N-Glycan Moieties in Neonatal Fc Receptor Determine Steady-state Membrane Distribution and Directional Transport of IgG

Timothy T Kuo,Eric J De Muinck,Steven M Claypool,Masaru Yoshida,Takashi Nagaishi,Victoria G Aveson,Wayne I Lencer,Richard S Blumberg

doi:10.1074/jbc.m805877200

Timothy T Kuo, Eric J De Muinck + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m805877200

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Abstract

The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I-related molecule known to protect IgG and albumin from catabolism and transport IgG across polarized epithelial cells in a bidirectional manner. Previous studies have shown species-specific differences in ligand binding, IgG transport direction, and steady-state membrane distribution when expressed in polarized epithelial cells. We hypothesized that these differences may be due to the additional N-glycans expressed on the rat FcRn, because N-glycans have been proposed to function as apical targeting signals, and that two of the N-glycan moieties have been shown to contribute to the IgG binding of rat FcRn. A panel of mutant human FcRn variants was generated to resemble the N-glycan expression of rat FcRn in various combinations and subsequently transfected into Madin-Darby canine kidney II cells together with human beta2-microglobulin. Mutant human FcRn clones that contained additional N-glycan side-chain modifications, including that which was fully rodentized, still exhibited specificity for human IgG and failed to bind to mouse IgG. At steady state, the mutant human FcRn with additional N-glycans redistributed to the apical cell surface similar to that of rat FcRn. Furthermore, the rodentized human FcRn exhibited a reversal of IgG transport with predominant transcytosis from an apical-to-basolateral direction, which resembled that of the rat FcRn isoform. These studies show that the N-glycans in FcRn contribute significantly to the steady-state membrane distribution and direction of IgG transport in polarized epithelia.

Highlights

Nothing is known with respect to FcRn. Most studies of both human and rat FcRn trafficking in epithelial cells have focused on the cytoplasmic tail, which has revealed the critical role of this domain in basolateral membrane targeting, endocytosis, and transcytosis
Using a similar mutagenesis method, hFcRn without any N-glycans was generated as an additional control (0-hFcRn). These human FcRn mutants and rat FcRn constructs were transfected into Madin-Darby canine kidney (MDCK) II cells that expressed the corresponding species of human or rat ␤2m, because we have previously found that this is substrate-limiting in MDCK II cells [22]
Little is known about the regulation of FcRn trafficking in epithelial cells with most of the knowledge deriving from an assessment of the role of the cytoplasmic tail in rodents and human

Summary

Introduction

To determine whether this apical redistribution of the rodentized hFcRn (4N-hFcRn) was observed with other hFcRn isoforms created (see supplemental Fig. S2), we performed the following series of studies on MDCK II cells expressing hFcRn containing one and two additional N-glycan carbohydrate modifications within the ␣1, ␣2, and/or ␣3 domains.

Results

Conclusion