Binding of Serpins to Immobilized Phospholipids and Phospholipids in Suspension.

Binding of serine protease inhibitors (serpins) to nonprotein ligands such as glycosaminoglycans or phospholipids has been shown to modify their inhibitory activity and-at least in the case of SERPINA5-to mediate serpin internalization into cells. Also phospholipid functions may be altered when bound to serpins or other proteins.By interacting with phospholipids, serpins might influence a variety of cellular functions. Binding of proteins to phospholipids can be studied by several methods. Here we describe solid-phase assays, in which pure phospholipids are immobilized on nitrocellulose membranes, PVDF membranes, or microtiter plates. Bound proteins are detected with specific antibodies and labeled secondary antibodies. We also describe a method visualizing binding of phospholipids in suspension by non-denaturing polyacrylamide gel electrophoresis (PAGE) followed by Western blotting.

Regulation of the Extracellular SERPINA5 (Protein C Inhibitor) Penetration Through Cellular Membranes.

It is generally accepted that the phospholipid bilayer of the cell membrane is impermeable for proteins and peptides and that these molecules require special mechanisms for their transport from the extra- to the intracellular space. Recently there is increasing evidence that certain proteins/peptides can also directly cross the phospholipid membrane. SERPINA5 (protein C inhibitor) is a secreted protease inhibitor with broad protease reactivity and wide tissue distribution. It binds glycosaminoglycans and certain phospoholipids, which can modulate its inhibitory activity. SERPINA5 has been shown to be internalized by platelets, granulocytes, HL-60 promyelocytic leukemia cells, and by Jurkat lymphoma cells. Once inside the cell it can translocate to the nucleus. There are several indications that SERPINA5 can directly cross the phospholipid bilayer of the cell membrane. In this review we will describe what is known so far about the conditions, as well as the cellular and molecular requirements for SERPINA5 translocation through the cell membrane and for its penetration of pure phospholipid vesicles.

Interaction of protein C inhibitor with the type II transmembrane serine protease enteropeptidase.

The serine protease inhibitor protein C inhibitor (PCI) is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP) is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4) M(-1) s(-1). Low molecular weight (LMWH) and unfractionated heparin (UFH) slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition) value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml). By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.