Glucosylceramide, a neutral glycosphingolipid anticoagulant cofactor, enhances the interaction of human- and bovine-activated protein C with negatively charged phospholipid vesicles.
The effect of glucosylceramide (GlcCer) on activated protein C (APC)-phospholipid interactions was examined using fluorescence resonance energy transfer. Human APC, labeled with either fluorescein (Fl-APC) or dansyl (DEGR-APC) donor, bound to phosphatidylcholine/phosphatidylserine (PC/PS, 9:1 w/w) vesicles containing octadecylrhodamine (OR) acceptor with a K(d) (app) = 16 micro g/ml, whereas Fl-APC (or DEGR-APC) bound to PC/PS/GlcCer(OR) (8:1:1) vesicles with a K(d) (app) = 3 micro g/ml. This 5-fold increase in apparent affinity was not species-specific since bovine DEGR-APC also showed a similar GlcCer-dependent enhancement of binding of APC to PC/PS vesicles. From the efficiency of fluorescence resonance energy transfer, distances of closest approach of approximately 63 and approximately 64 A were estimated between the dansyl on DEGR-APC and rhodamine in PC/PS/GlcCer(OR) and PC/PS(OR), respectively, assuming kappa(2) = 2/3. DEGR-APC bound to short chain C8-GlcCer with an apparent K(d) of 460 nm. The presence of C8-GlcCer selectively enhanced the binding of C16,6-NBD-phosphatidylserine but not C16,6-7-nitrobenz-2-oxa-1,3-diazole (NBD)-phosphatidylcholine to coumarin-labeled APC. These data suggest that APC binds to GlcCer, that PC/PS/GlcCer vesicles like PC/PS vesicles bind to the N-terminal gamma-carboxyglutamic acid domain of APC, and that one mechanism by which GlcCer enhances the activity of APC is by increasing its affinity for membrane surfaces containing negatively charged phospholipids.
Yegneswaran
Subramanian
S
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Deguchi
Hiroshi
H
Griffin
John H
JH
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology; D001665:Binding Sites; D002417:Cattle; D031541:Fluorescence Resonance Energy Transfer; D005963:Glucosylceramides / Q000494:pharmacology*; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011486:Protein C / Q000378:metabolism*; D011517:Prothrombin Time
D000925:Anticoagulants; D005963:Glucosylceramides; D008081:Liposomes; D010718:Phosphatidylserines; D010743:Phospholipids; D011486:Protein C