The distinct initiation sites and processing activities of TTLL4 and TTLL7 in glutamylation of brain tubulin.

Mammalian brain tubulins undergo a reversible posttranslational modification- polyglutamylation, which attaches a secondary polyglutamate chain to the primary sequence of proteins. Loss of its erasers can disrupt polyglutamylation homeostasis and cause neurodegeneration. Tubulin tyrosine ligase like 4 (TTLL4) and TTLL7 were known to modify tubulins both with preference for the β-isoform, but differently contribute to neurodegeneration. However, differences in their biochemical properties and functions remain largely unknown. Here, using an antibody-based method, we first characterized the properties of a purified recombinant TTLL4 and confirmed its sole role as an initiator, unlike TTLL7, which both initiates and elongates the side-chains. Unexpectedly, TTLL4 produced stronger glutamylation immunosignals for α-isoform than β-isoform in brain tubulins. Contrarily, the recombinant TTLL7 raised comparable glutamylation-immunoreactivity for two isoforms. Given the site selectivity of the glutamylation antibody, we analyzed modification sites of two enzymes. MS/MS analysis revealed their incompatible site selectivity on synthetic peptides mimicking carboxyl-termini of α1- and β2-tubulins and a recombinant tubulin. Particularly, in the recombinant α1A-tubulin, a novel region was found glutamylated by TTLL4 and TTLL7, that again at distinct sites. These results pinpoint different site specificities between two enzymes. Moreover, TTLL7 exhibits less efficiency to elongate microtubules (MTs) pre-modified by TTLL4, suggesting possible regulation of TTLL7 elongation activity by the initiation sites of TTLL4. Finally, we showed that kinesin behaves differentially on MTs modified by two enzymes. This study underpinned the different reactivity, site selectivity, and function of TTLL4 and TTLL7 on brain tubulins and shed light on their distinct role in vivo.