Identification of Tubulin Deglutamylase among Caenorhabditis elegans and Mammalian Cytosolic Carboxypeptidases (CCPs)

Worm Culture and Genetics

Nematodes were grown and maintained at 20 °C under standard conditions (13). All mutant and transgenic strains used in this study are listed in the supplemental Methods.

Immunohistochemical Analyses

Immunohistochemical analyses were performed as described in previous studies (14) with a slight modification. Full details are described in the supplemental Methods.

C. elegans Microscopy and Image Analysis

Immunostained worm samples were mounted on a slide glass with VECTASHIELD (Vector Laboratories) and analyzed under an FV1000 confocal microscope (Olympus). At least 20 adult worms for each strain were analyzed, and all images were collected using the same optical condition. Images were analyzed using FV10-ASW software (Olympus). Immunostained polyglutamylated (GT335) and α-tubulin signal intensities were measured in sensory cilia from the same optical slice. Following subtraction of background signals in non-ciliary regions, a GT335/α-tubulin signal intensity ratio was calculated for each image. GT335/α-tubulin ratios were averaged for each strain. The relative intensity of each strain was obtained from the average of GT335/ α-tubulin normalized by that of wild type and statistically analyzed by StatPlus (AnalystSoft).

Cell Culture and RNAi

The conventional methods of cell culture and gene knockdown by RNAi are described in the supplemental Methods.

In Vitro Deglutamylase Assay

Expression plasmids for CCP5 or -6 fused to a Myc tag (pCMV-tag3a-CCP5 and pCMV-tag3c-CCP6) were introduced into HEK293T cells using Lipofectamine 2000 (Invitrogen). Cells were lysed with PIPES-based buffer. For immunoprecipitation, the lysates were incubated with anti-Myc polyclonal antibody (Bethyl Laboratories) and subsequently precipitated with protein G-Sepharose beads (GE Healthcare). Tubulin deglutamylation assays were performed by adding 1.25 μg of porcine tubulin either to cell lysates or to immunoprecipitated Myc-CCP5 at 25 °C. Reactions were terminated by diluting mixtures with 2× SDS-PAGE sample buffer at 15 min. Samples were then subjected to Western blotting using GT335 (1:5000), anti-α-tubulin polyclonal (1:5000), and anti-Myc 9E10 (1:1000) antibodies.

C. elegans Sensory Ciliary MTs Are Strongly Polyglutamylated, Requiring ttll-4

We determined the distribution of polyglutamylation signals in C. elegans sensory cilia using antibodies that specifically recognize polyglutamylation. Using the monoclonal antibody (mAb) GT335, which specifically recognizes both monoglutamylation and polyglutamylation (10), robust signals were detected in the ciliary regions of the nematode head and tail (Fig. 1, A and B, left; supplemental Fig. 2A; and supplemental Movie 1). To ensure that the observed signals are indeed those of ciliary MTs, we employed strains expressing ciliated cell-specific transcriptional GFP transgenes (derived from promoter-GFP constructs), which illuminate the entire sensory neuron. In such strains, we found that the cilia of most amphid head neurons (ASE, ADF, ASH, ASK, AWA, AWC, and AFD) showed GT335 signals (Fig. 1B middle and right; supplemental Fig. 3). Strong signals were also seen in the ciliary structures of the inner (Fig. 1B, left panel, white arrows) and outer labial sensilla (Fig. 1B, left panel, yellow arrows; supplemental Movie 1). In contrast, no GT335 signals were detected in other ciliary neurons such as the neurons of the cephalic sensilla (supplemental Fig. 3, bottom row, arrow). In the tail, the ciliary structures of the phasmid neurons PHA, PHB, and PQR were also labeled with GT335 (supplemental Fig. 2B). Another mAb, B3, which specifically recognizes polyglutamylated tubulins (15), yielded similar signal staining patterns (supplemental Fig. 2C). Together, these data show that the MTs of C. elegans sensory cilia are highly polyglutamylated.

Tubulin polyglutamylation is mediated by enzymes of the TTLL family (8–10). The C. elegans genome contains six TTLL genes (ttll-4, -5, -9, -11, -12, and -15), and each gene belongs to a different clade (supplemental Fig. 1). Next, we studied the expression pattern of C. elegans ttll genes using transgenic worms carrying transcriptional GFP reporters (GFP directed by ttll promoter) or translational GFP reporters (GFP was fused to the entire open reading frame of the gene) under the control of the endogenous promoter of the gene (supplemental Figs. 4 and 5; summarized in supplemental Table 1). We found that three ttll genes, ttll-4, -9, and -11, are expressed in many ciliated sensory neurons in amphid (supplemental Fig. 5, D and F–H). C. elegans does not have any polyglycylase orthologs (TTLL3, -8, and -10) (16–18), which are found in other animals having motile cilia (18, 19), where polyglycylation exclusively occurs. Consistent with an absence of motile cilia and polyglycylase orthologs in C. elegans, immunostaining for polyglycylation in worms revealed no signals.³

To identify which ttll gene contributes to the polyglutamylation observed in the sensory cilia, we studied worm strains with mutations in the ttll genes (summarized in supplemental Table 1). The GT335 signals completely disappeared in ttll-4 worms (Fig. 1C, second row, asterisks, and D). To clearly evaluate the level of tubulin polyglutamylation, we quantified the signals of GT335 and of counterstained α-tubulin (Fig. 1C, middle column). The complete loss of GT335 signal was greater than a partial decrease of the α-tubulin signal in ttll-4 mutants (Fig. 1D). In contrast, only a slight reduction in GT335 signals was observed (Fig. 1, C, third row, and D, p < 0.01, Student's t test). We observed no overt decrease of GT335 immunoreactivities in a hypomorphic ttll-11(gk482) mutant (data not shown). The GT335 immunoreactivities were not altered in ttll-5(tm3360) and ttll-15(tm3871) mutants (data not shown), presumably because these genes are not expressed in ciliated sensory neurons (supplemental Fig. 5, E and J). The prominent loss of polyglutamylation observed in the ttll-4 mutant was reversed by overexpression of ttll-4::mCherry in sensory neurons (Fig. 1, C bottom row, and D). These findings demonstrate that TTLL-4 is a key enzyme that autonomously mediates tubulin polyglutamylation in sensory cilia.

CCPP-6 Is a Tubulin Deglutamylase in C. elegans

Previous reports suggest the presence of dynamic reversible regulation of tubulin polyglutamylation by deglutamylase activity (11, 12). Having identified TTLL-4 as a critical protein for tubulin polyglutamylation in C. elegans sensory cilia, we next turned our attention to identifying candidate deglutamylase genes in C. elegans. Cytosolic carboxypeptidases (CCPs) were recently identified as a subfamily of M14 metallocarboxypeptidases (20, 21). CCP1/Nna1, the first identified member of the CCP family, is mutated in Purkinje cell degeneration (pcd) mice (22); in these mice, the mitral cells in olfactory bulb show decreased signals of detyrosinated tubulins (20). C. elegans has only two CCP genes in its genome, F56H1.5 and EEED8.6, which we name here ccpp-1 and -6, respectively by their amino acid similarity and protein structure in comparison with six mammalian CCP genes (CCP1–6) (Fig. 2A; supplemental Fig. 6). CCPP-1 belongs to the M14D3 group with mammalian CCP1 and -4, and CCPP-6 belongs to the M14D2 group with mammalian CCP5 and -6 (Fig. 2A).

To determine whether ccpp-1 and -6 are involved in the regulation of tubulin polyglutamylation, we first examined their expression in sensory neurons. A transcriptional GFP reporter of ccpp-1 was expressed in amphid neurons (supplemental Fig. 7C). On the other hand, that of ccpp-6 was expressed only in the labial neurons but not in amphid neurons (data not shown).

A translational reporter mCherry fused to CCPP-1 protein showed the weak dotted signals around the pharynx (supplemental Fig. 7D, arrowheads). Meanwhile, a translational GFP reporter for ccpp-6 showed weak signals in the cell body of putative amphid neurons (supplemental Fig. 7, A and B, arrowheads), suggesting that expression in amphid cells was regulated by an enhancer element in a ccpp-6 intron(s). The weak signals observed for GFP/mCherry-tagged CCPP-1 and CCPP-6 transgenes indicate that both CCPP proteins are maintained at very low levels, possibly because they are rapidly degraded. Nevertheless, these results suggest that both CCPP-1 and CCPP-6 are expressed and function cell autonomously in amphid neurons.

Importantly, we found a robust reduction in the GT335 signals in the cilia of CCPP-6::GFP-expressing animals (p < 0.001, Student's t test), consistent with our hypothesis that CCPP-6 functions as a deglutamylase (Fig. 2, B, third row, asterisks, and D, upper panel). In contrast, the GT335 signals did not decrease in the CCPP-1::mCherry-expressing animals. Rather, the amphids of these animals showed increased GT335 signals along with the increase in the tubulin signals (Fig. 2, C, lower row, and D, lower panel). Hence, CCPP-1 might be involved in regulating tubulin function in the sensory cilia but does not likely act as a deglutamylase.

To determine whether endogenous CCPP-6 negatively regulates tubulin polyglutamylation, we analyzed tubulin polyglutamylation in ccpp-6 mutants. The tubulin GT335 signal significantly increased in ccpp-6 mutants (p < 0.05, Student's t test), indicating that CCPP-6 suppresses tubulin polyglutamylation (Fig. 2, B, second row, and D, upper panel). This enhanced signal is completely lost in the ccpp-6;ttll-4 double mutant background (Fig. 2, B, bottom row, and D, upper panel). These results indicate that ccpp-6 negatively regulates tubulin polyglutamylation in sensory cilia; this suggests that CCPP-6 is a tubulin deglutamylase.

Mammalian CCP5 Functions as a Tubulin Deglutamylase

We next examined whether mammalian CCPs (Fig. 2A) harbor tubulin deglutamylase(s). To this end, we exogenously overexpressed Myc-tagged CCP5 and -6, which are orthologs of C. elegans CCPP-6, and CCP2 as a control along with tubulin polyglutamylases, TTLL5 and -6, in HEK293T cells. Remarkably, GT335-detected tubulin bands completely disappeared when CCP5 was expressed (Fig. 3A). We could not detect such an effect for CCP2 and -6 at least in our system (Fig. 3A, data not shown). Strikingly, CCP5 did show no effect on the level of tyrosinated tubulin (Fig. 3B), indicating that CCP5 does not have detyrosinase activity. This deglutamylation activity was also detected for other proteins (supplemental Fig. 8, arrowheads). We further analyzed the effect of CCP5 in the endogenous polyglutamylation by the overexpression in mouse cortical neurons. The CCP5-expressing cells showed marked reduction in the GT335 signals (Fig. 3C, middle column).

To further examine whether mammalian CCP5 effectively counteracts tubulin polyglutamylation, we performed RNAi treatment of CCP5 in NIH3T3 cells. Two independent small interfering RNAs against CCP5 clearly increased intensities of GT335-detected bands at 50 kDa (Fig. 3D), which was overlapped with the β-tubulin band (Fig. 3D; green versus red). The two-dimensional electrophoresis revealed that the CCP5 knockdown caused an increase in the intensity of GT335-detected β-tubulin spots (Fig. 3E, top row), and more acidic β-tubulin spots were detected (Fig. 3E, bottom row). These results indicate that CCP5, one of the sequence orthologs of nematode CCPP-6 (Fig. 2A), is a tubulin deglutamylase in mammalian cells. The reason why β-tubulin polyglutamylation is effectively increased in CCP5 knockdown cells could be explained by the presence of endogenous glutamylation activity preferential to β-tubulin in non-neuronal cells (12).

We next carried out an in vitro enzyme assay of recombinant CCP5. We first tried to use nematode CCPP-6 and murine CCP5 expressed in bacteria. However, these recombinants did not show the enzyme activity or were hardly solubilized (see the supplemental Methods). This is probably because CCPP-6 and CCP5 fails to fold into the correct three-dimensional structure when expressed in bacteria. Thus, we decided to use Myc-tagged CCP5 expressed in HEK293T cells and successfully detected deglutamylase activity in vitro (Fig. 3, F and G; supplemental Fig. 9). The salt concentration drastically affected the CCP5 activity in the cell lysates; the enzyme was more active with 100 mm NaCl than with 0 or 50 mm NaCl (supplemental Fig. 9). CCP5 activity appeared to be saturated at 15 min as the decrease in GT335 signals of tubulin reached plateau (Fig. 3F). The faint decrease of GT335 signals in the mock transfection sample could be caused by the presence of endogenous deglutamylase in the cell lysates as reported previously (11, 12). The increase of α-tubulin signals in CCP5-containing samples could occur by the preferential binding of anti-α-tubulin antibody to less glutamylated tubulins (6, 23).

Despite the highest similarity between nematode CCPP-6 and mammalian CCP6, we were not able to detect deglutamylase activity of CCP6 in cultured cells. We were not able to detect the reduction of GT335 signal in the in vitro assay either (Fig. 3G). We would not, however, exclude possibilities that CCP6 functions as a deglutamylase. CCP6 might function under other experimental conditions or act on specific substrates, e.g. axonemal tubulins in cilia, as CCPP-6 works in the sensory cilia of C. elegans.

Finally, we challenged the enzyme activity assay by using Myc-CCP5 immobilized on antibody-protein G beads. In the immunoprecipitant of anti-Myc antibody, the antibody-purified CCP5 was detected in a major band (Fig. 3H). The broad bands with the faster migration than Myc-CCP5 (Fig. 3H, asterisk) were likely to be degraded Myc-CCP5 as the anti-Myc antibody detected some proteins at the same position (supplemental Fig. 10). Other minor bands seemed to be non-specifically precipitated proteins because they were also detected in the bead fraction of the empty vector-transfected cell lysates. Thus, Myc-CCP5 was the major and specific component of the immunoprecipitant. Incubation of substrate pig brain tubulin with this immobilized Myc-CCP5 showed clear reduction in the GT335 signal (Fig. 3I). The background activity detected in cell lysates (Fig. 3F) was completely eliminated in this assay (Fig. 3I). Taken together, these results clearly demonstrate that CCP5 has a tubulin deglutamylase activity.

Conclusion

By the genetic approach with C. elegans, we isolated CCPP-6 as a negative regulator of tubulin polyglutamylation in vivo. We also demonstrated that CCP5, a mammalian sequence ortholog of CCPP-6, had clear enzyme activity of tubulin deglutamylase by in vitro assay. In conclusion, we have identified C. elegans CCPP-6 and mammalian CCP5 as a tubulin deglutamylase by both in vitro and in vivo assay results.