Coagulation factor IX analysis in bioreactor cell culture supernatant predicts quality of the purified product

Zacchi, Lucía F.; Recinos, Dinora Roche; Pegg, Cassandra L.; Phung, Toan K.; Napoli, Mark; Aitken, Campbell; Sandford, Vanessa; Mahler, Stephen M.; Lee, Yih Yean; Schulz, Benjamin L.; Howard, Christopher B.

doi:10.1038/s42003-021-01903-x

Cited by 11 publications

(5 citation statements)

References 137 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For confirmation of sialic acid removal, each of the samples (+\− Neuraminidase A) were analyzed ( n = 1, total samples 18). LC-ESI-MS/MS was performed using a Prominence nanoLC system (Shimadzu) coupled to a TripleTof 5,600 instrument (SCIEX) using a Nanospray III interface using a data-dependent acquisition method as described ( Zacchi et al. 2021 ) with minor alterations.…”

Section: Methodsmentioning

confidence: 99%

The role of N-glycosylation in spike antigenicity for the SARS-CoV-2 gamma variant

Pegg,

Modhiran,

Parry

et al. 2023

Glycobiology

View full text Add to dashboard Cite

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 impact N-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 additional N-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 COVA2-17 binding. Glycan site-occupancy analyses revealed that the mutation resulted in N-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.

show abstract

Section: Methodsmentioning

confidence: 99%

The role of N-glycosylation in spike antigenicity for the SARS-CoV-2 gamma variant

Pegg,

Modhiran,

Parry

et al. 2023

Glycobiology

View full text Add to dashboard Cite

show abstract

“…The top 25 mL of supernatant were transferred to 3 kDa Amicon columns (Amicon Ultra-15 Centrifugal Filter Unit, UFC 900324) and concentrated following the manufacturer's recommendations. 100 mL of the concentrated sample were precipitated overnight in 4 volumes of methanol:acetone in a LoBind Eppendorf tube, as previously described (Zacchi et al, 2020a(Zacchi et al, , 2020b. The supernatant was eliminated after high speed centrifugation, and air-dried proteins were resuspended in 50 mM ammonium bicarbonate supplemented with 10 mM DTT and 1 mg of trypsin.…”

Section: Azocasein Assaymentioning

confidence: 99%

The histone chaperone HIR maintains chromatin states to control nitrogen assimilation and fungal virulence

et al. 2021

Self Cite

View full text Add to dashboard Cite

The histone chaperone HIR maintains chromatin states to control nitrogen assimilation and fungal virulence Graphical abstract Highlights d The HIR histone chaperone controls nutritional adaptation and fungal virulence d HIR1 modulates chromatin accessibility and transcription of metabolic genes d Innate immune cells show impaired recognition of fungal pathogens lacking HIR1 d Genetic ablation of HIR drives hypervirulence in systemic fungal infections

show abstract

“…One important secreted protein is coagulation factor IX (FIX), encoded by the F9 gene. Normal FIX secretion and post-translational modification, including γ-carboxylation, are necessary to stop bleeding and achieve hemostasis [11][12][13][14][15] . Genetic variation leading to loss of F9 function causes the bleeding disorder hemophilia B [16][17][18][19][20][21] .…”

Section: Introductionmentioning

confidence: 99%

Multiplex, multimodal mapping of variant effects in secreted proteins

Popp,

Powell,

Wheelock

et al. 2024

Preprint

View full text Add to dashboard Cite

Despite widespread advances in DNA sequencing, the functional consequences of most genetic variants remain poorly understood. Multiplexed Assays of Variant Effect (MAVEs) can measure the function of variants at scale, and are beginning to address this problem. However, MAVEs cannot readily be applied to the ~10% of human genes encoding secreted proteins. We developed a flexible, scalable human cell surface display method, Multiplexed Surface Tethering of Extracellular Proteins (MultiSTEP), to measure secreted protein variant effects. We used MultiSTEP to study the consequences of missense variation in coagulation factor IX (FIX), a serine protease where genetic variation can cause hemophilia B. We combined MultiSTEP with a panel of antibodies to detect FIX secretion and post-translational modification, measuring a total of 45,024 effects for 9,007 variants. 49.6% of possible F9 missense variants impacted secretion, post-translational modification, or both. We also identified functional constraints on secretion within the signal peptide and for nearly all variants that caused gain or loss of cysteine. Secretion scores correlated strongly with FIX levels in hemophilia B and revealed that loss of secretion variants are particularly likely to cause severe disease. Integration of the secretion and post-translational modification scores enabled reclassification of ~63% F9 variants of uncertain significance in the My Life, Our Future hemophilia genotyping project. Lastly, we showed that MultiSTEP can be applied to a wide variety of secreted proteins. Thus, MultiSTEP is a multiplexed, multimodal, and generalizable method for systematically assessing variant effects in secreted proteins at scale.

show abstract

Coagulation factor IX analysis in bioreactor cell culture supernatant predicts quality of the purified product

Cited by 11 publications

References 137 publications

The role of N-glycosylation in spike antigenicity for the SARS-CoV-2 gamma variant

The role of N-glycosylation in spike antigenicity for the SARS-CoV-2 gamma variant

The histone chaperone HIR maintains chromatin states to control nitrogen assimilation and fungal virulence

Multiplex, multimodal mapping of variant effects in secreted proteins

Contact Info

Product

Resources

About