Gillis S, Furie B C, Furie B, Patel H, Huberty M C, Switzer M, Foster W B, Scoble H A, Bond M D
Division of Hematology-Oncology, New England Medical Center, Boston, Massachusetts 02111, USA.
Protein Sci. 1997 Jan;6(1):185-96. doi: 10.1002/pro.5560060121.
The gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IX/gamma 40E is uncarboxylated at residue 40 and factor IX/gamma 36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully gamma-carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma-derived factor IX. As monitored by anti-factor IX:Ca (II)-specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX species underwent the Ca(II)-induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half-maximal inhibition of the binding of 125I-labeled plasma-derived factor IX at concentrations of 2-6 nM. Functionally, factor IX/gamma 36,40E and factor IX/gamma 40E were similar to fully gamma-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti-factor IX:Mg(II)-specific antibodies because these antibodies bound factor IX preferentially to factor IX/gamma 36,40E and factor IX/gamma 40E. These results demonstrate that the gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined.
维生素K依赖性凝血蛋白的γ-羧基谷氨酸（Gla）结构域在前33个残基内含有10个高度保守的Gla残基，但因子IX的独特之处在于在第36和40位还拥有另外2个Gla残基。为了确定它们的重要性，从异源表达的人因子IX中分离出缺乏这些Gla残基的因子IX种类。使用离子交换色谱法、肽图谱分析、质谱分析和N端测序，我们纯化并鉴定了两种部分羧化的重组因子IX种类；因子IX/γ40E在第40位残基未羧化，因子IX/γ36,40E在第36和40位残基均未羧化。将这些种类与完全γ-羧化的重组因子IX、未分级的重组因子IX和血浆来源的因子IX进行了比较。通过抗因子IX:Ca（II）特异性抗体监测以及通过内在荧光的猝灭监测，所有这些因子IX种类都经历了磷脂膜结合所需的Ca（II）诱导的构象转变，并且与由磷脂酰丝氨酸、磷脂酰胆碱和磷脂酰乙醇胺组成的磷脂囊泡等效结合。所有种类的内皮细胞结合也相似，125I标记的血浆来源的因子IX结合的半数最大抑制浓度为2 - 6 nM。在功能上，因子IX/γ36,40E和因子IX/γ40E在凝血活性以及与合成磷脂囊泡和活化血小板在凝血酶原酶复合物中参与因子X激活的能力方面，与完全γ-羧化的重组因子IX和血浆来源的因子IX相似。然而，Gla 36和Gla 40代表抗因子IX:Mg（II）特异性抗体靶向的表位的一部分，因为这些抗体优先结合因子IX而非因子IX/γ36,40E和因子IX/γ40E。这些结果表明，人因子IX中第36和40位谷氨酸残基的γ-羧化对于所检测的因子IX的任何功能都不是必需的。
