Exploring the N-glycosylation Pathway in Chlamydomonas reinhardtii Unravels Novel Complex Structures

Abstract

Chlamydomonas reinhardtii is a green unicellular eukaryotic model organism for studying relevant biological and biotechnological questions. The availability of genomic resources and the growing interest in C. reinhardtii as an emerging cell factory for the industrial production of biopharmaceuticals require an in-depth analysis of protein N-glycosylation in this organism. Accordingly, we used a comprehensive approach including genomic, glycomic, and glycoproteomic techniques to unravel the N-glycosylation pathway of C. reinhardtii. Using mass-spectrometry-based approaches, we found that both endogenous soluble and membrane-bound proteins carry predominantly oligomannosides ranging from Man-2 to Man-5. In addition, minor complex N-linked glycans were identified as being composed of partially 6-Omethylated Man-3 to Man-5 carrying one or two xylose residues. These findings were supported by results from a glycoproteomic approach that led to the identification of 86 glycoproteins. Here, a combination of in-source collision-induced dissodiation (CID) for glycan fragmentation followed by mass tag-triggered CID for peptide sequencing and PNGase F treatment of glycopeptides in the presence of 18 O-labeled water in conjunction with CID mass spectrometric analyses were employed. In conclusion, our data support the notion that the biosynthesis and maturation of N-linked glycans in the endoplasmic reticulum and Golgi apparatus occur via a GnT I-independent pathway yielding novel complex N-linked glycans that maturate differently from their counterparts in land plants. Molecular & Cellular

Exploring the Glycans of <em>Euglena gracilis</em>

“…Putative aminoethylphosphonate carrying N-glycans could only just be detected in the flagella samples, but at levels below the level of quantitation and these signals could also not be verified by tandem MS data. We did not find any indication for complex type N-glycans as they have been described for other algae, such as Chlamydomonas reinhardtii, where N-glycans were identified carrying both core and antenna xylose residues and some methylation [28]. Beta-elimination after PNGase F treatment did not result in any signals consistent with any O-or N-glycan-specific signatures.…”

N-Glycoproteomic Characterization of Mannosidase and Xylosyltransferase Mutant Strains of Chlamydomonasreinhardtii

“…These mutants were genetically analyzed, the number of N-glycosites harboring specific N-glycan compositions can be 149 determined for each strain. However, it should be noted that the abundance of N-glycans 150 released by peptide-N-glycosidase A/F, as described previously (Mathieu-Rivet et al 2013, 151 Vanier et al 2017, is not correlated to the number of different peptides bearing these glycans.…”

Identification of Haloferax volcanii Pilin N-Glycans with Diverse Roles in Pilus Biosynthesis, Adhesion, and Microcolony Formation

“…The mass spectrometer was operated in positive ion mode using two different methods. In the first method, in-source collision-induced dissociation (IS-CID) was performed similarly as described by Mathieu-Rivet et al (39) by applying an IS voltage of 80 V. Some samples were reanalyzed using 60, 70, and 90 V. MS1 scans were obtained from 400 to 2,000 m/z at a resolution of 70,000 full width at half-maximum, an AGC target of 10 6 , and a maximum ion injection time of 100 ms. IS-CID of N-glycopeptides leads to the fragmentation of glycosidic bonds, which results in a series of neutral losses on MS1 level. The "Mass Tags" option (accuracy of 5 ppm) was enabled to select ion pairs differing by the mass of a monosaccharide (hexose, 162.0528 Da; hexuronic acid, 176.0321 Da; charges 1-4) for further fragmentation by higher energy collisional dissociation (HCD).…”