Protein O-acylation is a post-translational modification that occurs through the covalent addition of fatty acids to protein serine and threonine residues. In eukaryotes, this PTM plays several vital roles in several aspects of embryogenesis. In 2010, it was discovered that in Corynebacterium glutamicum (Cg), some proteins are O-esterified on serine residues. This bacterium and related species, including tuberculosis-causing Mycobacterium tuberculosis (Mtb), have a unique outer membrane layer called the mycomembrane. The mycomembrane is typified by unusually long-chain fatty acids, known as mycolic acids, esterified to various acceptors, including (i) trehalose, forming trehalose monomycolate (TMM), and trehalose dimycolate (TDM), (ii) arabinogalactan, forming arabinogalactan-linked mycolate (AGM) and (iii) some mycomembrane resident-proteins, forming O-mycoloylated proteins. Despite this post-translational modification’s (PTM) uniqueness and the far-reaching importance of mycomembrane proteins in general, just a handful have been identified to date. Recently, we designed, synthesized, and applied O-AlkTMM-C7, a TMM analog with a terminal alkyne on its 7-carbon acyl chain, to metabolically label and identify a few O-mycoloylated proteins in an organic extract of Cg. Here, We applied this metabolic labeling tool, combined with quantitative proteomics, and identified over 20 putatively O-mycoloylated proteins in Cg. Including known O-mycoloylated porins PorB and PorH, which validated the method. The list also includes several secreted hydrolases and hypothetical proteins. We defined the site(s) of this PTM in some of the identified proteins, facilitated by a combination of cleavable linker-based enrichment strategy and quantitative mass spectrometry. This work sets the stage for functional characterization of the hypothetical proteins and this PTM in Cg and related pathogenic species, including Mtb and Corynebacterium diphtheriae.

Quantitative chemical proteomics strategies for identification and structural characterization of O-mycoloylated proteins in Corynebacterium glutamicum

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