Clustered O-Glycans of IgA1
2010; Elsevier BV; Volume: 9; Issue: 11 Linguagem: Inglês
10.1074/mcp.m110.001834
ISSN1535-9484
AutoresKazuo Takahashi, Stephanie Wall, Hitoshi Suzuki, Archer D. Smith, Stacy Hall, Knud Poulsen, Mogens Kilian, James A. Mobley, Bruce A. Julian, Jiří Městecký, Jan Novák, Matthew B. Renfrow,
Tópico(s)Renal Diseases and Glomerulopathies
ResumoIgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) O-glycans, plays a pivotal role in the pathogenesis of the disease. It is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. We have described the utility of activated ion-electron capture dissociation (AI-ECD) mass spectrometric analysis of IgA1 O-glycosylation. However, locating and characterizing the entire range of O-glycan attachment sites are analytically challenging due to the clustered serine and threonine residues in the HR of IgA1 heavy chain. To address this problem, we analyzed all glycoforms of the HR glycopeptides of a Gal-deficient IgA1 myeloma protein, mimicking the aberrant IgA1 in patients with IgAN, by use of a combination of IgA-specific proteases + trypsin and AI-ECD Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS). The IgA-specific proteases provided a variety of IgA1 HR fragments that allowed unambiguous localization of all O-glycosylation sites in the six most abundant glycoforms, including the sites deficient in Gal. Additionally, this protocol was adapted for on-line liquid chromatography (LC)-AI-ECD MS/MS and LC-electron transfer dissociation MS/MS analysis. Our results thus represent a new clinically relevant approach that requires ECD/electron transfer dissociation-type fragmentation to define the molecular events leading to pathogenesis of a chronic kidney disease. Furthermore, this work offers generally applicable principles for the analysis of clustered sites of O-glycosylation. IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) O-glycans, plays a pivotal role in the pathogenesis of the disease. It is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. We have described the utility of activated ion-electron capture dissociation (AI-ECD) mass spectrometric analysis of IgA1 O-glycosylation. However, locating and characterizing the entire range of O-glycan attachment sites are analytically challenging due to the clustered serine and threonine residues in the HR of IgA1 heavy chain. To address this problem, we analyzed all glycoforms of the HR glycopeptides of a Gal-deficient IgA1 myeloma protein, mimicking the aberrant IgA1 in patients with IgAN, by use of a combination of IgA-specific proteases + trypsin and AI-ECD Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS). The IgA-specific proteases provided a variety of IgA1 HR fragments that allowed unambiguous localization of all O-glycosylation sites in the six most abundant glycoforms, including the sites deficient in Gal. Additionally, this protocol was adapted for on-line liquid chromatography (LC)-AI-ECD MS/MS and LC-electron transfer dissociation MS/MS analysis. Our results thus represent a new clinically relevant approach that requires ECD/electron transfer dissociation-type fragmentation to define the molecular events leading to pathogenesis of a chronic kidney disease. Furthermore, this work offers generally applicable principles for the analysis of clustered sites of O-glycosylation. Glycosylation is one of the most common post-translational modifications of proteins. It is estimated that over half of mammalian proteins are glycosylated. Patients with several autoimmune disorders, chronic inflammatory diseases, and some infectious diseases exhibit abnormal glycosylation of serum immunoglobulins and other glycoproteins (1Mestecky J. Tomana M. Crowley-Nowick P.A. Moldoveanu Z. Julian B.A. Jackson S. Defective galactosylation and clearance of IgA1 molecules as a possible etiopathogenic factor in IgA nephropathy.Contrib. Nephrol. 1993; 104: 172-182Crossref PubMed Google Scholar, 2Moore J.S. Wu X. Kulhavy R. Tomana M. Novak J. Moldoveanu Z. Brown R. Goepfert P.A. Mestecky J. Increased levels of galactose-deficient IgG in sera of HIV-1-infected individuals.AIDS. 2005; 19: 381-389Crossref PubMed Scopus (89) Google Scholar, 3Rademacher T.W. Williams P. Dwek R.A. Agalactosyl glycoforms of IgG autoantibodies are pathogenic.Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 6123-6127Crossref PubMed Scopus (224) Google Scholar, 4Springer G.F. Immunoreactive T and Tn epitopes in cancer diagnosis, prognosis, and immunotherapy.J. Mol. Med. 1997; 75: 594-602Crossref PubMed Scopus (384) Google Scholar, 5Troelsen L.N. Garred P. Madsen H.O. Jacobsen S. Genetically determined high serum levels of mannose-binding lectin and agalactosyl IgG are associated with ischemic heart disease in rheumatoid arthritis.Arthritis Rheum. 2007; 56: 21-29Crossref PubMed Scopus (54) Google Scholar). The biological functions of these modifications in health and disease have become a significant area of interest in biomedical research (6Rudd P.M. Elliott T. Cresswell P. Wilson I.A. Dwek R.A. Glycosylation and the immune system.Science. 2001; 291: 2370-2376Crossref PubMed Scopus (1369) Google Scholar). A subset of these glycoproteins has clustered sites of O-glycosylation with serine- and threonine-rich stretches within the amino acid sequence. Mucins, such as membrane-associated MUC1, are perhaps the best known family of proteins that are heavily O-glycosylated. Their altered expression and aberrant glycosylation have made them potential targets as biomarkers for early detection of cancer (7Storr S.J. Royle L. Chapman C.J. Hamid U.M. Robertson J.F. Murray A. Dwek R.A. Rudd P.M. The O-linked glycosylation of secretory/shed MUC1 from an advanced breast cancer patient's serum.Glycobiology. 2008; 18: 456-462Crossref PubMed Scopus (115) Google Scholar). Immunoglobulin A1 (IgA1) 1The abbreviations used are:IgA1immunoglobulin A1IgANIgA nephropathyHRhinge regionECDelectron capture dissociationAIactivated ionETDelectron transfer dissociationSAsupplemental activationHAAGalNAc-specific lectin from Helix aspersaRPreversed phaseAGCautomatic gain controlLTQlinear quadrupole ion trap. 1The abbreviations used are:IgA1immunoglobulin A1IgANIgA nephropathyHRhinge regionECDelectron capture dissociationAIactivated ionETDelectron transfer dissociationSAsupplemental activationHAAGalNAc-specific lectin from Helix aspersaRPreversed phaseAGCautomatic gain controlLTQlinear quadrupole ion trap. contains both O- and N-glycans (Fig. 1). Aberrant O-glycosylation of IgA1 is involved in the pathogenesis of IgA nephropathy (IgAN) and the closely related Henoch-Schönlein purpura nephritis (1Mestecky J. Tomana M. Crowley-Nowick P.A. Moldoveanu Z. Julian B.A. Jackson S. Defective galactosylation and clearance of IgA1 molecules as a possible etiopathogenic factor in IgA nephropathy.Contrib. Nephrol. 1993; 104: 172-182Crossref PubMed Google Scholar, 8Novak J. Moldoveanu Z. Renfrow M.B. Yanagihara T. Suzuki H. Raska M. Hall S. Brown R. Huang W.Q. Goepfert A. Kilian M. Poulsen K. Tomana M. Wyatt R.J. Julian B.A. Mestecky J. IgA nephropathy and Henoch-Schoenlein purpura nephritis: aberrant glycosylation of IgA1, formation of IgA1-containing immune complexes, and activation of mesangial cells.Contrib. Nephrol. 2007; 157: 134-138Crossref PubMed Google Scholar). Interestingly, the aberrantly glycosylated molecules, IgA1 in IgAN and MUC1 in cancer, are recognized by the immune system as neoepitopes as evidenced by formation of specific antibodies (9Suzuki H. Fan R. Zhang Z. Brown R. Hall S. Julian B.A. Chatham W.W. Suzuki Y. Wyatt R.J. Moldoveanu Z. Lee J.Y. Robinson J. Tomana M. Tomino Y. Mestecky J. Novak J. Aberrantly glycosylated IgA1 in IgA nephropathy patients is recognized by IgG antibodies with restricted heterogeneity.J. Clin. Investig. 2009; 119: 1668-1677PubMed Google Scholar, 10Tomana M. Novak J. Julian B.A. Matousovic K. Konecny K. Mestecky J. Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies.J. Clin. Investig. 1999; 104: 73-81Crossref PubMed Scopus (382) Google Scholar, 11Vlad A.M. Kettel J.C. Alajez N.M. Carlos C.A. Finn O.J. MUC1 immunobiology: from discovery to clinical applications.Adv. Immunol. 2004; 82: 249-293Crossref PubMed Scopus (182) Google Scholar). Mucin-like bacterial surface proteins exhibit similar properties: the molecules have clustered bacterial O-glycans that mediate cellular adhesion, and blocking antibodies target these glycan-containing epitopes (12Stephenson A.E. Wu H. Novak J. Tomana M. Mintz K. Fives-Taylor P. 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. 2002; 43: 147-157Crossref PubMed Scopus (77) Google Scholar).An O-glycosylated protein from a single source contains a population of variably O-glycosylated isoforms that show a distinct distribution of microheterogeneity of the O-glycan chains in terms of number, sites of attachment, and composition. Characterizing these clustered sites and understanding how the distributions change under different biological conditions or disease states are an analytical challenge. Enzymatic or chemical release of O-glycans is not selective. The heterogeneity, composition, and quantitative aspects of different O-glycan chains can be assessed and quantified by gas chromatographic and/or mass spectrometric techniques. However, the site-specific information and context of location and composition of adjacent chains are lost. Carbohydrate-specific lectin analysis of O-glycoproteins can provide information on glycan composition and comparative differences between samples, such as those from healthy controls and patients with various disease states. We have successfully demonstrated this in the analysis of IgA1 O-glycans from patients with IgAN versus healthy controls and disease controls (13Moldoveanu Z. Wyatt R.J. Lee J.Y. Tomana M. Julian B.A. Mestecky J. Huang W.Q. Anreddy S.R. Hall S. Hastings M.C. Lau K.K. Cook W.J. Novak J. Patients with IgA nephropathy have increased serum galactose-deficient IgA1 levels.Kidney Int. 2007; 71: 1148-1154Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 14Suzuki H. Moldoveanu Z. Hall S. Brown R. Vu H.L. Novak L. Julian B.A. Tomana M. Wyatt R.J. Edberg J.C. Alarcón G.S. Kimberly R.P. Tomino Y. Mestecky J. Novak J. IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1.J. Clin. Investig. 2008; 118: 629-639PubMed Google Scholar, 15Tomana M. Matousovic K. Julian B.A. Radl J. Konecny K. Mestecky J. Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG.Kidney Int. 1997; 52: 509-516Abstract Full Text PDF PubMed Scopus (276) Google Scholar). This included proximal assessment of sites with galactose (Gal)-deficient O-glycans after digests with IgA-specific proteases (8Novak J. Moldoveanu Z. Renfrow M.B. Yanagihara T. Suzuki H. Raska M. Hall S. Brown R. Huang W.Q. Goepfert A. Kilian M. Poulsen K. Tomana M. Wyatt R.J. Julian B.A. Mestecky J. IgA nephropathy and Henoch-Schoenlein purpura nephritis: aberrant glycosylation of IgA1, formation of IgA1-containing immune complexes, and activation of mesangial cells.Contrib. Nephrol. 2007; 157: 134-138Crossref PubMed Google Scholar). Several studies have demonstrated the value of mass spectrometry (MS) in identifying Gal-deficient IgA1 in patients with IgAN (16Hiki Y. Odani H. Takahashi M. Yasuda Y. Nishimoto A. Iwase H. Shinzato T. Kobayashi Y. Maeda K. Mass spectrometry proves under-O-glycosylation of glomerular IgA1 in IgA nephropathy.Kidney Int. 2001; 59: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 17Hiki Y. Tanaka A. Kokubo T. Iwase H. Nishikido J. Hotta K. Kobayashi Y. Analyses of IgA1 hinge glycopeptides in IgA nephropathy by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.J. Am. Soc. Nephrol. 1998; 9: 577-582Crossref PubMed Google Scholar, 18Novak J. Tomana M. Kilian M. Coward L. Kulhavy R. Barnes S. Mestecky J. Heterogeneity of O-glycosylation in the hinge region of human IgA1.Mol. Immunol. 2000; 37: 1047-1056Crossref PubMed Scopus (59) Google Scholar, 19Odani H. Hiki Y. Takahashi M. Nishimoto A. Yasuda Y. Iwase H. Shinzato T. Maeda K. 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. 2000; 271: 268-274Crossref PubMed Scopus (80) Google Scholar, 20Renfrow M.B. Cooper H.J. Tomana M. Kulhavy R. Hiki Y. Toma K. Emmett M.R. Mestecky J. Marshall A.G. Novak J. Determination of aberrant O-glycosylation in the IgA1 hinge region by electron capture dissociation Fourier transform-ion cyclotron resonance mass spectrometry.J. Biol. Chem. 2005; 280: 19136-19145Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 21Tarelli E. Resistance to deglycosylation by ammonia of IgA1 O-glycopeptides: implications for the β-elimination of O-glycans linked to serine and threonine.Carbohydr. Res. 2007; 342: 2322-2325Crossref PubMed Scopus (9) Google Scholar), including our work that demonstrated the first direct localization of native sites of O-glycan chains in the hinge region (HR) of IgA1 by use of electron capture dissociation (ECD) (20Renfrow M.B. Cooper H.J. Tomana M. Kulhavy R. Hiki Y. Toma K. Emmett M.R. Mestecky J. Marshall A.G. Novak J. Determination of aberrant O-glycosylation in the IgA1 hinge region by electron capture dissociation Fourier transform-ion cyclotron resonance mass spectrometry.J. Biol. Chem. 2005; 280: 19136-19145Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 22Renfrow M.B. Mackay C.L. Chalmers M.J. Julian B.A. Mestecky J. Kilian M. Poulsen K. Emmett M.R. Marshall A.G. Novak J. Analysis of O-glycan heterogeneity in IgA1 myeloma proteins by Fourier transform ion cyclotron resonance mass spectrometry: implications for IgA nephropathy.Anal. Bioanal. Chem. 2007; 389: 1397-1407Crossref PubMed Scopus (78) Google Scholar). ECD and the more recently developed electron transfer dissociation (ETD) have been used to identify sites of O-glycosylation on a variety of proteins (23Chalkley R.J. Thalhammer A. Schoepfer R. Burlingame A.L. Identification of protein O-GlcNAcylation sites using electron transfer dissociation mass spectrometry on native peptides.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8894-8899Crossref PubMed Scopus (195) Google Scholar, 24Khidekel N. Ficarro S.B. Clark P.M. Bryan M.C. Swaney D.L. Rexach J.E. Sun Y.E. Coon J.J. Peters E.C. Hsieh-Wilson L.C. Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics.Nat. Chem. Biol. 2007; 3: 339-348Crossref PubMed Scopus (255) Google Scholar, 25Mirgorodskaya E. Roepstorff P. 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Localization of O-glycans in MUC1 glycoproteins using electron-capture dissociation fragmentation mass spectrometry.Glycobiology. 2009; 19: 375-381Crossref PubMed Scopus (33) Google Scholar, 27Vosseller K. Trinidad J.C. Chalkley R.J. Specht C.G. Thalhammer A. Lynn A.J. Snedecor J.O. Guan S. Medzihradszky K.F. Maltby D.A. Schoepfer R. Burlingame A.L. O-Linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry.Mol. Cell. Proteomics. 2006; 5: 923-934Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar).IgAN is the most common primary glomerulonephritis worldwide (28Julian B.A. Waldo F.B. Rifai A. Mestecky J. IgA nephropathy, the most common glomerulonephritis worldwide. A neglected disease in the United States?.Am. J. Med. 1988; 84: 129-132Abstract Full Text PDF PubMed Scopus (232) Google Scholar) with about 20–40% of patients developing end stage renal failure. It is characterized by mesangial deposits of IgA1-containing immune complexes (28Julian B.A. Waldo F.B. Rifai A. Mestecky J. IgA nephropathy, the most common glomerulonephritis worldwide. A neglected disease in the United States?.Am. J. Med. 1988; 84: 129-132Abstract Full Text PDF PubMed Scopus (232) Google Scholar). The distinctive O-glycan chains of IgA1 molecules play a pivotal role in the pathogenesis of IgAN (1Mestecky J. Tomana M. Crowley-Nowick P.A. Moldoveanu Z. Julian B.A. Jackson S. Defective galactosylation and clearance of IgA1 molecules as a possible etiopathogenic factor in IgA nephropathy.Contrib. Nephrol. 1993; 104: 172-182Crossref PubMed Google Scholar, 10Tomana M. Novak J. Julian B.A. Matousovic K. Konecny K. Mestecky J. Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies.J. Clin. Investig. 1999; 104: 73-81Crossref PubMed Scopus (382) Google Scholar, 14Suzuki H. Moldoveanu Z. Hall S. Brown R. Vu H.L. Novak L. Julian B.A. Tomana M. Wyatt R.J. Edberg J.C. Alarcón G.S. Kimberly R.P. Tomino Y. Mestecky J. Novak J. IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1.J. Clin. Investig. 2008; 118: 629-639PubMed Google Scholar, 15Tomana M. Matousovic K. Julian B.A. Radl J. Konecny K. Mestecky J. Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG.Kidney Int. 1997; 52: 509-516Abstract Full Text PDF PubMed Scopus (276) Google Scholar, 16Hiki Y. Odani H. Takahashi M. Yasuda Y. Nishimoto A. Iwase H. Shinzato T. Kobayashi Y. Maeda K. Mass spectrometry proves under-O-glycosylation of glomerular IgA1 in IgA nephropathy.Kidney Int. 2001; 59: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 29Allen A.C. Bailey E.M. Brenchley P.E. Buck K.S. Barratt J. Feehally J. Mesangial IgA1 in IgA nephropathy exhibits aberrant O-glycosylation: observation in three patients.Kidney Int. 2001; 60: 969-973Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 30Novak J. Julian B.A. Tomana M. Mesteck J. Progress in molecular and genetic studies of IgA nephropathy.J. Clin. Immunol. 2001; 21: 310-327Crossref PubMed Scopus (97) Google Scholar). IgA1 contains an HR between the first and second heavy chain constant region domains with a high content of Ser, Thr, and Pro. This segment usually has three to five O-glycan chains per HR (31Mattu 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 Fcα receptor interactions.J. Biol. Chem. 1998; 273: 2260-2272Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar) (see Fig. 1). Aberrantly glycosylated IgA1, deficient in Gal in some of the O-glycans in the HR, in serum is rare in healthy individuals but is present at elevated levels in IgAN patients (13Moldoveanu Z. Wyatt R.J. Lee J.Y. Tomana M. Julian B.A. Mestecky J. Huang W.Q. Anreddy S.R. Hall S. Hastings M.C. Lau K.K. Cook W.J. Novak J. Patients with IgA nephropathy have increased serum galactose-deficient IgA1 levels.Kidney Int. 2007; 71: 1148-1154Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 15Tomana M. Matousovic K. Julian B.A. Radl J. Konecny K. Mestecky J. Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG.Kidney Int. 1997; 52: 509-516Abstract Full Text PDF PubMed Scopus (276) Google Scholar). This distinctive IgA1 is in circulating immune complexes (8Novak J. Moldoveanu Z. Renfrow M.B. Yanagihara T. Suzuki H. Raska M. Hall S. Brown R. Huang W.Q. Goepfert A. Kilian M. Poulsen K. Tomana M. Wyatt R.J. Julian B.A. Mestecky J. IgA nephropathy and Henoch-Schoenlein purpura nephritis: aberrant glycosylation of IgA1, formation of IgA1-containing immune complexes, and activation of mesangial cells.Contrib. Nephrol. 2007; 157: 134-138Crossref PubMed Google Scholar, 10Tomana M. Novak J. Julian B.A. Matousovic K. Konecny K. Mestecky J. Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies.J. Clin. Investig. 1999; 104: 73-81Crossref PubMed Scopus (382) Google Scholar, 15Tomana M. Matousovic K. Julian B.A. Radl J. Konecny K. Mestecky J. Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG.Kidney Int. 1997; 52: 509-516Abstract Full Text PDF PubMed Scopus (276) Google Scholar) and in the glomerular deposits of IgAN patients (16Hiki Y. Odani H. Takahashi M. Yasuda Y. Nishimoto A. Iwase H. Shinzato T. Kobayashi Y. Maeda K. Mass spectrometry proves under-O-glycosylation of glomerular IgA1 in IgA nephropathy.Kidney Int. 2001; 59: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 29Allen A.C. Bailey E.M. Brenchley P.E. Buck K.S. Barratt J. Feehally J. Mesangial IgA1 in IgA nephropathy exhibits aberrant O-glycosylation: observation in three patients.Kidney Int. 2001; 60: 969-973Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). The absence of Gal apparently leads to the exposure of neoepitopes, including terminal and sialylated N-acetylgalactosamine (GalNAc) residues (9Suzuki H. Fan R. Zhang Z. Brown R. Hall S. Julian B.A. Chatham W.W. Suzuki Y. Wyatt R.J. Moldoveanu Z. Lee J.Y. Robinson J. Tomana M. Tomino Y. Mestecky J. Novak J. Aberrantly glycosylated IgA1 in IgA nephropathy patients is recognized by IgG antibodies with restricted heterogeneity.J. Clin. Investig. 2009; 119: 1668-1677PubMed Google Scholar, 10Tomana M. Novak J. Julian B.A. Matousovic K. Konecny K. Mestecky J. Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies.J. Clin. Investig. 1999; 104: 73-81Crossref PubMed Scopus (382) Google Scholar). These epitopes are recognized by naturally occurring anti-glycan IgG or IgA1 antibodies and, consequently, circulating immune complexes are formed (9Suzuki H. Fan R. Zhang Z. Brown R. Hall S. Julian B.A. Chatham W.W. Suzuki Y. Wyatt R.J. Moldoveanu Z. Lee J.Y. Robinson J. Tomana M. Tomino Y. Mestecky J. Novak J. Aberrantly glycosylated IgA1 in IgA nephropathy patients is recognized by IgG antibodies with restricted heterogeneity.J. Clin. Investig. 2009; 119: 1668-1677PubMed Google Scholar, 10Tomana M. Novak J. Julian B.A. Matousovic K. Konecny K. Mestecky J. Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies.J. Clin. Investig. 1999; 104: 73-81Crossref PubMed Scopus (382) Google Scholar, 15Tomana M. Matousovic K. Julian B.A. Radl J. Konecny K. Mestecky J. Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG.Kidney Int. 1997; 52: 509-516Abstract Full Text PDF PubMed Scopus (276) Google Scholar) that can deposit in the glomerular mesangia. To identify the pathogenic forms of IgA1, a thorough analysis of O-glycan microheterogeneity, including identification of the attachment sites, will be required.In this work, we demonstrate the complete analysis of O-glycoform microheterogeneity and site localization of the glycoforms in a naturally Gal-deficient IgA1 (Ale) myeloma protein that mimics the nephritogenic IgA1 in patients with IgAN (8Novak J. Moldoveanu Z. Renfrow M.B. Yanagihara T. Suzuki H. Raska M. Hall S. Brown R. Huang W.Q. Goepfert A. Kilian M. Poulsen K. Tomana M. Wyatt R.J. Julian B.A. Mestecky J. IgA nephropathy and Henoch-Schoenlein purpura nephritis: aberrant glycosylation of IgA1, formation of IgA1-containing immune complexes, and activation of mesangial cells.Contrib. Nephrol. 2007; 157: 134-138Crossref PubMed Google Scholar, 9Suzuki H. Fan R. Zhang Z. Brown R. Hall S. Julian B.A. Chatham W.W. Suzuki Y. Wyatt R.J. Moldoveanu Z. Lee J.Y. Robinson J. Tomana M. Tomino Y. Mestecky J. Novak J. Aberrantly glycosylated IgA1 in IgA nephropathy patients is recognized by IgG antibodies with restricted heterogeneity.J. Clin. Investig. 2009; 119: 1668-1677PubMed Google Scholar). Reversed phase (RP) LC FT-ICR MS successfully identified 10 distinct IgA1 HR fragments representing >99% of total IgA1. AI-ECD of the six most abundant IgA1 HR glycoforms (>95% of total IgA1) was accomplished with three distinct IgA-specific protease + trypsin digestions, identifying sites of Gal deficiency across four distinct IgA1 O-glycoforms. Based on the success of the ECD fragmentation of these IgA1 HR fragments, we adapted the analysis for on-line LC-MS/MS methods for both ECD and ETD. The variety of IgA1 HR proteolytic fragments provides a practical set of guidelines for the ECD/ETD analysis of clustered sites of O-glycosylation on this and other proteins. These results also provide insight into the order of attachment of the O-glycans in the IgA1 HR.DISCUSSIONIgA1 myeloma proteins frequently exhibit a high abundance of Gal-deficient O-glycans similar to those on nephritogenic IgA1 in patients with IgAN (8Novak J. Moldoveanu Z. Renfrow M.B. Yanagihara T. Suzuki H. Raska M. Hall S. Brown R. Huang W.Q. Goepfert A. Kilian M. Poulsen K. Tomana M. Wyatt R.J. Julian B.A. Mestecky J. IgA nephropathy and Henoch-Schoenlein purpura nephritis: aberrant glycosylation of IgA1, formation of IgA1-containing immune complexes, and activation of mesangial cells.Contrib. Nephrol. 2007; 157: 134-138Crossref PubMed Google Scholar, 9Suzuki H. Fan R. Zhang Z. Brown R. Hall S. Julian B.A. Chatham W.W. Suzuki Y. Wyatt R.J. Moldoveanu Z. Lee J.Y. Robinson J. Tomana M. Tomino Y. Mestecky J. Novak J. Aberrantly glycosylated IgA1 in IgA nephropathy patients is recognized by IgG antibodies with restricted heterogeneity.J. Clin. Investig. 2009; 119: 1668-1677PubMed Google Scholar, 10Tomana M. Novak J. Julian B.A. Matousovic K. Konecny K. Mestecky J. Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies.J. Clin. Investig. 1999; 104: 73-81Crossref PubMed Scopus (382) Google Scholar, 13Moldoveanu Z. Wyatt R.J. Lee J.Y. Tomana M. Julian B.A. Mestecky J. Huang W.Q. Anreddy S.R. Hall S. Hastings M.C. Lau K.K. Cook W.J. Novak J. Patients with IgA nephropathy have increased serum galactose-deficient IgA1 levels.Kidney Int. 2007; 71: 1148-1154Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 14Suzuki H. Moldoveanu Z. Hall S. Brown R. Vu H.L. Novak L. Julian B.A. Tomana M. Wyatt R.J. Edberg J.C. Alarcón G.S. Kimberly R.P. Tomino Y. Mestecky J. Novak J. IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1.J. Clin. Investig. 2008; 118: 629-639PubMed Google Scholar, 15Tomana M. Matousovic K. Julian B.A. Radl J. Konecny K. Mestecky J. Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG.Kidney Int. 1997; 52: 509-516Abstract Full Text PDF PubMed Scopus (276) Google Scholar), and because they are readily available in large amounts, they serve as convenient model proteins. 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