Characterization of a GDP-Fucose Transporter and a Fucosyltransferase Involved in the Fucosylation of Glycoproteins in the Diatom Phaeodactylum tricornutum.

Peiqing Zhang,Amelia Mak,Marie-Christine Kiefer-Meyer,Carole Plasson,Muriel Bardor,Carole Burel,Bruno Gügi,Azeddine Driouich,Clément Ovide,Gavin Teo,Zhiwei Song,Corrine Wan,Patrice Lerouge

doi:10.3389/fpls.2019.00610

Abstract

Although Phaeodactylum tricornutum is gaining importance in plant molecular farming for the production of high-value molecules such as monoclonal antibodies, little is currently known about key cell metabolism occurring in this diatom such as protein glycosylation. For example, incorporation of fucose residues in the glycans N-linked to protein in P. tricornutum is questionable. Indeed, such epitope has previously been found on N-glycans of endogenous glycoproteins in P. tricornutum. Meanwhile, the potential immunogenicity of the α(1,3)-fucose epitope present on plant-derived biopharmaceuticals is still a matter of debate. In this paper, we have studied molecular actors potentially involved in the fucosylation of the glycoproteins in P. tricornutum. Based on sequence similarities, we have identified a putative P. tricornutum GDP-L-fucose transporter and three fucosyltransferase (FuT) candidates. The putative P. tricornutum GDP-L-fucose transporter coding sequence was expressed in the Chinese Hamster Ovary (CHO)-gmt5 mutant lacking its endogenous GDP-L-fucose transporter activity. We show that the P. tricornutum transporter is able to rescue the fucosylation of proteins in this CHO-gmt5 mutant cell line, thus demonstrating the functional activity of the diatom transporter and its appropriate Golgi localization. In addition, we overexpressed one of the three FuT candidates, namely the FuT54599, in P. tricornutum and investigated its localization within Golgi stacks of the diatom. Our findings show that overexpression of the FuT54599 leads to a significant increase of the α(1,3)-fucosylation of the diatom endogenous glycoproteins.

Highlights

Diatoms are marine organisms that represent one of the most important source of biomass in the ocean (Nelson et al, 1995; Raven and Waite, 2004; Bowler et al, 2008)
We show that putative GDP-L-fucose transporter (PtGFT) is able to rescue the fucosylation of proteins in the Chinese Hamster Ovary (CHO)-gmt5 mutant cell line, demonstrating the functional activity of the diatom transporter
When expressed in CHO-gmt5 mutant lacking endogenous GDP-L-fucose transporter activity, the PtGFT candidate is efficiently addressed to Golgi membranes and is able to rescue the fucosylation of proteins in the CHO-gmt5 mutant cell line, demonstrating that the cDNA sequence registered under the NCBI accession number KT737477 codes a functional Golgi resident P. tricornutum transporter which is at least able to import GDP-L-fucose

Summary

Introduction

Diatoms are marine organisms that represent one of the most important source of biomass in the ocean (Nelson et al, 1995; Raven and Waite, 2004; Bowler et al, 2008). The mAb directed against the Hepatitis B was demonstrated to be of good quality, homogenous and glycosylated with oligomannosides (Vanier et al, 2015) This mAb is able to bind to human Fcγ receptors (FcγRI and FcγRIIIa in particular) which suggests that it could be efficiently used in human immunotherapy to induce phagocytosis and antibody dependent cell-mediated cytotoxicity response (Vanier et al, 2018). Such therapeutic application represents currently a multimillion dollar market sales (Walsh, 2014). It would be necessary in the future to engineer the N-glycosylation of diatom-produced mAb to favor the presence of complex-type and fine-tuned N-glycans as it is well established that glycosylation of mAbs and biopharmaceuticals in general influences their biological functionality and efficacy (Lingg et al, 2012; Buettner et al, 2018; Mimura et al, 2018)

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Conclusion