The conserved oligomeric Golgi complex is required for fucosylation of N-glycans in Caenorhabditis elegans

Abstract

The conserved oligomeric Golgi complex (COG) is a hetero-octomeric peripheral membrane protein required for retrograde vesicular transport and glycoconjugate biosynthesis within the Golgi. Mutations in subunits 1, 4, 5, 6, 7 and 8 are the basis for a rare inheritable human disease termed congenital disorders of glycosylation type-II. Defects to COG complex function result in aberrant glycosylation, protein trafficking and Golgi structure. The cellular function of the COG complex and its role in protein glycosylation are not completely understood. In this study, we report the first detailed structural analysis of N-glycans from a COG complex-deficient organism. We employed sequential ion trap mass spectrometry of permethylated N-glycans to demonstrate that the COG complex is essential for the formation of fucose-rich N-glycans, specifically antennae fucosylated structures in Caenorhabditis elegans. Our results support the supposition that disruption to the COG complex interferes with normal protein glycosylation in the medial and/or trans-Golgi.

Fig. 1.

MALDI-TOF profiles of reduced and permethylated N-glycans following hydrazinolysis from N2 Bristol and NF299 cogc-1 (k179) C. elegans strains. Fucose-rich glycans, which are absent in NF299, are marked with an asterisk and represent compositions with four fucose residues. N, N-acetylhexosamine (e.g. GlcNAc, N-acetylgalactosamine); H, hexose (e.g. galactose, mannose, glucose); F, deoxyhexose (e.g. fucose).

Fig. 2.

Relative abundance of N-linked glycans from N2 and NF299 strains by type. The y-axis represents the percent relative abundance of each composition, and the x-axis represents the molecular compositions (N, N-acetylhexosamine; H, hexose; F, fucose).

Fig. 3.

MS² spectra of N₂H₅F_(1-4) compositions from N2 and NF299. The decrease in terminal fucosylation (the absence in N₂H₅F₄) in NF299 is illustrated in the MS² spectrum. B-type ions with increased fucosylation are noticeable less in NF299 than N2. The presence in multiple structural isomers is also evident in both strains, but the extent of structural diversity cannot be resolved without further fragmentation of B- and Y-ions (i.e. MS³) (filled triangle, fucose; filled square, N-acetylhexosamine; filled circle, hexose).

Fig. 4.

MS³ spectra and fragmentation assignments of the m/z 1476 B-ion from GlcNAc₂Man₅Fuc (896²⁺) in N2 and NF299. Fragmentation profiles differ between the wild-type and COG-deficient strain illustrating changes in fucosylation. The wild-type N2 strain added a terminal fucose residue to the antenna and is evident from the m/z 1288, 1231, 707 and 433 ions. Fucose position in the NF299 strain was at the non-reducing core GlcNAc with an additional terminal hexose. This motif was apparent from the m/z 941, 871 and 646 ions as well as the absence of m/z 1288 ion, corresponding to the neutral loss of a terminal fucose.

Fig. 5.

N-Glycan structure and core fucose linkage assignment of GlcNAc₂Man₅Fuc₂ (983²⁺) from NF299. The MS³ profile of the 1272 B-type ion from the difucosylated precursor illustrates that fucosylation is at the non-reducing-end GlcNAc of the chitobiose core and not at the antenna. The MS⁴ spectrum of the 646 ion (983²⁺ → 1272 → 646) shows that the topology is Hex-Fuc-GlcNAc and the fragmentation assignment is consistent with a 1-4 linkage.

Fig. 6.

N-Glycan structure and antenna fucose linkage assignment of GlcNAc₂Man₅Fuc₂ (983²⁺) from N2. The wild-type structural isomer of the 1272 B-type ion differs from the NF299, where fucose is a terminal residue on the antenna and not at the chitobiose core. MS⁴ of the 433 ion, which is the Fuc-Hex C-type disaccharide, shows the linkage to be both 1-2 and 1-4 with fucose at the terminus.

Fig. 7.

Assignment of the Hex-Hex disaccharide on the antenna from N2 GlcNAc₂Man₅Fuc₂ (983²⁺). The topology and linkage of the Hex-Hex on the opposite arm of the Fuc-Hex disaccharide from that illustrated in Figure 6 was determined to be 1-6 and 1-4.

Fig. 8.

MS³ spectra and fragmentation assignments of the m/z 1272 B-ions from the MS² of GlcNAc₂Man₅Fuc₃ (1070²⁺) in N2 and NF299. The structures detected in both spectra from NF299 contain fucose at the core and the location of fucose in N2 was terminal and on the antenna. The NF299 strain did not add fucose to the antenna.

Fig. 9.

MS³ spectra and fragmentation assignments of the m/z 1476 B-ions from the MS² of GlcNAc₂Man₅Fuc₃ (1070²⁺) in N2 and NF299. Similar to the m/z 1272 structure in Figure 8, the structures from NF299 contain fucose at the core. The location of fucose in N2 was terminal and on the antenna.

Fig. 10.

Changes in fucosylation of the Hex₅Fuc_(1-4) N-glycan isomers. Glycan structures are annotated using the Consortium for Functional Glycomics representation. ✓ represents a detected structure, an ↓ or ↑ indicates a decrease or increase and “nd” denotes the glycans that were not detected. The corresponding m/z is also shown.