Lactadherin binds selectively to membranes containing phosphatidyl-L-serine and increased curvature.
Lactadherin, a milk protein, contains discoidin-type lectin domains with homology to the phosphatidylserine-binding domains of blood coagulation factor VIII and factor V. We have found that lactadherin functions, in vitro, as a potent anticoagulant by competing with blood coagulation proteins for phospholipid binding sites [J. Shi and G.E. Gilbert, Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid binding sites, Blood 101 (2003) 2628-2636]. We wished to characterize the membrane-binding properties that correlate to the anticoagulant capacity. We labeled bovine lactadherin with fluorescein and evaluated binding to membranes of composition phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine, 4:20:76 supported by 2 mum diameter glass microspheres. Lactadherin bound saturably with an apparent KD of 3.3+/-0.4 nM in a Ca++ -independent manner. The number of lactadherin binding sites increased proportionally to the phosphatidylserine content over a range 0-2% and less rapidly for higher phosphatidylserine content. Inclusion of phosphatidylethanolamine in phospholipid vesicles did not enhance the apparent affinity or number of lactadherin binding sites. The number of sites was at least 4-fold higher on small unilamellar vesicles than on large unilamellar vesicles, indicating that lactadherin binding is enhanced by membrane curvature. Lactadherin bound to membranes with synthetic dioleoyl phosphatidyl-L-serine but not dioleoyl phosphatidyl-D-serine indicating stereoselective recognition of phosphatidyl-L-serine. We conclude that lactadherin resembles factor VIII and V with stereoselective preference for phosphatidyl-L-serine and preference for highly curved membranes.
Shi
Jialan
J
Department of Medicine, VA Boston Healthcare System, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02132, USA.
Heegaard
Christian W
CW
Rasmussen
Jan T
JT
Gilbert
Gary E
GE
D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D048430:Cell Shape; D005165:Factor V; D005169:Factor VIII; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D008894:Milk Proteins / Q000737:chemistry* / Q000378:metabolism; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding
D000954:Antigens, Surface; D008563:Membrane Lipids; D008894:Milk Proteins; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D005165:Factor V; D005169:Factor VIII