Identification of N-glycan oligomannoside isomers in the diatom Phaeodactylum tricornutum.

Microalgae are emerging production systems for recombinant proteins like monoclonal antibodies. In this context, the characterization of the host cell N-glycosylation machinery and of the microalgae-made biopharmaceuticals, which are mainly glycoprotein-based products, requires efficient analytical methodologies dedicated to the profiling of the N-glycans. Herein, in order to gain knowledge regarding its N-glycosylation pathway, we profile the protein N-linked oligosaccharides isolated from the diatom Phaeodactylum tricornutum that has been used successfully to produce functional monoclonal antibodies. The combination of ion mobility spectrometry-mass Spectrometry and electrospray ionization-multistage tandem mass spectrometry allows us to decipher the detailed structure of the oligomannoside isomers and to demonstrate that the processing of the oligomannosides N-linked to proteins occurs in this diatom as reported in mammals. Therefore, P. tricornutum synthesizes human-like oligomannosides in contrast to other microalgae species. This represent an advantage as an alternative ecofriendly expression system to produce biopharmaceuticals used for human therapy.

User-friendly extraction and multistage tandem mass spectrometry based analysis of lipid-linked oligosaccharides in microalgae.

BACKGROUND: Protein 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. METHODS: In 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. RESULTS: The 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. CONCLUSION: The 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.