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.
Similar content being viewed by others
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
References
Di Scipio, R.G., Kurachi, K., Davie, E.W.: Activation of human factor IX (Christmas factor). J. Clin. Invest. 61(6), 1528–1538 (1978). https://doi.org/10.1172/JCI109073
Bond, M., Jankowski, M., Patel, H., Karnik, S., Strang, A., Xu, B., Rouse, J., Koza, S., Letwin, B., Steckert, J., Amphlett, G., Scoble, H.: Biochemical characterization of recombinant factor IX. Semin. Hematol. 35(2 Suppl 2), 11–17 (1998)
Arruda, V.R., Hagstrom, J.N., Deitch, J., Heiman-Patterson, T., Camire, R.M., Chu, K., Fields, P.A., Herzog, R.W., Couto, L.B., Larson, P.J., High, K.A.: Posttranslational modifications of recombinant myotube-synthesized human factor IX. Blood. 97(1), 130–138 (2001). https://doi.org/10.1182/blood.V97.1.130
Mathur, A., Zhong, D., Sabharwal, A.K., Smith, K.J., Bajaj, S.P.: Interaction of factor IXa with factor VIIIa. Effects of protease domain Ca2+ binding site, proteolysis in the autolysis loop, phospholipid, and factor X. J. Biol. Chem. 272(37), 23418–23426 (1997). https://doi.org/10.1074/jbc.272.37.23418
Goodeve, A.C.: Hemophilia B: molecular pathogenesis and mutation analysis. J. Thromb. Haemost. 13(7), 1184–1195 (2015). https://doi.org/10.1111/jth.12958
Lyseng-Williamson, K.A.: Coagulation factor IX (recombinant), albumin fusion protein (Albutrepenonacog Alfa; Idelvion®): a review of its use in haemophilia B. Drugs. 77(1), 97–106 (2016). https://doi.org/10.1007/s40265-016-0679-8
Turecek, P.L., Abbuhl, B., Tangada, S.D., Chapman, M., Gritsch, H., Rottensteiner, H., Schrenk, G., Mitterer, A., Dietrich, B., Hollriegl, W., Schiviz, A., Horling, F., Reipert, B.M., Muchitsch, E.M., Pavlova, B.G., Scheiflinger, F.: Nonacog gamma, a novel recombinant factor IX with low factor IXa content for treatment and prophylaxis of bleeding episodes. Expert. Rev. Clin. Pharmacol. 8(2), 163–177 (2015). https://doi.org/10.1586/17512433.2015.1011126
Cooley, B., Funkhouser, W., Monroe, D., Ezzell, A., Mann, D.M., Lin, F.C., Monahan, P.E., Stafford, D.W.: Prophylactic efficacy of BeneFIX vs Alprolix in hemophilia B mice. Blood. 128(2), 286–292 (2016). https://doi.org/10.1182/blood-2016-01-696104
Franchini, M.: Current management of hemophilia B: recommendations, complications and emerging issues. Expert. Rev. Hematol. 7(5), 573–581 (2014). https://doi.org/10.1586/17474086.2014.947955
Johansson, L., Karpf, D.M., Hansen, L., Pelzer, H., Persson, E.: Activation peptides prolong the murine plasma half-life of human factor VII. Blood. 117(12), 3445–3452 (2011). https://doi.org/10.1182/blood-2010-06-290098
Stanley, T.B., Wu, S.-M., Houben, R.J.T.J., Mutucumarana, V.P., Stafford, D.W.: Role of the propeptide and γ-glutamic acid domain of factor ix for in vitro carboxylation by the vitamin k-dependent carboxylase. Biochemistry. 37(38), 13262–13268 (1998). https://doi.org/10.1021/bi981031y
Jorgensen, M.J., Cantor, A.B., Furie, B.C., Brown, C.L., Shoemaker, C.B., Furie, B.: Recognition site directing vitamin K-dependent gamma-carboxylation resides on the propeptide of factor IX. Cell. 48(2), 185–191 (1987). https://doi.org/10.1016/0092-8674(87)90422-3
Huang, M., Rigby, A.C., Morelli, X., Grant, M.A., Huang, G., Furie, B., Seaton, B., Furie, B.C.: Structural basis of membrane binding by Gla domains of vitamin K-dependent proteins. Nat. Struct. Biol. 10(9), 751–756 (2003). https://doi.org/10.1038/nsb971
Hansson, K., Stenflo, J.: Post-translational modifications in proteins involved in blood coagulation. J. Thromb. Haemost. 3(12), 2633–2648 (2005). https://doi.org/10.1111/j.1538-7836.2005.01478.x
Derian, C.K., VanDusen, W., Przysiecki, C.T., Walsh, P.N., Berkner, K.L., Kaufman, R.J., Friedman, P.A.: Inhibitors of 2-ketoglutarate-dependent dioxygenases block aspartyl beta-hydroxylation of recombinant human factor IX in several mammalian expression systems. J. Biol. Chem. 264(12), 6615–6618 (1989)
Fernlund, P., Stenflo, J.: Beta-hydroxyaspartic acid in vitamin K-dependent proteins. J. Biol. Chem. 258(20), 12509–12512 (1983)
Harris R.J., P.D.I., Truong L., Smith K.J.: Partial Phosphorylation of Serine-68 in EGF-1 of Human Factor IX. In: Proceedings of XIth international conference on methods in protein structure analysis 1996
Nishimura, H., Kawabata, S., Kisiel, W., Hase, S., Ikenaka, T., Takao, T., Shimonishi, Y., Iwanaga, S.: Identification of a disaccharide (Xyl-Glc) and a trisaccharide (Xyl2-Glc) O-glycosidically linked to a serine residue in the first epidermal growth factor-like domain of human factors VII and IX and protein Z and bovine protein Z. J. Biol. Chem. 264(34), 20320–20325 (1989)
Harris, R.J., van Halbeek, H., Glushka, J., Basa, L.J., Ling, V.T., Smith, K.J., Spellman, M.W.: Identification and structural analysis of the tetrasaccharide NeuAcα (2→ 6) Galβ (1→ 4) GlcNAcβ (1→ 3) Fucα1→ O-linked to serine 61 of human factor IX. Biochemistry. 32(26), 6539–6547 (1993). https://doi.org/10.1021/bi00077a007
Nishimura, H., Takao, T., Hase, S., Shimonishi, Y., Iwanaga, S.: Human factor IX has a tetrasaccharide O-glycosidically linked to serine 61 through the fucose residue. J. Biol. Chem. 267(25), 17520–17525 (1992)
King, S.L., Joshi, H.J., Schjoldager, K.T., Halim, A., Madsen, T.D., Dziegiel, M.H., Woetmann, A., Vakhrushev, S.Y., Wandall, H.H.: Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells. Blood Adv. 1(7), 429–442 (2017). https://doi.org/10.1182/bloodadvances.2016002121
Kurachi, K., Davie, E.W.: Isolation and characterization of a cDNA coding for human factor IX. Proc. Natl. Acad. Sci. U. S. A. 79(21), 6461–6464 (1982)
Agarwala, K.L., Kawabata, S., Takao, T., Murata, H., Shimonishi, Y., Nishimura, H., Iwanaga, S.: Activation peptide of human factor IX has oligosaccharides O-glycosidically linked to threonine residues at 159 and 169. Biochemistry. 33(17), 5167–5171 (1994). https://doi.org/10.1021/bi00183a021
Huang, L.J., Lin, J.H., Tsai, J.H., Chu, Y.Y., Chen, Y.W., Chen, S.L., Chen, S.H.: Identification of protein O-glycosylation site and corresponding glycans using liquid chromatography-tandem mass spectrometry via mapping accurate mass and retention time shift. J. Chromatogr. A 1371(Supplement C), 136–145 (2014). https://doi.org/10.1016/j.chroma.2014.10.046
Moremen, K.W., Tiemeyer, M., Nairn, A.V.: Vertebrate protein glycosylation: diversity, synthesis and function. Nat. Rev. Mol. Cell Biol. 13(7), 448–462 (2012). https://doi.org/10.1038/nrm3383
Thaysen-Andersen, M., Packer, N.H., Schulz, B.L.: Maturing glycoproteomics technologies provide unique structural insights into the N-glycoproteome and its regulation in health and disease. Mol. Cell. Proteomics. 15(6), 1773–1790 (2016). https://doi.org/10.1074/mcp.O115.057638
Bertozzi, C.R., Rabuka, D.: Structural basis of glycan diversity. In: Varki, A., Cummings, R.D., Esko, J.D., Freeze, H.H., Stanley, P., Bertozzi, C.R., Hart, G.W., Etzler, M.E. (eds.) Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, New York (2009)
Chevreux, G., Faid, V., Andre, M.H., Tellier, Z., Bihoreau, N.: Differential investigations from plasma-derived and recombinant factor IX revealed major differences in post-translational modifications of activation peptides. Vox Sang. 104(2), 171–174 (2013). https://doi.org/10.1111/j.1423-0410.2012.01649.x
Lee, L.Y., Moh, E.S., Parker, B.L., Bern, M., Packer, N.H., Thaysen-Andersen, M.: Toward automated N-glycopeptide identification in glycoproteomics. J. Proteome Res. 15(10), 3904–3915 (2016). https://doi.org/10.1021/acs.jproteome.6b00438
Schulz, B.L., Aebi, M.: Analysis of glycosylation site occupancy reveals a role for Ost3p and Ost6p in site-specific N-glycosylation efficiency. Mol. Cell. Proteomics. 8(2), 357–364 (2009). https://doi.org/10.1074/mcp.M800219-MCP200
Makino, Y., Omichi, K., Kuraya, N., Ogawa, H., Nishimura, H., Iwanaga, S., Hase, S.: Structural analysis of N-linked sugar chains of human blood clotting factor IX. J. Biochem. 128(2), 175–180 (2000). https://doi.org/10.1093/oxfordjournals.jbchem.a022738
McGraw, R.A., Davis, L.M., Noyes, C.M., Lundblad, R.L., Roberts, H.R., Graham, J.B., Stafford, D.W.: Evidence for a prevalent dimorphism in the activation peptide of human coagulation factor IX. Proc. Natl. Acad. Sci. U. S. A. 82(9), 2847–2851 (1985). https://doi.org/10.1073/pnas.82.9.2847
Perez-Riverol, Y., Csordas, A., Bai, J., Bernal-Llinares, M., Hewapathirana, S., Kundu, D.J., Inuganti, A., Griss, J., Mayer, G., Eisenacher, M., Perez, E., Uszkoreit, J., Pfeuffer, J., Sachsenberg, T., Yilmaz, S., Tiwary, S., Cox, J., Audain, E., Walzer, M., Jarnuczak, A.F., Ternent, T., Brazma, A., Vizcaino, J.A.: The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 47(D1), D442–D450 (2019). https://doi.org/10.1093/nar/gky1106
Sievers, F., Wilm, A., Dineen, D., Gibson, T.J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Soding, J., Thompson, J.D., Higgins, D.G.: Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal omega. Mol. Syst. Biol. 7(1), 539 (2011). https://doi.org/10.1038/msb.2011.75
Soding, J.: Protein homology detection by HMM-HMM comparison. Bioinformatics. 21(7), 951–960 (2005). https://doi.org/10.1093/bioinformatics/bti125
Varki, A., Cummings, R.D., Aebi, M., Packer, N.H., Seeberger, P.H., Esko, J.D., Stanley, P., Hart, G., Darvill, A., Kinoshita, T., Prestegard, J.J., Schnaar, R.L., Freeze, H.H., Marth, J.D., Bertozzi, C.R., Etzler, M.E., Frank, M., Vliegenthart, J.F., Lutteke, T., Perez, S., Bolton, E., Rudd, P., Paulson, J., Kanehisa, M., Toukach, P., Aoki-Kinoshita, K.F., Dell, A., Narimatsu, H., York, W., Taniguchi, N., Kornfeld, S.: Symbol nomenclature for graphical representations of glycans. Glycobiology. 25(12), 1323–1324 (2015). https://doi.org/10.1093/glycob/cwv091
Samis, J.A., Kam, E., Nesheim, M.E., Giles, A.R.: Neutrophil elastase cleavage of human factor IX generates an activated factor IX-like product devoid of coagulant function. Blood. 92(4), 1287–1296 (1998)
Zhurov, K.O., Fornelli, L., Wodrich, M.D., Laskay, U.A., Tsybin, Y.O.: Principles of electron capture and transfer dissociation mass spectrometry applied to peptide and protein structure analysis. Chem. Soc. Rev. 42(12), 5014–5030 (2013). https://doi.org/10.1039/c3cs35477f
Gil, G.C., Velander, W.H., Van Cott, K.E.: Analysis of the N-glycans of recombinant human factor IX purified from transgenic pig milk. Glycobiology. 18(7), 526–539 (2008). https://doi.org/10.1093/glycob/cwn035
Dutta, D., Mandal, C., Mandal, C.: Unusual glycosylation of proteins: beyond the universal sequon and other amino acids. Biochim. Biophys. Acta. 1861(12), 3096–3108 (2017). https://doi.org/10.1016/j.bbagen.2017.08.025
Faid, V., Denguir, N., Chapuis, V., Bihoreau, N., Chevreux, G.: Site-specific N-glycosylation analysis of human factor XI: identification of a noncanonical NXC glycosite. Proteomics. 14(21–22), 2460–2470 (2014). https://doi.org/10.1002/pmic.201400038
Miletich, J.P., Broze, G.J.: Beta protein C is not glycosylated at asparagine 329. The rate of translation may influence the frequency of usage at asparagine-X-cysteine sites. J. Biol. Chem. 265(19), 11397–11404 (1990)
Liu, T., Qian, W.J., Gritsenko, M.A., Camp 2nd, D.G., Monroe, M.E., Moore, R.J., Smith, R.D.: Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry. J. Proteome Res. 4(6), 2070–2080 (2005). https://doi.org/10.1021/pr0502065
Grinnell, B.W., Walls, J.D., Gerlitz, B.: Glycosylation of human protein C affects its secretion, processing, functional activities, and activation by thrombin. J. Biol. Chem. 266(15), 9778–9785 (1991)
Kolkman, J.A., Mertens, K.: Insertion loop 256−268 in coagulation factor IX restricts enzymatic activity in the absence but not in the presence of factor VIII. Biochemistry. 39(25), 7398–7405 (2000). https://doi.org/10.1021/bi992735q
Ponder, K.P.: FIXing factor VIII inhibitors. Blood. 119(2), 325–326 (2012). https://doi.org/10.1182/blood-2011-11-389486
Brandstetter, H., Bauer, M., Huber, R., Lollar, P., Bode, W.: X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B. Proc. Natl. Acad. Sci. 92(21), 9796–9800 (1995). https://doi.org/10.1073/pnas.92.21.9796
Chen, S.W.W., Pellequer, J.L., Schved, J.F., Giansily-Blaizot, M.: Model of a ternary complex between activated factor VII, tissue factor and factor IX. Thromb. Haemost. 88(1), 74–82 (2002)
Elliott, S., Lorenzini, T., Asher, S., Aoki, K., Brankow, D., Buck, L., Busse, L., Chang, D., Fuller, J., Grant, J., Hernday, N., Hokum, M., Hu, S., Knudten, A., Levin, N., Komorowski, R., Martin, F., Navarro, R., Osslund, T., Rogers, G., Rogers, N., Trail, G., Egrie, J.: Enhancement of therapeutic protein in vivo activities through glycoengineering. Nat. Biotechnol. 21(4), 414–421 (2003). https://doi.org/10.1038/nbt799
Hallgren, K.W., Zhang, D., Kinter, M., Willard, B., Berkner, K.L.: Methylation of gamma-carboxylated Glu (Gla) allows detection by liquid chromatography-mass spectrometry and the identification of Gla residues in the gamma-glutamyl carboxylase. J. Proteome Res. 12(6), 2365–2374 (2013). https://doi.org/10.1021/pr3003722
Gillis, S., Furie, B.C., Furie, B., Patel, H., Huberty, M.C., Switzer, M., Foster, W.B., Scoble, H.A., Bond, M.D.: Gamma-carboxyglutamic acids 36 and 40 do not contribute to human factor IX function. Protein Sci. 6(1), 185–196 (1997). https://doi.org/10.1002/pro.5560060121
Good, D.M., Wirtala, M., McAlister, G.C., Coon, J.J.: Performance characteristics of electron transfer dissociation mass spectrometry. Mol. Cell. Proteomics. 6(11), 1942–1951 (2007). https://doi.org/10.1074/mcp.M700073-MCP200
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.
Author information
Authors and Affiliations
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.
Corresponding author
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10719-020-09922-2