Abstract

Nna1 (CCP1) defines a subfamily of M14 metallocarboxypeptidases (CCP1-6) and is mutated in pcd (Purkinje cell degeneration) mice. Nna1, CCP4, and CCP6 are involved in the post-translational process of polyglutamylation, where they catalyze the removal of polyglutamate side chains. However, it is unknown whether these three cytosolic carboxypeptidases share identical enzymatic properties and redundant biological functions. We show that like Nna1, purified recombinant CCP4 and CCP6 deglutamylate tubulin, but unlike Nna1, neither rescues Purkinje cell degeneration in pcd mice, indicating that they do not have identical functions. Using biotin-based synthetic substrates, we established that the three enzymes are distinguishable based upon individual preferences for glutamate chain length, the amino acid immediately adjacent to the glutamate chain, and whether their activity is enhanced by nearby acidic amino acids. Nna1 and CCP4 remove the C-terminal glutamate from substrates with two or more glutamates, whereas CCP6 requires four or more glutamates. CCP4 behaves as a promiscuous glutamase, with little preference for chain length or neighboring amino acid composition. Besides glutamate chain length dependence, Nna1 and CCP6 exhibit higher k(cat)/K(m) when substrates contain nearby acidic amino acids. All cytosolic carboxypeptidases exhibit a monoglutamase activity when aspartic acid precedes a single glutamate, which, together with their other individual preferences for flanking amino acids, greatly increases the potential substrates for these enzymes and the biological processes in which they act. Additionally, Nna1 metabolized substrates mimicking the C terminus of tubulin in a way suggesting that the tyrosinated form of tubulin will accumulate in pcd mice.

Highlights

  • All cytosolic carboxypeptidase (CCP) family members catalyze deglutamylation of tubulin

  • Recombinant Nna1, CCP4, and CCP6 Metabolize the Polyglutamate Side Chain of Tubulin—Previous enzymatic studies of Nna1 family members were performed using tubulin as substrate and lysates of cells transfected with CCP4 and CCP6 as a source of enzyme with detection using an antibody to the polyglutamate chain of tubulin [10]

  • Nna1 consistently showed the highest activity in this assay, with CCP4 and CCP6 being roughly equipotent (Fig. 1B and data not shown)

Read more

Summary

Introduction

All cytosolic carboxypeptidase (CCP) family members catalyze deglutamylation of tubulin. Significance: The results expand the range of substrates and biological processes influenced by CCPs. Nna (CCP1) defines a subfamily of M14 metallocarboxypeptidases (CCP1– 6) and is mutated in pcd (Purkinje cell degeneration) mice. CCP4, and CCP6 are involved in the posttranslational process of polyglutamylation, where they catalyze the removal of polyglutamate side chains. It is unknown whether these three cytosolic carboxypeptidases share identical enzymatic properties and redundant biological functions. All cytosolic carboxypeptidases exhibit a monoglutamase activity when aspartic acid precedes a single glutamate, which, together with their other individual preferences for flanking amino acids, greatly increases the potential substrates for these enzymes and the biological processes in which they act. Nna metabolized substrates mimicking the C terminus of tubulin in a way suggesting that the tyrosinated form of tubulin will accumulate in pcd mice

Objectives
Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call