肝素辅助因子II糖胺聚糖结合位点精氨酸103和赖氨酸185的定点突变。
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Blinder MA, Tollefsen DM
肝素辅助因子II糖胺聚糖结合位点精氨酸103和赖氨酸185的定点突变。
生物化学学报，2009,29(1):391 - 391。
- PubMed ID
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2104620 (PubMed视图］
- 摘要
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肝素辅助因子(HCII)对凝血酶的抑制作用被肝素或硫酸皮丹加速约1000倍。我们最近发现突变Arg189----His降低了HCII对硫酸皮肤素的亲和力，但对肝素没有(Blinder, m.a.， Andersson, t.r.， Abildgaard, U, and Tollefsen, d.m.， 1989)。化学，264,5128-5133)。其他研究者认为抗凝血酶的Arg47和Lys125(与HCII的Arg103和Lys185同源)参与肝素的结合。为了研究HCII中相应的残基，我们构建了氨基酸取代(Arg103----Leu, Gln或Trp;Lys185----Met, Asn, or Thr)通过寡核苷酸定向诱变cDNA，并在大肠杆菌中表达产物。检测重组HCII变体与肝素- sepharose的结合以及在不同浓度的肝素或硫酸皮丹存在下对凝血酶的抑制作用。所有的Arg103变体都以正常的亲和力与肝素结合。此外，在缺乏糖胺聚糖的情况下，Arg103----Leu变体对凝血酶的抑制率正常，并且在正常浓度的肝素和硫酸皮肤聚糖的作用下加速。这些结果表明，与抗凝血酶不同，HCII不需要在该位置与肝素或硫酸皮聚糖相互作用带正电荷。 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.