Tuesday, April 29, 2025
4/29/2025
Chemoenzymatic Semisynthesis of Proteoglycan and Its Cofactor Activities - Project Summary/Abstract Thrombomodulin (TM) is a proteoglycan expressed on the surface of vascular endothelial cells and contributes to vascular haemostatic balance. TM serves as a cofactor for thrombin-mediated activation of both protein C and thrombin activatable fibrinolysis inhibitor (TAFI), which alter thrombin specificity from procoagulant toward anticoagulant and antifibrinolytic pathways. It is acritical question whether protein C and TAFI compete for TM/thrombin complex. Earlier study with soluble TM found that protein C and TAFI competitively bind to TM/thrombin complex in solution, but later study of endothelial cells found that protein C and TAFI do not compete each other. Interestingly, our recent study with liposomal TM found a lipid-dependent competition between protein C and TAFI for the thrombin/TM complex. These results indicate that membrane lipids affect protein C and TAFI activation. Therefore, whether and how these two physiologically important events are either independently or cooperatively regulated in the vasculature deserve to be filly elucidated. TM is a proteoglycan with chondroitin sulfate (CS) glycosaminoglycan (GAG) on its serine-threonine rich region. TM CSGAG can be involved in thrombin binding via it’s exosite II and protein C activation. However, whether the TM CSGAG is involved in TM’s TAFI activation and its role in regulation of protein C and TAFI activation are remain uninvestigated so far. TM is a membrane proteoglycan, membrane lipids in TM cofactor activities is of significant interest. Liposomes, in which lipid composition closely resembles that of cell membranes, have been extensively studied as cell membrane model for studying membrane protein function and drug delivery application. Therefore, we propose a recombinant TM/CSGAGA-liposome conjugate, which mimics the native endothelial TM and lipid components and will be useful tool to study TM’s protein C and TAFI activation. Three specific aims will be investigated: Aim 1. Synthesizing recombinant TM-CSGAG conjugates and investigating CSGAG’s role in protein C and TAFI activation, Aim 2. Synthesizing recombinant TM- liposome conjugates and investigating lipid effect on protein C and TAFI activation, Aim 3. Synthesize recombinant TM/CSGG-liposome conjugates and investigating CSGAG and lipid effect on protein C and TAFI activation. The engineered membrane mimetic platform with TM domains and defined CSGAG will permit a renewed understanding of both CSGAG’s and lipid’s role in TM cofactor activities. Broadly, this strategy will provide a new tool for functional studies of membrane proteoglycan and glycoprotein of any kind.
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