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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.


Related in: MedlinePlus

The H36 and AD7.5 antibodies recognize the same protein bands in reducing and non-reducing conditions.A total cell extract of D. discoideum cells grown in liquid HL5 medium was prepared in the (A) absence or (B) presence of 2-mercaptoethanol (2-Me). The extract was separated by SDS-PAGE, transferred to nitrocellulose membranes, and two tracks of each membrane were blotted with the H36 or AD7.5 antibody. This experiment was repeated twice with identical results.
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pone.0158270.g004: The H36 and AD7.5 antibodies recognize the same protein bands in reducing and non-reducing conditions.A total cell extract of D. discoideum cells grown in liquid HL5 medium was prepared in the (A) absence or (B) presence of 2-mercaptoethanol (2-Me). The extract was separated by SDS-PAGE, transferred to nitrocellulose membranes, and two tracks of each membrane were blotted with the H36 or AD7.5 antibody. This experiment was repeated twice with identical results.

Mentions: An immunoprecipitation approach performed on total cell lysates combined with a mass spectrometry analysis was used to identify the nature of the antigen recognized by the H36 antibody. The main protein detected by this analysis was cysteine proteinase 7 (CP7), or proteinase-1, which is a 47-kDa protein encoded by the cprG gene [29]. However, CP7 was never detected by the mass spectrometric analyses performed on purified MLBs (Table 1). CP7 is the principal proteolytic enzyme in D. discoideum vegetative cells [18, 30] and is also the main protein recognized by the AD7.5 monoclonal antibody, which binds to N-acetylglucosamine-1-phosphate [18]. Since the H36 antibody also recognizes many proteins, a Western blot analysis of a total extract of D. discoideum cells was performed in parallel using both antibodies. Interestingly, the migration profiles of the proteins detected by the AD7.5 antibody were different in reducing and non-reducing conditions [31]. In non-reducing condition, the antibody recognized two major bands (47 and 55 kDa) while, in reducing conditions, multiple smaller molecular weight bands were detected (Fig 4), as previously described [29, 31], suggesting that the two bands detected in non-reducing conditions may contain many different proteins. As shown in Fig 4, the H36 and AD7.5 antibodies both generated the same band profiles in reducing and non-reducing conditions, confirming that the antigen recognized by the H36 antibody is also N-acetylglucosamine-1-phosphate, a post-translational modification that can be found on many proteins. These results also suggested that a protein other than CP7 was detected on the MLBs by the H36 antibody, which would explain why CP7 was not identified on MLBs by mass spectrometry. Interestingly, the CDD protein, which was one of the four major proteins associated with purified MLBs, was also detected by immunoprecipitation with the H36 antibody.


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

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

The H36 and AD7.5 antibodies recognize the same protein bands in reducing and non-reducing conditions.A total cell extract of D. discoideum cells grown in liquid HL5 medium was prepared in the (A) absence or (B) presence of 2-mercaptoethanol (2-Me). The extract was separated by SDS-PAGE, transferred to nitrocellulose membranes, and two tracks of each membrane were blotted with the H36 or AD7.5 antibody. This experiment was repeated twice with identical results.
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Related In: Results  -  Collection

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

pone.0158270.g004: The H36 and AD7.5 antibodies recognize the same protein bands in reducing and non-reducing conditions.A total cell extract of D. discoideum cells grown in liquid HL5 medium was prepared in the (A) absence or (B) presence of 2-mercaptoethanol (2-Me). The extract was separated by SDS-PAGE, transferred to nitrocellulose membranes, and two tracks of each membrane were blotted with the H36 or AD7.5 antibody. This experiment was repeated twice with identical results.
Mentions: An immunoprecipitation approach performed on total cell lysates combined with a mass spectrometry analysis was used to identify the nature of the antigen recognized by the H36 antibody. The main protein detected by this analysis was cysteine proteinase 7 (CP7), or proteinase-1, which is a 47-kDa protein encoded by the cprG gene [29]. However, CP7 was never detected by the mass spectrometric analyses performed on purified MLBs (Table 1). CP7 is the principal proteolytic enzyme in D. discoideum vegetative cells [18, 30] and is also the main protein recognized by the AD7.5 monoclonal antibody, which binds to N-acetylglucosamine-1-phosphate [18]. Since the H36 antibody also recognizes many proteins, a Western blot analysis of a total extract of D. discoideum cells was performed in parallel using both antibodies. Interestingly, the migration profiles of the proteins detected by the AD7.5 antibody were different in reducing and non-reducing conditions [31]. In non-reducing condition, the antibody recognized two major bands (47 and 55 kDa) while, in reducing conditions, multiple smaller molecular weight bands were detected (Fig 4), as previously described [29, 31], suggesting that the two bands detected in non-reducing conditions may contain many different proteins. As shown in Fig 4, the H36 and AD7.5 antibodies both generated the same band profiles in reducing and non-reducing conditions, confirming that the antigen recognized by the H36 antibody is also N-acetylglucosamine-1-phosphate, a post-translational modification that can be found on many proteins. These results also suggested that a protein other than CP7 was detected on the MLBs by the H36 antibody, which would explain why CP7 was not identified on MLBs by mass spectrometry. Interestingly, the CDD protein, which was one of the four major proteins associated with purified MLBs, was also detected by immunoprecipitation with the H36 antibody.

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.


Related in: MedlinePlus