Identification of novel glycosylation events on human serum-derived factor IX

Pegg, Cassandra L.; Zacchi, Lucía F.; Recinos, Dinora Roche; Howard, Christopher B.; Schulz, Benjamin L.

doi:10.1007/s10719-020-09922-2

Cited by 11 publications

(9 citation statements)

References 51 publications

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“…Spike protein samples (10 µg of protein) were prepared in triplicate (n=27). Proteins were denatured, reduced, alkylated, quenched and methanol/acetone precipitated overnight as previously described 61 . Protein pellets were resuspended in 50 µL of 50 mM NH4HCO3 and digested for 16 h with trypsin (Sigma-Aldrich, Product code T6567) with an enzyme to protein ratio of 1:20.…”

Section: Proteomic and Glycoproteomic Sample Preparationmentioning

confidence: 99%

The role ofN-glycosylation in spike antigenicity for the SARS-CoV-2 Gamma variant

Pegg

Modhiran

Parry

et al. 2023

Preprint

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The emergence of SARS-CoV-2 variants alters the efficacy of existing immunity towards the viral spike protein, whether acquired from infection or vaccination. Mutations that impactN-glycosylation of spike may be particularly important in influencing antigenicity, but their consequences are difficult to predict. Here, we compare the glycosylation profiles and antigenicity of recombinant viral spike of ancestral Wu-1 and the Gamma strain, which has two additionalN-glycosylation sites due to amino acid substitutions in the N-terminal domain (NTD). We found that a mutation at residue 20 from threonine to asparagine within the NTD caused the loss of NTD-specific antibody binding. Glycan site-occupancy analyses revealed that the mutation resulted inN-glycosylation switching to the new sequon at N20 from the native N17 site. Site-specific glycosylation profiles demonstrated distinct glycoform differences between Wu-1, Gamma, and selected NTD variant spike proteins, but these did not affect antibody binding. Finally, we evaluated the specificity of spike proteins against convalescent COVID-19 sera and found reduced cross-reactivity against some mutants, but not Gamma spike compared to Wuhan spike. Our results illustrate the impact of viral divergence on spike glycosylation and SARS-CoV-2 antibody binding profiles.

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Section: Proteomic and Glycoproteomic Sample Preparationmentioning

confidence: 99%

The role ofN-glycosylation in spike antigenicity for the SARS-CoV-2 Gamma variant

Pegg

Modhiran

Parry

et al. 2023

Preprint

Self Cite

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“…Among them, CID is most commonly used. For example, a recent study used LC-MS/MS, based on CID, to identify enriched ADP-ribosylated proteomes, and it investigated their relevance in living cells, noting the effects of environmental changes and drugs [ 104 ]; however, sometimes CID tend to yield MS/MS spectra that are dominated by the fragment ion that has lost the original modifications, therefore, other collisional activation methods have been used either alone or in combination with CID (e.g., EthcD [ 105 ], EtciD [ 106 ], etc.) as alternative ways to study PTM structures.…”

Section: Applications Of Mass Spectrometry-based Techniques For Struc...mentioning

confidence: 99%

“…A comprehensive proteoform identification was achieved by combining all three MS/MS techniques [ 108 ]. Hybrid fragmentation techniques, such as EThcD and ETciD, have been broadly used for structural characterizations of glycoproteins, one of the most common and important PTMs [ 50 , 105 , 106 ]. Structural elucidations of glycans will be discussed further in Section 3.3 .…”

Section: Applications Of Mass Spectrometry-based Techniques For Struc...mentioning

confidence: 99%

Recent Advances in Mass Spectrometry-Based Structural Elucidation Techniques

2022

Molecules

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Mass spectrometry (MS) has become the central technique that is extensively used for the analysis of molecular structures of unknown compounds in the gas phase. It manipulates the molecules by converting them into ions using various ionization sources. With high-resolution MS, accurate molecular weights (MW) of the intact molecular ions can be measured so that they can be assigned a molecular formula with high confidence. Furthermore, the application of tandem MS has enabled detailed structural characterization by breaking the intact molecular ions and protonated or deprotonated molecules into key fragment ions. This approach is not only used for the structural elucidation of small molecules (MW < 2000 Da), but also crucial biopolymers such as proteins and polypeptides; therefore, MS has been extensively used in multiomics studies for revealing the structures and functions of important biomolecules and their interactions with each other. The high sensitivity of MS has enabled the analysis of low-level analytes in complex matrices. It is also a versatile technique that can be coupled with separation techniques, including chromatography and ion mobility, and many other analytical instruments such as NMR. In this review, we aim to focus on the technical advances of MS-based structural elucidation methods over the past five years, and provide an overview of their applications in complex mixture analysis. We hope this review can be of interest for a wide range of audiences who may not have extensive experience in MS-based techniques.

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“…The hFIX gene is composed of eight exons that encode a pre-pro-protein of 461 amino acids. The pre-pro-protein is subsequently processed into a 415residue mature hFIX (Goodeve, 2015;Pegg et al, 2020;Yoshitake et al, 1985).…”

Section: Introductionmentioning

confidence: 99%

“…The GLA domain is composed of an N-terminal ω-loop (residues 1-11), followed by three short α-helices: helix A (residues [14][15][16][17][18][19], helix B (residues [24][25][26][27][28][29][30][31][32] and helix C (residues 35-45) (Huang et al, 2004;Ohkubo & Tajkhorshid, 2008). A 35-residue activation peptide connects the EGF2 domain to the C-terminal serine protease (SP) domain.…”

Section: Introductionmentioning

confidence: 99%

The Impacts of G4S Mutation on N-glycosylation and conformation of the Human Coagulation Factor IX GLA domain: In silico and In vitro Analysis

Ghasemi

Maddah

Kalhor

et al. 2021

Preprint

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Missense mutations are the most prevalent form of mutation in hemophilia B patients. These alterations may result in the creation of novel and non-native N-glycosylation sites (Asn-X-Ser/Thr) through single amino acid substitutions. The pathogenic mechanisms of N-glycosylation mutations in hemophilia B patients have not been extensively studied yet. By survey among known missense mutations, we found only one N-glycosylation mutation in the γ-carboxyglutamic-rich (GLA) domain of the human coagulation factor IX (hFIX). This mutation that was reported in patients with mild and moderate hemophilia B, is caused by G4S amino acid substitution. To investigate the possibility of glycan attachment to the novel N-glycosylation site in G4S-mutant hFIX and the occurrence of hyperglycosylation, site-directed mutagenesis was applied to introduce the selected mutation into the coding sequence of the hFIX. The nucleotide sequences of the both native and G4S-mutant hFIX were separately cloned into the pcDNA3.1 expression plasmid and transiently expressed in HEK293T cells. Our results from gradient SDS-PAGE and western blotting analysis of the both recombinant native and mutant hFIX demonstrated no glycan attachment to the new N-glycosylation site in the G4S-mutant hFIX. Molecular dynamics (MD) simulation was also conducted to provide atomistic insights into structure and behavior of the native and G4S-mutant GLA domains in the both free and membrane-bound states. The results revealed that the mutation slightly affected the dynamic behavior of the mutant GLA domain. The conformational analysis proved that the native GLA domain had less fluctuation and more stability than the mutant GLA domain. The slight conformational changes may influence the binding capacity and interaction of the mutant GLA domain to phospholipid bilayer which is necessary for coagulation activity of the hFIX. These findings were in accordance with the nature of the G4S mutation which causes mild hemophilia B.

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Identification of novel glycosylation events on human serum-derived factor IX

Cited by 11 publications

References 51 publications

The role ofN-glycosylation in spike antigenicity for the SARS-CoV-2 Gamma variant

The role ofN-glycosylation in spike antigenicity for the SARS-CoV-2 Gamma variant

Recent Advances in Mass Spectrometry-Based Structural Elucidation Techniques

The Impacts of G4S Mutation on N-glycosylation and conformation of the Human Coagulation Factor IX GLA domain: In silico and In vitro Analysis

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