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 kcat/KM. 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.
Highlights
The neuronal ceroid lipofuscinoses (NCLs)2 are a group of hereditary neurodegenerative diseases primarily affecting children and adolescents who share similar clinical features, including visual loss, seizures, men
Without detailed understanding of tripeptidyl-peptidase I (TPP I) substrate specificity and the combined actions of other lysosomal proteinases, it remains difficult to distinguish whether subunit c accumulation in LINCL is a primary or secondary effect of TPP I deficiency
Construction of fluorogenic peptides was based on a procedure where we sequentially evaluated each P1, P2, and P3 position, using the results of the previous analysis to design subsequent libraries
Summary
Reagents—Rink amide 4-methylbenzhydrylamine resin and PyBOP were from Novabiochem. Fmoc-His(t-butyloxycarbonyl)-OH was from Bachem. Because the concentration of each component in the peptide library during incubation was ϳ5 M, which is well below the KM value of the corresponding substrate (Ն30 M), the enzyme kinetics can be considered as a first order reaction with respect to substrate and follows the relationship -ds/dt ϭ [E]0 ϫ [S]t ϫ kcat/KM, where [E]0 is the total TPP I concentration, and [S]t is the substrate concentration after digestion for time t. Integrated peak areas for the two most intense product ions for each peptide were obtained and used for calculation of kcat/KM based on one time point for the P1Ј-P2Ј pentapeptide library or on a time course of digestion for the P1Ј tetrapeptide library as described above for the P2 libraries
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