Department of Medical Gastroenterology, Odense University Hospital, University of Southern Denmark, Odense, Denmark.
Pancreas. 2011 May;40(4):540-6. doi: 10.1097/MPA.0b013e3182152fdf.
In a hereditary pancreatitis family from Denmark, we identified a novel intragenic duplication of 9 nucleotides in exon-2 of the human cationic trypsinogen (PRSS1) gene (c.63_71dup) which at the amino-acid level resulted in the insertion of 3 amino acids within the activation peptide of cationic trypsinogen (p.K23_I24insIDK). The aim of the present study was to characterize the effect of this unique genetic alteration on the function of human cationic trypsinogen.
Wild-type and mutant cationic trypsinogens were produced recombinantly and purified to homogeneity. Trypsinogen activation was followed by enzymatic assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Trypsinogen secretion was measured from transfected HEK 293T cells.
Recombinant cationic trypsinogen carrying the p.K23_I24insIDK mutation exhibited greater than 10-fold increased autoactivation. Activation by human cathepsin B also was accelerated by 10-fold. Secretion of the p.K23_I24insIDK mutant from transfected cells was diminished, consistent with intracellular autoactivation.
This is the first report of an intragenic duplication within the PRSS1 gene causing hereditary pancreatitis. The accelerated activation of p.K23_I24insIDK by cathepsin B is a unique biochemical property not found in any other pancreatitis-associated trypsinogen mutant. In contrast, the robust autoactivation of the novel mutant confirms the notion that increased autoactivation is a disease-relevant mechanism in hereditary pancreatitis.
在一个来自丹麦的遗传性胰腺炎家系中，我们在人类阳离子胰蛋白酶原（PRSS1）基因的exon-2 中发现了一个新的 9 个核苷酸的基因内重复（c.63_71dup），导致阳离子胰蛋白酶原激活肽内插入 3 个氨基酸（p.K23_I24insIDK）。本研究的目的是研究这种独特的遗传改变对人类阳离子胰蛋白酶原功能的影响。
重组产生野生型和突变型阳离子胰蛋白酶原，并进行纯化至均一性。通过酶促测定和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳来监测胰蛋白酶原的激活。通过转染的 HEK 293T 细胞来测量胰蛋白酶原的分泌。
携带 p.K23_I24insIDK 突变的重组阳离子胰蛋白酶原表现出超过 10 倍的自动激活增加。人组织蛋白酶 B 的激活也被加速了 10 倍。突变体 p.K23_I24insIDK 从转染细胞中的分泌减少，与细胞内自动激活一致。
这是 PRSS1 基因内基因内重复导致遗传性胰腺炎的首例报道。p.K23_I24insIDK 被组织蛋白酶 B 加速激活是一种独特的生化特性，在任何其他胰腺炎相关胰蛋白酶原突变体中都未发现。相比之下，新型突变体的强大自动激活证实了增加自动激活是遗传性胰腺炎的一种相关疾病机制的观点。
