Glycosaminoglycan-binding properties and kinetic characterization of human heparin cofactor II expressed in Escherichia coli.
Glycosaminoglycan-binding properties and kinetic characterization of human heparin cofactor II expressed in Escherichia coli.
复制标题
大肠杆菌中表达的人肝素辅因子 II 的糖胺聚糖结合特性和动力学特征。
DOI：
10.1016/j.ab.2010.07.024
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发表时间：
2010
影响因子：
2.9
通讯作者：
Verhamme,IngridM
中科院分区：
文献类型：
--
作者：
Sarilla,Suryakala;Habib,SallyY;Tollefsen,DouglasM;Friedman,DavidB;Arnett,DianaR;Verhamme,IngridM
肝素辅因子II(αCofactorII，HCII)通过与糖胺多聚糖(GAG)、肝素和硫酸皮肤素(DS)形成三元络合物，加速了肝素辅因子II(HcII)对凝血酶(T)的不可逆灭活作用。通过对基因中27个稀有密码子和5个二级Shine-Dalgarno序列进行沉默突变，优化了人HCII在大肠杆菌中的低表达。比较了重组HCII、天然和脱糖血浆HCII的抑制活性及其对肝素和DS的亲和力。重组和去糖基化的HCII分别与肝素结合，解离常数(Kd)分别为6±1和7±1μM，∼比血浆高6倍，KD40±4μM。重组和去糖化的HCII与DS的结合，KD4±1μM，比血浆高4倍，KD15±4∼M，缺乏N-糖基化和酪氨酸硫化的重组HCII，1：1化学计量比灭活的α-凝血酶，与血浆HCII相似。重组和脱糖HCII灭活凝血酶的二级速率常数相似，最佳GAG浓度低于血浆HCII，与其较弱的GAG结合一致。这种较弱的结合可能归因于Asn169N-葡聚糖与HCII肝素结合位点的干扰。
Irreversible inactivation of α-thrombin (T) by the serpin, heparin cofactor II (HCII), is accelerated by ternary complex formation with the glycosaminoglycans (GAGs) heparin and dermatan sulfate (DS). Low expression of human HCII in Escherichia coli was optimized by silent mutation of 27 rare codons and five secondary Shine–Dalgarno sequences in the cDNA. The inhibitory activities of recombinant HCII, and native and deglycosylated plasma HCII, and their affinities for heparin and DS were compared. Recombinant and deglycosylated HCII bound heparin with dissociation constants (KD) of 6±1 and 7±1μM, respectively, ∼6-fold tighter than plasma HCII, with KD40±4μM. Binding of recombinant and deglycosylated HCII to DS, both with KD4±1μM, was ∼4-fold tighter than for plasma HCII, with KD15±4μM. Recombinant HCII, lacking N-glycosylation and tyrosine sulfation, inactivated α-thrombin with a 1:1 stoichiometry, similar to plasma HCII. Second-order rate constants for thrombin inactivation by recombinant and deglycosylated HCII were comparable, at optimal GAG concentrations that were lower than those for plasma HCII, consistent with its weaker GAG binding. This weaker binding may be attributed to interference of the Asn169N-glycan with the HCII heparin-binding site.