Enhanced Aromatic Sequons Increase Oligosaccharyltransferase Glycosylation Efficiency and Glycan Homogeneity

Murray, Amber N.; Chen, Wentao; Antonopoulos, Aristotelis; Hanson, Sarah R.; Wiseman, R. Luke; Dell, Anne; Haslam, Stuart M.; Powers, David L.; Powers, Evan T.

doi:10.1016/j.chembiol.2015.06.017

Cited by 42 publications

(45 citation statements)

References 61 publications

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“…Indeed, molecular dynamics analyses on our model protein had suggested a direct interaction of the glycan with surrounding amino acids (16) and recent studies characterized the CH-p interaction between the CH groups of the first GlcNAc of the glycan core and aromatic amino acid side chains of the glycoproteins (46). The presence of phenylalanine at the 22 position of the N-glycosylation sequon localized in a reverse turn increases the stability of the protein, but also of glycan homogeneity (47)(48)(49). We thus hypothesized that, in PDI, the glycan structures observed on site 4 are the result of the change in the glycan torsion caused by the interaction of the core GlcNAc with the tyrosine, itself favoring the interaction of the whole glycan with the protein surface and the domain a of PDI.…”

Section: Discussionmentioning

confidence: 86%

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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Section: Discussionmentioning

confidence: 86%

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

Losfeld¹,

Scibona²,

Lin³

et al. 2017

FASEB j.

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show abstract

“…Residues outside of the traditional N-X-S/T (where X ≠ P) sequon can profoundly affect both the rate of glycosylation by oligosaccharyltransferase and the predominant glycoform observed (119). Further, mechanisms that govern intracellular proteostasis have been observed to impact the N -glycosylation pattern of secreted glycoproteins (120).…”

Section: Resultsmentioning

confidence: 99%

Integrated Omics and Computational Glycobiology Reveal Structural Basis for Influenza A Virus Glycan Microheterogeneity and Host Interactions

Khatri

Klein

White

et al. 2016

Molecular & Cellular Proteomics

114

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Despite sustained biomedical research effort, influenza A virus remains an imminent threat to the world population and a major healthcare burden. The challenge in developing vaccines against influenza is the ability of the virus to mutate rapidly in response to selective immune pressure. Hemagglutinin is the predominant surface glycoprotein and the primary determinant of antigenicity, virulence and zoonotic potential. Mutations leading to changes in the number of HA glycosylation sites are often reported. Such genetic sequencing studies predict at best the disruption or creation of sequons for N-linked glycosylation; they do not reflect actual phenotypic changes in HA structure. Therefore, combined analysis of glycan micro and macro-heterogeneity and bioassays will better define the relationships among glycosylation, viral bioactivity and evolution. We present a study that integrates proteomics, glycomics and glycoproteomics of HA before and after adaptation to innate immune system pressure. We combined this information with glycan array and immune lectin binding data to correlate the phenotypic changes with biological activity. Underprocessed glycoforms predominated at the glycosylation sites found to be involved in viral evolution in response to selection pressures and interactions with innate immune-lectins. To understand the structural basis for site-specific glycan microheterogeneity at these sites, we performed structural modeling and molecular dynamics simulations. We observed that the presence of immature, high-mannose type glycans at a particular site correlated with reduced accessibility to glycan remodeling enzymes. Further, the high mannose glycans at sites implicated in immune lectin recognition were predicted to be capable of forming trimeric interactions with the immune-lectin surfactant protein-D.

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“…Thus, the high-density glycan clustering within the mannose patch presumably impaired steric access to cleavage by the GH47 α-mannosidases. Finally, the impact of proteinglycan interactions on glycan maturation was examined as a part of a larger study on residues that flank glycosylation site acceptor sequons (48). Aromatic residues at the n-2 positions relative to glycosylation sites on the polypeptide backbone resulted in increased glycan occupancy but also reduced the efficiency of glycan trimming.…”

Section: Implications For Processing Of Oligomannose Glycans In the Smentioning

confidence: 99%

Substrate recognition and catalysis by GH47 α-mannosidases involved in Asn-linked glycan maturation in the mammalian secretory pathway

Xiang

Karaveg

Moremen

2016

Proc. Natl. Acad. Sci. U.S.A.

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Maturation of Asn-linked oligosaccharides in the eukaryotic secretory pathway requires the trimming of nascent glycan chains to remove all glucose and several mannose residues before extension into complex-type structures on the cell surface and secreted glycoproteins. Multiple glycoside hydrolase family 47 (GH47) α-mannosidases, including endoplasmic reticulum (ER) α-mannosidase I (ERManI) and Golgi α-mannosidase IA (GMIA), are responsible for cleavage of terminal α1,2-linked mannose residues to produce uniquely trimmed oligomannose isomers that are necessary for ER glycoprotein quality control and glycan maturation. ERManI and GMIA have similar catalytic domain structures, but each enzyme cleaves distinct residues from tribranched oligomannose glycan substrates. The structural basis for branch-specific cleavage by ERManI and GMIA was explored by replacing an essential enzyme-bound Ca 2+ ion with a lanthanum (La 3+) ion. This ion swap led to enzyme inactivation while retaining high-affinity substrate interactions. Cocrystallization of La 3+ -bound enzymes with Man 9 GlcNAc 2 substrate analogs revealed enzyme-substrate complexes with distinct modes of glycan branch insertion into the respective enzyme active-site clefts. Both enzymes had glycan interactions that extended across the entire glycan structure, but each enzyme engaged a different glycan branch and used different sets of glycan interactions. Additional mutagenesis and time-course studies of glycan cleavage probed the structural basis of enzyme specificity. The results provide insights into the enzyme catalytic mechanisms and reveal structural snapshots of the sequential glycan cleavage events. The data also indicate that full steric access to glycan substrates determines the efficiency of mannose-trimming reactions that control the conversion to complex-type structures in mammalian cells.Asn-linked glycan processing | α-mannosidase | glycosidase mechanism | glycoside hydrolase | bioinorganic chemistry

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Enhanced Aromatic Sequons Increase Oligosaccharyltransferase Glycosylation Efficiency and Glycan Homogeneity

Cited by 42 publications

References 61 publications

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

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

Integrated Omics and Computational Glycobiology Reveal Structural Basis for Influenza A Virus Glycan Microheterogeneity and Host Interactions

Substrate recognition and catalysis by GH47 α-mannosidases involved in Asn-linked glycan maturation in the mammalian secretory pathway

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