TTLL7 Is a Mammalian β-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 ␤-tubulin polyglutamylation as well as their physiological functions in the neuronal tissue remain elusive. We report identification of a mammalian polyglutamylase with specificity for ␤-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 ␤-tubulin in vitro. When expressed in HEK293T cells, TTLL7 demonstrated specificity for ␤-tubulin and not for ␣-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 ␤-tubulin. Following stimulation of PC12 cells with nerve growth factor to differentiate, the level of TTLL7 increased concomitantly with polyglutamylation of ␤-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 ␤-tubulin. Anti-TTLL7 antibody revealed that TTLL7 was distributed in a somatodendritic compartment in the mouse brain. These findings indicate that TTLL7 is a ␤-tubulin polyglutamylase and is required for the growth of MAP2-positive neurites in PC12 cells.Microtubules have important functions in a variety of dynamic activities within the cell including intracellular transport, cell motility, and cell division. They provide structural support for the cell and are an important component of the cytoskeletal framework that generates cell morphology. In neurons microtubules are required for formation of specialized processes, i.e. dendrites and axons. Structural microtubule-associated proteins (MAPs), 2 such as MAP2 and tau, regulate the stability of microtubules in those processes.Microtubules are predominantly composed of tubes of polymerized dimers of ␣-and ␤-tubulin. A central question in cell biology is how functional heterogeneity is imparted to different types (e.g. dendritic, axonal, and centriolar) of microtubules. Two basic mechanisms have been proposed to explain this issue. The first involves duplication and divergence of genes encoding tubulin. The second mechanism involves the addition of a variety of post-translational modifications (PTM) to microtubules that further increases the tubulin heterogeneity (1). These PTMs are asso...

Replacement of microglia in the aged brain reverses cognitive, synaptic, and neuronal deficits in mice

“…In a specific way, genes associated with either actin cytoskeleton remodeling or synaptic vesicle release were downregulated with aging and subsequently restored to young adult levels with microglial replacement. Several of these genes are associated with microtubule ( Map1a , Map1b) and cytoskeleton dynamics ( Prepl; Morawski et al, 2013), and neurite ( Ttll7, Nfasc; Ikegami et al, 2006; Wang & Bixby, 1999) as well as axon/dendrite outgrowth ( Clasp2 ; Beffert et al, 2012). In addition, several genes play a role in endocytosis ( Cltc) and microtubule transport ( Kif1a , Kif1b , Kif21a, Dync1h1, and Dnm3) —all important in synaptic vesicle release.…”

TTLL10 is a protein polyglycylase that can modify nucleosome assembly protein 1

“…The anti-poly-glycine polyclonal (R-polygly) antibody, which was generated by using Cys-(Gly) 9 as a immunogen [14], was the generous gift of Dr. Gorovsky (U. Rochester, NY). The monoclonal antibody (mAb) 4A8 for NAP1 was the kind gift of Dr. Ishimi (Ibaraki Univ., Mito, Japan) [15].…”

“…Immunoblotting and immunohistochemistry were performed as described [9]. Total testicular protein was isolated from C57BL/6J mice of the indicated age using standard methods.…”

“…Genetic and biochemical approaches enabled identification of tubulin polyglutamylases (i.e., tubulin glutamate ligases), as well as functions for this activity in vivo [6,7]. More recently, tubulin polyglutamylases have been identified as members of the tubulin tyrosine ligase-like (TTLL) family [8][9][10][11]. Knockdown of TTLL7, a polyglutamylase for b-tubulin, demonstrated the essential role of the b-tubulin glutamylation in neurite growth [9].…”

“…More recently, tubulin polyglutamylases have been identified as members of the tubulin tyrosine ligase-like (TTLL) family [8][9][10][11]. Knockdown of TTLL7, a polyglutamylase for b-tubulin, demonstrated the essential role of the b-tubulin glutamylation in neurite growth [9]. In addition, analysis of ROSA22 mice [6] that lack PGs1, a non-catalytic subunit of a polyglutamylase protein complex with specificity for a-tubulin [8], demonstrated that a-tubulin glutamylation is required for intracellular targeting of KIF1 kinesin motor and its cargo synaptic vesicles, as well as for continuous synaptic transmission [12].…”