Pancreatic lipase (PNLIP) is the major lipolytic enzyme secreted by the pancreas. A recent study identified human PNLIP variants P245A, I265R, F300L, S304F, and F314L in European cohorts with chronic pancreatitis. Functional analyses indicated that the variants were normally secreted but exhibited reduced stability when exposed to pancreatic proteases. Proteolysis of the PNLIP variants yielded an intact C-terminal domain, while the N-terminal domain was degraded. The protease-sensitive PNLIP phenotype was strongly correlated with chronic pancreatitis, suggesting a novel pathological pathway underlying the disease. To facilitate preclinical mouse modeling, here we investigated how the human mutations affected the secretion and proteolytic stability of mouse PNLIP. We found that variants I265R, F300L, S304F, and F314L were secreted at high levels, while P245A had a secretion defect and accumulated inside the cells. Proteolysis experiments indicated that wild-type mouse PNLIP was resistant to cleavage, while variant I265R was readily degraded by mouse trypsin and chymotrypsin C. Variants F300L, S304F, and F314L were unaffected by trypsin but were slowly proteolyzed by chymotrypsin C. The proteases degraded the N-terminal domain of variant I265R, leaving the C-terminal domain intact. Structural analyses suggested that changes in stabilizing interactions around the I265R mutation site contribute to the increased proteolytic susceptibility of this variant. The results demonstrate that variant I265R is the best candidate for modeling the protease-sensitive PNLIP phenotype in mice.

中文翻译：

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在小鼠直系同源物中模拟蛋白酶敏感的人胰脂肪酶突变


胰脂肪酶 （PNLIP） 是胰腺分泌的主要脂解酶。最近的一项研究在欧洲慢性胰腺炎队列中发现了人类 PNLIP 变体 P245A、I265R、F300L、S304F 和 F314L。功能分析表明，这些变体正常分泌，但在暴露于胰蛋白酶时表现出稳定性降低。PNLIP 变体的蛋白水解产生了完整的 C 端结构域，而 N 端结构域被降解。蛋白酶敏感的 PNLIP 表型与慢性胰腺炎密切相关，表明该疾病存在新的病理途径。为了促进临床前小鼠建模，我们在这里研究了人类突变如何影响小鼠 PNLIP 的分泌和蛋白水解稳定性。我们发现变体 I265R 、 F300L 、 S304F 和 F314L 分泌水平较高，而 P245A 存在分泌缺陷并在细胞内积累。蛋白水解实验表明，野生型小鼠 PNLIP 对切割具有抗性，而变体 I265R 很容易被小鼠胰蛋白酶和胰凝乳蛋白酶 C 降解。变体 F300L、S304F 和 F314L 不受胰蛋白酶的影响，但被胰凝乳蛋白酶 C 缓慢蛋白水解。蛋白酶降解了变体 I265R 的 N 端结构域，使 C 端结构域保持完整。结构分析表明，I265R 突变位点周围稳定相互作用的变化导致该变体的蛋白水解敏感性增加。结果表明，变体 I265R 是模拟小鼠蛋白酶敏感 PNLIP 表型的最佳候选者。