Patients with IgA nephropathy (IgAN) have elevated circulating levels of IgA1 with some O-glycans consisting of galactose (Gal)-deficient N-acetylgalactosamine (GalNAc) with or without N-acetylneuraminic acid (NeuAc). We have analyzed O-glycosylation heterogeneity of naturally asialo-IgA1 (Ale) myeloma protein that mimics Gal-deficient IgA1 (Gd-IgA1) of patients with IgAN, except that IgA1 O-glycans of IgAN patients are frequently sialylated. Specifically, serum IgA1 of healthy controls has more α2,3-sialylated O-glycans (NeuAc attached to Gal) than α2,6-sialylated O-glycans (NeuAc attached to GalNAc). As IgA1-producing cells from IgAN patients have an increased activity of α2,6-sialyltransferase (ST6GalNAc), we hypothesize that such activity may promote premature sialylation of GalNAc and, thus, production of Gd-IgA1, as sialylation of GalNAc prevents subsequent Gal attachment. Distribution of NeuAc in IgA1 O-glycans may play an important role in the pathogenesis of IgAN. To better understand biological functions of NeuAc in IgA1, we established protocols for enzymatic sialylation leading to α2,3- or α2,6-sialylation of IgA1 O-glycans. Sialylation of Gal-deficient asialo-IgA1 (Ale) myeloma protein by an ST6GalNAc enzyme generated sialylated IgA1 that mimics the Gal-deficient IgA1 glycoforms in patients with IgAN, characterized by α2,6-sialylated Gal-deficient GalNAc. In contrast, sialylation of the same myeloma protein by an α2,3-sialyltransferase yielded IgA1 typical for healthy controls, characterized by α2,3-sialylated Gal. The GalNAc-specific lectin from Helix aspersa (HAA) is used to measure levels of Gd-IgA1. We assessed HAA binding to IgA1 sialylated at Gal or GalNAc. As expected, α2,6-sialylation of IgA1 markedly decreased reactivity with HAA. Notably, α2,3-sialylation also decreased reactivity with HAA. Neuraminidase treatment recovered the original HAA reactivity in both instances. These results suggest that binding of a GalNAc-specific lectin is modulated by sialylation of GalNAc as well as Gal in the clustered IgA1 O-glycans. Thus, enzymatic sialylation offers a useful model to test the role of NeuAc in reactivities of the clustered O-glycans with lectins.

Department of Biomedicine Aarhus University Aarhus Denmark

Department of Microbiology University of Alabama at Birmingham Birmingham Alabama United States of America

Department of Microbiology University of Alabama at Birmingham Birmingham Alabama United States of America; Department of Medicine University of Alabama at Birmingham Birmingham Alabama United States of America

Department of Microbiology University of Alabama at Birmingham Birmingham Alabama United States of America; Department of Nephrology Fujita Health University School of Medicine Toyoake Japan

Department of Microbiology University of Alabama at Birmingham Birmingham Alabama United States of America; Faculty of Medicine and Dentistry Department of Immunology Palacky University in Olomouc Olomouc Czech Republic

Department of Nephrology Fujita Health University School of Medicine Toyoake Japan

Fujita Health University School of Health Sciences Toyoake Japan

UAB Biomedical FT ICR MS Laboratory Department of Biochemistry and Molecular Genetics University of Alabama at Birmingham Birmingham Alabama United States of America

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Mestecky J, Tomana M, Crowley-Nowick PA, Moldoveanu Z, Julian BA, et al. (1993) Defective galactosylation and clearance of IgA1 molecules as a possible etiopathogenic factor in IgA nephropathy. Contrib Nephrol 104: 172–182. PubMed

Moore JS, Wu X, Kulhavy R, Tomana M, Novak J, et al. (2005) Increased levels of galactose-deficient IgG in sera of HIV-1-infected individuals. AIDS 19: 381–389. PubMed

Rademacher TW, Williams P, Dwek RA (1994) Agalactosyl glycoforms of IgG autoantibodies are pathogenic. Proc Natl Acad Sci U S A 91: 6123–6127. PubMed PMC

Springer GF (1984) T and Tn, general carcinoma autoantigens. Science 224: 1198–1206. PubMed

Troelsen LN, Garred P, Madsen HO, Jacobsen S (2007) Genetically determined high serum levels of mannose-binding lectin and agalactosyl IgG are associated with ischemic heart disease in rheumatoid arthritis. Arthritis Rheum 56: 21–29. PubMed

Mestecky J, Raska M, Julian BA, Gharavi AG, Renfrow MB, et al. (2013) IgA Nephropathy: Molecular Mechanisms of the Disease. Annu Rev Pathol Mech Dis 8: 217–240. PubMed

Xue J, Zhu LP, Wei Q (2013) IgG-Fc N-glycosylation at Asn297 and IgA O-glycosylation in the hinge region in health and disease. Glycoconj J 30: 735–745. PubMed

Novak J, Renfrow MB, Gharavi AG, Julian BA (2013) Pathogenesis of immunoglobulin A nephropathy. Curr Opin Nephrol Hypertens 22: 287–294. PubMed

Scott DW, Patel RP (2013) Endothelial heterogeneity and adhesion molecules N-glycosylation: implications in leukocyte trafficking in inflammation. Glycobiology 23: 622–633. PubMed

Stuchlova Horynova M, Raska M, Clausen H, Novak J (2013) Aberrant O-glycosylation and anti-glycan antibodies in an autoimmune disease IgA nephropathy and breast adenocarcinoma. Cell Mol Life Sci 70: 829–839. PubMed PMC

Rudd PM, Elliott T, Cresswell P, Wilson IA, Dwek RA (2001) Glycosylation and the immune system. Science 291: 2370–2376. PubMed

Varki A (2008) Sialic acids in human health and disease. Trends Mol Med 14: 351–360. PubMed PMC

Varki A, Gagneux P (2012) Multifarious roles of sialic acids in immunity. Ann N Y Acad Sci 1253: 16–36. PubMed PMC

Schmitt FC, Figueiredo P, Lacerda M (1995) Simple mucin-type carbohydrate antigens (T, sialosyl-T, Tn and sialosyl-Tn) in breast carcinogenesis. Virchows Archiv 427: 251–258. PubMed

Sewell R, Backstrom M, Dalziel M, Gschmeissner S, Karlsson H, et al. (2006) The ST6GalNAc-I sialyltransferase localizes throughout the Golgi and is responsible for the synthesis of the tumor-associated sialyl-Tn O-glycan in human breast cancer. J Biol Chem 281: 3586–3594. PubMed

Brockhausen I (2006) Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions. EMBO Rep 7: 599–604. PubMed PMC

Pinho S, Marcos NT, Ferreira B, Carvalho AS, Oliveira MJ, et al. (2007) Biological significance of cancer-associated sialyl-Tn antigen: modulation of malignant phenotype in gastric carcinoma cells. Cancer Lett 249: 157–170. PubMed

Storr SJ, Royle L, Chapman CJ, Hamid UM, Robertson JF, et al. (2008) The O-linked glycosylation of secretory/shed MUC1 from an advanced breast cancer patient’s serum. Glycobiology 18: 456–462. PubMed

Picco G, Julien S, Brockhausen I, Beatson R, Antonopoulos A, et al. (2010) Over-expression of ST3Gal-I promotes mammary tumorigenesis. Glycobiology 20: 1241–1250. PubMed PMC

Mungul A, Cooper L, Brockhausen I, Ryder K, Mandel U, et al. (2004) Sialylated core 1 based O-linked glycans enhance the growth rate of mammary carcinoma cells in MUC1 transgenic mice. Int J Oncol 25: 937–943. PubMed

Bresalier RS, Ho SB, Schoeppner HL, Kim YS, Sleisenger MH, et al. (1996) Enhanced sialylation of mucin-associated carbohydrate structures in human colon cancer metastasis. Gastroenterology 110: 1354–1367. PubMed

Schindlbeck C, Jeschke U, Schulze S, Karsten U, Janni W, et al. (2005) Characterisation of disseminated tumor cells in the bone marrow of breast cancer patients by the Thomsen-Friedenreich tumor antigen. Histochem Cell Biol 123: 631–637. PubMed

Julien S, Lagadec C, Krzewinski-Recchi MA, Courtand G, Le Bourhis X, et al. (2005) Stable expression of sialyl-Tn antigen in T47-D cells induces a decrease of cell adhesion and an increase of cell migration. Breast Cancer Res Treat 90: 77–84. PubMed

Mestecky J, Russell MW (1986) IgA subclasses. Monogr Allergy 19: 277–301. PubMed

Baenziger J, Kornfeld S (1974) Structure of the carbohydrate units of IgA1 immunoglobulin. II. Structure of the O-glycosidically linked oligosaccharide units. J Biol Chem 249: 7270–7281. PubMed

Mattu TS, Pleass RJ, Willis AC, Kilian M, Wormald MR, et al. (1998) The glycosylation and structure of human serum IgA1, Fab, and Fc regions and the role of N-glycosylation on Fcα receptor interactions. J Biol Chem 273: 2260–2272. PubMed

Tarelli E, Smith AC, Hendry BM, Challacombe SJ, Pouria S (2004) 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: 2329–2335. PubMed

Renfrow MB, Cooper HJ, Tomana M, Kulhavy R, Hiki Y, et al. (2005) Determination of aberrant O-glycosylation in the IgA1 hinge region by electron capture dissociation Fourier transform-ion cyclotron resonance mass spectrometry. J Biol Chem 280: 19136–19145. PubMed

Renfrow MB, Mackay CL, Chalmers MJ, Julian BA, Mestecky J, et al. (2007) Analysis of O-glycan heterogeneity in IgA1 myeloma proteins by Fourier transform ion cyclotron resonance mass spectrometry: implications for IgA nephropathy. Anal Bioanal Chem 389: 1397–1407. PubMed

Takahashi K, Wall SB, Suzuki H, Smith AD, Hall S, et al. (2010) Clustered O-glycans of IgA1: Defining macro- and micro-heterogeneity by use of electron capture/transfer dissociation. Mol Cell Proteomics 9: 2545–2557. PubMed PMC

Takahashi K, Smith AD, Poulsen K, Kilian M, Julian BA, et al. (2012) Naturally occurring structural isomers in serum IgA1 O-glycosylation. J Proteome Res 11: 692–702. PubMed PMC

Berger J, Hinglais N (1968) [Intercapillary deposits of IgA-IgG]. Urol Nephrol (Paris) 74: 694–695. PubMed

Conley ME, Cooper MD, Michael AF (1980) Selective deposition of immunoglobulin A1 in immunoglobulin A nephropathy, anaphylactoid purpura nephritis, and systemic lupus erythematosus. J Clin Invest 66: 1432–1436. PubMed PMC

Julian BA, Waldo FB, Rifai A, Mestecky J (1988) IgA nephropathy, the most common glomerulonephritis worldwide. A neglected disease in the United States? Am J Med 84: 129–132. PubMed

Donadio JV, Grande JP (2002) IgA nephropathy. N Engl J Med 347: 738–748. PubMed

Wyatt RJ, Julian BA (2013) IgA nephropathy. N Engl J Med 368: 2402–2414. PubMed

Czerkinsky C, Koopman WJ, Jackson S, Collins JE, Crago SS, et al. (1986) Circulating immune complexes and immunoglobulin A rheumatoid factor in patients with mesangial immunoglobulin A nephropathies. J Clin Invest 77: 1931–1938. PubMed PMC

Coppo R, Basolo B, Piccoli G, Mazzucco G, Bulzomi MR, et al. (1984) IgA1 and IgA2 immune complexes in primary IgA nephropathy and Henoch-Schonlein nephritis. Clin Exp Immunol 57: 583–590. PubMed PMC

Schena FP, Pastore A, Ludovico N, Sinico RA, Benuzzi S, et al. (1989) Increased serum levels of IgA1-IgG immune complexes and anti-F(ab’)2 antibodies in patients with primary IgA nephropathy. Clin Exp Immunol 77: 15–20. PubMed PMC

Tomana M, Matousovic K, Julian BA, Radl J, Konecny K, et al. (1997) Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG. Kidney Int 52: 509–516. PubMed

Tomana M, Novak J, Julian BA, Matousovic K, Konecny K, et al. (1999) Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies. J Clin Invest 104: 73–81. PubMed PMC

Novak J, Tomana M, Matousovic K, Brown R, Hall S, et al. (2005) IgA1-containing immune complexes in IgA nephropathy differentially affect proliferation of mesangial cells. Kidney Int 67: 504–513. PubMed

Suzuki H, Fan R, Zhang Z, Brown R, Hall S, et al. (2009) Aberrantly glycosylated IgA1 in IgA nephropathy patients is recognized by IgG antibodies with restricted heterogeneity. J Clin Invest 119: 1668–1677. PubMed PMC

Suzuki H, Kiryluk K, Novak J, Moldoveanu Z, Herr AB, et al. (2011) The pathophysiology of IgA nephropathy. J Am Soc Nephrol 22: 1795–1803. PubMed PMC

Berthoux F, Suzuki H, Thibaudin L, Yanagawa H, Maillard N, et al. (2012) Autoantibodies targeting galactose-deficient IgA1 associate with progression of IgA nephropathy. J Am Soc Nephrol 23: 1579–1587. PubMed PMC

Novak J, Julian BA, Tomana M, Mestecky J (2001) Progress in molecular and genetic studies of IgA nephropathy. J Clin Immunol 21: 310–327. PubMed

Suzuki H, Moldoveanu Z, Hall S, Brown R, Vu HL, et al. (2008) IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1. J Clin Invest 118: 629–639. PubMed PMC

Suzuki H, Raska M, Yamada K, Moldoveanu Z, Julian BA, et al. (2014) Cytokines alter IgA1 O-glycosylation by dysregulating C1GalT1 and ST6GalNAc-II enzymes. J Biol Chem 289: 5330–5339. PubMed PMC

Leung JC, Poon PY, Lai KN (1999) Increased sialylation of polymeric immunoglobulin A1: mechanism of selective glomerular deposition in immunoglobulin A nephropathy? J Lab Clin Med 133: 152–160. PubMed

Amore A, Cirina P, Conti G, Brusa P, Peruzzi L, et al. (2001) Glycosylation of circulating IgA in patients with IgA nephropathy modulates proliferation and apoptosis of mesangial cells. J Am Soc Nephrol 12: 1862–1871. PubMed

Leung JC, Tang SC, Chan DT, Lui SL, Lai KN (2002) Increased sialylation of polymeric lambda-IgA1 in patients with IgA nephropathy. J Clin Lab Anal 16: 11–19. PubMed PMC

Odani H, Hiki Y, Takahashi M, Nishimoto A, Yasuda Y, et al. (2000) Direct evidence for decreased sialylation and galactosylation of human serum IgA1 Fc O-glycosylated hinge peptides in IgA nephropathy by mass spectrometry. Biochem Biophys Res Commun 271: 268–274. PubMed

Horie A, Hiki Y, Odani H, Yasuda Y, Takahashi M, et al. (2003) IgA1 molecules produced by tonsillar lymphocytes are under-O-glycosylated in IgA nephropathy. Am J Kidney Dis 42: 486–496. PubMed

Xu LX, Zhao MH (2005) Aberrantly glycosylated serum IgA1 are closely associated with pathologic phenotypes of IgA nephropathy. Kidney Int 68: 167–172. PubMed

Ding JX, Xu LX, Lv JC, Zhao MH, Zhang H, et al. (2007) Aberrant sialylation of serum IgA1 was associated with prognosis of patients with IgA nephropathy. Clin Immunol 125: 268–274. PubMed

Maenuma K, Yim M, Komatsu K, Hoshino M, Tachiki-Fujioka A, et al. (2009) A library of mutated Maackia amurensis hemagglutinin distinguishes putative glycoforms of immunoglobulin A1 from IgA nephropathy patients. J Proteome Res 8: 3617–3624. PubMed

Leung JC, Tang SC, Lam MF, Chan TM, Lai KN (2001) Charge-dependent binding of polymeric IgA1 to human mesangial cells in IgA nephropathy. Kidney Int 59: 277–285. PubMed

Coppo R, Amore A, Gianoglio B, Reyna A, Peruzzi L, et al. (1993) Serum IgA and macromolecular IgA reacting with mesangial matrix components. Contrib Nephrol 104: 162–171. PubMed

Kokubo T, Hiki Y, Iwase H, Tanaka A, Toma K, et al. (1998) Protective role of IgA1 glycans against IgA1 self-aggregation and adhesion to extracellular matrix proteins. J Am Soc Nephrol 9: 2048–2054. PubMed

Sano T, Hiki Y, Kokubo T, Iwase H, Shigematsu H, et al. (2002) Enzymatically deglycosylated human IgA1 molecules accumulate and induce inflammatory cell reaction in rat glomeruli. Nephrol Dial Transplant 17: 50–56. PubMed

Hiki Y (2009) O-linked oligosaccharides of the IgA1 hinge region: roles of its aberrant structure in the occurrence and/or progression of IgA nephropathy. Clin Exp Nephrol 13: 415–423. PubMed

Hiki Y, Odani H, Takahashi M, Yasuda Y, Nishimoto A, et al. (2001) Mass spectrometry proves under-O-glycosylation of glomerular IgA1 in IgA nephropathy. Kidney Int 59: 1077–1085. PubMed

Allen AC, Bailey EM, Brenchley PE, Buck KS, Barratt J, et al. (2001) Mesangial IgA1 in IgA nephropathy exhibits aberrant O-glycosylation: observations in three patients. Kidney Int 60: 969–973. PubMed

Moldoveanu Z, Wyatt RJ, Lee JY, Tomana M, Julian BA, et al. (2007) Patients with IgA nephropathy have increased serum galactose-deficient IgA1 levels. Kidney Int 71: 1148–1154. PubMed

Horynova M, Takahashi K, Hall S, Renfrow MB, Novak J, et al. (2012) Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells. Protein Expr Purif 81: 175–180. PubMed PMC

Moore JS, Kulhavy R, Tomana M, Moldoveanu Z, Suzuki H, et al. (2007) Reactivities of N-acetylgalactosamine-specific lectins with human IgA1 proteins. Mol Immunol 44: 2598–2604. PubMed PMC

Raska M, Moldoveanu Z, Suzuki H, Brown R, Kulhavy R, et al. (2007) Identification and characterization of CMP-NeuAc:GalNAc-IgA1 α2,6-sialyltransferase in IgA1-producing cells. J Mol Biol 369: 69–78. PubMed PMC

Monteiro RC, Halbwachs-Mecarelli L, Roque-Barreira MC, Noel LH, Berger J, et al. (1985) Charge and size of mesangial IgA in IgA nephropathy. Kidney Int 28: 666–671. PubMed

Lai KN, Chui SH, Lewis WH, Poon AS, Lam CW (1994) Charge distribution of IgA-lambda in IgA nephropathy. Nephron 66: 38–44. PubMed

Tomana M, Kulhavy R, Mestecky J (1988) Receptor-mediated binding and uptake of immunoglobulin A by human liver. Gastroenterology 94: 762–770. PubMed

Stockert RJ (1995) The asialoglycoprotein receptor: relationships between structure, function, and expression. Physiol Rev 75: 591–609. PubMed

Basset C, Devauchelle V, Durand V, Jamin C, Pennec YL, et al. (1999) Glycosylation of immunoglobulin A influences its receptor binding. Scand J Immunol 50: 572–579. PubMed

Steirer LM, Park EI, Townsend RR, Baenziger JU (2009) The asialoglycoprotein receptor regulates levels of plasma glycoproteins terminating with sialic acid α2,6-galactose. J Biol Chem 284: 3777–3783. PubMed PMC

Moldoveanu Z, Epps JM, Thorpe SR, Mestecky J (1988) The sites of catabolism of murine monomeric IgA. J Immunol 141: 208–213. PubMed

Mestecky J, Moldoveanu Z, Tomana M, Epps JM, Thorpe SR, et al. (1989) The role of the liver in catabolism of mouse and human IgA. Immunol Invest 18: 313–324. PubMed

Novak J, Julian BA, Tomana M, Mestecky J (2008) IgA glycosylation and IgA immune complexes in the pathogenesis of IgA nephropathy. Semin Nephrol 28: 78–87. PubMed PMC

Stephenson AE, Wu H, Novak J, Tomana M, Mintz K, et al. (2002) The Fap1 fimbrial adhesin is a glycoprotein: antibodies specific for the glycan moiety block the adhesion of Streptococcus parasanguis in an in vitro tooth model. Mol Microbiol 43: 147–157. PubMed

Mistry D, Stockley RA (2006) IgA1 protease. Int J Biochem Cell Biol 38: 1244–1248. PubMed PMC

Kilian M, Reinholdt J, Lomholt H, Poulsen K, Frandsen EV (1996) Biological significance of IgA1 proteases in bacterial colonization and pathogenesis: critical evaluation of experimental evidence. APMIS 104: 321–338. PubMed

Reinholdt J, Tomana M, Mortensen SB, Kilian M (1990) Molecular aspects of immunoglobulin A1 degradation by oral streptococci. Infect Immun 58: 1186–1194. PubMed PMC

Corfield T (1992) Bacterial sialidases–roles in pathogenicity and nutrition. Glycobiology 2: 509–521. PubMed

Severi E, Hood DW, Thomas GH (2007) Sialic acid utilization by bacterial pathogens. Microbiology 153: 2817–2822. PubMed

King SJ (2010) Pneumococcal modification of host sugars: a major contributor to colonization of the human airway? Mol Oral Microbiol 25: 15–24. PubMed

Lewis AL, Lewis WG (2012) Host sialoglycans and bacterial sialidases: a mucosal perspective. Cell Microbiol 14: 1174–1182. PubMed

Hastings MC, Moldoveanu Z, Suzuki H, Berthoux F, Julian BA, et al. (2013) Biomarkers in IgA nephropathy: relationship to pathogenetic hits. Expert Opin Med Diagn 7: 615–627. PubMed PMC

GalNAc-T14 may Contribute to Production of Galactose-Deficient Immunoglobulin A1, the Main Autoantigen in IgA Nephropathy

Quantitative assessment of successive carbohydrate additions to the clustered O-glycosylation sites of IgA1 by glycosyltransferases

Galactose-deficient IgA1 and the corresponding IgG autoantibodies predict IgA nephropathy progression

The Origin and Activities of IgA1-Containing Immune Complexes in IgA Nephropathy

N-acetylgalactosaminide α2,6-sialyltransferase II is a candidate enzyme for sialylation of galactose-deficient IgA1, the key autoantigen in IgA nephropathy