Felderbauer P, Schnekenburger J, Lebert R, Bulut K, Parry M, Meister T, Schick V, Schmitz F, Domschke W, Schmidt W E
Department of Internal Medicine I, St Josef Hospital, Ruhr-University of Bochum, Gudrunstr. 54, 44791 Bochum, Germany.
J Med Genet. 2008 Aug;45(8):507-12. doi: 10.1136/jmg.2007.056481. Epub 2008 May 29.
The understanding of genetic risk factors for chronic pancreatitis increased in the last decade with the discovery of mutations in the cationic trypsinogen gene (PRSS1). The first mutation was detected at the R122 autocleavage site of the protein (R122H) and subsequently two other mutations in this region, R122C and V123M, were described that resulted in a similar phenotype of hereditary pancreatitis. This study reports a novel A121T mutation within this region and characterises the resulting molecular properties at the autocleavage site.
Blood samples of a PRSS1 A121T carrier family were analysed for PRSS1 mutations using melting point curve analysis, restriction endonucleases and DNA sequencing. Conformation dependent properties of the mutated sequence were analysed by molecular modelling. The autodegradation kinetic of the mutated trypsin sequence was measured by a novel fluorescence resonance energy transfer (FRET) assay using designed 11 amino acid peptides from PRSS1 aa 118-aa 127 containing the trypsin cleavage site at aa 122 coupled to a Dabcyl/EDANS FRET system. The kinetic of tryptic peptide cleavage was measured in a fluorescence enzyme linked immunosorbent assay (ELISA) reader.
DNA sequencing revealed a novel G to A transition at position 133279 of the published genomic sequence (#U66061 GenBank). The mutation results in an amino acid substitution of alanine by threonine at position 121 (A121T) of the cationic trypsinogen. Four additional mutation carriers could be identified among the relatives while only the first patient developed chronic pancreatitis. Molecular modelling of PRSS1 A121T revealed a change in the bond pattern between the R122 region and the calcium binding loop, whereas FRET assays showed an increased trypsin cleavage rate with a reaction kinetic elevated by more than 80%.
The novel PRSS1 A121T mutation highlights the surface exposed region PRSS1 A121-R122-V123 as a hotspot for hereditary pancreatitis associated trypsinogen mutations. Molecular modelling and FRET assays provide evidence for an A121T mutation dependent increase in susceptibility to trypsin digestion at the R122 cleavage site suggesting an enhanced autodegradation and a loss-of-function at the autocleavage site.
随着阳离子胰蛋白酶原基因（PRSS1）突变的发现，过去十年中对慢性胰腺炎遗传危险因素的认识有所增加。首次在该蛋白的R122自切割位点检测到突变（R122H），随后在该区域描述了另外两个突变，即R122C和V123M，它们导致了遗传性胰腺炎的相似表型。本研究报告了该区域内一个新的A121T突变，并对自切割位点产生的分子特性进行了表征。
使用熔点曲线分析、限制性内切酶和DNA测序对PRSS1 A121T携带者家族的血样进行PRSS1突变分析。通过分子建模分析突变序列的构象依赖性特性。使用来自PRSS1第118位氨基酸至第127位氨基酸的设计的11个氨基酸肽，其在第122位氨基酸处含有胰蛋白酶切割位点，并与Dabcyl/EDANS荧光共振能量转移（FRET）系统偶联，通过一种新的FRET测定法测量突变型胰蛋白酶序列的自降解动力学。在荧光酶联免疫吸附测定（ELISA）读数仪中测量胰蛋白酶肽切割的动力学。
DNA测序显示，已发表的基因组序列（#U66061 GenBank）第133279位发生了一个新的从G到A的转变。该突变导致阳离子胰蛋白酶原第121位氨基酸由丙氨酸替换为苏氨酸（A121T）。在亲属中又鉴定出另外四名突变携带者，而只有第一名患者发展为慢性胰腺炎。PRSS1 A121T的分子建模显示R122区域与钙结合环之间的键型发生了变化，而FRET测定显示胰蛋白酶切割速率增加，反应动力学提高了80%以上。
新的PRSS1 A121T突变突出了PRSS1 A121-R122-V123表面暴露区域是遗传性胰腺炎相关胰蛋白酶原突变的热点。分子建模和FRET测定为R122切割位点处A121T突变依赖性的胰蛋白酶消化敏感性增加提供了证据，表明自降解增强且自切割位点功能丧失。
