Scrutiny of Mycobacterium tuberculosis 19 kDa antigen proteoforms provides new insights in the lipoglycoprotein biogenesis paradigm

Abstract

Post-translational modifications (PTMs) are essential processes conditioning the biophysical properties and biological activities of the vast majority of mature proteins. However, occurrence of several distinct PTMs on a same protein dramatically increases its molecular diversity. The comprehensive understanding of the functionalities resulting from any particular PTM association requires a highly challenging full structural description of the PTM combinations. Here, we report the in-depth exploration of the natural structural diversity of the M. tuberculosis (Mtb) virulence associated 19 kDa lipoglycoprotein antigen (LpqH) using intact protein high-resolution mass spectrometry (HR-MS) coupled to liquid chromatography. Combined top-down and bottom-up HR-MS analyses of the purified Mtb LpqH protein allow, for the first time, to uncover a complex repertoire of about 130 molecular species resulting from the intrinsically heterogeneous combination of lipidation and glycosylation together with some truncations. Direct view on the co-occurring PTMs stoichiometry reveals the presence of functionally distinct LpqH lipidation states and indicates that glycosylation is independent from lipidation. This work allowed the identification of a novel unsuspected phosphorylated form of the unprocessed preprolipoglycoprotein totally absent from the current lipoglycoprotein biogenesis pathway and providing new insights into the biogenesis and functional determinants of the mycobacterial lipoglycoprotein interacting with the host immune PRRs.

Lipoprotein Glycosylation by Protein-O-Mannosyltransferase (MAB_1122c) Contributes to Low Cell Envelope Permeability and Antibiotic Resistance of Mycobacterium abscessus

“…Consequently, lipoprotein glycosylation in M. abscessus Δ pmt is abolished completely. All glycosyl-residues in M. abscessus were detected in the N -terminal region of the model lipoproteins as described for lipoproteins expressed in other mycobacteria as well ( Herrmann et al, 1996 ; Michell et al, 2003 ; Sartain and Belisle, 2009 ; Mohiman et al, 2012 ; Sethi et al, 2016 ; Parra et al, 2017 ). This observation indicates the existence of a conserved motif which is recognized by Pmt from various mycobacterial species and directs site-specific glycosylation of lipoproteins in fast-and slow-growing mycobacteria ( Sartain and Belisle, 2009 ; Smith et al, 2014 ).…”

“…In this study we investigate the effects of protein glycosylation deficiency on the mycobacterial cell envelope and therefore we focus on the lipoprotein glycosylating function of Pmt. In M. tuberculosis , Mycobacterium smegmatis or the closely related Corynebacterium glutamicum the lipoproteins LppX, Mpt83, LprI, LpqH, LprG, and SodC have been found with glycosyl-residues ( Michell et al, 2003 ; Sartain and Belisle, 2009 ; Mohiman et al, 2012 ; Sethi et al, 2016 ; Alonso et al, 2017 ; Parra et al, 2017 ). The emerging pathogen Mycobacterium abscessus , a highly drug resistant fast-growing mycobacterium which infects the lungs of patients with chronic pulmonary disease or cystic fibrosis, also localizes lipoproteins in its cell envelope ( Ripoll et al, 2009 ; Nessar et al, 2012 ).…”

Integrative proteomic and glycoproteomic profiling of Mycobacterium tuberculosis culture filtrate

“…Due to their relevance in M. tuberculosis virulence and immune modulation [62], manual validation of peptide spectra of Mpt83 and LpqH, including peptide ions fragment matches, are presented in Fig 3A and 3B, respectively. Although both proteins are largely evidenced as being O-glycosylated due to their interaction with ConA, as native proteins [18] or after heterologous expression in M. smegmatis [63][64][65], to our knowledge this is the first direct glycoproteomic identification in culture filtrate of MTB, of Mpt83 and LpqH derived O-glycosylated peptides. In both cases, O-glycosylation site assignation is coincident with the evidence in M. smegmatis model [63,65].…”

The N-terminal peptide moiety of the Mycobacterium tuberculosis 19 kDa lipoprotein harbors RP105-agonistic properties

“…Furthermore, the remaining 122 amino acids of the native M. tuberculosis 19 kDa lipoprotein may contain motifs that similarly contribute to its RP105 agonism. O‐glycosylation of threonine‐rich motifs near the N‐terminus of the native 19 kDa lipoprotein contribute to its biological functions, and it remains to be determined whether posttranslational modifications shape RP105 agonism in native proteins. Nevertheless, direct comparison of the synthetic lipopeptide and the native M. tuberculosis 19 kDa lipoprotein in our study suggests that the 16 N‐terminal amino acids of the 19 kDa lipoprotein are a dominant contributor to RP105‐mediated macrophage cytokine production.…”