Limits...
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

Examination of posttranslational modifications of archaellins and pilins in various mutant backgrounds. (A) Western blot detection of archaellin FlaB2 in various M.maripaludis mutant strains. Whole cell lysates were separated by SDS-PAGE (15% gel), transferred to Immobilon membrane and the blot developed with antibodies to FlaB2. (B) Western blot detection of EpdE in various M. maripaludis mutant strains expressing a plasmid borne C-terminal FLAG-tagged version of EpdE. (C) Western blot detection of EpdD in various M. maripaludis mutant strains expressing a plasmid borne C-terminal FLAG-tagged version of EpdD. For panels (B) and (C), whole cell lysates were separated by SDS-PAGE (17.5% gel), transferred to Immobilon membrane and developed with antibodies to the FLAG-tag.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4390842&req=5

life-05-00085-f004: Examination of posttranslational modifications of archaellins and pilins in various mutant backgrounds. (A) Western blot detection of archaellin FlaB2 in various M.maripaludis mutant strains. Whole cell lysates were separated by SDS-PAGE (15% gel), transferred to Immobilon membrane and the blot developed with antibodies to FlaB2. (B) Western blot detection of EpdE in various M. maripaludis mutant strains expressing a plasmid borne C-terminal FLAG-tagged version of EpdE. (C) Western blot detection of EpdD in various M. maripaludis mutant strains expressing a plasmid borne C-terminal FLAG-tagged version of EpdD. For panels (B) and (C), whole cell lysates were separated by SDS-PAGE (17.5% gel), transferred to Immobilon membrane and developed with antibodies to the FLAG-tag.

Mentions: FlaB2 was detected in Western blots of whole cell lysates of wildtype, ∆flaK, ∆aglB and the ∆flaK∆aglB double mutant strains (Figure 4A). FlaB2 runs as a slightly larger protein in the ∆flaK mutant compared to wildtype, as a result of it retaining the 12 amino acid signal peptide [18,19]. In a ∆aglB mutant, where N-glycosylation at four sequons is now prevented [34], the protein migrates much faster than in the wildtype cells as expected from the lack of attached glycan. In the ∆flaK∆aglB double mutant, the FlaB2 detected is slightly larger in molecular mass than that observed in the ∆aglB mutant. This band represents the non-glycosylated FlaB2 with its signal peptide intact, meaning that the smaller molecular mass band observed in the ∆aglB mutant would be the non-glycosylated protein but with its signal peptide removed. Thus, for archaellins, the two posttranslational modifications can occur independently of the other; N-linked glycosylation of the protein can occur whether the signal peptide is removed or not and it is also possible for the protein to have its signal peptide removed whether the protein is first glycosylated or not.


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

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

Examination of posttranslational modifications of archaellins and pilins in various mutant backgrounds. (A) Western blot detection of archaellin FlaB2 in various M.maripaludis mutant strains. Whole cell lysates were separated by SDS-PAGE (15% gel), transferred to Immobilon membrane and the blot developed with antibodies to FlaB2. (B) Western blot detection of EpdE in various M. maripaludis mutant strains expressing a plasmid borne C-terminal FLAG-tagged version of EpdE. (C) Western blot detection of EpdD in various M. maripaludis mutant strains expressing a plasmid borne C-terminal FLAG-tagged version of EpdD. For panels (B) and (C), whole cell lysates were separated by SDS-PAGE (17.5% gel), transferred to Immobilon membrane and developed with antibodies to the FLAG-tag.
© Copyright Policy
Related In: Results  -  Collection

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

life-05-00085-f004: Examination of posttranslational modifications of archaellins and pilins in various mutant backgrounds. (A) Western blot detection of archaellin FlaB2 in various M.maripaludis mutant strains. Whole cell lysates were separated by SDS-PAGE (15% gel), transferred to Immobilon membrane and the blot developed with antibodies to FlaB2. (B) Western blot detection of EpdE in various M. maripaludis mutant strains expressing a plasmid borne C-terminal FLAG-tagged version of EpdE. (C) Western blot detection of EpdD in various M. maripaludis mutant strains expressing a plasmid borne C-terminal FLAG-tagged version of EpdD. For panels (B) and (C), whole cell lysates were separated by SDS-PAGE (17.5% gel), transferred to Immobilon membrane and developed with antibodies to the FLAG-tag.
Mentions: FlaB2 was detected in Western blots of whole cell lysates of wildtype, ∆flaK, ∆aglB and the ∆flaK∆aglB double mutant strains (Figure 4A). FlaB2 runs as a slightly larger protein in the ∆flaK mutant compared to wildtype, as a result of it retaining the 12 amino acid signal peptide [18,19]. In a ∆aglB mutant, where N-glycosylation at four sequons is now prevented [34], the protein migrates much faster than in the wildtype cells as expected from the lack of attached glycan. In the ∆flaK∆aglB double mutant, the FlaB2 detected is slightly larger in molecular mass than that observed in the ∆aglB mutant. This band represents the non-glycosylated FlaB2 with its signal peptide intact, meaning that the smaller molecular mass band observed in the ∆aglB mutant would be the non-glycosylated protein but with its signal peptide removed. Thus, for archaellins, the two posttranslational modifications can occur independently of the other; N-linked glycosylation of the protein can occur whether the signal peptide is removed or not and it is also possible for the protein to have its signal peptide removed whether the protein is first glycosylated or not.

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