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. 2007 Jun;148(3):507-14.
doi: 10.1111/j.1365-2249.2007.03374.x. Epub 2007 Mar 26.

Differential binding characteristics of native monomeric and polymeric immunoglobulin A1 (IgA1) on human mesangial cells and the influence of in vitro deglycosylation of IgA1 molecules

Y-H Gao  1 , L-X Xu, J-J Zhang, Y Zhang, M-H Zhao, H-Y Wang
Affiliations

Differential binding characteristics of native monomeric and polymeric immunoglobulin A1 (IgA1) on human mesangial cells and the influence of in vitro deglycosylation of IgA1 molecules

Y-H Gao et al. Clin Exp Immunol. 2007 Jun.

Abstract

Recent studies had demonstrated that serum and mesangial immunoglobulin A1 (IgA1) in patients with IgA nephropathy (IgAN) were polymeric and deglycosylated. The current study was to investigate the binding characteristics of monomeric and polymeric normal human IgA1 on mesangial cells and the influence of in vitro deglycosylation of IgA1 molecules. The normal human IgA1 was desialylated and degalactosylated with specific enzymes, respectively. The monomeric IgA1 (mIgA1) and polymeric IgA1 (pIgA1) were separated by Sephacryl S-300 chromatography. The binding capacities of the mIgA1 and pIgA1 to primary human mesangial cells (HMC) were evaluated by classical radioligand assay. Both the native mIgA1 and pIgA1 could bind to HMC in a dose-dependent and saturable manner. The maximal binding capacity of the native pIgA1 were significantly higher than that of the native mIgA1 (P < 0.05). However, the affinity of the native mIgA1 was almost 100 times higher than that of the native pIgA1. After deglycosylation, binding of the two deglycosylated mIgA1 to HMC could not be detected. However, the maximal binding capacities of the two deglycosylated pIgA1 to HMC were increased significantly compared with that of native pIgA1. The affinity of the two deglycosylated pIgA1 was similar to that of native pIgA1 (P > 0.05). The current study suggests differential binding characteristics of native monomeric and polymeric IgA1 on mesangial cells. Glycosylation of IgA1 molecules could significantly affect the binding of IgA1 on HMC.

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Figures

Fig. 1
Fig. 1
Reactivity of the native immunoglobulin A1 (IgA1), desialylated (deSIgA1) and further degalactosylated IgA1 (deS/deGal IgA1) with Sambuscus nigra agglutinin (SNA) and Vicia vilosa lectin (VVL). SNA could specifically bind the terminal sialic acid. The terminal sialic acid of O-glycan in deSIgA1 and deS/deGalIgA1 was effectively removed. The sialic acid level of native mIgA1 was significantly higher than that of deS mIgA1 and deS/deGal mIgA1 (P < 0·05) (a). The sialic acid level of native pIgA1 was significantly higher than that of deS pIgA1 and deS/deGal pIgA1 (P < 0·05) (b). VVL could specifically bind the terminal GalNAc. The sialic acid and galactose were removed and the terminal GalNAc of O-glycan in deSIgA1/deGalIgA1 was highly exposed. The GalNAc level of deS/deGal mIgA1 was significantly higher than that of native mIgA1 and deS mIgA1 (P < 0·05) (c). The GalNAc level of deS/deGal pIgA1 was significantly higher than that of native pIgA1 and deS pIgA1 (P < 0·05) (d). Native mIgA1: monomeric native IgA1; deS mIgA1: monomeric deSIgA1; deS/deGal mIgA1: monomeric deS/deGalIgA1; native pIgA1: polymeric native IgA1; deS pIgA1: polymeric deSIgA1 and deS/deGal pIgA1: polymeric deS/deGalIgA1.
Fig. 2
Fig. 2
The purity of the six immunoglobulin A1 (IgA1) fractions and changes of the size of the IgA heavy chain after deglycosylation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the six IgA1 fractions were highly pure and the size of the IgA1 heavy chain was slightly decreased after deglycosylation. Lane 1: native monomeric IgA1 (mIgA1); lane 2: desialylated (deS) mIgA1; lane 3: deS/degalactosylated (deGal) IgA1; lane 4: native polymeric IgA1 (pIgA1); lane 5: deS pIgA1; lane 6: deS/deGal pIgA1.
Fig. 3
Fig. 3
The binding characteristics and binding capacities of the native monomeric immunoglobulin A1 (mIgA1) and polymeric IgA1 (pIgA1) to human mesangial cells (HMC). The binding of native mIgA1 and native pIgA1 to HMC was in a dose-dependent and saturable manner. The binding capacity of native pIgA1 was significantly higher than that of native mIgA1 (P < 0·05).
Fig. 4
Fig. 4
Scatchard analysis of Kd values of the native monomeric immunoglobulin A1 (mIgA1) and polymeric IgA1 (pIgA1) binding to human mesangial cells. Kd of native mIgA1 and native pIgA1 were 6·25 ± 4·33 × 10−9mol/l and 1·12 ± 0·54 × 10−7mol/l, respectively. The affinity of native mIgA1 was almost 100 times higher than that of native pIgA1.
Fig. 5
Fig. 5
The binding characteristics of deglycosylated monomeric immunoglobulin A1 (mIgA1) to human mesangial cells (HMC). The binding of desialylated (deS) mIgA1 and deS/degalactosylated (deGal) mIgA1 to HMC was linear.
Fig. 6
Fig. 6
The binding characteristics and binding capacities of three polymeric immunoglobulin A1 (IgA1) to human mesangial cells (HMC). The binding of the three polymeric IgA1 to HMC was in a dose-dependent and saturable manner. The binding capacities of the two deglycosylated polymeric IgA1 were significantly higher than that of the native polymeric IgA1 (P < 0·05, respectively).
Fig. 7
Fig. 7
Scatchard analysis of Kd values of three polymeric immunoglobulin A1 (pIgA1) binding to human mesangial cells (HMC). The affinity to HMC of the three polymeric IgA1 was similar. Kd of native pIgA1, desialylated (deS) pIgA1 and deS/degalactosylated (deGal) pIgA1 were 1·12 ± 0·54 × 10−7mol/l, 3·10 ± 2·00 × 10−7mol/l and 3·44 ± 2·70 × 10−7mol/l, respectively (P > 0·05).
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