Abstract
BackgroundProtein N-glycosylation is initiated within the endoplasmic reticulum through the synthesis of a lipid-linked oligosaccharides (LLO) precursor. This precursor is then transferred en bloc on neo-synthesized proteins through the action of the oligosaccharyltransferase giving birth to glycoproteins. The N-linked glycans bore by the glycoproteins are then processed into oligomannosides prior to the exit of the glycoproteins from the endoplasmic reticulum and its entrance into the Golgi apparatus. In this compartment, the N-linked glycans are further maturated in complex type N-glycans. This process has been well studied in a lot of eukaryotes including higher plants. In contrast, little information regarding the LLO precursor and synthesis of N-linked glycans is available in microalgae.MethodsIn this report, a user-friendly extraction method combining microsomal enrichment and solvent extractions followed by purification steps is described. This strategy is aiming to extract LLO precursor from microalgae. Then, the oligosaccharide moiety released from the extracted LLO were analyzed by multistage tandem mass spectrometry in two models of microalgae namely the green microalgae, Chlamydomonas reinhardtii and the diatom, Phaeodactylum tricornutum.ResultsThe validity of the developed method was confirmed by the analysis of the oligosaccharide structures released from the LLO of two xylosyltransferase mutants of C. reinhardtii confirming that this green microalga synthesizes a linear Glc3Man5GlcNAc2 identical to the one of the wild-type cells. In contrast, the analysis of the oligosaccharide released from the LLO of the diatom P. tricornutum demonstrated for the first time a Glc2Man9GlcNAc2 structure.ConclusionThe method described in this article allows the fast, non-radioactive and reliable multistage tandem mass spectrometry characterization of oligosaccharides released from LLO of microalgae including the ones belonging to the Phaeodactylaceae and Chlorophyceae classes, respectively. The method is fully adaptable for extracting and characterizing the LLO oligosaccharide moiety from microalgae belonging to other phyla.
Highlights
Protein N-glycosylation is initiated within the endoplasmic reticulum through the synthesis of a lipidlinked oligosaccharides (LLO) precursor
Optimization of a protocol for the linked oligosaccharide precursor (LLO) precursor extraction and characterization in microalgae Nowadays, most of the protocols reported in the literature for LLO extractions and analyses are based on radioactivity labelling
The microsomal fraction containing the LLO anchored in the endoplasmic reticulum (ER) were recovered from the other cell components by successive centrifugation steps at 20,000×g followed by an ultracentrifugation step at 100,000×g
Summary
Protein N-glycosylation is initiated within the endoplasmic reticulum through the synthesis of a lipidlinked oligosaccharides (LLO) precursor. The second step involves the transfer of the oligosaccharide part of the LLO from the PP-Dol to an asparagine residue of a neosynthesized protein by the oligosaccharyltransferase complex (OST) [10, 11] This is directly followed by the quality control of the protein folding involving, the removal of the three terminal glucose residues as a final step, leading to the formation of oligomannosidic N-linked glycans [12, 13]. This nascent folded glycoprotein is transferred to the Golgi apparatus where N-linked glycans will undergo specific maturation by glycosyltransferases and glycosidases which are different among species and lead to different complex-type N-glycans. The N-glycosylation maturation process generates complex-type N-glycans GlcNAc2Man3GlcNAc2 bearing alpha(1,3)core fucose and beta(1,2)-core xylose and eventually Lewis a epitopes [7, 14, 15]
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