Hogg P J, Owensby D A, Chesterman C N
Centre for Thrombosis and Vascular Research, Prince of Wales Hospital, University of New South Wales, Sydney, Australia.
J Biol Chem. 1993 Oct 15;268(29):21811-8.
Thrombospondin 1 was recently shown to bind to and inhibit the activity of neutrophil elastase (Hogg, P. J., Owensby, D. A., Mosher, D. F., Misenheimer, T. M., and Chesterman, C. N. (1993) J. Biol. Chem. 268, 7139-7146). This finding led us to question whether thrombospondin 1 also binds and inhibits the other major serine proteinase of neutrophils, cathepsin G. In a competitive binding assay, cathepsin G bound to thrombospondin 1 reversibly and saturably with a dissociation constant in the low nanomolar range. The kinetic mechanism of inhibition of cathepsin G activity by thrombospondin 1 was determined using the synthetic cathepsin G substrate, Suc-Ala-Ala-Pro-Phe-p-nitroanilide, and is consistent with hyperbolic tight-binding inhibition in which thrombospondin 1 binds cathepsin G and the Michaelis cathepsin G-substrate complex and weakens, but does not abolish, the efficiency of hydrolysis of Suc-Ala-Ala-Pro-Phe-p-nitroanilide. In the presence of 2 mM calcium ions, 2.9 +/- 0.4 mol of cathepsin G interacted with 1 mol of thrombospondin 1 trimer with a site-binding constant of 7.0 +/- 3.5 nM, which reduced the efficiency of hydrolysis of Suc-Ala-Ala-Pro-Phe-p-nitroanilide 8.5 +/- 1.4-fold. A lower limit for the on rate constant of 5 x 10(6) M-1 S-1 was established. The affinity of binding and stoichiometry for the interaction between cathepsin G and thrombospondin 1 was enhanced in the absence of calcium ions. In the presence of EDTA, 5.3 +/- 0.5 mol of cathepsin G interacted with 1 mol of thrombospondin 1 with a site-binding constant of 2.1 +/- 1.6 nM, implying the existence of two binding sites for cathepsin G on each subunit of thrombospondin 1, one or both of which is variably exposed and sensitive to calcium ions. Thrombospondin 1 protected fibronectin from cleavage by cathepsin G and blocked cathepsin G-mediated platelet aggregation. In summary, the binding of cathepsin G to thrombospondin 1 is tight, reversible, and close enough to the active site of cathepsin G to perturb the interactions of a small synthetic substrate and exclude a macromolecular protein substrate and platelets. Using defined proteolytic fragments and different conformers of thrombospondin 1, the binding sites for cathepsin G have been localized to the thrombospondin 1 type 3 repeats.
血小板反应蛋白1最近被证明可结合并抑制中性粒细胞弹性蛋白酶的活性（霍格，P.J.，欧文斯比，D.A.，莫舍，D.F.，米森海默，T.M.，和切斯特曼，C.N.（1993年）《生物化学杂志》268卷，7139 - 7146页）。这一发现使我们质疑血小板反应蛋白1是否也能结合并抑制中性粒细胞的另一种主要丝氨酸蛋白酶——组织蛋白酶G。在竞争性结合试验中，组织蛋白酶G与血小板反应蛋白1可逆且饱和结合，解离常数在低纳摩尔范围内。使用合成的组织蛋白酶G底物Suc - Ala - Ala - Pro - Phe - p - 硝基苯胺，确定了血小板反应蛋白1抑制组织蛋白酶G活性的动力学机制，这与双曲线紧密结合抑制一致，即血小板反应蛋白1结合组织蛋白酶G和米氏组织蛋白酶G - 底物复合物，减弱但不消除Suc - Ala - Ala - Pro - Phe - p - 硝基苯胺的水解效率。在2 mM钙离子存在下，2.9±0.4摩尔的组织蛋白酶G与1摩尔的血小板反应蛋白1三聚体相互作用，位点结合常数为7.0±3.5 nM，这使Suc - Ala - Ala - Pro - Phe - p - 硝基苯胺的水解效率降低了8.5±1.4倍。确定了结合速率常数的下限为5×10⁶ M⁻¹ s⁻¹。在没有钙离子的情况下，组织蛋白酶G与血小板反应蛋白1相互作用的结合亲和力和化学计量比增强。在EDTA存在下，5.3±0.5摩尔的组织蛋白酶G与1摩尔的血小板反应蛋白1相互作用，位点结合常数为2.1±1.6 nM，这意味着在血小板反应蛋白1的每个亚基上存在两个组织蛋白酶G结合位点，其中一个或两个位点可变地暴露并对钙离子敏感。血小板反应蛋白1保护纤连蛋白不被组织蛋白酶G切割，并阻断组织蛋白酶G介导的血小板聚集。总之，组织蛋白酶G与血小板反应蛋白1的结合紧密、可逆，且与组织蛋白酶G的活性位点足够接近，从而干扰小合成底物的相互作用，并排除大分子蛋白质底物和血小板。使用确定的蛋白水解片段和不同构象的血小板反应蛋白1，已将组织蛋白酶G的结合位点定位到血小板反应蛋白1的3型重复序列。
