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Pilin Processing Follows a Different Temporal Route than That of Archaellins in Methanococcus maripaludis.

Nair DB, Jarrell KF - Life (Basel) (2015)

Bottom Line: In contrast, pilins are not glycosylated unless they have been acted on by EppA to have the signal peptide removed.However, EppA can still remove signal peptides from non-glycosylated pilins.These findings indicate that there is a difference in the order of the posttranslational modifications of pilins and archaellins even though both are type IV pilin-like proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada. 7ndb@queensu.ca.

ABSTRACT
Methanococcus maripaludis has two different surface appendages: type IV-like pili and archaella. Both structures are believed to be assembled using a bacterial type IV pilus mechanism. Each structure is composed of multiple subunits, either pilins or archaellins. Both pilins and archaellins are made initially as preproteins with type IV pilin-like signal peptides, which must be removed by a prepilin peptidase-like enzyme. This enzyme is FlaK for archaellins and EppA for pilins. In addition, both pilins and archaellins are modified with N-linked glycans. The archaellins possess an N-linked tetrasaccharide while the pilins have a pentasaccharide which consists of the archaellin tetrasaccharide but with an additional sugar, an unidentified hexose, attached to the linking sugar. In this report, we show that archaellins can be processed by FlaK in the absence of N-glycosylation and N-glycosylation can occur on archaellins that still retain their signal peptides. In contrast, pilins are not glycosylated unless they have been acted on by EppA to have the signal peptide removed. However, EppA can still remove signal peptides from non-glycosylated pilins. These findings indicate that there is a difference in the order of the posttranslational modifications of pilins and archaellins even though both are type IV pilin-like proteins.

No MeSH data available.


Related in: MedlinePlus

Western blot detection of native EpdE in various mutant backgrounds. (A) Western blot analysis to detect EpdE in various M. maripaludis mutant strains using anti-EpdE antibody. (B) Detection of EpdE in Western blots of the ∆eppA mutant and in the ∆eppA mutant following complementation with a plasmid borne copy of eppA. Shown are samples after the complemented cells were grown for three transfers in N-free medium supplemented with alanine (promoter on conditions) and the same cells grown in N-free medium supplemented with NH4Cl (promoter off-conditions). C-terminal histagged EpdE was expressed in E.coli and purified by a Ni-affinity column is used for size comparison. In both (A) and (B), whole cell lysates were separated by SDS-PAGE (17.5% gel), transferred to Immobilon membrane and developed with antibodies to EpdE. Arrows point to a nonspecific cross-reactive band that can be observed in all whole lysate lanes, including the ∆epdE lane.
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life-05-00085-f003: Western blot detection of native EpdE in various mutant backgrounds. (A) Western blot analysis to detect EpdE in various M. maripaludis mutant strains using anti-EpdE antibody. (B) Detection of EpdE in Western blots of the ∆eppA mutant and in the ∆eppA mutant following complementation with a plasmid borne copy of eppA. Shown are samples after the complemented cells were grown for three transfers in N-free medium supplemented with alanine (promoter on conditions) and the same cells grown in N-free medium supplemented with NH4Cl (promoter off-conditions). C-terminal histagged EpdE was expressed in E.coli and purified by a Ni-affinity column is used for size comparison. In both (A) and (B), whole cell lysates were separated by SDS-PAGE (17.5% gel), transferred to Immobilon membrane and developed with antibodies to EpdE. Arrows point to a nonspecific cross-reactive band that can be observed in all whole lysate lanes, including the ∆epdE lane.

Mentions: Western blots using the purified IgY were not specific, however, requiring a subsequent affinity purification step. Antibodies eluted from this step were used in subsequent Western blots. The anti-EpdE antibodies, with their affinity purified from EpdE protein expressed in E. coli where it would not be glycosylated, may react more strongly to non-processed pilin compared to the wildtype pilin glycoprotein. As seen initially with the EpdE FLAG-tagged protein expressed in trans, native EpdE in eppA deletion strains also migrated as a much smaller protein than in wildtype cells or the flaK deletion strain (Figure 3A). When the ∆eppA strain was complemented with a plasmid borne copy of eppA, the EpdE detected in Western blots returned to the size observed in the wildtype cells (Figure 3B), proving that EpdE is, in fact, an EppA substrate, as initially predicted by Szabo et al. [21] and that signal peptide removal was necessary for the dramatic shift of the pilin to the higher apparent molecular mass. For a size comparison, we also included the purified C-terminal histagged version of EpdE expressed in E. coli and used to generate antibodies. This version of EpdE would be non-glycosylated and still have its signal peptide attached, as E. coli lacks the genes necessary for the two posttranslational modifications. It would however be larger than the native EpdE protein by virtue of the extra six C-terminal histidine residues. The size of EpdE as detected by Western blots using anti-EpdE antibodies is smaller in the ∆eppA and ∆eppA ∆flaK strains than the E. coli-produced histagged version. This would be consistent with the ∆eppA and ∆eppA ∆flaK strain versions being non-glycosylated and with their signal peptide attached, as in the E. coli produced version but without the hexahistidine C-terminal extension.


Pilin Processing Follows a Different Temporal Route than That of Archaellins in Methanococcus maripaludis.

Nair DB, Jarrell KF - Life (Basel) (2015)

Western blot detection of native EpdE in various mutant backgrounds. (A) Western blot analysis to detect EpdE in various M. maripaludis mutant strains using anti-EpdE antibody. (B) Detection of EpdE in Western blots of the ∆eppA mutant and in the ∆eppA mutant following complementation with a plasmid borne copy of eppA. Shown are samples after the complemented cells were grown for three transfers in N-free medium supplemented with alanine (promoter on conditions) and the same cells grown in N-free medium supplemented with NH4Cl (promoter off-conditions). C-terminal histagged EpdE was expressed in E.coli and purified by a Ni-affinity column is used for size comparison. In both (A) and (B), whole cell lysates were separated by SDS-PAGE (17.5% gel), transferred to Immobilon membrane and developed with antibodies to EpdE. Arrows point to a nonspecific cross-reactive band that can be observed in all whole lysate lanes, including the ∆epdE lane.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4390842&req=5

life-05-00085-f003: Western blot detection of native EpdE in various mutant backgrounds. (A) Western blot analysis to detect EpdE in various M. maripaludis mutant strains using anti-EpdE antibody. (B) Detection of EpdE in Western blots of the ∆eppA mutant and in the ∆eppA mutant following complementation with a plasmid borne copy of eppA. Shown are samples after the complemented cells were grown for three transfers in N-free medium supplemented with alanine (promoter on conditions) and the same cells grown in N-free medium supplemented with NH4Cl (promoter off-conditions). C-terminal histagged EpdE was expressed in E.coli and purified by a Ni-affinity column is used for size comparison. In both (A) and (B), whole cell lysates were separated by SDS-PAGE (17.5% gel), transferred to Immobilon membrane and developed with antibodies to EpdE. Arrows point to a nonspecific cross-reactive band that can be observed in all whole lysate lanes, including the ∆epdE lane.
Mentions: Western blots using the purified IgY were not specific, however, requiring a subsequent affinity purification step. Antibodies eluted from this step were used in subsequent Western blots. The anti-EpdE antibodies, with their affinity purified from EpdE protein expressed in E. coli where it would not be glycosylated, may react more strongly to non-processed pilin compared to the wildtype pilin glycoprotein. As seen initially with the EpdE FLAG-tagged protein expressed in trans, native EpdE in eppA deletion strains also migrated as a much smaller protein than in wildtype cells or the flaK deletion strain (Figure 3A). When the ∆eppA strain was complemented with a plasmid borne copy of eppA, the EpdE detected in Western blots returned to the size observed in the wildtype cells (Figure 3B), proving that EpdE is, in fact, an EppA substrate, as initially predicted by Szabo et al. [21] and that signal peptide removal was necessary for the dramatic shift of the pilin to the higher apparent molecular mass. For a size comparison, we also included the purified C-terminal histagged version of EpdE expressed in E. coli and used to generate antibodies. This version of EpdE would be non-glycosylated and still have its signal peptide attached, as E. coli lacks the genes necessary for the two posttranslational modifications. It would however be larger than the native EpdE protein by virtue of the extra six C-terminal histidine residues. The size of EpdE as detected by Western blots using anti-EpdE antibodies is smaller in the ∆eppA and ∆eppA ∆flaK strains than the E. coli-produced histagged version. This would be consistent with the ∆eppA and ∆eppA ∆flaK strain versions being non-glycosylated and with their signal peptide attached, as in the E. coli produced version but without the hexahistidine C-terminal extension.

Bottom Line: In contrast, pilins are not glycosylated unless they have been acted on by EppA to have the signal peptide removed.However, EppA can still remove signal peptides from non-glycosylated pilins.These findings indicate that there is a difference in the order of the posttranslational modifications of pilins and archaellins even though both are type IV pilin-like proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada. 7ndb@queensu.ca.

ABSTRACT
Methanococcus maripaludis has two different surface appendages: type IV-like pili and archaella. Both structures are believed to be assembled using a bacterial type IV pilus mechanism. Each structure is composed of multiple subunits, either pilins or archaellins. Both pilins and archaellins are made initially as preproteins with type IV pilin-like signal peptides, which must be removed by a prepilin peptidase-like enzyme. This enzyme is FlaK for archaellins and EppA for pilins. In addition, both pilins and archaellins are modified with N-linked glycans. The archaellins possess an N-linked tetrasaccharide while the pilins have a pentasaccharide which consists of the archaellin tetrasaccharide but with an additional sugar, an unidentified hexose, attached to the linking sugar. In this report, we show that archaellins can be processed by FlaK in the absence of N-glycosylation and N-glycosylation can occur on archaellins that still retain their signal peptides. In contrast, pilins are not glycosylated unless they have been acted on by EppA to have the signal peptide removed. However, EppA can still remove signal peptides from non-glycosylated pilins. These findings indicate that there is a difference in the order of the posttranslational modifications of pilins and archaellins even though both are type IV pilin-like proteins.

No MeSH data available.


Related in: MedlinePlus