Calnexin and ERp57 Facilitate the Assembly of the Neonatal Fc Receptor for IgG with β2-Microglobulin in the Endoplasmic Reticulum

Abstract

The neonatal FcR (FcRn) consists of an MHC class I-like H chain in nonconvalent association with β2-microglobulin (β2m). The proper folding of FcRn in the endoplasmic reticulum is essential for FcRn function. Using a low stringency immunoprecipitation of human FcRn, we observed the coprecipitation of an 88-kDa band. Mass spectrometry analysis revealed that this band was identical with calnexin (CNX). This association was verified by Western blotting the CNX or FcRn immunoprecipitates with either an anti-FcRn or anti-CNX Ab. In the β2m-null FO-1 cell transfected with FcRn H chain alone or both FcRn H chain and β2m, CNX bound to the FcRn H chain before the FcRn H chain association with β2m. However, calreticulin only bound to the FcRn H chain-β2m complex. Furthermore, the thiol oxidoreductase ERp57 was detected in FcRn-CNX complexes, suggesting its role in disulfide bond formation of the FcRn H chain. Removal of the N-linked glycosylation site from the FcRn H chain resulted in a decreased association of the FcRn H chain for β2m. However, the absence of CNX did not significantly affect FcRn assembly as defined by the ability of FcRn to bind IgG and exit to the cell surface. This suggests that other chaperones compensate for the function of CNX in FcRn assembly. In addition, we found that tapasin and TAP were not involved in FcRn assembly, as shown by coimmunoprecipitation in THP-1 cells and IgG-binding assays in 721.220 (tapasin-deficient) and 721.174 (TAP-deficient) cells transfected with FcRn. These findings show the importance of chaperones in FcRn assembly.