Detection of Tripeptidyl Peptidase I Activity in Living Cells by Fluorogenic Substrates

Abstract

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.

Activatable Optical Probes for the Detection of Enzymes

“…Serine proteases [271] Plasmin [272], Thrombin [272], Subtilisin [37], Tripeptidyl peptidase I [273], Hepatitis C viral protease [274], chymotrypsin [274] Metalloproteases Aminopeptidases [275,276] Cysteine proteases Caspases [269,270], Cathepsin B [274], Cathepsin C [277], Cathepsin K [278], SARS main protease [279], papain [274] Glycosidases β-glucosidase [213] Phosphodiesterase/Phosphatases [225,280] Lipase/esterase [86] Other hydrolases β-N-Acetylglucoaminidase [281],…”

Interactions of the proteins of neuronal ceroid lipofuscinosis: clues to function

“…Prosegment binding to TPP1 outside of the lysosome could permit activity at alkaline pH, in other regions of the cell; however, TPP1 activity has been examined mostly in vitro. In cultured hippocampal neurons, TPP1 is contained in lyso-somes, shown by colocalization with lysosomal membrane protein, LAMP1, and is not found in synpatophysin-positive synaptic vesicles in neurons [86]. …”