Reduced immunoglobulin A transcytosis associated with immunoglobulin A nephropathy and nasopharyngeal carcinoma

Abstract

Polymeric IgA (pIgA) is transcytosed by the pIgA receptor (pIgR) across mucosal epithelial cells. After transcytosis to the apical surface, the extracellular, ligand-binding portion of the pIgR is proteolytically cleaved. A missense mutation in human pIgR, A580V, is associated with IgA nephropathy and nasopharyngeal carcinoma. We report that this mutation reduces the rate of transcytosis of pIgR and pIgA, and seemingly the rate of pIgR cleavage. We propose that the defects in pIgR trafficking caused by the A580V mutation may underlie the pathogenesis of both diseases.

FIGURE 1.

pIgR-WT and pIgR-A580V expression in MDCK cells. A, similar expression level of pIgR is shown in pIgR-WT and pIgR-A580V cells by immunofluorescent (IF) staining. B, immunoblot (IB) shows duplicate pIgR-WT- and pIgR-A580V-expressing MDCK samples, with actin as a loading control, showing equivalent WT and A580V expression. Cropped boxes represent different bands from the same gel and exposure.

FIGURE 2.

A580V mutation in human pIgR decreases pIgR/pIgA transcytosis in pIgR-expressing MDCK cells. A, pIgR/pIgA transcytosis was significantly decreased in pIgR-A580V-expressing cells, compared with pIgR-WT. Cells were basolaterally labeled with biotin at 17 °C for 30 min and washed at 17 °C for 30 min. Cells were then chased with MEM/BSA at 37 °C for 60 min. Top, apically released SC (Ap-SC) is expressed as a percentage of total labeled pIgR in all fractions (i.e. SC in AP and BL media, and pIgR). Middle, basolaterally released SC (BL-SC) is expressed as a percentage of total labeled pIgR in all fractions. Bottom, pIgR remaining in the cells is expressed as a percentage of total pIgR in all fractions. More AP-SC is seen in the pIgR-WT-expressing cells than for pIgR-A580V (n = 12). B, transcytosis of biotinylated pIgA was also significantly decreased in pIgR-A580V-expressing cells compared with pIgR-WT after 40 and 80 min of transcytosis (n = 14).

FIGURE 3.

A580V mutation in human pIgR increases pIgA endocytosis in pIgR-expressing MDCK cells. A, filter-grown pIgR-WT- and pIgR-A580V-expressing cells were treated apically with biotinylated pIgA at 4 °C. Cells were warmed to 37 °C for the indicated period to allow pIgR-pIgA internalization. Finally, cells were cooled to 4 °C, and internalized pIgA levels were determined by resistance to exogenous protease addition. AP endocytosis is expressed as a percentage of total labeled pIgA. AP pIgA endocytosis rates were increased significantly in plgR-A580V-expressing cells compared with plgR-WT from 5 to 30 min (n = 4 at 5 min, n = 6 at 10 and 30 min). B, representative Western blotting (IB) image from A. Samples with equal amount of protein were blotted for biotinylated pIgA shown as pIgA-HC (heavy chain) and pIgA-LC (light chain). Cropped boxes represent different bands from the same gel and exposure.

FIGURE 4.

A580V mutation in human pIgR decreases pIgR AP cleavage in pIgR-expressing MDCK cells. A, filter-grown pIgR cells were apically biotinylated at 4 °C. Samples were precipitated by rabbit anti-human pIgR antibodies and then blotted for biotinylated SC and pIgR. AP pIgR cleavage (presented as a percentage of total AP-labeled pIgR) was measured at 5 min at 37 °C. A decrease in pIgR-A580V cleavage over pIgR-WT occurred (n = 8). B, representative Western blotting (IB) image from A is shown. Cropped boxes represent different bands from the same gel and exposure.