Whinna H C, Blinder M A, Szewczyk M, Tollefsen D M, Church F C
Department of Pathology, University of North Carolina, Chapel Hill 27599.
J Biol Chem. 1991 May 5;266(13):8129-35.
Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits alpha-thrombin in a reaction that is dramatically enhanced by heparin and other glycosaminoglycans/polyanions. We investigated the glycosaminoglycan binding site in HC by: (i) chemical modification with pyridoxal 5'-phosphate (PLP) in the absence and presence of heparin and dermatan sulfate; (ii) molecular modeling; and (iii) site-directed oligonucleotide mutagenesis. Four lysyl residues (173, 252, 343, and 348) were protected from modification by heparin and to a lesser extent by dermatan sulfate. Heparin-protected PLPHC retained both heparin cofactor and dermatan sulfate cofactor activity while dermatan sulfate-protected PLPHC retained some dermatan sulfate cofactor activity and little heparin cofactor activity. Molecular modeling studies revealed that Lys173 and Lys252 are within a region previously shown to contain residues involved in glycosaminoglycan binding. Lys343 and Lys348 are distant from this region, but protection by heparin and dermatan sulfate might result from a conformational change following glycosaminoglycan binding to the inhibitor. Site-directed mutagenesis of Lys173 and Lys343 was performed to further dissect the role of these two regions during HC-heparin and HC-dermatan sulfate interactions. The Lys343----Asn or Thr mutants had normal or only slightly reduced heparin or dermatan sulfate cofactor activity and eluted from heparin-Sepharose at the same ionic strength as native recombinant HC. However, the Lys173----Gln or Leu mutants had greatly reduced heparin cofactor activity and eluted from heparin-Sepharose at a significantly lower ionic strength than native recombinant HC but retained normal dermatan sulfate cofactor activity. Our results demonstrate that Lys173 is involved in the interaction of HC with heparin but not with dermatan sulfate, whereas Lys343 is not critical for HC binding to either glycosaminoglycan. These data provide further evidence for the determinants required for glycosaminoglycan binding to HC.
肝素辅因子II（HC）是一种血浆丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂），在肝素和其他糖胺聚糖/聚阴离子显著增强的反应中抑制α-凝血酶。我们通过以下方法研究了HC中的糖胺聚糖结合位点：（i）在不存在和存在肝素及硫酸皮肤素的情况下用5'-磷酸吡哆醛（PLP）进行化学修饰；（ii）分子建模；（iii）定点寡核苷酸诱变。四个赖氨酰残基（173、252、343和348）在肝素存在时受到保护而不被修饰，在硫酸皮肤素存在时受到的保护程度较小。肝素保护的PLP-HC保留了肝素辅因子和硫酸皮肤素辅因子活性，而硫酸皮肤素保护的PLP-HC保留了一些硫酸皮肤素辅因子活性，肝素辅因子活性则很低。分子建模研究表明，Lys173和Lys252位于先前显示包含参与糖胺聚糖结合的残基的区域内。Lys343和Lys348远离该区域，但肝素和硫酸皮肤素的保护可能是由于糖胺聚糖与抑制剂结合后发生的构象变化所致。对Lys173和Lys343进行定点诱变，以进一步剖析这两个区域在HC-肝素和HC-硫酸皮肤素相互作用中的作用。Lys343→Asn或Thr突变体的肝素或硫酸皮肤素辅因子活性正常或仅略有降低，并且在与天然重组HC相同的离子强度下从肝素-琼脂糖凝胶上洗脱。然而，Lys173→Gln或Leu突变体的肝素辅因子活性大大降低，并且在比天然重组HC低得多的离子强度下从肝素-琼脂糖凝胶上洗脱，但保留了正常的硫酸皮肤素辅因子活性。我们的结果表明，Lys173参与HC与肝素的相互作用，但不参与与硫酸皮肤素的相互作用，而Lys343对于HC与任何一种糖胺聚糖的结合都不是关键的。这些数据为糖胺聚糖与HC结合所需的决定因素提供了进一步的证据。
