Determination of the Substrate Specificity of Tripeptidyl-peptidase I Using Combinatorial Peptide Libraries and Development of Improved Fluorogenic Substrates

Abstract

Classical late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease caused by mutations in CLN2, the gene encoding the lysosomal protease tripeptidyl-peptidase I (TPP I). The natural substrates for TPP I and the pathophysiological processes associated with lysosomal storage and disease progression are not well understood. Detailed characterization of TPP I substrate specificity should provide insights into these issues and also aid in the development of improved clinical and biochemical assays. To this end, we constructed fluorogenic and standard combinatorial peptide libraries and analyzed them using fluorescence and mass spectrometry-based activity assays. The fluorogenic group 7-amino-4-carbamoylmethylcoumarin was incorporated into a series of 7-amino-4-carbamoylmethylcoumarin tripeptide libraries using a design strategy that allowed systematic evaluation of the P1, P2, and P3 positions. TPP I digestion of these substrates liberates the fluorescence group and results in a large increase in fluorescence that can be used to calculate kinetic parameters and to derive the substrate specificity constant k cat /K M . In addition, we implemented a mass spectrometry-based assay to measure the hydrolysis of individual peptides in peptide pools and thus expand the scope of the analysis. Nonfluorogenic tetrapeptide and pentapeptide libraries were synthesized and analyzed to evaluate P1 and P2 residues. Together, this analysis allowed us to predict the relative specificity of TPP I toward a wide range of potential biological substrates. In addition, we evaluated a variety of new fluorogenic peptides with a P3 Arg residue, and we demonstrated their superiority compared with the widely used substrate Ala-Ala-Phe-AMC for selectively measuring TPP I activity in biological specimens.

Crystal Structure and Autoactivation Pathway of the Precursor Form of Human Tripeptidyl-peptidase 1, the Enzyme Deficient in Late Infantile Ceroid Lipofuscinosis

“…7D. This is entirely consistent with the known exopeptidase specificity of TPP1, where peptides with prolines at either P1 or P1Ј are extremely poor substrates (20), thus preventing further exopeptidyl digestion past Arg 175 . The Leu 182 N terminus produced by cleavage at pH 4.5 ( Fig.…”

“…TPP1 exhibits broad substrate specificity (20) and is the only mammalian member of the S53 sedolisin family (reviewed in Ref. 21), which includes a number of unusual bacterial serine peptidases (22).…”

Residual levels of tripeptidyl-peptidase I activity dramatically ameliorate disease in late-infantile neuronal ceroid lipofuscinosis

“…TPPI was measured in the H and L fractions after dilution with cold 0.15M NaCl-0.1% Triton X-100 using 200 µM Arg-Ala-Phe-ACC (generously provided by Dr. John W. Taylor, Rutgers University) as substrate using the kinetic assay described previously [15]. For determination of K M , TPPI was measured using Arg-Ala-Phe-ACC and Ala-Ala-Phe-AMC (Sigma, St. Louis, MO).…”

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

“…Recently, several other fluorescent substrates have been developed that are specifically cleaved by TPP-I in cell homogenates (Oyama et al 2005;Tian et al 2006) or used for histochemical TPP-I detection (Dikov et al 2004). To study TPP-I activity in living cells, we have designed two new fluorogenic substrates, [Ala-Ala-Phe] 2 -rhodamine 110 and [Arg-Nle-Nle] 2 -rhodamine 110.…”