Shi Jialan, Heegaard Christian W, Rasmussen Jan T, Gilbert Gary E
Department of Medicine, VA Boston Healthcare System, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02132, USA.
Biochim Biophys Acta. 2004 Nov 17;1667(1):82-90. doi: 10.1016/j.bbamem.2004.09.006.
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.
乳粘连蛋白是一种乳蛋白，含有盘状结构域凝集素结构域，与凝血因子VIII和因子V的磷脂酰丝氨酸结合结构域具有同源性。我们发现，在体外，乳粘连蛋白通过与凝血蛋白竞争磷脂结合位点发挥强效抗凝剂的作用[J. Shi和G.E. Gilbert，乳粘连蛋白通过竞争磷脂结合位点抑制凝血酶复合物，《血液》101 (2003) 2628 - 2636]。我们希望表征与抗凝能力相关的膜结合特性。我们用荧光素标记牛乳粘连蛋白，并评估其与由直径2μm的玻璃微球支撑的磷脂酰丝氨酸/磷脂酰乙醇胺/磷脂酰胆碱组成比例为4:20:76的膜的结合。乳粘连蛋白以3.3±0.4 nM的表观解离常数（KD）饱和结合，且与Ca++无关。在0 - 2%的范围内，乳粘连蛋白结合位点的数量与磷脂酰丝氨酸含量成比例增加，对于更高的磷脂酰丝氨酸含量增加较慢。在磷脂囊泡中加入磷脂酰乙醇胺不会增强乳粘连蛋白结合位点的表观亲和力或数量。小单层囊泡上的位点数量比大单层囊泡上至少高4倍，表明膜曲率增强了乳粘连蛋白的结合。乳粘连蛋白与合成的二油酰磷脂酰-L-丝氨酸结合，但不与二油酰磷脂酰-D-丝氨酸结合，表明对磷脂酰-L-丝氨酸具有立体选择性识别。我们得出结论，乳粘连蛋白与因子VIII和V相似，对磷脂酰-L-丝氨酸具有立体选择性偏好，对高度弯曲的膜具有偏好。
