Study Of Binding Dynamic Characteristic Of Deglycosylated IgA1 With Human Mesangial Cells

[Background and Objective] IgA nephropathy (IgAN) is the most common glomerular disease characterized by the predominant deposition of IgAl subclass in mesangial area, with a prevalence of 20-40% in primary glomerular disease. More important, over one third of patients with IgAN could progress to end-stage renal failure after 20-25 years and eventually required renal replacement therapy.Human IgA is highly glycosylated and contains two subclasses of IgAl and IgA2. IgAl subclass bears 5 O-linked glycans that are linked to serine or threonine residues between the CHI and CH2 domains in the hinge region of each heavy chain. N-acetylgalactosamine (GalNAc) is linked with the amino acid residues, and galactose (Gal) is linked in 6-1, 3 to GalNAc. Sialic acid (Neu5Ac) has an a-2, 6 linkage with GalNAc and a-2, 3 linkage with Gal residues. Recent studies have demonstrated that in IgAN the O-glycosylation pattern of serum and mesangial IgAl was abnormal, with reduced galactosylation and/or sialylation, leading to increased frequency of truncated glycan chains consisting of GalNAc and/or Gal alone by binding to specific lectins or mass spectrometry.Our previous study had demonstrated that aggregated-IgA1 (algA1) from patients with IgAN and healthy controls could bind to human mesangial cells (HMC), the binding capacity and pathophysiological effects of aIgA from patients with IgAN were much stronger than algAl from healthy controls. algAl from patients with IgANcould bind to cultured HMC with a higher affinity compared with algAl from healthy controls, and could induce enhanced HMC proliferation, cytokine expression, and extracellular matrix production. Our speculation was that glycosylation of IgAl might affect the binding capacity of IgAl with HMC and might play an important role in renal injury of IgAN.The purpose of the current study is to investigate the binding capacities of different in vitro deglycosylated IgAl on HMC with standard radio-ligand binding assay.[Methods] Serum IgAl was purified by jacalin affinity chromatography andthen were desialylated and/or degalactosylated with neuraminidase and/or 6-galactosidase. The efficacy of deglycosylations was assessed by Peanut agglutinin and Vicia villosa lectin. The sizes of normal IgAl and deglycosylated IgAl were determined by Sephacryl S-300 chromatography and binding capacities to primary HMC were evaluated by radioligand binding assays. The results are expressed as mean ± SD. Comparisons among means were performed using one-way ANOVA and S-N-K test in SPSS (SPSS Inc. Chicago, IL, USA) version 11.0 statistical analysis program. Take "a=0. 05" as test standard.[ Results ] The average molecular size of IgAl, DesIgAl andDesIgAl/DeGallgAl were 268kd, 373kd and 314kd respectively. Normal IgAl and deglycosylated IgAlcould bind to HMC in a dose-dependent, saturable manner and the maximal binding capacities of normal IgAl, DesIgAl and Des/DeGallgAl were 161.08±61.18fmol/105cell, 297.26±93.45fmol/105cell and 317.92±19.27fmol/105cell (normal IgAl vs DesIgAl, P=0.044, normal IgAl vs Des/DeGallgAl, P=0.026) respectively. The binding sites/cell were 0.97±0.37xl06/cell, 1.77±0.56xl06/cell and 2.56±0.22xl06/cell (normal IgAl vs DesIgAl, normal IgAl vs Des/DeGallgAl, ZM).O5) respectively based on a condation that one IgAl receptor could bind one IgAl molecule only. However, more aggregated IgAl were found in DesIgAl and Des/DeGallgAl. Scatchard analysis revealed a Kd of 7.43±3.23xl0~7mol/L, 8.43±4.44xl0"7mol/Land 11.3±0.56xlO'7mol/L (P>0.05) respectively.[Conclusions] The binding capacities of DesIgAl and Des/DeGallgAl to HMC were significantly higher than that of normal IgAl, which at least in part was due to more macromolecular IgAl in deglycoslated IgAl. However, there were nosignificant differences in the affinities of normal IgAl, DesIgAl and Des/DeGallgAl with HMC. Deglycosylated IgAl might play an important role in the pathogenesis of IgAN.