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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 DolP is highly saturated.A. The MS spectrum of the NPLC fraction with a retention time of 14–14.5 min contains [M-H]- ion peaks corresponding to N-acetylhexosamine-linked C6o, C65 and C70 DolP (m/z 1126.871, 1194.934 and 1262.995, respectively). Each DolP species includes 7–11 unsaturated isoprene subunits. B. Zoomed-in spectrum of N-acetylhexosamine-charged C65 DolP [M-H]- ion peaks showing the varying degree of dolichol saturation. Peaks corresponding to species containing dolichol that includes 2–6 saturated bonds are indicated accordingly. C. MS/MS spectrum of the [M-H]- ion of C65 DolP at m/z 1194.934. The fragmentation scheme (inset) shows the distribution of the saturated bonds (mostly near the ω-terminus).
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pone.0130482.g003: P. furiosus DolP is highly saturated.A. The MS spectrum of the NPLC fraction with a retention time of 14–14.5 min contains [M-H]- ion peaks corresponding to N-acetylhexosamine-linked C6o, C65 and C70 DolP (m/z 1126.871, 1194.934 and 1262.995, respectively). Each DolP species includes 7–11 unsaturated isoprene subunits. B. Zoomed-in spectrum of N-acetylhexosamine-charged C65 DolP [M-H]- ion peaks showing the varying degree of dolichol saturation. Peaks corresponding to species containing dolichol that includes 2–6 saturated bonds are indicated accordingly. C. MS/MS spectrum of the [M-H]- ion of C65 DolP at m/z 1194.934. The fragmentation scheme (inset) shows the distribution of the saturated bonds (mostly near the ω-terminus).

Mentions: To better characterize the lipid carrier to which the heptasaccharide and its precursors are bound, NPLC-ESI MS was performed. With a retention time of 14–14.5 min, [M-H]- peaks corresponding to monosaccharide (N-acetylhexosamine)-charged C6o, C65 and C70 DolP (m/z 1126.871, 1194.934 and 1262.995, respectively) were observed (Fig 3A). Each species comprises a series of DolP variants showing differing degrees of isoprene subunit saturation (Fig 3B). When MS/MS analysis was performed on the [M-H]- ion of C65 DolP at m/z 1194.934 (Fig 3C), a fragmentation pattern consistent with a DolP molecule presenting a saturated isoprene subunit at the α-position, as well as multiple saturated isoprene subunits close to the ω-end of the polyisoprenoid chain, was obtained The results show that some isoprene units are only partially saturated. For example, the m/z 846 and 844 fragments suggest that isoprene-6 (from the ω-end) is only partially saturated. Such heterogeneity makes complete characterization difficult, even with the most advanced MS/MS techniques. Indeed, to the best of our knowledge, no better characterization of dolichols has been previously reported. In addition, a peak at m/z 78.950, corresponding to a phosphate group, was noted.


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 DolP is highly saturated.A. The MS spectrum of the NPLC fraction with a retention time of 14–14.5 min contains [M-H]- ion peaks corresponding to N-acetylhexosamine-linked C6o, C65 and C70 DolP (m/z 1126.871, 1194.934 and 1262.995, respectively). Each DolP species includes 7–11 unsaturated isoprene subunits. B. Zoomed-in spectrum of N-acetylhexosamine-charged C65 DolP [M-H]- ion peaks showing the varying degree of dolichol saturation. Peaks corresponding to species containing dolichol that includes 2–6 saturated bonds are indicated accordingly. C. MS/MS spectrum of the [M-H]- ion of C65 DolP at m/z 1194.934. The fragmentation scheme (inset) shows the distribution of the saturated bonds (mostly near the ω-terminus).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130482.g003: P. furiosus DolP is highly saturated.A. The MS spectrum of the NPLC fraction with a retention time of 14–14.5 min contains [M-H]- ion peaks corresponding to N-acetylhexosamine-linked C6o, C65 and C70 DolP (m/z 1126.871, 1194.934 and 1262.995, respectively). Each DolP species includes 7–11 unsaturated isoprene subunits. B. Zoomed-in spectrum of N-acetylhexosamine-charged C65 DolP [M-H]- ion peaks showing the varying degree of dolichol saturation. Peaks corresponding to species containing dolichol that includes 2–6 saturated bonds are indicated accordingly. C. MS/MS spectrum of the [M-H]- ion of C65 DolP at m/z 1194.934. The fragmentation scheme (inset) shows the distribution of the saturated bonds (mostly near the ω-terminus).
Mentions: To better characterize the lipid carrier to which the heptasaccharide and its precursors are bound, NPLC-ESI MS was performed. With a retention time of 14–14.5 min, [M-H]- peaks corresponding to monosaccharide (N-acetylhexosamine)-charged C6o, C65 and C70 DolP (m/z 1126.871, 1194.934 and 1262.995, respectively) were observed (Fig 3A). Each species comprises a series of DolP variants showing differing degrees of isoprene subunit saturation (Fig 3B). When MS/MS analysis was performed on the [M-H]- ion of C65 DolP at m/z 1194.934 (Fig 3C), a fragmentation pattern consistent with a DolP molecule presenting a saturated isoprene subunit at the α-position, as well as multiple saturated isoprene subunits close to the ω-end of the polyisoprenoid chain, was obtained The results show that some isoprene units are only partially saturated. For example, the m/z 846 and 844 fragments suggest that isoprene-6 (from the ω-end) is only partially saturated. Such heterogeneity makes complete characterization difficult, even with the most advanced MS/MS techniques. Indeed, to the best of our knowledge, no better characterization of dolichols has been previously reported. In addition, a peak at m/z 78.950, corresponding to a phosphate group, was noted.

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