Abstract 1456: Proteomic Analysis Of Proteins Bound By D-4F In Plasma From C57bl/6 And ApoA-I KO Mice
D-4F, an 18 amino acid apolipoprotein A-I (ApoA-I) mimetic, has been shown to be highly effective at improving vascular function in murine models of atherosclerosis. It has been hypothesized that D-4F inhibits atherosclerosis and increases vasodilation by interacting with ApoA-I to improve HDL function. However, the mechanisms by which D-4F protects vascular function remain unclear. We hypothesize that D-4F binds proteins involved in atherosclerosis. To test this hypothesis, we incubated HA-tagged D-4F with plasma from C57bl/6 and ApoA-I knockout mice (ApoA-I KO) on a C57bl/6 background and identified the proteins that were bound. Plasma from C57bl/6 mice or ApoA-I KO mice (n=6/group) was incubated with HA-GG-D-4F (Ac-YPYDVPDYA-GG-DWFKAFYDKVAEKFKEAF-NH2) or HA-GG-scramble-D-4F (Ac-YPYDVPDYA-GG-DWFAKDYFKKAFVEEFAK-NH2) at 10μg/ml. Next an anti-HA antibody was used to immunoprecipitate the HA-tagged peptides and associated proteins. The proteins were then analyzed by ESI-nano-LC/MS/MS. Analysis revealed that D-4F bound several proteins involved in lipid metabolism (ApoA-I, -II, -IV, ApoE, ApoC-I, -II and -III, -IV); complement regulatory protein (Complement C3); several kinases (PKC and Serine/threonine-protein kinase); and Serpins (Serine protease inhibitor 1 and A3K) which inhibit serine endopeptidases in C57bl/6 mice plasma. Although ApoA-I KO mice do not express ApoA-I, D-4F bound several lipoproteins associated with lipid metabolism (ApoE, ApoA-II, -IV, and ApoC-II, -III, -IV) in plasma from ApoA-I KO mice. D-4F also associated with the complement regulatory protein, Complement C3 in plasma of ApoA-I KO mice. Interestingly, D-4F bound serum paraoxonase and bradykinin only in the plasma of ApoA-I KO mice. These data show that D-4F associates, either directly or indirectly through lipoproteins, with a unique array of proteins involved in lipid metabolism; and second; that D-4F’s ability to associate with Complement C3, serum paraoxonase, bradykinin and Serpins suggest that D-4F may be able to modify the atherogenic properties of proteins involved in inflammation.