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

Abstract

Mutations in either cubilin (CUBN) or amnionless (AMN) genes cause Imerslund–Gräsbeck syndrome (IGS), a hereditary disease characterised by anaemia attributed to selective intestinal malabsorption of cobalamin and low-molecular weight proteinuria. Although cubilin protein 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 mutations and all previously reported AMN missense mutations resulted in endoplasmic reticulum (ER) retention and completely inhibited amnionless-dependent plasma membrane expression of cubilin. The ER retention of cubilin and amnionless was confirmed in renal proximal tubular cells of a patient with IGS. Notably, the interaction between cubilin and amnionless was not sufficient, but amnionless-mediated glycosylation of cubilin was necessary for their surface expression. Quantitative mass spectrometry and mutagenesis demonstrated that N-linked glycosylation of at least 4 residues of cubilin protein was required for its surface targeting. These results delineated the molecular mechanisms of membrane trafficking of cubilin in renal and intestinal cells.

In Vivo Expression of NUP93 and Its Alteration by NUP93 Mutations Causing Focal Segmental Glomerulosclerosis

“…To confirm the expression of NUP93 in endothelial cells, double-staining with CD31, an endothelial marker, was performed, and revealed expression of NUP93 in endothelial cells both within and outside the glomeruli (Figure 2b, A, B, 1, and 2). For the renal tubules, we performed double-staining with NUP93 and amnionless, as a marker of proximal tubules, 33 and confirmed that NUP93 was expressed in the form of dots in the nuclei of the proximal tubules (Figure 2b, C, D, and 3)and distal tubules (Figure 2b, C, D, and 4) of the human kidney sections.

Figure 3Clinical course and kidney histology of the affected individual with NUP93 mutations. Clinical course of the patient with focal segmental glomerulosclerosis carrying NUP93 mutations (a).

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Structural assembly of the megadalton-sized receptor for intestinal vitamin B12 uptake and kidney protein reabsorption

“…The AMN IGS mutations L59P 27 , M69K 31 , C234F 32 and G254E 27 are all retained in the ER when co-expressed with cubilin in HEK293 cells 16 , which explains why these mutations impair cubam receptor function and cause IGS. Introducing the individual mutations in our E. coli expression system does not yield any soluble AMN or AMN–cubilin complex (Supplementary Figure 5 ).…”

“…Cubam and megalin co-localise in the endocytic apparatus and appear to be functionally dependent on each other, at least in the kidney 43 , 44 . However, megalin is not required for cubam surface expression 16 and the fact that megalin seems less expressed in the intestine 45 suggests that cubam is capable of functioning independently of megalin. The extraordinary sizes of receptor ecto-domains in both megalin and cubam are remarkable and may be advantageous for optimal catching and endocytosis of multiple ligands from the extracellular fluids.…”

“…AMN is formed by an extracellular domain, a transmembrane helix and a short cytoplasmic domain harbouring two adaptor protein-2-binding signals (Phe-X-Asn-Pro-X-Phe) for ligand-independent internalisation in clathrin-coated pits 13 , 14 . As cubilin and AMN depend on each other for proper processing and translocation to the apical membrane, disrupted interaction of the two proteins leads to retention of both in the endoplasmic reticulum (ER) 13 , 15 , 16 . Accordingly, defects in the genes encoding either cubilin or AMN both give rise to IGS.…”

Proteinuria as a presenting sign of combined methylmalonic acidemia and homocysteinemia: case report

“…The latter shows high expression in the distal intestinal tract and proximal renal tubules, and has important roles in the absorption of Cbl within the intestine and reabsorption of urinary protein in the kidney. Hence, a CUBN mutation on chromosome 10 and/or AMN on chromosome 14 can lead to IGS [ 18 – 20 ]. Genetic analyses of peripheral blood-derived DNA in Case 2 revealed two heterozygous variants of AMN , c.742C > T (p.Q248*, from his mother) and c.761G > A (p.G254E, from his father), in accordance with IGS.…”