Ikuko Nakamura

Detection of gender difference and epitope specificity of IgG antibody activity against IgA1 hinge portion in IgA nephropathy patients by using synthetic hinge peptide and glycopeptide probes.

BACKGROUND AND AIMS There are many reports on the presence of an incompletely glycosylated O-linked oligosaccharide(s) on the IgA1 hinge region in some immunoglobulin (IgA) nephropathy patients. Furthermore, the production of an antibody against the naked hinge peptide portion was reported in an IgA nephropathy patient. In this report, characterization of the IgG antibody against the hinge portion was carried out by using synthetic hinge glycopeptide probes. METHODS AND RESULTS The following synthetic hinge peptide and glycopeptides were prepared: 19mer peptide, V-P-S-T-P-P-T-P-S-P-S-T-P-P-T-P-S-P-S (designated HP), the peptide having a single alpha-linked GalNAc residue at positions 4, 7, 9, 11 and 15 (4 GN - 15 GN, respectively) and the same peptide having five GalNAc residues at all five positions (GN5). The mean value of the antibody activity against these probes was compared with each other. The highest activity against the naked hinge peptide (HP) and lowest activity against the fully glycosylated hinge peptide (GN5) were obvious. As attachment of GalNAc to position 4 or 11 on the peptide brought about a significant reduction of the activity against the naked hinge peptide, the P-S-T-P sequence included in both positions was thought to be the most probable site recognized by these antibodies. As an additional unexpected observation, a gender difference in this antibody activity against all the probes was found. The antibody activity in a female was significantly higher compared with that in a male. CONCLUSION Because the frequency of incidence of IgA nephropathy is known to be slightly higher in males, this gender difference might indicate a protective meaning to remove aberrantly glycosylated molecules from the patients serum.

Quantitative analysis of IgA1 binding protein prepared from human serum by hypoglycosylated IgA1/Sepharose affinity chromatography

The binding protein to a hypoglycosylated IgA1/Sepharose (IgA1-BP) could be prepared from human sera. IgG was a major component in the IgA1-BP. A Protein A column was used to remove the IgG; however, about half of the IgA1-BP was passed from the column [Biochem. Biophys. Res. Commun., 264 (1999) 424]. Quantitative analysis of the passed fraction (PAP) by laser nepherometry indicated that it was composed of a fairly large amount of IgA, IgM and complement C3 besides IgG. The relative content of IgG:IgA:IgM:C3:C4 was 25:10:41:22:2 in the PAP fraction. Meanwhile, the Protein A bound-fraction was essentially composed of IgG (78%) and IgM (19%). The total amount of IgA1-BP was not different between the sera from IgA nephropathy patients and other nephropathy patients. With respect to the IgA content in the IgA1-BP from IgA nephropathy patients, it was significantly higher than that from other nephropathy patients. It was found that the IgA1-BP from some IgA nephropathy patients contained a few micrograms of aberrant IgA per ml of serum. Thus, the obtained results suggested the preferential deposition of the self-aggregated IgA composed of hypoglycosylated IgA1 and co-deposition of IgG, IgM and C3 in the glomeruli in an IgA nephropathy patient.

The structural alteration of O‐glycosylated hinge peptides in serum IgA1 before and after tonsillectomy in IgA nephropathy

The human IgA1 molecule has exceptional O-glycans in its hinge region. The fundamental structure of the Oglycans is the linkage between the a-anomeric carbon atom in N-acetylgalactosamine (GalNAc) and the hydroxy group of serine or threonine (GalNAc a1-O-Ser (Thr)). A wide structural variety of the sugar side-chains in glycoproteins is observed under physiological conditions. The phenomenon is called ‘the microheterogeneity of carbohydrates’. In several previous studies analysing the structural varieties of the O-glycans in the hinge region of serum IgA1 and the deposited IgA1 in glomeruli (glomerular IgA1) in IgA nephropathy (IgAN), it was found that the hinge glycopeptides in IgA1 were under-glycosylated. Recently, we investigated that the O-glycan structure of IgA1 molecules produced by tonsillar lymphocytes (tonsillar IgA1) were also under-glycosylated in IgAN patients. These findings suggested that the underglycosylated IgA1 might play a role in the cause of IgAN. Upper respiratory infections and tonsillitis often precede increases in haematuria and proteinuria in patients with IgAN. Béné et al. reported that tonsillectomy might be an efficient treatment for preventing the progression of IgAN. Recently, Hotta et al. reported that tonsillectomy combined with steroid pulse therapy (combination therapy) had a strong effect on IgAN. Therefore, tonsillectomy and combination therapy might have an influence upon the structural varieties of O-glycans of IgA1 in IgAN.

Quantitative and qualitative analysis of tonsillar IgA in IgA nephropathy patients

There are many reports of the presence of an incompletely glycosylated O-linked oligosaccharides in the IgA1 hinge region in some IgA nephropathy patients. Meanwhile, there are also some reports on the relationship of tonsillectomy and IgA nephropathy. In this experiment, immunoglobulins in the extract of tonsillectomized tissue and other sources were analysed by the isoelectoric focusing (IEF) and enzymelinked immunosorbent assay (ELISA). The IEF profile of the tonsillar IgA differed from that in the pooled serum and it was enriched in cationic IgA. The profiles were very similar in the extracts from chronic tonsillitis controls and IgA nephropathy patients. Similarly, the IEF profiles of serum IgA from controls and IgA nephropathy patients were also similar. Enzymatic removal of sialic acid induced a shift of the cathode side. Both the profiles of IgA from the treated tonsillar extract and the treated serum were approximately overlapped. On the other hand, the presence of asialo Galβ1,3GalNAc in the cationic IgA from tonsillar extract and aberrant IgA1 prepared from serum (IgA1 binding protein,) became clear with the enzymatic transfer of sialic acid to IgA1. Serum IgA also contained partly sialylated IgA1. Quantitative analysis of IgA and IgG in the extracts indicated that the IgA was significantly higher, but the IgG was significantly lower in IgA nephropathy patients. It was found that the IgA1 produced in tonsillar tissue differed from the serum IgA1. Furthermore, overproduction of asialo IgA1 due to the disordered balance between IgAand IgG-producing cells in the tonsil from the IgA nephropathy patient became obvious. Although the transfer of such an asialo IgA1 from tonsil tissue to the serum is still not completely clear, there is a possibility of a tonsillar source of a few micrograms of the aberrant IgA1 in the serum. REFERENCE