Mass spectrometry analysis of IgA1 hinge region in patients with IgA nephropathy.
BACKGROUND
Physicochemical alterations of the IgA molecule are supposed to play a pathogenetic role in IgA nephropathy (IgAN). The present study was carried out to analyze the structural variety of O-glycans on the IgA1 hinge region in IgAN. Sera from 9 IgAN patients and 9 healthy controls were individually examined to evaluate the IgA1 content and binding lectins (jacalin and Helix aspersa), using enzyme-linked immunosorbent assay (ELISA) techniques. The IgA1 from pooled sera were separated by affinity chromatography (jacalin), and the fragment containing the hinge region was prepared by pyridylethylation and trypsin treatment. The IgA fragments containing the hinge glycopeptide (33-mer hinge peptide core (HP) + O-glycans) were separated by jacalin affinity chromatography. Because we used jacalin, we only analyzed the Gal-3GalNAc residue containing IgA. The molecular weight (MW) of the IgA1 fragments was estimated using an ion trap mass spectrometer equipped with an electrospray ion source (ESI/MS).


RESULTS
IgA1 concentration in pathological sera was higher than in the control serum (p<0.01). Compared with controls, serum IgA1 from IgAN patients showed significantly greater binding to the 2 lectins, jacalin (p<0.01) and Helix aspersa (HA, p<0.001), which are specific for O-linked Gal-beta1,3-GalNAc and GalNAc, respectively. Analyses of pooled sera showed that the number of O-glycosidic chains was comparable in IgAN and normal sera. With regards to the individual residues, we found that IgAN sera contained less sugar and galactose and sialic acid moieties than sera from control subjects, was reduced in IgAN sera, while terminal N-acetylgalactosamine levels were higher when compared with normal serum.


CONCLUSIONS
Abnormalities of hinge region O-linked glycans were confirmed using advanced spectrometry technology. The pathogenetic implications for aggregation and defective removal of IgA1 are discussed.
Department of Analytical Chemistry, University of Turin, Turin - Italy.
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D
Gastaldi
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Paradisi
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Baiocchi
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Medana
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Lo Duca
L M
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Sena
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Roccatello
D000328:Adult; D000368:Aged; D020816:Amino Acid Motifs; D016022:Case-Control Studies; D002853:Chromatography, Liquid; D005260:Female; D005922:Glomerulonephritis, IGA / Q000503:physiopathology*; D006801:Humans; D007070:Immunoglobulin A / Q000737:chemistry*; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D011134:Polysaccharides / Q000737:chemistry*
D007070:Immunoglobulin A; D011134:Polysaccharides