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.

Fig. 1.

MALDI-TOF mass spectra of N-glycans borne by soluble and membrane proteins from two different strains of C. reinhardtii, CC-503 and CC-1036. The N-glycans were released using PNGase A and labeled with the 2-AB fluorescent tag prior to MALDI-TOF-MS analysis. Mass spectra of the 2-AB-labeled N-glycans released from CC-503 soluble (A) and membrane-bound (B) proteins and from CC-1036 soluble (C) and membrane-bound (D) proteins are shown. Ions observed are sodium adducts. Oligomannoside N-glycans are annotated with their proposed carbohydrate structures according to the symbolic nomenclature adopted by the Consortium for Functional Glycomics (101). ■, N-acetylglucosamine; ●, mannose. The alphanumeric code indicates complex-type N-glycan structures (letters) and the number of methyl groups present on the structure (digits). The asterisks indicate ions that have been identified but not annotated in the spectra.

Fig. 2.

MALDI-TOF mass spectra of 2-AB-labeled N-glycans released from CC-503 soluble proteins using PNGase A. A, mass spectrum of the 2-AB-labeled N-glycans released from CC-503 soluble proteins. B, zoomed view of the outlined area in panel A. C, mass spectrum of the 2-AB-labeled N-glycans released from CC-503 soluble proteins after permethylation. D, zoomed view of the outlined area in panel C. Ions observed are sodium adducts and are annotated with their proposed carbohydrate structures according to the symbolic nomenclature from the Consortium for Functional Glycomics (101). ■, N-acetylglucosamine; ●, mannose; ☆, xylose; ▴, fucose; Me, methyl group. The alphanumeric code indicates complex N-glycan structures (letters) and the number of methyl groups present on the structure (digits). The asterisks indicate ions that have been identified but not annotated in the spectra.

Fig. 3.

Fragmentation by MALDI-TOF-TOF-MS of the Man₅Pen₂GlcNAc₂N-glycan revealing the linkage information for the pentose residues. A, MS2 spectrum of permethylated 2-AB-labeled Man₅Pen₂GlcNAc₂. B, MS2 spectrum of native 2-AB-labeled Man₅Pen₂GlcNAc₂ indicating the positions of the methyl groups. C, assignment of the product ions observed in MS2 for the indicated N-glycan structure. ■, N-acetylglucosamine; ●, mannose; ☆, xylose; Me, methyl group. The asterisks indicate fragment ions that have been identified but not annotated on the structure in panel C.

Fig. 4.

Mass spectrometric analysis of the PKHD1–1 peptide TITVANN*GTHSTATILK. A, MS1 spectrum of doubly charged, N-glycosylated peptide ions (P) differing in glycan composition and chain length. Distinctive peak clustering suggests multiple branching of a complex-type glycan and/or the co-elution of glycoforms. The presence of p + HexNAc + Hex and similar species indicates concomitant O-glycosylation or glycan rearrangement during fragmentation. Only major peaks are annotated. For detailed annotation of cluster 7 ranging from m/z 1389 to 1419, please refer to the supplemental material. Possible glycan compositions: X₁: Hex+MeHex₂+Pent; X₂: Hex₂+MeHex₂+Pent; X₃: Hex₂+MeHex₂+DeHex₂ or Hex+MeHex₃+DeHex+Pent or MeHex₄+Pent₂; X₄: Hex₂+MeHex₂+DeHex₂+Pent or Hex+MeHex₃+DeHex+Pent₂ or MeHex₄+Pent₃; X₅: Hex₃+MeHex₂+DeHex₂+Pent or Hex₂+MeHex₃+DeHex+Pent₂ or Hex+MeHex₄+Pent₃; X₆: Hex₂+MeHex₃+DeHex₂+Pent or Hex+MeHex₄+DeHex+Pent₂ or MeHex₅+Pent₃; X₇: Hex₂+MeHex₄+DeHex₂+Pent or Hex+MeHex₅+DeHex+Pent₂ or MeHex₆+Pent₃; X₈: Hex₃+MeHex₄+DeHex₂+Pent or Hex₂+MeHex₅+DeHex+Pent₂ or Hex+MeHex₆+Pent₃. B, multistage-activation (MSA)-CID spectrum of TITVANN*GTHSTATILK (precursor m/z 973.0197(MH²⁺)) modified by one HexNAc residue. C, MSA-CID spectrum of the same peptide as in B differing by one additional core HexNAc residue (precursor m/z 1074.5597 (MH²⁺)). The majority of ions consistent with those in B are not annotated. ■, HexNAc; *, loss of water/ammonia.

Fig. 5.

Mass spectrometric analysis of the HSP70G peptide IIEVPVN*ETDTATGAEGAGADADTKAEK modified by a HexNAc₂Hex₅ glycan. A, in-source CID MS1 spectrum of several doubly and triply charged peptide ions (P) differing in glycan chain length. B, CID spectrum of IIEVPVN*ETDTATGAEGAGADADTKAEK (precursor m/z: 1488.7174 (MH²⁺)) modified by one HexNAc residue. Fragmentation was carried out by MSA of precursor and neutral loss ions arising from the cleavage of the HexNAc residue (−203 Da). The resulting composite spectrum contains several b- and y-type ions showing modifications by HexNAc. ■, HexNAc; *, loss of water or ammonia.

Fig. 6.

Proposed N-glycosylation biosynthesis pathway in C. reinhardtii. The proposed pathway is based on the major N-glycan structures found according to the in silico analysis. N-glycan structures have been drawn using the symbolic nomenclature adopted by the Consortium for Functional Glycomics (101). ■, N-acetylglucosamine; ●, mannose; ☆, xylose.