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

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Abstract

Human Factor IX is a highly post-translationally modified protein that is an important clotting factor in the blood coagulation cascade. Functional deficiencies in Factor IX result in the bleeding disorder haemophilia B, which is treated with plasma-derived or recombinant Factor IX concentrates. Here, we investigated the post-translational modifications of human serum-derived Factor IX and report previously undescribed O-linked monosaccharide compositions at serine 141 and a novel site of glycosylation. At serine 141 we observed two monosaccharide compositions, with HexNAc1Hex1NeuAc2 dominant and a low level of HexNAc1Hex1NeuAc1. This O-linked site lies N-terminal to the first cleavage site for the activation peptide, an important region of the protein that is removed to activate Factor IX. The novel site is an N-linked site in the serine protease domain with low occupancy in a non-canonical consensus motif at asparagine 258, observed with a HexNAc4Hex5NeuAc2 monosaccharide composition attached. This is the first reported instance of a site of modification in the serine protease domain. The description of these glycosylation events provides a basis for future functional studies and contributes to structural characterisation of native Factor IX for the production of effective therapeutic biosimilars and biobetters.

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Abbreviations

AP:

activation peptide

CID:

collision-induced dissociation

EGF:

epidermal growth factor-like

ER:

endoplasmic reticulum

ETD:

electron-transfer dissociation

Fuc:

fucose

Gal:

galactose

Gla:

γ-glutamic acid

Glc:

glucose

GlcNAc:

N-acetylglucosamine

HCD:

higher-energy C-trap dissociation or beam-type collision-induced dissociation

Hex:

hexose

HexNAc:

N-acetylhexosamine

Hya:

β-hydroxyaspartate

NeuAc:

N-acetylneuraminic acid

Pent:

pentose

Phos:

phosphorylation

PNGase F:

Peptide-N-Glycosidase F

PP:

propeptide

PTMs:

post-translantional modifications

PSMs:

peptide-to-spectrum matches

SP:

signal peptide

Sulf:

sulfation

Xyl:

xylose

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Acknowledgments

We thank Dr. Amanda Nouwens and Peter Josh at The University of Queensland, School of Chemistry and Molecular Biosciences Mass Spectrometry Facility for their assistance and expertise.

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Authors and Affiliations

  1. School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, 4072, Australia

    Cassandra L. Pegg & Benjamin L. Schulz

  2. Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD, 4072, Australia

    Lucia F. Zacchi, Dinora Roche Recinos, Christopher B. Howard & Benjamin L. Schulz

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  1. Cassandra L. Pegg
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Contributions

BLS, CLP, CBH, and LFZ conceived and coordinated this study. All authors designed experiments. CLP performed the experiments and analysed the data. BLS, CLP, LFZ, and DRR reviewed and interpreted the results. CLP and BLS wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript. The authors declare that they have no conflicts of interest with the contents of this article.

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Correspondence to Benjamin L. Schulz.

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Tables S1, S2 and S3 Byonic Glycan database, Sequest HT and Byonic Results (XLSX 1568 kb)

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Pegg, C.L., Zacchi, L.F., Recinos, D.R. et al. Identification of novel glycosylation events on human serum-derived factor IX. Glycoconj J 37, 471–483 (2020). https://doi.org/10.1007/s10719-020-09922-2

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