title: 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-O-methylated 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.
authors.affiliation: Université de Rouen, Laboratoire Glyco-MEV, EA 4358, Institut de Recherche et d'Innovation Biomédicale (IRIB), 76821 Mont-Saint-Aignan Cedex, France;
authors.forename: Elodie
authors.initials: E
authors.lastname: Mathieu-Rivet
authors.forename: Martin
authors.initials: M
authors.lastname: Scholz
authors.forename: Carolina
authors.initials: C
authors.lastname: Arias
authors.forename: Flavien
authors.initials: F
authors.lastname: Dardelle
authors.forename: Stefan
authors.initials: S
authors.lastname: Schulze
authors.forename: François
authors.initials: F
authors.lastname: Le Mauff
authors.forename: Gavin
authors.initials: G
authors.lastname: Teo
authors.forename: Ana Karina
authors.initials: AK
authors.lastname: Hochmal
authors.forename: Amaya
authors.initials: A
authors.lastname: Blanco-Rivero
authors.forename: Corinne
authors.initials: C
authors.lastname: Loutelier-Bourhis
authors.forename: Marie-Christine
authors.initials: MC
authors.lastname: Kiefer-Meyer
authors.forename: Christian
authors.initials: C
authors.lastname: Fufezan
authors.forename: Carole
authors.initials: C
authors.lastname: Burel
authors.forename: Patrice
authors.initials: P
authors.lastname: Lerouge
authors.forename: Flor
authors.initials: F
authors.lastname: Martinez
authors.forename: Muriel
authors.initials: M
authors.lastname: Bardor
authors.forename: Michael
authors.initials: M
authors.lastname: Hippler
mesh_terms: D020418:Algal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D023281:Genomics; D054794:Glycomics; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D008745:Methylation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014994:Xylose / Q000737:chemistry
chemical_list: D020418:Algal Proteins; D006023:Glycoproteins; D011134:Polysaccharides; D014994:Xylose; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I
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