Robust autoactivation, chymotrypsinC independence and diminished secretion define a subset of hereditary pancreatitis-associated cationic trypsinogen mutants
Robust autoactivation, chymotrypsinC independence and diminished secretion define a subset of hereditary pancreatitis-associated cationic trypsinogen mutants
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DOI：
10.1111/febs.12292
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发表时间：
2013-06-01
期刊：
影响因子：
5.4
通讯作者：
Sahin-Toth, Miklos
中科院分区：
文献类型：
--
作者：
Geisz, Andrea;Hegyi, Peter;Sahin-Toth, Miklos
人阳离子胰蛋白酶原突变通过改变胰凝乳蛋白酶C（CTRC）激活和降解的蛋白水解调节引起遗传性胰腺炎。CTRC通过将活化肽加工成较短形式来刺激胰蛋白酶原自活化，但也通过切割胰蛋白酶原中的钙结合环来促进降解。突变使胰蛋白酶原对CTRC介导的降解具有抗性和/或增加CTRC对活化肽的加工。在这里，我们证明了激活肽突变D19 A，D22 G，K23 R和K23_I24insIDK强烈增加胰蛋白酶原自激活的速率，在CTRC的存在和不存在下。CTRC降解的突变体是不变的，和加工的活化肽增加了四倍，仅在D19 A突变体。然而，令人惊讶的是，这种增加的处理对自动激活的影响很小。胰蛋白酶原激活肽中的四天冬氨酸基序结合钙（KD类似于1.6 mm），其刺激自激活。出乎意料的是，钙结合不受任何激活肽突变的影响。尽管正常的结合，自激活的突变体D22 G和K23_I24insIDK不刺激钙。最后，激活肽突变体表现出减少分泌从转染细胞，分泌的胰蛋白酶原水平与自激活率成反比。我们得出结论，D19 A，D22 G，K23 R和K23_I24insIDK形成遗传性胰腺炎相关突变的一个机制不同的子集，主要通过直接刺激自激活发挥其作用，独立于CTRC。显著增加的自激活的潜在严重临床影响被分泌减少所抵消，导致与典型遗传性胰腺炎难以区分的临床表型。
Mutations in human cationic trypsinogen cause hereditary pancreatitis by altering its proteolytic regulation of activation and degradation by chymotrypsinC (CTRC). CTRC stimulates trypsinogen autoactivation by processing the activation peptide to a shorter form, but also promotes degradation by cleaving the calcium-binding loop in trypsinogen. Mutations render trypsinogen resistant to CTRC-mediated degradation and/or increase processing of the activation peptide by CTRC. Here we demonstrate that the activation peptide mutations D19A, D22G, K23R and K23_I24insIDK robustly increased the rate of trypsinogen autoactivation, both in the presence and absence of CTRC. Degradation of the mutants by CTRC was unchanged, and processing of the activation peptide was increased fourfold in the D19A mutant only. Surprisingly, however, this increased processing had only a minimal effect on autoactivation. The tetra-aspartate motif in the trypsinogen activation peptide binds calcium (KD of similar to 1.6 mm), which stimulates autoactivation. Unexpectedly, calcium binding was not compromised by any of the activation peptide mutations. Despite normal binding, autoactivation of mutants D22G and K23_I24insIDK was not stimulated by calcium. Finally, the activation peptide mutants exhibited reduced secretion from transfected cells, and secreted trypsinogen levels were inversely proportional with autoactivation rates. We conclude that D19A, D22G, K23R and K23_I24insIDK form a mechanistically distinct subset of hereditary pancreatitis-associated mutations that exert their effect primarily through direct stimulation of autoactivation, independently of CTRC. The potentially severe clinical impact of the markedly increased autoactivation is offset by diminished secretion, resulting in a clinical phenotype that is indistinguishable from typical hereditary pancreatitis.