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Originally published as JHC exPRESS on June 16, 2006.
doi:10.1369/jhc.5A6900.2006
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Journal of Histochemistry and Cytochemistry
Volume 54 (9): 991-996, 2006
Copyright ©The Histochemical Society, Inc.

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

Robert Steinfeld, Jens C. Fuhrmann and Jutta Gärtner

Department of Pediatrics and Pediatric Neurology, University of Göttingen, Göttingen, Germany (RS,JG), and Centre for Molecular Neurobiology, University of Hamburg, Hamburg, Germany(JCF)

Correspondence to: Dr. Robert Steinfeld, Pädiatrie II, Zentrum Kinderheilkunde und Jugendmedizin, Universitätsklinikum Göttingen, Robert-Koch-Str. 40, D-37075 Göttingen, Germany. E-mail: rsteinfeld{at}med.uni-goettingen.de

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. (J Histochem Cytochem 54:991–996, 2006)

Key Words: neuronal ceroid lipofuscinosis • tripeptidyl peptidase I • neurodegeneration • living cell cytochemistry • protease


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