Polyglutamylation: biology and analysis

Abstract

Polyglutamylation is a posttranslational modification (PTM) that adds several glutamates on glutamate residues in the form of conjugated peptide chains by a family of enzymes known as polyglutamylases. Polyglutamylation is well documented in microtubules. Polyglutamylated microtubules consist of different α- and β-tubulin subunits with varied number of added glutamate residues. Kinetic control and catalytic rates of tubulin modification by polyglutamylases influence the polyglutamylation pattern of functional microtubules. The recent studies uncovered catalytic mechanisms of the glutamylation enzymes family, particularly tubulin tyrosine ligase-like (TTLL). Variable length polyglutamylation of primary sequence glutamyl residues have been mapped with a multitude of protein chemistry and proteomics approaches. Although polyglutamylation was initially considered a tubulin-specific modification, the recent studies have uncovered a calmodulin-dependent glutamylase, SidJ. Nano-electrospray ionization (ESI) proteomic approaches have identified quantifiable polyglutamylated sites in specific substrates. Indeed, conjugated glutamylated peptides were used in nano-liquid chromatography gradient delivery due to their relative hydrophobicity for their tandem mass spectrometry (MS/MS) characterization. The recent polyglutamylation characterization has revealed three major sites: E445 in α-tubulin, E435 in β-tubulin, and E860 in SdeA. In this review, we have summarized the progress made using proteomic approaches for large-scale detection of polyglutamylated peptides, including biology and analysis.

Keywords: Mass spectrometry; NanoESI; Polyglutamylation; Protein chemistry; Proteomics; Tubulin.

Fig. 1

Tubulin heterodimer consisting of α- and β-tubulin with various posttranslational modifications and representative enzymes. Adapted from Magiera and Janke (2014) and modified to include newly discovered PTMs

Fig. 2

Electron ionization (EI) mass spectrum of nonderivatized L-glutamic acid. The EI mass spectrum was generated on a 75-eV double-focusing mass spectrometer by direct introduction. From: Spectral Database for Organic Compounds SDBS, compound: MS-NW-2511, SDBS NO. 1097. https://sdbs.db.aist.go.jp/sdbs/cgi-bin/landingpage?sdbsno=1097

Fig. 3

Gradient profile of acetonitrile (ACN/min) in nanoLC–MS/MS (lower panel) of both subtilisin-digested (upper left panel) and trypsin-digested (upper right panel) unmodified tubulin peptides. The graphs illustrate the utility of calculated hydrophobic index (HI calc) as an indicator of the percentage ACN/min gradient in nanoLC–MS/MS. The hydrophobicity of the glutamylated peptides was validated from the reproducibility of the ordered elution of synthetic peptide standards. The line slope for hydrophobic index in relation to retention time of standards reflects the organic solvent gradient programmed for HPLC separation. A similar slope value can be determined from the analysis of tubulin peptides digested by subtilisin. Once established the elution of nonspecific digested peptides mirrored the separation parameters, that would allow one to compare the relative hydrophobicity of peptides with expected glutamyl residue values from the tryptic standard peptides

Fig. 4

a Representation of a di-glutamylated SdeA peptide and its unmodified species from the dataset by Bhogaraju et al. (2019). The data were re-analyzed with TPP/COMET. Color-coded PeptideProphet probability values are represented on the peptide map. Unmodified TMT-labeled peptide QVGRHGEGTESEFSVYLPEDVALVPVK (m/z 850.9593, [M + H]⁺ 3400.8153) eluted at 73.06 min and the di-glutamylated TMT-labeled peptide QVGRHGEGTESEFSVYLPEDVALVPVK (m/z 915.4806, [M + H]⁺ 3658.9005) at 74.9 min. b MS/MS spectrum of di-glutamylated TMT-labeled peptide QVGRHGEGTESEFSVYLPEDVALVPVK (m/z 915.4806, [M + H]⁺ 3658.9005) re-analyzed from the dataset published previously (Bhogaraju et al. 2019)