N-glycans of the microalga Chlorella vulgaris are of the oligomannosidic type but highly methylated

Abstract

Microalgae of the genus Chlorella vulgaris are candidates for the production of lipids for biofuel production. Besides that, Chlorella vulgaris is marketed as protein and vitamin rich food additive. Its potential as a novel expression system for recombinant proteins inspired us to study its asparagine-linked oligosaccharides (N-glycans) by mass spectrometry, chromatography and gas chromatography. Oligomannosidic N-glycans with up to nine mannoses were the structures found in culture collection strains as well as several commercial products. These glycans co-eluted with plant N-glycans in the highly shape selective porous graphitic carbon chromatography. Thus, Chlorella vulgaris generates oligomannosidic N-glycans of the structural type known from land plants and animals. In fact, Man5 (Man5GlcNAc2) served as substrate for GlcNAc-transferase I and a trace of an endogenous structure with terminal GlcNAc was seen. The unusual more linear Man5 structure recently found on glycoproteins of Chlamydomonas reinhardtii occurred - if at all - in traces only. Notably, a majority of the oligomannosidic glycans was multiply O-methylated with 3-O-methyl and 3,6-di-O-methyl mannoses at the non-reducing termini. This modification has so far been neither found on plant nor vertebrate N-glycans. It’s possible immunogenicity raises concerns as to the use of C. vulgaris for production of pharmaceutical glycoproteins.

Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae

“…The combination of genomic annotation and experimental evidence has revealed some details about N-glycans in some species; however, more information is needed. Analysis of five different microalgal species [94,[107][108][109][110][111] showed two different glycosylation pathways based on presence or absence of the GnT I enzyme. The green microalgae C. reinhardtii and C. vulgaris, and the red microalga Porphyridium purpureum lack GnT I, which has been validated experimentally [94,107,108,111].…”

“…In this pathway, the 5 Man and 2 GlcNAc N-linked protein is subjected to the action of xylosyltransferases (XyT) and methyltransferases (MeT), leading to unique N-linked structures containing methylated mannoses linked to one or two xyloses ( Figure 1). The structures vary slightly among these microalgae, with different possible locations of the xylose residues [94,107,108,111]. On the other hand, Baïet and colleagues [109] demonstrated that GnT I is present and active in the diatom P. tricornutum.…”

“…However, performing a KI of the GnT I enzyme in a GnT I-independent species lacking FuT, XyT, and MeT (based on computational analysis) might result in non-immunogenic "hybrid" and "complex" glycans. A recent study pursued this strategy in C. vulgaris and characterised "hybrid" N-glycans lacking fucose or xylose residues after overexpression of recombinant GnT I [111]. Alternatively, GnT I-dependent species already present "hybrid" and "complex" glycans.…”

Multiple xylosyltransferases heterogeneously xylosylate proteinN‐linked glycans inChlamydomonas reinhardtii

“…With regards to microalgae, studies have demonstrated that the Golgi maturation of N ‐glycans in microalgae like Volvox carteri , Porphyridium sp., Phaeodactylum tricornutum , Botryococcus braunii , Euglena gracilius , Chlorella vulgaris , also give rise to a large variety of oligosaccharides ranging from oligomannosidic to complex‐type N ‐glycans decorating with GlcNAc, xylose, fucose, aminoethylphosphonate moieties and/or O ‐methylated mannose residues (Balshüsemann and Jaenicke, ; Baïet et al , ; Levy‐Ontman et al , , ; O’Neill et al , ; Schulze et al , ; Mócsai et al , ). Among microalgae, Chlamydomonas reinhardtii is the best studied so far as it has been chosen as a model laboratory system for many biological processes ranging from photosynthesis over flagellar movement including acclimation to nutrient deficiency (Rochaix, ; Merchant et al , ; Marshall, ; Harris, ).…”

Bioengineering of Microalgae: Recent Advances, Perspectives, and Regulatory Challenges for Industrial Application

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