Fine-tuning the N-glycosylation of recombinant human erythropoietin using Chlamydomonas reinhardtii mutants

Abstract

Microalgae are considered as attractive expression systems for the production of biologics. As photosynthetic unicellular organisms, they do not require costly and complex media for growing and are able to secrete proteins and perform protein glycosylation. Some biologics have been successfully produced in the green microalgae Chlamydomonas reinhardtii. However, post-translational modifications like glycosylation of these Chlamydomonas-made biologics have poorly been investigated so far. Therefore, in this study, we report on the first structural investigation of glycans linked to human erythropoietin (hEPO) expressed in a wild-type C. reinhardtii strain and mutants impaired in key Golgi glycosyltransferases. The glycoproteomic analysis of recombinant hEPO (rhEPO) expressed in the wild-type strain demonstrated that the three N-glycosylation sites are 100% glycosylated with mature N-glycans containing four to five mannose residues and carrying core xylose, core fucose and O-methyl groups. Moreover, expression in C. reinhardtii insertional mutants defective in xylosyltransferases A and B and fucosyltransferase resulted in drastic decreases of core xylosylation and core fucosylation of glycans N-linked to the rhEPOs, thus demonstrating that this strategy offers perspectives for humanizing the N-glycosylation of the Chlamydomonas-made biologics.

Keywords: Chlamydomonas reinhardtii; biologics; erythropoietin; glycoengineering; glycosylation.

Figure 1

rhEPO is secreted in the culture medium in C. reinhardtii transformants and is N‐glycosylated. (a) Construct used for the expression rhEPO in C. reinhardtii. (b) Western blot analyses using an anti‐hEPO antibody performed on total proteins from the cell pellets (upper panel) and TCA precipitated proteins from the culture medium (bottom panel) of CC5325, IM _XTA  × IM _XTB and IM _XTA  × IM _XTB  × IM _FUT mutants. (−) Proteins from non‐transformed strains used as negative controls. (c) rhEPOs were purified from the culture media, submitted to a deglycosylation with Endo H or PNGase F and then analysed by Western blot using an anti‐hEPO antibody. (−) and (+): Samples non‐treated or treated by Endo H or PNGase F. C: positive control, corresponding to commercial rhEPO. The black arrows point out on intact commercial rhEPO.

Figure 2

Distribution of N‐glycans on the three N‐glycosylation sites of rhEPOs produced in C. reinhardtii CC5325, IM _XTA  × IM _XTB and IM _XTA  × IM _XTB  × IM _FUT mutants on rhEPOs. For structures containing one xylose residue, this xylose residue is represented as β(1,2) linked to the core mannose but it may also be linked to the trimannnosyl linear branch (Lucas et al., 2020). Other N‐glycans were identified either lacking Fuc on the proximal GlcNAc or with additional O‐methylations. Blue square: GlcNAc; green circle: Man; blue circle: Glc; yellow star: xylose, red triangle: α(1,3)‐fucose, red triangle with a star: O‐methyl‐fucose, Me: methyl group on Man.

Figure 3

MS/MS spectra of [M + 3H/3]³⁺ ion at m/z 1154.144 (a), 952.750 (b) and 948.738 (c) that were assigned to rhEPO peptide HCSLNENITVPDTK (Pep) N‐linked to G2M5XF* in CC5325, M4F** in IM _XTA  × IM _XTB and M5 in IM _XTA  × IM _XTB  × IM _FUT . Main doubly charged ions were assigned to the glycopeptide fragments. Blue square: GlcNAc; green circle: Man; blue circle: Glc, yellow star: β(1,2)‐xylose and red triangle: α(1,3)‐fucose. *O‐methylation.