Steinfeld Robert, Fuhrmann Jens C, Gärtner Jutta
Department of Pediatrics and Pediatric Neurology, University of Göttingen, Germany.
J Histochem Cytochem. 2006 Sep;54(9):991-6. doi: 10.1369/jhc.5A6900.2006. Epub 2006 Jun 16.
Tripeptidyl peptidase I (TPP-I) is a lysosomal peptidase with unclear physiological function. TPP-I deficiency is associated with late-infantile neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative disease of childhood that is characterized by loss of neurons and photoreceptor cells. We have developed two novel fluorogenic substrates, [Ala-Ala-Phe]2-rhodamine 110 and [Arg-Nle-Nle]2-rhodamine 110, that are cleaved by TPP-I in living cells. Fluorescence of liberated rhodamine 110 was detected by flow cytometry and was dependent on the level of TPP-I expression. Rhodamine-related fluorescence could be suppressed by preincubation with a specific inhibitor of TPP-I. When investigated by fluorescent confocal microscopy, rhodamine signals colocalized with lysosomal markers. Thus, cleavage of these rhodamide-derived substrates is a marker for mature enzymatically active TPP-I. In addition, TPP-I-induced cleavage of [Ala-Ala-Phe]2-rhodamine 110 could be visualized in primary neurons. We conclude that [Ala-Ala-Phe]2-rhodamine 110 and [Arg-Nle-Nle]2-rhodamine 110 are specific substrates for determining TPP-I activity and intracellular localization in living cells. Further, these substrates could be a valuable tool for studying the neuronal pathology underlying classical late-infantile NCL. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
三肽基肽酶I（TPP-I）是一种溶酶体肽酶，其生理功能尚不清楚。TPP-I缺乏与晚期婴儿神经元蜡样脂褐质沉积症（NCL）相关，NCL是一种儿童期致命的神经退行性疾病，其特征是神经元和光感受器细胞丧失。我们开发了两种新型荧光底物，[丙氨酸-丙氨酸-苯丙氨酸]2-罗丹明110和[精氨酸-正亮氨酸-正亮氨酸]2-罗丹明110，它们在活细胞中被TPP-I切割。通过流式细胞术检测释放的罗丹明110的荧光，其依赖于TPP-I的表达水平。罗丹明相关荧光可通过与TPP-I的特异性抑制剂预孵育来抑制。当通过荧光共聚焦显微镜检查时，罗丹明信号与溶酶体标记物共定位。因此，这些罗丹明衍生底物的切割是成熟的具有酶活性的TPP-I的标志物。此外，在原代神经元中可以观察到TPP-I诱导的[Ala-Ala-Phe]2-罗丹明110的切割。我们得出结论，[Ala-Ala-Phe]2-罗丹明110和[Arg-Nle-Nle]2-罗丹明110是用于确定活细胞中TPP-I活性和细胞内定位的特异性底物。此外，这些底物可能是研究经典晚期婴儿型NCL潜在神经病理学的有价值工具。本文包含在线补充材料，网址为http://www.jhc.org。请在线访问本文以查看这些材料。
