Comparison of the Enzymatic and Functional Properties of Three Cytosolic Carboxypeptidase Family Members

• Published in: The Journal of biological chemistry 2015-01, Vol.290 (2), p.1222-1232
• Main Authors: Wu, Hui-Yuan, Rong, Yongqi, Correia, Kristen, Min, Jaeki, Morgan, James I.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Carboxypeptidases - chemistry; Carboxypeptidases - genetics; Carboxypeptidases - metabolism; Cerebellum; Cytosolic Carboxypeptidase; Enzyme Kinetics; Enzymology; GTP-Binding Proteins - genetics; GTP-Binding Proteins - metabolism; Mice; Mice, Transgenic; Nerve Degeneration - metabolism; Nerve Degeneration - pathology; Neurodegeneration; Polyglutamic Acid - metabolism; Polyglutamylation; Post-translational Modification (PTM); Purkinje Cells - metabolism; Purkinje Cells - pathology; Serine-Type D-Ala-D-Ala Carboxypeptidase - genetics; Serine-Type D-Ala-D-Ala Carboxypeptidase - metabolism; Substrate Specificity; Transgenic Mice; Tubulin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.604850

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 kcat/Km 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.All cytosolic carboxypeptidase (CCP) family members catalyze deglutamylation of tubulin. Nna1 (CCP1), CCP4, and CCP6 exhibit distinct sequence preferences and kinetics for synthetic polyglutamate substrates. CCP4 and CCP6 fail to rescue Purkinje cell degeneration in Nna1-dysfunctional mice. CCPs are not biologically equivalent in Purkinje neurons. The results expand the range of substrates and biological processes influenced by CCPs.