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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.
Highlights
Modulatory effects on the anti-microbial immune response of the infected host, promoting the activation of neutrophils[8] and CD4+T cells[9] or the differentiation of dendritic cells[10]
With the aim of recovering the broadest range of molecular species, histidine tagged recombinant Mtb LpqH was cloned in an M. smegmatis expression system[21] and purified from the cell protein extract by nickel-affinity chromatography
We report here the thorough characterization of the proteoform repertoire of recombinant LpqH, a lipoglycoprotein antigen from Mtb, using a combination of up to date top-down and bottom-up mass spectrometry
Summary
Modulatory effects on the anti-microbial immune response of the infected host, promoting the activation of neutrophils[8] and CD4+T cells[9] or the differentiation of dendritic cells[10]. Structural features of the mycobacterial lipo and/or glycoproteins have been mostly inferred from tandem mass spectrometry analysis of modified peptides issued from proteolytic digestion of either purified proteins[27,28,29,30] or complex secretomes[2,31] While these bottom-up approaches are determinant to evidence and characterize the mycobacterial lipo-glycoprotein PTMs, they only provide partial and theoretical combinatorial views of the parent proteins that do not allow reconstructing unambiguously the genuine proteoform repertoires. CZE-MS analysis of intact proteins secreted by M. marinum[35] enabled the detection of hundreds of different molecular weights, 58 of which were assigned to proteoforms issued from the combination of N-terminal acetylation, signal peptide maturation and methionine excision These secretome proteomic studies identified some lipoproteins but without any experimental evidence concerning the nature or localization of the acylation. This new phosphorylated LpqH molecular species is absent from the current models of the bacterial lipoglycoprotein biosynthesis pathways, and questions about the influence of this modification on the downstream glycosylation-lipidation of the mature LpqH
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