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

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.

and with 6-O-methyl mannose 19 . A glycopeptide based study on Botryococcus brauni -a green algae belonging to the class of Trebouxiophyceae just as Chlorella -discovered N-glycopeptides with up to three GlcNAc residues indicating action of GlcNAc-transferase I (GnTI) 20 . Furthermore, methyl-hexose and pentose were found by CID-MS/MS of glycopeptides. This small number of papers on glycoprotein structures of microalgae (including just a few green algae) can be rated as a sign of ignorance given their ecological significance and their growing role as biofactories. In particular, C. reinhardtii and Chlorella species are regarded as promising production hosts for proteins and glycoproteins 14,21 and the diatom microalgae Phaeodactylum tricornutum has recently been demonstrated to produce a fully functional anti-hepatitis antibody with high-mannose glycans 22 . However, in the green alga C. reinhardtii the Man 5 GlcNAc 2 N-glycan assumed to represent a substrate for recombinant GnTI turned out as having an unusual, more linear structure inaccessible for GnTI 23 .
In this work we investigated the N-glycosylation of Chlorella vulgaris strains from culture collections as well as of commercial products. MALDI-TOF MS, chromatography on graphitic carbon and amide silica, gas chromatography of constituent sugars and action of GlcNAc-transferase I (GnTI) were applied to characterize the N-glycans of C. vulgaris.

MALDI-TOF MS profiles of Chlorella vulgaris N-glycans. N-glycans from the live strain SAG 211-11b
were isolated from the complete bulk material by a succession of pepsin digestion, cation exchange, PNGase A digestion and another cation exchange step. MALDI-TOF MS of the resulting oligosaccharides revealed a rather complex pattern with five prominent groups of peaks. The smallest masses within each group had compositions from Man 5 GlcNA 2 to Man 9 GlcNAc 2 (Man5 to Man9) (Fig. 1A). These well-known compounds were followed by peaks spaced by 14.018 Da indicating series of methyl groups (Fig. 1B). Essentially the same profiles were obtained when samples were repeatedly (>four times) analyzed as in the case of SAG 211-11b and GreenGem.
The same compounds albeit in different proportions were found in the C. vulgaris strains UTEX 395 (Fig. 1C) and SAG 211-8 l (Fig. 1D). Very similar MALDI-TOF MS patterns were found for several commercial Chlorella products ( Fig. 1E-H). Remarkably, some of these strains were designated as C. pyrenoidosa, although this taxonomic name has been dismissed some time ago and the respective strains have been assigned as other Chlorella species and lines or even as other genera 8 . Many commercial products nevertheless bear this species name and it still occurs in the scientific literature. It shall not be concealed that other "C. pyrenoidosa" products exhibited different N-glycan profiles that apparently contained pentoses, O-methyl groups and possibly deoxyhexoses but these shall be subject of a future study.
Location of methyl groups. To characterize the nature of glycan methylation, N-glycans from SAG211-11b and from GreenGem tablets were hydrolyzed. The monosaccharides were reduced, peracetylated and subjected to GC-MS together with suitable partially methylated standards 24 . This revealed the presence of 3-O-methyl mannose (24% compared to the mannose peak) and smaller amounts of 3,6-di-O-methyl mannose (4%) (Fig. 2) in C. vulgaris 211-11b. Similar values were found for the GreenGem sample, whereby the only semiquantitative nature of this figures shall be conceded as reference compounds for quantitative analysis of the methyl hexoses were not available. MALDI-TOF LIFT MS/MS spectra of underivatized glycans showed little useful detail about the location of the methyl groups. In order to obtain ESI-MS/MS spectra without the risk of hybrid spectra with precursors of differing degree of methylation (7 Th mass difference at charge state 2), we attempted HILIC fractionation, which led to a preparation of suitably isolated trimethylated Man9. The ESI-MS/MS spectrum of Me 3 Man 9 GlcNAc 2 was in perfect agreement with the assumption that all methyl groups were attached to terminal mannose residues (Fig. 3). Y-ions or GlcNAc-truncated y-ions showed +14 Da increments only at a size range that possibly contained terminal Man residues. The pattern is particularly consistent with the assumption of a major fraction with all terminal residues being mono-methylated and a minor part with a non-, mono-and dimethylated mannose each. In fact, the presence of at least three isomers of Me 3 Man 9 GlcNAc 2 is indicated by PGC chromatography (Fig. 4A). As a detail out of many, the large dimethylated Man9 peak (Fig. 4A) generates B-ions for a mono-methyl-mannose m/z = 339.1), whereas the later eluting, smaller one that for a di-methyl-mannose m/z = 353.1).
Then again, the smaller glycans down to Man5 also were decorated with up to 4 methyl-groups. I.e. residues other than those modified in Man9 must have born the methyl groups. This insight has an interesting repercussion as to the biosynthesis of these glycans: The methyl groups are transferred to the mature N-glycans.   25 ]. Further support for this conclusion came from the accessilibity of the Man5Gn product to core α1,6-fucosyl transferase ( Supplementary Fig. 2). The earlier eluting peak Man5 that was not converted by GnTI may have the unusual structure with two α1,2-linked mannoses on the 3-arm but no branching mannoses on the 6-arm recently described as the only Man5 glycan in Chlamydomonas reinhardtii 23 . This MM 2 isomer would elute rather early on PGC 25 . To verify its nature, we prepared glycans from an alg3 and also mannosidase (mns123) mutant plant that contained by necessity the MM 2 isomer 26 . Surprisingly, this isomer did not coelute with the strange Chlorella Man5 (Fig. 4).
A last point worth mentioning is that the control sample already contained a small amount of a compound with just the same mass and elution position as Man5Gn (Fig. 4).
Influence of methylation on chromatographic behavior. On the PGC columns, methylation increased retention in accordance with the view of graphitic carbon operating -at least in part -by a reversed-phase mechanism. Isobaric methyl isomers were remarkably well separated (Fig. 4). The effect on the HILIC column was opposite and more uniform. Methylation resulted in a strong forward shift roughly equivalent to one mannose residue (Supplementary Fig. 3).

Discussion
Oligomannosidic glycans with zero to five methyl groups on terminal mannose residue constitute the N-glycomes of Chlorella vulgaris type strains 211-11b, SAG 211-8 l and UTEX 235. A number of commercially available algal preparations also exhibited this pattern irrespective of the species name declared by the suppliers. It must be emphasized that other products as well as other species exhibited different N-glycan patterns ( Supplementary  Fig. 4). These differences may harbor a valuable means for strain or species differentiation but plumbing this option would by far exceed the scope of the present study.
On a Man9 N-glycan of SAG 211-11b, MS/MS showed the methyl groups to reside on the terminal mannose residues (Fig. 4). As methylation affected glycans of all size, we propose that it takes place after the mannosidase trimming as a finishing touch of glycan maturation. The idea of incorporation of already methylated mannose residues during precursor synthesis would require that both cytosolic GDP-mannose and ER luminal dolichol-P-mannose would exist in part in a methylated version and that the respective transferases would accept these donors. Notably, C. vulgaris features 3-O-methyl rather than 6-O-methyl mannose as found for C. reinhardtii 19 . A possible purpose of methylation might be to confer resistance to unwelcome mannosidase trimming by competing organisms. In fact, it did confer resistance to jack bean α-mannosidase ( Supplementary Fig. 5).
A recent work on Chlamydomonas reinhardtii revealed the surprising fact that the N-glycan Man5 (Man 5 GlcNAc 2 ) did not have the common and expected structure (M 6 M 3 )M (see reference 25 for explanation) but rather the isomer MM 2-2 with an untruncated 3-arm 23 . This isomer is formed in the absence of ALG3 = Dol-P-Ma n:Man 5 GlcNAc 2 -PP-Dol α1,3-mannosyltransferase. As a consequence, heterologous expression of GnTI did not  glycans this strongly supports the idea of conserved pathway up to Man5 and very probably even Man5Gn. Highly homologous genes have recently been identified even in a red microalga 27 and a diatom species 15 .
The current work certainly shows that microalgae, i.e. Chlorellas, can harbor the complete Glc 3 Man 9 pathway as Man5 to Man9 structures indistinguishable from plant N-glycans were found. No ALG3 bottle neck exists in Chlorella as is the case in C. reinhardtii 23 , but C. vulgaris would have been but a slightly better choice for heterologous expression of GnTI, as only about 1% of the total N-glycome existed as the GnTI substrate (M 6 M 3 )M. The occurrence of traces of Man5Gn, i.e. the initial product of GnTI in C. vulgaris is backed up by glycoproteomic evidence for the occurrence of terminal HexNAc in an other Trebouxiophyceae species 20 .
Utilization of C. vulgaris as a production host for glycoproteins would at first require to identify the O-methyltransferase acting on terminal mannose residues. While the immunogenicity of methylated N-glycans has not yet been demonstrated, it is arguable that only methyltransferase knock-out microalgae could be considered for the expression of therapeutic glycoproteins. Such knock-out lines could also answer the scientific question as to the biological purpose of N-glycan methylation.

Sources of biological samples. Culture collection strains and commercial tablets as collected in 2016
to 2017 are listed in (Table 1) HILIC on a TSK Amide80 column (4 × 250 mm, 5 µm; Tosoh Bioscience GmbH, Griesheim, Germany) was performed on underivatized glycans for preparative purposes 28 . Fractions of 0.5 mL were analyzed by MALDI-TOF MS. This led inter alia to a fraction of Me 3 Man 9 GlcNAc 2 that could be used for ESI-MS/MS without any danger of interference by adjacent peaks with more or less methyl groups ( Supplementary Fig. 3).
Mass spectrometric methods. MALDI-TOF MS of glycan pools was performed with dihydroxybenzoic acid as the matrix on a Bruker Autoflex MALDI-TOF instrument in the positive ion reflectron mode. Usually, unreduced samples were analyzed, but in some cases reduction with 1% sodium borohydride was done to readily discriminate glycan from non-glycan peaks.
Reduced glycans were analyzed by LC-ESI-MS with a porous graphitic carbon (PGC) column (0.32 µm x 150 mm) operated by an Ultimate RSLC (Thermo Scientific, Vienna) connected to a Maxis 4 G Q-TOF MS (Bruker, Bremen, Germany) 25 . N-glycans from white kidney beans were used as reference 25 . MS/MS was performed in positive mode.
The monosaccharide constituents were analyzed after hydrolysis of glycan pools of fractions with 4 M trifluoroacetic acid at 100° for 4 h. Sugars were reduced with NaBD 4 , peracetylated and analyzed on an Agilent J&W HP-5ms GC Column (30 m x 0.25 mm, 0.25 µm) installed in a GC-MS system (GC 7820 A & MSD 5975, Agilent, Waldbronn, Germany). Partially methylated alditol acetates were available from a previous study 29 and their relative retention times were additionally confirmed by literature data 30 . GlcNAc-transferase reaction. Man5 substrates were prepared from the N-glycan pools of either Green Gem tablets or kidney beans by size separation on an amide column as described 31 . Rabbit GlcNAc-transferase I (GnTI) lacking the N-terminal 105 amino acids 32 was expressed with an N-terminal His 6 -tag using a pVT-Bac vector and the baculovirus insect cell system 33 . The enzyme was purified by metal chelate chromatography 34 . The purified enzyme was added to 0.45 nmol of Man5 from GreenGem tablets or kidney beans in 50 mM MES buffer (pH 7.0) containing 500 nmol MnCl 2 and 10 nmol UDP-GlcNAc (Kyowa Hakko, Tokyo) and incubated overnight at 37 °C. The glycans in the mixtures were purified using carbon solid phase cartridges (Multi-Sep Hypercarb 10 mg, Thermo Scientific, Vienna) as described 35 . The eluate was dried, taken up in pure water and analyzed by PGC-LC-ESI-MS as described above.

Data Availability
The datasets (MS files) generated during the current study are available from the corresponding author on request.