TTLL7 is a mammalian beta-tubulin polyglutamylase required for growth of MAP2-positive neurites

Abstract

Microtubules form a cytoskeletal framework that influences cell shape and provides structural support for the cell. Microtubules in the nervous system undergo a unique post-translational modification, polyglutamylation of the C termini of their tubulin subunits. The mammalian enzymes that perform beta-tubulin polyglutamylation as well as their physiological functions in the neuronal tissue remain elusive. We report identification of a mammalian polyglutamylase with specificity for beta-tubulin as well as its distribution and function in neurite growth. To identify putative tubulin polyglutamylases, we searched tubulin tyrosine ligase-like (TTLL) proteins for those predominantly expressed in the nervous system. Of 13 TTLL proteins, TTLL7 was transcribed at the highest level in the nervous system. Recombinant TTLL7 catalyzed tubulin polyglutamylation with high preference to beta-tubulin in vitro. When expressed in HEK293T cells, TTLL7 demonstrated specificity for beta-tubulin and not for alpha-tubulin or nucleosome assembly protein 1. Consistent with these findings, knockdown of TTLL7 in a primary culture of superior cervical ganglion neurons caused a loss of polyglutamylated beta-tubulin. Following stimulation of PC12 cells with nerve growth factor to differentiate, the level of TTLL7 increased concomitantly with polyglutamylation of beta-tubulin. Short interference RNA-mediated knockdown of TTLL7 repressed nerve growth factor-stimulated MAP (microtubule-associated protein) 2-positive neurite growth in PC12 cells. Consistent with having a role in the growth of MAP2-positive neurites, TTLL7 accumulated within a MAP2-enriched somatodendritic portion of superior cervical ganglion, as did polyglutamylated beta-tubulin. Anti-TTLL7 antibody revealed that TTLL7 was distributed in a somatodendritic compartment in the mouse brain. These findings indicate that TTLL7 is a beta-tubulin polyglutamylase and is required for the growth of MAP2-positive neurites in PC12 cells.

FIGURE 1. TTLL7 is predominantly expressed in neuronal tissues

A, schematic view of mouse TTLL proteins. B, phylogenic tree of mouse TTLL proteins based on similarity of TTL domain. The phylogenic tree of mTTLL proteins was generated with NJPlot program. C, expression profile of human TTLL7. hTTLL7 is predominantly expressed in neuronal tissues, such as spinal cord, thalamus, hippocampus, and hypothalamus. D, in situ hybridization analysis of mouse TTLL7 in mouse adult brain. mTTLL7 transcripts were strongly detected in olfactory bulb (ob), hippocampus (h), thalamus (th), midbrain (m), cerebral cortex (cx), and cerebellum (ce). Scale bar, 5 mm.

FIGURE 2. TTLL7 is a β-tubulin polyglutamylase

A, in vitro enzyme activity of recombinant mouse TTLL7. Recombinant mTTLL7 incorporated [³H]glutamate into microtubules with a high preference to β-tubulin in vitro. These activities were lost when ATP was omitted from reaction mixture (–ATP) or when a point mutation causing failure of the enzyme to use ATP was inserted in mTTLL7 (E349V). Enzyme activities are expressed as radioactivity incorporated into microtubules (disintegration per minute; dpm). The result is representative of three independent experiments. B, in vivo enzyme activity of a subset of mouse TTLL proteins. Only mTTLL7 displayed tubulin polyglutamylase activity in HEK293T cells. Polyglutamylated tubulins were detected with a mAb, GT335. The blot was reprobed with DM1A and Tub2.1 to verify migrating positions of both tubulins (displayed on the right of GT335-labeled blot image). Note that the band marked with an asterisk, detected with GT335 in all samples, does not co-migrate with α-tubulin. Amounts of total tubulins were confirmed by reprobing with DM1A and DM1B. Amount of glyceraldehyde-3-phosphate dehydrogenase was monitored as a loading control (Ctrl). The result is representative of three independent experiments. Expression efficacies were shown in supplemental Fig. S2. C, immunocytochemical analysis of effect of overexpressed mTTLL7 in PtK2 cells. Overexpression of EGFP-mTTLL7 in PtK2 cells resulted in polyglutamylation of microtubules. Scale bar, 20 μm. D, in vivo enzyme activity of mTTLL7 on NAP1. To visualize signals other than tubulin labeled by GT335, x-ray film was overexposed. To efficiently detect polyglutamylated NAP1 signal, we overexpressed FLAG-NAP1 with or without mTTLL7. mTTLL7 did not enhance signal intensities of bands other than β-tubulin. Ectopic NAP1 also was not polyglutamylated by mTTLL7. E, knockdown of endogenous TTLL7 and loss of β-tubulin polyglutamylation in primary cultures of SCG neurons. Antiserum raised against TTLL7 detected a protein of ~90 kDa (arrowhead) that disappeared following treatment with siRNA targeted to mTTLL7. Polyglutamylated (PG-) tubulins were detected with mAb GT335. Total α-tubulin and β-tubulin were detected with mAb DM1A and DM1B, respectively.

FIGURE 3. TTLL7 and polyglutamylated β-tubulin are involved in neurite growth

A, expression of TTLL7 in neuronally differentiated PC12 cells. Neuronal PC12 cells expressed TTLL7 at 14 days following NGF stimulation. The amount of TTLL7 was diminished by rTTLL7-specific siRNA treatment. B, effect of TTLL7 knockdown on β-tubulin polyglutamylation in PC12 cells. Neuronal PC12 cells contained both polyglutamylated α- and β-tubulin. Polyglutamylated β-tubulin was lost by rTTLL7-specific siRNA treatment. C, effect of the knockdown of TTLL7 on neurite growth of PC12 cells. PC12 cells treated with siRNA for 24 h were differentiated to neuronal cells by application of NGF for 48 h. TTLL7-targeted siRNA suppressed neurite formation and extension. Scale bar, 10 μm. D and E, quantification of neurite number in PC12 cells. Neurite number was quantified at 24 h after NGF-stimulated differentiation. The data were from three independent experiments. ***, p < 0.001 with Mann-Whitney U test. F and G, quantification of neurite length in PC12 cells. Neurite length was quantified at 24 h after NGF-stimulated differentiation. The data were from three independent experiments. ***, p < 0.001 with Mann-Whitney U test. H, immunocytochemical staining of differentiated PC12 cell. Neuronally differentiated PC12 cells were stained with FITC-DM1A (green) and with anti-MAP2 antibodies (red). Scale bar, 20 μm. Ctrl, control.

FIGURE 4. TTLL7 and polyglutamylated β-tubulin are located in MAP2-positive subcellular compartments

A, intracellular distribution of TTLL7 in explant culture of SCG neurons. TTLL7 was exclusively located in ganglion bodies (GB), where MAP2 accumulated. Equal amounts of protein were loaded in GB and neuritic portion (N). B, distribution of polyglutamylated tubulins in explant culture of SCG neurons. Polyglutamylated β-tubulin concentrated in ganglion bodies (GB), whereas polyglutamylated α-tubulin was dominant in neurite compartment (N). To obtain equal amounts of tubulin, the samples from the ganglion body contained three times the amount of protein compared with samples from neurites. C, immunocytochemical analysis of TTLL7 distribution in SCG neurons. TTLL7 was exclusively accumulated in cell bodies where MAP2 was enriched. Scale bar, 20 μm. D, distribution of TTLL7 in mouse brain. TTLL7 was concentrated in soma and dendrites of Purkinje neurons in cerebellum (left panels), in apical dendrites of pyramidal neurons in cerebral cortex (middle panels), and in apical dendrites of pyramidal neurons in hippocampal CA1 region (right panels). The signals of TTLL7 and MAP2 partially overlapped. The nuclei were stained with TOTO-3 (cyan). Staining with preimmune serum was performed as a negative control for the TTLL7 staining. Scale bar, 50 μm.