Elafin and its precursor trappin-2 still inhibit neutrophil serine proteinases when they are covalently bound to extracellular matrix proteins by tissue transglutaminase
Elafin and its precursor trappin-2 still inhibit neutrophil serine proteinases when they are covalently bound to extracellular matrix proteins by tissue transglutaminase
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DOI：
10.1021/bi051418i
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发表时间：
2005-11-29
期刊：
影响因子：
2.9
通讯作者：
Moreau, T
中科院分区：
文献类型：
--
作者：
Guyot, N;Zani, ML;Moreau, T
Elafin 及其前体 trappin-2（也称为 pre-elafin）是中性粒细胞丝氨酸蛋白酶（例如白细胞弹性蛋白酶和蛋白酶 3）的有效蛋白质抑制剂。Trappin-2 具有富含 Gln 和 Lys 残基的独特保守序列基序。这些基序是转谷氨酰胺酶的底物，可以使 trappin-2 与细胞外基质蛋白交联，从而将抑制剂锚定在其作用位点。我们使用蛋白质印迹和基于 ELISA 的测定来证明 elafin 和 trappin-2 都可以通过 2 型转谷氨酰胺酶在体外与各种细胞外基质蛋白缀合。交联的elafin和trappin-2仍然抑制它们的目标蛋白酶。表面等离子体共振研究可以确定控制纤连蛋白结合的弹性蛋白和 trappin-2 与中性粒细胞弹性蛋白酶和蛋白酶 3 相互作用的动力学常数。当通过转谷氨酰胺作用与纤连蛋白交联时，两种抑制剂都是有效的抑制剂，其与目标蛋白酶相互作用的平衡解离常数 K-i 为 0.3 nM（弹性蛋白酶-弹性蛋白）、20 nM（蛋白酶） 3-elafin)、0.3 nM (弹性蛋白酶-trappin-2) 和 12 nM (蛋白酶 3-trappin-2)。缀合抑制剂与其靶酶的反应比可溶性抑制剂更慢，可能是由于它们的固定化，弹性蛋白酶的结合速率常数为 2-7 x 10(5) M-1 s(-1)，蛋白酶 3 的结合速率常数为 1-4 x 10(4) M-1 s(-1)。我们相信这是转谷氨酰胺酶介导的丝氨酸蛋白酶抑制剂与蛋白质交联的首次证明保留其抑制能力。
Elafin and its precursor trappin-2 (also called pre-elafin) are potent protein inhibitors of neutrophil serine proteases such as leukocyte elastase and proteinase 3. Trappin-2 has unique conserved sequence motifs rich in Gln and Lys residues. These motifs are substrates for transglutaminases that may enable trappin-2 to be cross-linked to extracellular matrix proteins, thus anchoring the inhibitor at its site of action. We have used Western blotting and ELISA-based assays to demonstrate that both elafin and trappin-2 can be conjugated to various extracellular matrix proteins in vitro by a type 2 transglutaminase. Cross-linked elafin and trappin-2 still inhibited their target proteases. Surface plasmon resonance studies allowed the determination of the kinetic constants governing the interaction of fibronectin-bound elafin and trappin-2 with neutrophil elastase and proteinase 3. Both inhibitors were potent inhibitors when crosslinked to fibronectin by transglutamination, with equilibrium dissociation constants K-i for their interaction with target proteases of 0.3 nM (elastase-elafin), 20 nM (proteinase 3-elafin), 0.3 nM (elastase-trappin-2), and 12 nM (proteinase 3-trappin-2). The conjugated inhibitors reacted more slowly with their target enzymes than did the soluble inhibitors, perhaps due to their immobilization, with association rate constants of 2-7 x 10(5) M-1 s(-1) for elastase and 1-4 x 10(4) M-1 s(-1) for proteinase 3. We believe this is the first demonstration that transglutaminase-mediated cross-linking of serine protease inhibitors to proteins preserves their inhibitory capacities.