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N-Linked Glycans Are Assembled on Highly Reduced Dolichol Phosphate Carriers in the Hyperthermophilic Archaea Pyrococcus furiosus.

Chang MM, Imperiali B, Eichler J, Guan Z - PLoS ONE (2015)

Bottom Line: In this study, glycan-charged dolichol phosphate from the hyperthermophile Pyrococcus furiosus was identified and structurally characterized.As with other described archaeal dolichol phosphates, the α- and ω-terminal isoprene subunits of the P. furiosus lipid are saturated, in contrast to eukaryal phosphodolichols that present only a saturated α-position isoprene subunit.Interestingly, an additional 1-4 of the 12-14 isoprene subunits comprising P. furiosus dolichol phosphate are saturated, making this lipid not only the longest archaeal dolichol phosphate described to date but also the most highly saturated.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States of America.

ABSTRACT
In all three domains of life, N-glycosylation begins with the assembly of glycans on phosphorylated polyisoprenoid carriers. Like eukaryotes, archaea also utilize phosphorylated dolichol for this role, yet whereas the assembled oligosaccharide is transferred to target proteins from dolichol pyrophosphate in eukaryotes, archaeal N-linked glycans characterized to date are derived from a dolichol monophosphate carrier, apart from a single example. In this study, glycan-charged dolichol phosphate from the hyperthermophile Pyrococcus furiosus was identified and structurally characterized. Normal and reverse phase liquid chromatography-electrospray ionization mass spectrometry revealed the existence of dolichol phosphate charged with the heptasaccharide recently described in in vitro studies of N-glycosylation on this species. As with other described archaeal dolichol phosphates, the α- and ω-terminal isoprene subunits of the P. furiosus lipid are saturated, in contrast to eukaryal phosphodolichols that present only a saturated α-position isoprene subunit. Interestingly, an additional 1-4 of the 12-14 isoprene subunits comprising P. furiosus dolichol phosphate are saturated, making this lipid not only the longest archaeal dolichol phosphate described to date but also the most highly saturated.

No MeSH data available.


Related in: MedlinePlus

P. furiosus contains heptasaccharide-charged DolP.A. The mass spectrum of the RPLC fraction with a retention time of 8.5–9.5 min contains [M-2H]2- peaks of m/z 1046.592, 1081.120 and 1114.637, corresponding to heptasaccharide-charged C60, C65 and C70 DolP, respectively. The 13C1-containing isotopic and monoisotopic [M-2H]2- peaks of heptasaccharide-charged C65 DolP are indicated. The inset shows [M-2H]2- peaks of m/z 914.551, 948.575 and 979.583, corresponding to C60, C65 and C70 DolP attached to the pentasaccharide precursor of the complete heptasaccharide, as well as [M-2H]2- peaks of m/z 980.585, 1014.089 and 1048.116, corresponding to C60, C65 and C70 DolP attached to the hexasaccharide precursor of the same heptasaccharide. B. The chemical structure (based on the N-linked glycan) and MS/MS fragmentation scheme are shown in the panel top. The MS/MS spectrum of the [M-2H]2- peak of m/z 1081.12 corresponding to heptasaccharide-charged C65 DolP is presented in the panel bottom. The arrows indicating x10 and x15 reflect magnification of the ion peaks in the corresponding region of m/z values on the spectrum.
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pone.0130482.g001: P. furiosus contains heptasaccharide-charged DolP.A. The mass spectrum of the RPLC fraction with a retention time of 8.5–9.5 min contains [M-2H]2- peaks of m/z 1046.592, 1081.120 and 1114.637, corresponding to heptasaccharide-charged C60, C65 and C70 DolP, respectively. The 13C1-containing isotopic and monoisotopic [M-2H]2- peaks of heptasaccharide-charged C65 DolP are indicated. The inset shows [M-2H]2- peaks of m/z 914.551, 948.575 and 979.583, corresponding to C60, C65 and C70 DolP attached to the pentasaccharide precursor of the complete heptasaccharide, as well as [M-2H]2- peaks of m/z 980.585, 1014.089 and 1048.116, corresponding to C60, C65 and C70 DolP attached to the hexasaccharide precursor of the same heptasaccharide. B. The chemical structure (based on the N-linked glycan) and MS/MS fragmentation scheme are shown in the panel top. The MS/MS spectrum of the [M-2H]2- peak of m/z 1081.12 corresponding to heptasaccharide-charged C65 DolP is presented in the panel bottom. The arrows indicating x10 and x15 reflect magnification of the ion peaks in the corresponding region of m/z values on the spectrum.

Mentions: When the MS profile of the RPLC fraction with a retention time of 8.5–9.5 min was examined, a doubly de-protonated [M-2H]2- ion peak of m/z 1081.120 was observed (Fig 1A). The mass of this ion (observed mass 2164.256 Da) matches with C65 DolP linked to the heptasaccharide that P. furiosus AglB was previously shown to add to sequon-containing peptides (13C1-containing isotopic exact mass 2164.250 Da). In the same window of retention time, monoisotopic [M-2H]2- peaks of m/z 1046.592 and 1114.637, corresponding to heptasaccharide-charged C60 and C70 DolP, were also observed, albeit at lesser intensities. The same profile also included 13C1-containing isotopic [M-2H]2- peaks of m/z 914.551, 948.575 and 979.583, corresponding to C60, C65 and C70 DolP attached to the pentasaccharide precursor of the complete heptasaccharide, as well as [M-2H]2- peaks of m/z 980.585, 1014.089 and 1048.16, corresponding to C60, C65 and C70 DolP attached to the hexasaccharide precursor of the same heptasaccharide (Fig 1A, inset). Note that the m/z values reported correspond to either the monoisotopic or 13C1-containing isotopic peak, depending on which appeared as the highest peak in the mass spectrum of each species.


N-Linked Glycans Are Assembled on Highly Reduced Dolichol Phosphate Carriers in the Hyperthermophilic Archaea Pyrococcus furiosus.

Chang MM, Imperiali B, Eichler J, Guan Z - PLoS ONE (2015)

P. furiosus contains heptasaccharide-charged DolP.A. The mass spectrum of the RPLC fraction with a retention time of 8.5–9.5 min contains [M-2H]2- peaks of m/z 1046.592, 1081.120 and 1114.637, corresponding to heptasaccharide-charged C60, C65 and C70 DolP, respectively. The 13C1-containing isotopic and monoisotopic [M-2H]2- peaks of heptasaccharide-charged C65 DolP are indicated. The inset shows [M-2H]2- peaks of m/z 914.551, 948.575 and 979.583, corresponding to C60, C65 and C70 DolP attached to the pentasaccharide precursor of the complete heptasaccharide, as well as [M-2H]2- peaks of m/z 980.585, 1014.089 and 1048.116, corresponding to C60, C65 and C70 DolP attached to the hexasaccharide precursor of the same heptasaccharide. B. The chemical structure (based on the N-linked glycan) and MS/MS fragmentation scheme are shown in the panel top. The MS/MS spectrum of the [M-2H]2- peak of m/z 1081.12 corresponding to heptasaccharide-charged C65 DolP is presented in the panel bottom. The arrows indicating x10 and x15 reflect magnification of the ion peaks in the corresponding region of m/z values on the spectrum.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4476749&req=5

pone.0130482.g001: P. furiosus contains heptasaccharide-charged DolP.A. The mass spectrum of the RPLC fraction with a retention time of 8.5–9.5 min contains [M-2H]2- peaks of m/z 1046.592, 1081.120 and 1114.637, corresponding to heptasaccharide-charged C60, C65 and C70 DolP, respectively. The 13C1-containing isotopic and monoisotopic [M-2H]2- peaks of heptasaccharide-charged C65 DolP are indicated. The inset shows [M-2H]2- peaks of m/z 914.551, 948.575 and 979.583, corresponding to C60, C65 and C70 DolP attached to the pentasaccharide precursor of the complete heptasaccharide, as well as [M-2H]2- peaks of m/z 980.585, 1014.089 and 1048.116, corresponding to C60, C65 and C70 DolP attached to the hexasaccharide precursor of the same heptasaccharide. B. The chemical structure (based on the N-linked glycan) and MS/MS fragmentation scheme are shown in the panel top. The MS/MS spectrum of the [M-2H]2- peak of m/z 1081.12 corresponding to heptasaccharide-charged C65 DolP is presented in the panel bottom. The arrows indicating x10 and x15 reflect magnification of the ion peaks in the corresponding region of m/z values on the spectrum.
Mentions: When the MS profile of the RPLC fraction with a retention time of 8.5–9.5 min was examined, a doubly de-protonated [M-2H]2- ion peak of m/z 1081.120 was observed (Fig 1A). The mass of this ion (observed mass 2164.256 Da) matches with C65 DolP linked to the heptasaccharide that P. furiosus AglB was previously shown to add to sequon-containing peptides (13C1-containing isotopic exact mass 2164.250 Da). In the same window of retention time, monoisotopic [M-2H]2- peaks of m/z 1046.592 and 1114.637, corresponding to heptasaccharide-charged C60 and C70 DolP, were also observed, albeit at lesser intensities. The same profile also included 13C1-containing isotopic [M-2H]2- peaks of m/z 914.551, 948.575 and 979.583, corresponding to C60, C65 and C70 DolP attached to the pentasaccharide precursor of the complete heptasaccharide, as well as [M-2H]2- peaks of m/z 980.585, 1014.089 and 1048.16, corresponding to C60, C65 and C70 DolP attached to the hexasaccharide precursor of the same heptasaccharide (Fig 1A, inset). Note that the m/z values reported correspond to either the monoisotopic or 13C1-containing isotopic peak, depending on which appeared as the highest peak in the mass spectrum of each species.

Bottom Line: In this study, glycan-charged dolichol phosphate from the hyperthermophile Pyrococcus furiosus was identified and structurally characterized.As with other described archaeal dolichol phosphates, the α- and ω-terminal isoprene subunits of the P. furiosus lipid are saturated, in contrast to eukaryal phosphodolichols that present only a saturated α-position isoprene subunit.Interestingly, an additional 1-4 of the 12-14 isoprene subunits comprising P. furiosus dolichol phosphate are saturated, making this lipid not only the longest archaeal dolichol phosphate described to date but also the most highly saturated.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States of America.

ABSTRACT
In all three domains of life, N-glycosylation begins with the assembly of glycans on phosphorylated polyisoprenoid carriers. Like eukaryotes, archaea also utilize phosphorylated dolichol for this role, yet whereas the assembled oligosaccharide is transferred to target proteins from dolichol pyrophosphate in eukaryotes, archaeal N-linked glycans characterized to date are derived from a dolichol monophosphate carrier, apart from a single example. In this study, glycan-charged dolichol phosphate from the hyperthermophile Pyrococcus furiosus was identified and structurally characterized. Normal and reverse phase liquid chromatography-electrospray ionization mass spectrometry revealed the existence of dolichol phosphate charged with the heptasaccharide recently described in in vitro studies of N-glycosylation on this species. As with other described archaeal dolichol phosphates, the α- and ω-terminal isoprene subunits of the P. furiosus lipid are saturated, in contrast to eukaryal phosphodolichols that present only a saturated α-position isoprene subunit. Interestingly, an additional 1-4 of the 12-14 isoprene subunits comprising P. furiosus dolichol phosphate are saturated, making this lipid not only the longest archaeal dolichol phosphate described to date but also the most highly saturated.

No MeSH data available.


Related in: MedlinePlus