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Determination of the substrate specificity of tripeptidyl-peptidase I using combinatorial peptide libraries and
Yu Tian1, Istvan Sohar, John W Taylor
1Center for Advanced Biotechnology and Medicine, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.
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.
Insights
Researchers characterized tripeptidyl-peptidase I (TPP I) substrate specificity using peptide libraries. This work advances understanding of neuronal ceroid lipofuscinosis and improves TPP I activity assays.
Area of Science:
- Biochemistry
- Neuroscience
- Genetics
Background:
- Late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease.
- Mutations in CLN2, encoding tripeptidyl-peptidase I (TPP I), cause this disease.
- The natural substrates and disease mechanisms of TPP I are poorly understood.
Purpose of the Study:
- To characterize TPP I substrate specificity.
- To gain insights into lysosomal storage and disease progression.
- To develop improved clinical and biochemical assays for TPP I.
Main Methods:
- Construction and analysis of fluorogenic and standard combinatorial peptide libraries.
- Utilized fluorescence and mass spectrometry-based activity assays.
- Systematic evaluation of peptide positions (P1, P2, P3, P1', P2') and kinetic parameter determination (kcat/KM).
Main Results:
- Predicted the relative specificity of TPP I toward various potential biological substrates.
- Identified superior fluorogenic peptides with a P3 Arg residue for selective TPP I activity measurement.
- Demonstrated improved selectivity compared to the standard substrate Ala-Ala-Phe-AMC.
Conclusions:
- Detailed characterization of TPP I substrate specificity provides crucial insights into neuronal ceroid lipofuscinosis.
- Developed novel, more selective substrates for TPP I activity assays.
- Findings facilitate better understanding of TPP I function and disease pathogenesis.
