealed that in mice PCI is almost exclusively expressed in the reproductive tract. Therefore, the biological role of PCI in humans is still unclear. We have shown in a previous project that PCI specifically binds retinoids (derivatives of Vitamin A, which are involved in the regulation of differentiation and development). Furthermore we have obtained data indicating that PCI can be internalized by leukocytes and translocated to the nucleus, suggesting that PCI might also have intracellular functions, which may by related to signaling by retinoids and possibly other PCI-binding lipids. In this project we therefore want to analyze the interaction of PCI with retinoids and other lipids, especially fatty acids, and determine the biological role of this interaction. We will determine the retinoid-binding site within the PCI molecule and analyze whether or not other lipids can bind to the same site. Furthermore we want to determine, if PCI influences the uptake of or signaling by retinoids, or if retinoids influence the internalization and nuclear translocation of PCI. We will furthermore study effects of PCI-binding lipids (e.g. retinoids) in mouse organ explants using organs from wild-type (containing PCI) and PCI-knockout mice. Alltogether, results of these studies should clarify the biological relevance of retionid/lipid-binding by PCI.

Protein C inhibitor (PCI) is a secreted non-specific protease inhibitor and belongs to the serpin (serine protease inhibitor) family. PCI is a heparin-binding serpin, and heparin modulates its activity and target enzyme specificity. The physiological role of PCI has not been defined so far. In previous projects we have shown that PCI binds retinoids. It was therefore the aim of the present project to study the interaction of PCI with lipids in general. We were able to show that PCI specifically binds certain phospholipids [i.e. oxidized phosphatidylethanolamine (OxPE), phosphatidylserine (PS), and oxidized phosphatidylserine (OxPS)]. Furthermore we have shown that the inhibitory activity of PCI towards its target proteases activated protein C, thrombin, and urokinase is stimulated by these phospholipids. The inactivation of tissue kallikrein by PCI, on the other hand, is inhibited in a dose-dependent manner by OxPE, PS, and OxPS. Therefore the effect of phospholipids on PCI activity is very similar to the effect of heparin. Due to these observations we analyzed the hypothesis, if the heparin-binding site of PCI (H-helix) is involved in phospholipid binding. We expressed and purified a recombinant PCI mutant, in which the amino acids responsible for heparin binding were deleted. This mutant did not bind to immobilized phospholipids. It inhibited activated protein C, but its activity was neither stimulated by heparin nor by phospholipids. Furthermore we could show that a peptide corresponding to the heparin-binding site of PCI competed not only with the stimulatory effect of heparin, but also with the stimulatory effect of phospholipids. Therefore we conclude that the heparin-binding site of PCI is also involved in phospholipid binding. The identified PCI binding phospholipids OxPE, PS, and OxPS are normally not accessible for PCI in vivo. However, they can be exposed, when cells undergo apoptosis and/or at sites of inflammation. To determine, if binding of PCI to phospholipids also occurs in vivo, we analyzed atherosclerotic plaques by immunohistochemistry. We can show colocalization of PCI and the PS-binding protein Annexin V. We were also interested in analyzing the effect of PCI on the phagocytosis of PS-exposing, apoptotic cells by macrophages. Using flow cytometry we can show that cells, which expose PS, also bind PCI. Preliminary data furthermore suggest that PCI stimulates phagocytosis. Future studies should clarify the biological role of the interaction of PCI with phospholipids.