O-GlcNAcylation, a single attachment of N-acetylglucosamine (GlcNAc) on serine and threonine residues, plays important roles in normal and pathobiological states of many diseases. Aberrant expression of O-GlcNAc modification was found in many types of cancer including colorectal cancer (CRC). This modification mainly occurs in nuclear-cytoplasmic proteins; however, it can exist in some extracellular and secretory proteins. In this study, we investigated whether O-GlcNAc-modified proteins are present in serum of patients with CRC. Serum glycoproteins of CRC patients and healthy controls were enriched by wheat germ agglutinin, a glycan binding protein specifically binds to terminal GlcNAc and sialic acid. Two-dimensional gel electrophoresis, RL2 O-GlcNAc immunoblotting, affinity purification, and mass spectrometry were performed. The results showed that RL2 O-GlcNAc antibody predominantly reacted against serum immunoglobulin A1 (IgA1). The levels of RL2-reacted IgA were significantly increased while total IgA were not different in patients with CRC compared to those of healthy controls. Analyses by ion trap mass spectrometry using collision-induced dissociation and electron-transfer dissociation modes revealed one O-linked N-acetylhexosamine modification site at Ser268 located in the heavy constant region of IgA1; unfortunately, it cannot be discriminated whether it was N-acetylglucosamine or N-acetylgalactosamine because of their identical molecular mass. Although failed to demonstrate unequivocally it was O-GlcNAc, these data indicated that serum-IgA had an aberrantly increased reactivity against RL2 O-GlcNAc antibody in CRC patients. This specific glycosylated form of serum-IgA1 will expand the spectrum of aberrant glycosylation which provides valuable information to cancer glycobiology.
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two dimensional gel electrophoresis
β-Elimination/Michael Addition with DTT
coomassie brilliant blue R-250
EGF repeat-specific O-GlcNAc-transferase
higher energy collision dissociation
- IPG strip:
immobilized pH gradient strip
liquid chromatography tandem mass spectrometry
anti O-GlcNAc antibody
Tris-Buffered Saline + 0.1 % TWEEN20
wheat germ agglutinin
Reis, C.A., Osorio, H., Silva, L., Gomes, C., David, L.: Alterations in glycosylation as biomarkers for cancer detection. J. Clin. Pathol. 63(4), 322–329 (2010). https://doi.org/10.1136/jcp.2009.071035
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
Pinho, S.S., Reis, C.A.: Glycosylation in cancer: mechanisms and clinical implications. Nat. Rev. Cancer. 15(9), 540–555 (2015). https://doi.org/10.1038/nrc3982
Hart, G.W., Housley, M.P., Slawson, C.: Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins. Nature 446(7139), 1017–1022 (2007)
Zachara, N.E., Vosseller, K., Hart, G.W.: Detection and analysis of proteins modified by o-linked N-acetylglucosamine. Curr. Protoc. Mol. Biol. Chap. 17, Unit17 16 (2011). https://doi.org/10.1002/0471142727.mb1706s95
Ma, Z., Vosseller, K.: O-GlcNAc in cancer biology. Amino Acids. 45(4), 719–733 (2013). https://doi.org/10.1007/s00726-013-1543-8
Chaiyawat, P., Chokchaichamnankit, D., Lirdprapamongkol, K., Srisomsap, C., Svasti, J., Champattanachai, V.: Alteration of O-GlcNAcylation affects serine phosphorylation and regulates gene expression and activity of pyruvate kinase M2 in colorectal cancer cells. Oncol. Rep. 34(4), 1933–1942 (2015). https://doi.org/10.3892/or.2015.4178
Phueaouan, T., Chaiyawat, P., Netsirisawan, P., Chokchaichamnankit, D., Punyarit, P., Srisomsap, C., Svasti, J., Champattanachai, V.: Aberrant O-GlcNAc-modified proteins expressed in primary colorectal cancer. Oncol. Rep. 30(6), 2929–2936 (2013). https://doi.org/10.3892/or.2013.2794
Chaiyawat, P., Weeraphan, C., Netsirisawan, P., Chokchaichamnankit, D., Srisomsap, C., Svasti, J., Champattanachai, V.: Elevated O-GlcNAcylation of extracellular vesicle proteins derived from metastatic colorectal cancer cells. Cancer Genomics Proteomics 13(5), 387–398 (2016)
Varshney, S., Stanley, P.: EOGT and O-GlcNAc on secreted and membrane proteins. Biochem. Soc. Trans. 45(2), 401–408 (2017). https://doi.org/10.1042/BST20160165
Sakaidani, Y., Nomura, T., Matsuura, A., Ito, M., Suzuki, E., Murakami, K., Nadano, D., Matsuda, T., Furukawa, K., Okajima, T.: O-linked-N-acetylglucosamine on extracellular protein domains mediates epithelial cell-matrix interactions. Nat. Commun. 2, 583 (2011). https://doi.org/10.1038/ncomms1591
Champattanachai, V., Netsirisawan, P., Chaiyawat, P., Phueaouan, T., Charoenwattanasatien, R., Chokchaichamnankit, D., Punyarit, P., Srisomsap, C., Svasti, J.: Proteomic analysis and abrogated expression of O-GlcNAcylated proteins associated with primary breast cancer. Proteomics. 13(14), 2088–2099 (2013). https://doi.org/10.1002/pmic.201200126
Cao, W., Cao, J., Huang, J., Yao, J., Yan, G., Xu, H., Yang, P.: Discovery and confirmation of O-GlcNAcylated proteins in rat liver mitochondria by combination of mass spectrometry and immunological methods. PLoS One. 8(10), e76399 (2013). https://doi.org/10.1371/journal.pone.0076399
Charoensuksai, P., Kuhn, P., Wang, L., Sherer, N., Xu, W.: O-GlcNAcylation of co-activator-associated arginine methyltransferase 1 regulates its protein substrate specificity. Biochem. J. 466(3), 587–599 (2015). https://doi.org/10.1042/BJ20141072
Di Domenico, F., Owen, J.B., Sultana, R., Sowell, R.A., Perluigi, M., Cini, C., Cai, J., Pierce, W.M., Butterfield, D.A.: The wheat germ agglutinin-fractionated proteome of subjects with Alzheimer’s disease and mild cognitive impairment hippocampus and inferior parietal lobule: Implications for disease pathogenesis and progression. J. Neurosci. Res. 88(16), 3566–3577 (2010). https://doi.org/10.1002/jnr.22528
Meillour, N.-L., Vercoutter-Edouart, P., Hilliou, A.S., Le Danvic, F., Levy, C.: Proteomic analysis of pig (Sus scrofa) olfactory soluble proteome reveals O-Linked-N-Acetylglucosaminylation of secreted odorant-binding proteins. Front. Endocrinol. (Lausanne) 5, 202 (2014). https://doi.org/10.3389/fendo.2014.00202
Kerr, M.A.: The structure and function of human IgA. Biochem. J. 271(2), 285–296 (1990)
Tanaka, A., Iwase, H., Hiki, Y., Kokubo, T., Ishii-Karakasa, I., Toma, K., Kobayashi, Y., Hotta, K.: Evidence for a site-specific fucosylation of N-linked oligosaccharide of immunoglobulin A1 from normal human serum. Glycoconj. J. 15(10), 995–1000 (1998)
Lehoux, S., Mi, R., Aryal, R.P., Wang, Y., Schjoldager, K.T., Clausen, H., van Die, I., Han, Y., Chapman, A.B., Cummings, R.D., Ju, T.: Identification of distinct glycoforms of IgA1 in plasma from patients with immunoglobulin A (IgA) nephropathy and healthy individuals. Mol. Cell. Proteomics 13(11), 3097–3113 (2014). https://doi.org/10.1074/mcp.M114.039693
Zauner, G., Selman, M.H., Bondt, A., Rombouts, Y., Blank, D., Deelder, A.M., Wuhrer, M.: Glycoproteomic analysis of antibodies. Mol. Cell. Proteomics 12(4), 856–865 (2013). https://doi.org/10.1074/mcp.R112.026005
Pillebout, E., Jamin, A., Ayari, H., Housset, P., Pierre, M., Sauvaget, V., Viglietti, D., Deschenes, G., Monteiro, R.C., Berthelot, L., group, H.S.: Biomarkers of IgA vasculitis nephritis in children. PLoS One 12(11), e0188718 (2017). https://doi.org/10.1371/journal.pone.0188718
Halim, A., Westerlind, U., Pett, C., Schorlemer, M., Ruetschi, U., Brinkmalm, G., Sihlbom, C., Lengqvist, J., Larson, G., Nilsson, J.: Assignment of saccharide identities through analysis of oxonium ion fragmentation profiles in LC-MS/MS of glycopeptides. J. Proteome Res. 13(12), 6024–6032 (2014). https://doi.org/10.1021/pr500898r
Greis, K.D., Hayes, B.K., Comer, F.I., Kirk, M., Barnes, S., Lowary, T.L., Hart, G.W.: Selective detection and site-analysis of O-GlcNAc-modified glycopeptides by beta-elimination and tandem electrospray mass spectrometry. Anal. Biochem. 234(1), 38–49 (1996). https://doi.org/10.1006/abio.1996.0047
Takahashi, K., Wall, S.B., Suzuki, H., Smith, ADt., Hall, S., Poulsen, K., Kilian, M., Mobley, J.A., Julian, B.A., Mestecky, J., Novak, J., Renfrow, M.B.: Clustered O-glycans of IgA1: defining macro- and microheterogeneity by use of electron capture/transfer dissociation. Mol. Cell. Proteomics 9(11), 2545–2557 (2010). https://doi.org/10.1074/mcp.M110.001834
Mattu, T.S., Pleass, R.J., Willis, A.C., Kilian, M., Wormald, M.R., Lellouch, A.C., Rudd, P.M., Woof, J.M., Dwek, R.A.: The glycosylation and structure of human serum IgA1, Fab, and Fc regions and the role of N-glycosylation on Fcalpha receptor interactions. J. Biol. Chem. 273(4), 2260–2272 (1998). https://doi.org/10.1074/jbc.273.4.2260
Tarelli, E., Smith, A.C., Hendry, B.M., Challacombe, S.J., Pouria, S.: Human serum IgA1 is substituted with up to six O-glycans as shown by matrix assisted laser desorption ionisation time-of-flight mass spectrometry. Carbohydr. Res. 339(13), 2329–2335 (2004). https://doi.org/10.1016/j.carres.2004.07.011
Zachara, N., Akimoto, Y., Hart, G.W.: The O-GlcNAc Modification. In: rd, Varki, A., Cummings, R.D., Esko, J.D., Stanley, P., Hart, G.W., Aebi, M., Darvill, A.G., Kinoshita, T., Packer, N.H., Prestegard, J.H., Schnaar, R.L., Seeberger, P.H. (eds.) Essentials of Glycobiology, pp. 239–251. Cold Spring Harbor, New York (2015)
The authors would like to thank Dr. Carlito B Lebrilla, Professor of Department of Chemistry, University of California, Davis, CA, USA, for his suggestions and revision of the Manuscript. This work was supported by the National Science and Technology Development Agency (Grant no. P-12-01487) and the Chulabhorn Research Institute (Grant no. BC-2020-02), Thailand.
This work was financially supported by the National Science and Technology Development Agency (Grant no. P-12-01487) and the Chulabhorn Research Institute (Grant no. BC-2020-02), Thailand.
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All authors declare no conflict of interest.
Ethic was approved by the Institutional Review Board of the Royal Thai Army Medical Department, Thailand (S012h/56), Bangkok, Thailand.
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The CRC patient and the healthy control participants gave informed consent at Phramongkutklao Hospital, Bangkok, Thailand.
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The MS/MS database search of eleven protein spots by in-gel tryptic digestion (XLSX 692 kb)
The MS/MS database search of DTT-peptides by in-solution tryptic digestion (XLSX 488 kb)
The MS/MS searches of O-HexNAc peptide of IgA1 by Compass Data Analysis of Bruker (XLSX 809 kb)
The treatment of purified serum-IgA samples digested with O-GlcNAcase (OGA) (DOC 207 kb)
The extracted ion chromatogram (EIC) of the peptides with and without HexNAc modification (DOC 280 kb)
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Verathamjamras, C., Sriwitool, Te., Netsirisawan, P. et al. Aberrant RL2 O-GlcNAc antibody reactivity against serum-IgA1 of patients with colorectal cancer. Glycoconj J (2021). https://doi.org/10.1007/s10719-021-09978-8
- Colorectal cancer
- Immunoglobulin A1
- RL2 antibody
- Wheat germ agglutinin