Detection of tripeptidyl peptidase I activity in living cells by fluorogenic substrates.
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.
Steinfeld
Robert
R
Department of Pediatrics and Pediatric Neurology, University of Göttingen, Germany. rsteinfeld@med.uni-goettingen.de
Fuhrmann
Jens C
JC
Gärtner
Jutta
J
D000626:Aminopeptidases; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D010450:Endopeptidases / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D020014:K562 Cells; D008214:Lymphocytes / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology; D051379:Mice; D018613:Microscopy, Confocal; D009842:Oligopeptides / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism*; D057057:Serine Proteases; D000091346:Tripeptidyl-Peptidase 1
D015415:Biomarkers; D005456:Fluorescent Dyes; D009842:Oligopeptides; D012235:Rhodamines; C000717268:Tpp1 protein, mouse; D000091346:Tripeptidyl-Peptidase 1; C513502:alanyl-alanyl-phenylalanyl-alanyl-alanyl-phenylalanyl-rhodamine 110; C513503:arginyl-norleucyl-norleucyl-arginyl-norleucyl-norleucyl-rhodamine 110; D010450:Endopeptidases; D057057:Serine Proteases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C000717267:TPP1 protein, human