N-glycoprotein macroheterogeneity: biological implications and proteomic characterization

Zacchi, Lucía F.; Schulz, Benjamin L.

doi:10.1007/s10719-015-9641-3

Cited by 79 publications

(80 citation statements)

References 182 publications

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“…The analysis of site-specific microheterogeneity on a protein with multiple N-glycosylation sites in cell or tissue-derived extracts requires more elaborate analytical methods that have become available only recently (16,17,(34)(35)(36). Few studies have shown the relation between protein primary sequence and the observed glycan profile.…”

mentioning

confidence: 99%

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Losfeld¹,

Scibona²,

Lin³

et al. 2017

FASEB j.

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To study how the interaction between N-linked glycans and the surrounding amino acids influences oligosaccharide processing, we used protein disulfide isomerase (PDI), a glycoprotein bearing 5 N-glycosylation sites, as a model system and expressed it transiently in a Chinese hamster ovary (CHO)-S cell line. PDI was produced as both secreted Sec-PDI and endoplasmic reticulum-retained glycoprotein (ER)-PDI, to study glycan processing by ER and Golgi resident enzymes. Quantitative site-specific glycosylation profiles were obtained, and flux analysis enabled modeling site-specific glycan processing. By altering the primary sequence of PDI, we changed the glycan/ protein interaction and thus the site-specific glycoprofile because of the improved enzymatic fluxes at enzymatic bottlenecks. Our results highlight the importance of direct interactions between N-glycans and surface-exposed amino acids of glycoproteins on processing in the ER and the Golgi and the possibility of changing a site-specific N-glycan profile by modulating such interactions and thus the associated enzymatic fluxes. Altering the primary protein sequence can therefore be used to glycoengineer recombinant proteins.-Losfeld, M.-E., Scibona, E., Lin, C.-W., Villiger, T. K., Gauss, R., Morbidelli, M., Aebi, M. Influence of protein/glycan interaction on site-specific glycan heterogeneity. FASEB J. 31, 4623-4635 (2017). www.fasebj.org KEY WORDS: glycoengineering • glycobiology • glycoprotein maturation • Golgi processing • enzymatic flux N-glycosylation is a major posttranslational modification of proteins that modulates folding, maturation, trafficking, and secretion, as well as specific protein functions (1-3). Contrary to many other posttranslational modifications, N-glycans are composed of many monosaccharide units, creating a large diversity of glycan structures (4, 5). In eukaryotic cells, the N-glycosylation occurs in the endoplasmic reticulum (ER) after a conserved pathway. The oligosaccharide GlcNAc 2 Man 9 Glc 3 is assembled on the lipid dolicholpyrophosphate at the membrane of the ER and then is covalently linked to the side-chain amide of the asparagine residue in the sequon N-X-S/T by oligosaccharyltransferase (OST) (1, 6, 7). Multiple sites on one glycoprotein can be modified by the OST complex and with the increasing complexity of multicellular organism, an increasing number of N-glycosylation sites in the glycoproteome are observed. It is estimated that up to 6000 different sites are modified by OST in mammalian cells (8). In the second phase of the N-glycosylation pathway, the protein-linked glycan is trimmed by glucosidases and mannosidases (1, 9) and is subsequently modified by different glycosyltransferases spatially distributed along the secretory pathway. N-glycan processing enzymes have defined substrate specificities and locations in the secretory pathway (9, 10), and their expression level can be regulated upon internal and external signals.Glycan maturation in the secretory pathway is not a template-driven process. The...

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mentioning

confidence: 99%

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Losfeld¹,

Scibona²,

Lin³

et al. 2017

FASEB j.

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“…The use of such nonconsensus sequons seems to be more frequent in rodents, where even glutamine-linked glycosylation has been reported (14). Low efficiency N-glycosylation in these noncanonical sequons is consistent with a role of the canonical sequon in recognition and high-affinity binding to OST to promote glycan transfer (15). Mammalian N-glycans share a common trimannosylchitobiose core comprised of three mannose (Man) and two N-acetylglucosamine (GlcNAc) residues, extended with a variety of monosaccharides including Man, GlcNAc, galactose (Gal), fucose (Fuc), N-acetylgalactosamine (GalNAc) and sialic acids such as N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc).…”

mentioning

confidence: 54%

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

Thaysen‐Andersen

Packer

Schulz

2016

Molecular & Cellular Proteomics

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“…Glycoforms of a single protein can differ in the glycan structures attached at a specific site (defined as microheterogeneity) or in the proportion of sites that are occupied (defined as macroheterogeneity) (13). The complex nature of protein glycosylation and subsequent structural diversity of glycoproteins present significant challenges when defining the glycosylation profile of glycoproteins.…”

Section: Protein Glycosylationmentioning

confidence: 99%

“…This may be due to the small size of the theoretical peptide 446 KNISIQD 452 . There are conflicting reports on occupancy at N447 using site-directed mutagenesis combined with gel-electrophoresis (13,14). However, the crystal structure of NDV V4-VAR F revealed up to three residues (GlcNAc 2 Man 1 ) at N447 in trimers of F, indicting this site can be occupied.…”

Section: Supplemental Collisional Activation Of Non-dissociated Peptimentioning

confidence: 99%

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Structural characterisation of glycosylation of paramyxovirus attachment and fusion proteins by mass spectrometry

Pegg¹

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The family Paramyxoviridae (paramyxovirus) contains several significant human and animal pathogens.Represented within this family are human respiratory syncytial virus (hRSV) and Newcastle disease virus (NDV). The former contributes significantly to severe respiratory tract disease in infants, children and immunocompromised individuals. At present, highly efficacious therapeutics or safe and effective vaccines are not available for hRSV. NDV is the causative agent of Newcastle disease, afflicting a wide range of avian species. The desire to study NDV is due not only to the significant economic impact it has on the poultry industry worldwide, but also its potential use as an oncolytic agent and vaccine vector in humans and animals. Additionally, findings on NDV may be translated to closely related viruses that cause disease in humans, such as parainfluenza viruses.As outlined in Chapter 1 of this thesis, the infectious processes of all members of this family are driven by two major membrane glycoproteins whose ectodomains project from the viral envelope: the fusion (F) and attachment glycoproteins. The F glycoprotein is responsible for viral entry by means of fusion with host cell membranes while the variable attachment glycoproteins, haemagglutinin, haemagglutininneuraminidase (HN) and major surface glycoprotein (G), are involved in viral attachment to host cells.Previous studies have shown that altering the glycosylation profile of these proteins can modulate the ability of the virus to infect host cells and stimulate the host immune system. As yet, site-specific glycan heterogeneity of hRSV and NDV surface glycoproteins has not been defined at a chemical level. As described in Chapter 2, reverse-phase liquid chromatography tandem mass spectrometry (MS) strategies were implemented to characterise intact glycopeptides from the attachment and F glycoproteins of hRSV and NDV. Site-occupancy and monosaccharide compositions at a given site were determined using collision-induced dissociation, higher-energy collision dissociation, electron-transfer dissociation and electron-transfer dissociation combined with collision dissociation. As described in Chapter 3, a spectral processing program was developed called OxoExtract to aid identification of intact glycopeptides that were fragmented with higher-energy collision dissociation.The F and HN proteins of NDV were derived from virions propagated in embryonic eggs. Analyses of HN in Chapter 4 revealed high mannose N-linked glycans and complex or hybrid N-linked glycans that were variably fucosylated, sialylated and sulfated or phosphorylated. In total 63, 58, and 37 glycans were identified at sites N341, N433 and N481, respectively. In addition, a previously undocumented Olinked glycosylation site was identified in the stalk domain of the protein. Observed glycans from NDV F described in Chapter 5 were mainly high mannose, containing variations of five to nine mannose ii residues across four sites, N85, N191, N366 and N471. There was also evidence of fucosylated c...

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N-glycoprotein macroheterogeneity: biological implications and proteomic characterization

Cited by 79 publications

References 182 publications

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

Structural characterisation of glycosylation of paramyxovirus attachment and fusion proteins by mass spectrometry

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