Session: Parallel session 9 - Top-down and Structural analysis

Deciphering the tubulin code with top-down proteomics

Megan GANT¹, Carsten JANKE², Julia CHAMOT-ROOKE¹, Marija NISAVIC¹, Maria MAGIERA², Mathieu DUPRÉ¹, Mariya GENOVA², Thibault CHAZE¹, Sinda KHANFIR², Philippe BASTIN¹, Mariette MATONDO¹

¹Insitut Pasteur, Paris, France
²Institut Curie, Orsay, France

Introduction

Microtubules (MT) are essential cytoskeletal elements, composed of α- and β-tubulins. Tubulin post-translational modifications (PTMs) are regulators for MT functions and recently the concept of “tubulin code” has emerged. Two enzymes, TTLL1 and TTLL7, catalyze α- and β-tubulin glutamylation in humans and mice, but the site location, and number of glutamate residues added per site, are unknown. We show that top-down proteomics (TDP) is required to decode tubulin proteoforms and attribute biological significance

Methods

Tubulin was purified from WT and TTLL7-modified HeLa cells and the brains of WT, TTLL1-/-, TTLL7-/- and TTLL1/7-/- mice by polymerization/depolymerization cycles1. TDP samples were analyzed using a Vanquish Horizon LC with a C4 column and an Orbitrap Tribrid Eclipse mass spectrometer (Thermo) using several fragmentation modes. Data were analyzed using BioPharma Finder and ProSight Lite. Bottom-up (BU)samples were digested with trypsin, neprosin and thermolysin and desalted using C18 Empore stage tips before MS analysis

Results & Conclusion

We record the intact masses of a wide variety of tubulin proteoforms which arise from different gene isoforms or from a combination of PTMs. We found novel polyglutamylated proteoforms with up to seven glutamylations and a C-terminal detyrosination. Polyglutamylation sites were mapped on several tubulin species, and we show the loss of polyglutamylated proteoforms in TTLL1-/-, TTLL7-/- and TTLL1/7-/- tubulin and an increase in β-tubulin polyglutamylation on tubulin from human cells which overexpress glutamylating enzyme TTLL7. We compared levels of polyglutamylation for α- and β-tubulins from WT, TTLL1-/-, TTLL7-/- and TTLL1/7-/- mouse brains and show changes in sites for polyglutamylation between WT and KO tubulin. We also performed BUP analysis using several digestion enzymes, presenting a multi-faceted analysis of tubulin polyglutamylation and the roles played by the glutamylating enzymes TTLL1 and TTLL7.