Amnionless-mediated glycosylation is crucial for cell surface targeting of cubilin in renal and intestinal cells

Mutations in either cubilin(CUBN) or amnionless(AMN) genes cause Imerslund–Grasbeck syndrome(IGS), a hereditary disease characterised by anaemia attributed to selective intestinal malabsorptionof cobalaminand low-molecular weight proteinuria. Although cubilinprotein does not have a transmembrane segment, it functions as a multi-ligand receptor by binding to the transmembrane protein, amnionless. We established a system to quantitatively analyse membrane targeting of the protein complex in cultured renal and intestinal cells and analysed the pathogenic mechanisms of mutations found in IGS patients. A novel CUBN mutation, several previously reported CUBN missense mutationsand all previously reported AMN missense mutationsresulted in endoplasmic reticulum ( ER) retentionand completely inhibited amnionless-dependent plasma membrane expression of cubilin. The ER retentionof cubilinand amnionlesswas confirmed in renal proximal tubular cells of a patient with IGS. Notably, the interaction between cubilinand amnionlesswas not sufficient, but amnionless-mediated glycosylation of cubilinwas necessary for their surface expression. Quantitative mass spectrometry and mutagenesis demonstrated that N-linked glycosylationof at least 4 residues of cubilinprotein was required for its surface targeting. These results delineated the molecular mechanisms of membrane trafficking of cubilinin renal and intestinal cells.