Blinder M A, Tollefsen D M
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1990 Jan 5;265(1):286-91.
Inhibition of thrombin by heparin cofactor (HCII) is accelerated approximately 1000-fold by heparin or dermatan sulfate. We found recently that the mutation Arg189----His decreases the affinity of HCII for dermatan sulfate but not for heparin (Blinder, M. A., Andersson, T. R., Abildgaard, U., and Tollefsen, D. M. (1989) J. Biol. Chem. 264, 5128-5133). Other investigators have implicated Arg47 and Lys125 of anti-thrombin (homologous to Arg103 and Lys185 of HCII) in heparin binding. To investigate the corresponding residues in HCII, we have constructed amino acid substitutions (Arg103----Leu, Gln, or Trp; Lys185----Met, Asn, or Thr) by oligonucleotide-directed mutagenesis of the cDNA and expressed the products in Escherichia coli. The recombinant HCII variants were assayed for binding to heparin-Sepharose and for inhibition of thrombin in the presence of various concentrations of heparin or dermatan sulfate. All of the Arg103 variants bound to heparin with normal affinity. Furthermore, inhibition of thrombin by the Arg103----Leu variant occurred at a normal rate in the absence of a glycosaminoglycan and was accelerated by normal concentrations of heparin and dermatan sulfate. These results indicate that HCII, unlike anti-thrombin, does not require a positive charge at this position for the interaction with heparin or dermatan sulfate. The Arg103----Gln and Arg103----Trp variants inhibited thrombin at about one-third of the normal rate in the absence of a glycosaminoglycan, suggesting that these mutations exert an effect on the reactive site (Leu444-Ser445) of HCII. All of the Lys185 variants bound to heparin with decreased affinity but inhibited thrombin at approximately the normal rate in the absence of a glycosaminoglycan. These variants required greater than 10-fold higher concentrations of heparin to accelerate inhibition of thrombin and were not stimulated significantly by dermatan sulfate, suggesting that heparin and dermatan sulfate interact with Lys185 of HCII. These results provide evidence that the glycosaminoglycan-binding site in HCII includes Lys185 but not Arg103, both of which were predicted to be involved by homology to anti-thrombin.
肝素辅因子（HCII）对凝血酶的抑制作用可被肝素或硫酸皮肤素加速约1000倍。我们最近发现，Arg189→His突变降低了HCII对硫酸皮肤素的亲和力，但不影响其对肝素的亲和力（布林德，M.A.，安德森，T.R.，阿比尔德加德，U.，和托勒夫森，D.M.（1989年）《生物化学杂志》264卷，5128 - 5133页）。其他研究人员指出抗凝血酶的Arg47和Lys125（与HCII的Arg103和Lys185同源）参与肝素结合。为了研究HCII中相应的残基，我们通过对cDNA进行寡核苷酸定向诱变构建了氨基酸取代（Arg103→Leu、Gln或Trp；Lys185→Met、Asn或Thr），并在大肠杆菌中表达产物。对重组HCII变体进行了与肝素 - 琼脂糖结合的检测以及在不同浓度肝素或硫酸皮肤素存在下对凝血酶抑制作用的检测。所有Arg103变体都以正常亲和力与肝素结合。此外，在没有糖胺聚糖的情况下，Arg103→Leu变体对凝血酶的抑制以正常速率发生，并且在正常浓度的肝素和硫酸皮肤素作用下加速。这些结果表明，与抗凝血酶不同，HCII在该位置与肝素或硫酸皮肤素相互作用时不需要正电荷。在没有糖胺聚糖的情况下，Arg103→Gln和Arg103→Trp变体以约正常速率的三分之一抑制凝血酶，这表明这些突变对HCII的活性位点（Leu444 - Ser445）产生了影响。所有Lys185变体与肝素结合的亲和力降低，但在没有糖胺聚糖的情况下以大致正常的速率抑制凝血酶。这些变体需要超过10倍浓度的肝素才能加速对凝血酶的抑制，并且硫酸皮肤素对其刺激不明显，这表明肝素和硫酸皮肤素与HCII的Lys185相互作用。这些结果提供了证据，表明HCII中的糖胺聚糖结合位点包括Lys185但不包括Arg103，通过与抗凝血酶的同源性预测这两个残基都应参与其中。
