Teaching dolichol-linked oligosaccharides more tricks with alternatives to metabolic radiolabeling

Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390-9041, USA.
Glycobiology (Impact Factor: 3.15). 09/2007; 17(8):75R-85R. DOI: 10.1093/glycob/cwm029
Source: PubMed


The dolichol cycle involves synthesis of the lipid-linked oligosaccharide (LLO) Glc(3)Man(9)GlcNAc(2)-P-P-dolichol (G(3)M(9)Gn(2)-P-P-Dol), transfer of G(3)M(9)Gn(2) to asparaginyl residues of nascent endoplasmic reticulum (ER) polypeptides by oligosaccharyltransferase (OT), and recycling of the resultant Dol-P-P to Dol-P for new rounds of LLO synthesis. The importance of the dolichol cycle in secretory and membrane protein biosynthesis, ER function, and human genetic disease is now widely accepted. Elucidation of the fundamental properties of the dolichol cycle in intact cells was achieved through the use of radioactive sugar precursors, typically [(3)H]-labeled or [(14)C]-labeled d-mannose, d-galactose, or d-glucosamine. However, difficulties were encountered with cells or tissues not amenable to metabolic labeling, or in experiments influenced by isotope dilution, variable rates of LLO turnover, or special culture conditions required for the use of radioactive sugars. This article will review recently developed alternatives for LLO analysis that do not rely upon metabolic labeling with radioactive precursors, and thereby circumvent these problems. New information revealed by these methods with regard to regulation, genetic disorders, and evolution of the dolichol cycle, as well as caveats of radiolabeling techniques, will be discussed.

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    • "The analysis of LLO precursors of N-glycans is regarded a delicate task as it traditionally involves biphasic extractions of the dolichol-linked sugars and very often metabolic labeling to allow for detection (Lehrman 2007). Non-radioactive methods based on fluorescent labeling of free oligosaccharides have recently been introduced (Gao and Lehrman 2002; Grubenmann et al. 2004; O'Reilly et al. 2006; Hosokawa et al. 2010b). "
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    ABSTRACT: Oligomannosidic (OM) N-glycans occur as a mixture of isomers, which at early stages of glycosidase trimming also comprise structures with one to three glucose residues. A complementary set of isomers is generated during the biosynthesis of the lipid-linked precursor. Here, we demonstrate the remarkable capacity of liquid chromatography (LC) with porous graphitic carbon and mass spectrometric detection for the determination of OM isomers. Protein-linked N-glycans were released enzymatically from samples with known isomer composition such as kidney bean proteins and ribonuclease B. Lipid-linked oligosaccharides were obtained by a direct mild acid hydrolysis of microsomes thus avoiding biphasic partitioning. A parallel analysis of pyridylaminated glycans by amide-silica and reversed-phase high-performance LC, the application of branch-specific α-mannosidases and work with ALG mutant plants led to the assignment of the relative retention times of the isomers occurring during the degradation of the Glc(3)Man(9)GlcNAc(2) precursor oligosaccharide to Man(5)GlcNAc(2) and beyond. A tightly woven net of evidence supports these assignments. Noteworthy, this isomer assignment happens in the course of a comprehensive analysis of all types of a sample's N-glycans.
    Glycobiology 03/2012; 22(3):389-99. DOI:10.1093/glycob/cwr138 · 3.15 Impact Factor
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    • "These data showed that MEFs contain the M6P-responsive component(s) needed for LLO cleavage, and that ER stress can increase M6P in intact MEFs in the required concentration range. To directly assess whether the elevated M6P might have caused LLO cleavage and therefore provide an explanation for the generation of Dol-P (Figures 2 and 4A) in intact stressed MEFs, we used a variation of the FACE technique in which the glycan portions of LLOs were released from the lipid carrier with mild acid, and then labeled with the fluorophore 7-amino-1,3-naphthalenedisulfonic acid (ANDS) (Gao and Lehrman, 2006;Lehrman, 2007). Interestingly, both DTT and TG stresses resulted in a statistically significant suppression of G 3 M 9 Gn 2 -P-P-Dol levels in PERK -/- MEFs (Figure 4B). "
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    ABSTRACT: Mannose-6-phosphate (M6P) is an essential precursor for mannosyl glycoconjugates, including lipid-linked oligosaccharides (LLO; glucose(3)mannose(9)GlcNAc(2)-P-P-dolichol) used for protein N-glycosylation. In permeabilized mammalian cells, M6P also causes specific LLO cleavage. However, the context and purpose of this paradoxical reaction are unknown. In this study, we used intact mouse embryonic fibroblasts to show that endoplasmic reticulum (ER) stress elevates M6P concentrations, leading to cleavage of the LLO pyrophosphate linkage with recovery of its lipid and lumenal glycan components. We demonstrate that this M6P originates from glycogen, with glycogenolysis activated by the kinase domain of the stress sensor IRE1-α. The apparent futility of M6P causing destruction of its LLO product was resolved by experiments with another stress sensor, PKR-like ER kinase (PERK), which attenuates translation. PERK's reduction of N-glycoprotein synthesis (which consumes LLOs) stabilized steady-state LLO levels despite continuous LLO destruction. However, infection with herpes simplex virus 1, an N-glycoprotein-bearing pathogen that impairs PERK signaling, not only caused LLO destruction but depleted LLO levels as well. In conclusion, the common metabolite M6P is also part of a novel mammalian stress-signaling pathway, responding to viral stress by depleting host LLOs required for N-glycosylation of virus-associated polypeptides. Apparently conserved throughout evolution, LLO destruction may be a response to a variety of environmental stresses.
    Molecular biology of the cell 07/2011; 22(17):2994-3009. DOI:10.1091/mbc.E11-04-0286 · 4.47 Impact Factor

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