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Identification of Proteins Associated with Multilamellar Bodies Produced by Dictyostelium discoideum.

Denoncourt AM, Paquet VE, Sedighi A, Charette SJ - PLoS ONE (2016)

Bottom Line: The presence of SctA on MLBs was confirmed by immunofluorescence and Western blotting using a specific anti-SctA antibody.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.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Intégrative et des Systèmes, Pavillon Charles-Eugène-Marchand, Université Laval, Quebec City, QC, Canada.

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.

No MeSH data available.


Protein profile of purified MLBs.Proteins were extracted from purified MLBs, solubilized with denaturation solutions, and run on an SDS-PAGE gel. The gel was stained with Coomassie blue to reveal the proteins. Most of the proteins had low molecular weights. Only a few major bands are visible on the gel. Based on mass spectrometric results, the 18-kDa band was a mix of the SctA and PonC proteins, while the 46 and 56-kDa bands corresponded to the CDD and PhoPQ proteins, respectively. The 10-kDa band gave inconclusive results.
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pone.0158270.g001: Protein profile of purified MLBs.Proteins were extracted from purified MLBs, solubilized with denaturation solutions, and run on an SDS-PAGE gel. The gel was stained with Coomassie blue to reveal the proteins. Most of the proteins had low molecular weights. Only a few major bands are visible on the gel. Based on mass spectrometric results, the 18-kDa band was a mix of the SctA and PonC proteins, while the 46 and 56-kDa bands corresponded to the CDD and PhoPQ proteins, respectively. The 10-kDa band gave inconclusive results.

Mentions: The protein profile of purified MLBs is shown in Fig 1. The molecular weights of the most abundant proteins were less than 25 kDa. Four bands were excised from the gels for identification by mass spectrometry. The band at ~10 kDa could not be identified while the band at ~18 kDa was composed of SctA and PonC. The CDD protein was identified in the 46 kDa band. The slice at 56 kDa was also excised because it corresponded to the molecular weight of one of the major proteins detected in the total MLB extract. As expected, the PhoPQ protein was identified in the 56 kDa band. The molecular weights of proteins did not necessarily correspond to the molecular weights of the associated band. This may have been due to post-translational modifications of the proteins (such as glycosylation or cleavage), which would have affected their migration on the gel.


Identification of Proteins Associated with Multilamellar Bodies Produced by Dictyostelium discoideum.

Denoncourt AM, Paquet VE, Sedighi A, Charette SJ - PLoS ONE (2016)

Protein profile of purified MLBs.Proteins were extracted from purified MLBs, solubilized with denaturation solutions, and run on an SDS-PAGE gel. The gel was stained with Coomassie blue to reveal the proteins. Most of the proteins had low molecular weights. Only a few major bands are visible on the gel. Based on mass spectrometric results, the 18-kDa band was a mix of the SctA and PonC proteins, while the 46 and 56-kDa bands corresponded to the CDD and PhoPQ proteins, respectively. The 10-kDa band gave inconclusive results.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920372&req=5

pone.0158270.g001: Protein profile of purified MLBs.Proteins were extracted from purified MLBs, solubilized with denaturation solutions, and run on an SDS-PAGE gel. The gel was stained with Coomassie blue to reveal the proteins. Most of the proteins had low molecular weights. Only a few major bands are visible on the gel. Based on mass spectrometric results, the 18-kDa band was a mix of the SctA and PonC proteins, while the 46 and 56-kDa bands corresponded to the CDD and PhoPQ proteins, respectively. The 10-kDa band gave inconclusive results.
Mentions: The protein profile of purified MLBs is shown in Fig 1. The molecular weights of the most abundant proteins were less than 25 kDa. Four bands were excised from the gels for identification by mass spectrometry. The band at ~10 kDa could not be identified while the band at ~18 kDa was composed of SctA and PonC. The CDD protein was identified in the 46 kDa band. The slice at 56 kDa was also excised because it corresponded to the molecular weight of one of the major proteins detected in the total MLB extract. As expected, the PhoPQ protein was identified in the 56 kDa band. The molecular weights of proteins did not necessarily correspond to the molecular weights of the associated band. This may have been due to post-translational modifications of the proteins (such as glycosylation or cleavage), which would have affected their migration on the gel.

Bottom Line: The presence of SctA on MLBs was confirmed by immunofluorescence and Western blotting using a specific anti-SctA antibody.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.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Intégrative et des Systèmes, Pavillon Charles-Eugène-Marchand, Université Laval, Quebec City, QC, Canada.

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.

No MeSH data available.