Identification of specific posttranslational O -mycoloylations mediating protein targeting to the mycomembrane

Abstract

The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum, a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum, we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O-mycoloylation, pyroglutamylation, and N-formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O-acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment.Corynebacteriales | O-acylation | sequence motif | top-down proteomics | NMR

Top-Down Proteomics: Ready for Prime Time?

“…198 Subsequent to the expression and affinity purification of recombinant OMPs, PorA, ProH, PorB, and Por C of bacteria C. glutamicum (Figure 7a), top-down LC-MS/MS analysis of the OMPs associated with mycoloyl-arabinogalactan-peptidoglycan (mAGP) complex and secreted in the extracellular medium revealed well-conserved PTMs (Figure 7b-f), including O -mycoloylatoin, pyroglutaminatoin, and N-formylation. 198 Among these modifications, in particular, O -mycoloylatoin was only found in the mAGP-associated proteoforms, indicating that the presence of mycoloyl residues is essential for targeting OMPs to the mycolic acid-containing lipid bilayer. 198 The top-down approach plays a particularly important role in this case, because these OMPs from C. glutamicum are relatively hydrophobic with the presence of PTMs containing C32–C36 mycolyl residues and lack arginine and lysine residues for generating adequate tryptic peptides.…”

How many human proteoforms are there?

“…In heart disease, proteoform dynamics have been observed on proteins such as cardiac troponin I 64 , apolipoprotein C-III 65 , and B-type natriuretic peptide, the latter a key regulator of blood pressure and also the gold standard biomarker for clinically assessing heart failure 66 . Within the field of infectious diseases, proteoform-resolved approaches have been instrumental for understanding infectivity and dissemination of Salmonella typhimurium 67 , Corynebacterium glutamicum 68 and Neisseria meningitidis 69 . Finally, the clinically deployed use of whole-protein MALDI–TOF MS for rapid identification of the species and strain of pathogenic bacteria has been adopted by thousands of hospitals and clinics worldwide 70,71 .…”

Development of a Potential Protein Display Platform in Corynebacterium glutamicum Using Mycolic Acid Layer Protein, NCgl1337, as an Anchoring Motif

“…: total six proteins including NCgl0329, NCgl0776, NCgl0933, NCgl2375, NCgl2430, and NCgl2779, were excluded because it was predicted by SignalP program that those proteins do not have signal peptide. Next, it is recently reported that O ‐mycoloylation of protein is a key post‐translational modification for localization of protein on mycolic acid layer . Proteins are transported and acylated with one or several mycolic acids by the cMytC on Ser residues located within short linear motifs (SS or SG), and through mycoloylation, protein has enhanced hydrophobicity which is the main driving force for mycomembrane localization.…”

“…We found that both proteins were well expressed and present at the cell surface (membrane fraction) (Figure A). If the protein are not correctly acylated, proteins cannot be anchored to mycolic acid layer and subsequently are secreted into culture medium . To determine the amount of proteins in extracellular medium, protein samples in culture medium were analyzed by SDS–PAGE.…”