The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids.
Bottom Line: Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency.In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues.The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process.
Affiliation: Institut de Biotecnologia i de Biomedicina, Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain Institut Curie, 91405 Orsay, France.Show MeSH
Mentions: Glycylation is a posttranslational modification similar to polyglutamylation, performed by members of the TTLL family (Rogowski et al., 2009). Nothing is known about the enzymes responsible for shortening or removing posttranslationally generated glycine chains from tubulin. The unique deglycylase identified so far is an M20 peptidase family member in Giardia duodenalis (Lalle et al., 2011). Because this enzyme deglycylates the 14-3-3 proteins, and because M20 peptidases are not found in the mammalian genome, other candidates were considered as potential deglyclases in mammals, among them CCP2 and CCP3. Glycylation is typically enriched on the axonemes of motile cilia and flagella (Redeker et al., 1994). Thus the presence of CCP2 and CCP3 genes in ciliated organisms (Rodríguez de la Vega Otazo et al., 2013) and their increased expression levels in mouse tissues with motile cilia such as testis and trachea (Figure 4A) provided a potential link to glycylation. Although the structural model already suggested that both enzymes are rather specific to acidic amino acids (Figure 1), we wanted to obtain additional experimental proof to confirm this. Our attempts to directly test deglycylating activity in cells with glycylated MTs, which could be generated by expression of glycylating enzymes, failed due to the toxicity of this treatment for the cells. Thus we developed an alternative test for deglycylating activity, in which we coexpressed a chimeric telokin containing four Gly residues on the very C-terminus. This construct is specifically detected with the polyclonal antibody polyG, and removal of only one Gly residue completely abolished detection (Supplemental Figure S3B). The chimeric telokin-Gly was coexpressed together with CCP2_Z1703 or CCP3_Z1670, and a similar experiment was performed with a polyE antibody and a telokin with three Glu residues as positive control (Supplemental Figure S3C). Whereas both CCP2_Z1703 and CCP3_Z1670 were perfectly able to remove C-terminal glutamates, the unchanged polyG signals strongly suggest that C-terminally located Gly residues cannot be hydrolyzed by these enzymes. Thus CCP2 and CCP3 do not carry an intrinsic deglycylase activity.
Affiliation: Institut de Biotecnologia i de Biomedicina, Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain Institut Curie, 91405 Orsay, France.