Abstract
Dictyostelium discoideum amoebae produce and secrete multilamellar bodies (MLBs) when fed digestible bacteria. The aim of the present study was to elucidate the proteic content of MLBs. The lipid composition of MLBs is mainly amoebal in origin, suggesting that MLB formation is a protozoa-driven process that could play a significant role in amoebal physiology. We identified four major proteins on purified MLBs using mass spectrometry in order to better understand the molecular mechanisms governing MLB formation and, eventually, to elucidate the true function of MLBs. These proteins were SctA, PhoPQ, PonC and a protein containing a cytidine/deoxycytidylate deaminase (CDD) zinc-binding region. SctA is a component of pycnosomes, which are membranous materials that are continuously secreted by amoebae. The presence of SctA on MLBs was confirmed by immunofluorescence and Western blotting using a specific anti-SctA antibody. The CDD protein may be one of the proteins recognized by the H36 antibody, which was used as a MLB marker in a previous study. The function of the CDD protein is unknown. Immunofluorescence and flow cytometric analyses confirmed that the H36 antibody is a better marker of MLBs than the anti-SctA antibody. This study is an additional step to elucidate the potential role of MLBs and revealed that only a small set of proteins appeared to be present on MLBs.
Highlights
Multilamellar bodies (MLBs) are structures of lysosomal origin composed of multiple concentric membrane layers [1]
The extraction and solubilization of the MLB-associated proteins require the use of solutions containing high concentrations of chaotropic agents such as urea because they were embedded in compact multilamellar lipidic layers
The H36 and AD7.5 antibodies both generated the same band profiles in reducing and nonreducing conditions, confirming that the antigen recognized by the H36 antibody is N-acetylglucosamine-1-phosphate, a post-translational modification that can be found on many proteins. These results suggested that a protein other than cysteine proteinase 7 (CP7) was detected on the MLBs by the H36 antibody, which would explain why CP7 was not identified on MLBs by mass spectrometry
Summary
Multilamellar bodies (MLBs) are structures of lysosomal origin composed of multiple concentric membrane layers [1]. They are produced by various types of eukaryotic cells, including protozoa such as Dictyostelium discoideum, which are soil organisms that feed mainly on bacteria by phagocytosis. They produce MLBs ranging in size from 0.5 to 2 μm and secrete them into the environment [2,3,4]. Proteins Associated with D. discoideum MLBs (FRQS, http://www.frqs.gouv.qc.ca), and a grant from the Fonds de Recherche du Québec - Nature et Technologies (FRQNT, http://www.frqnt.gouv.qc.ca). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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