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

Abstract

The endocytic receptor cubam formed by the 460-kDa protein cubilin and the 45-kDa transmembrane protein amnionless (AMN), is essential for intestinal vitamin B12 (B12) uptake and for protein (e.g. albumin) reabsorption from the kidney filtrate. Loss of function of any of the two components ultimately leads to serious B12 deficiency and urinary protein loss in humans (Imerslund-Gräsbeck’s syndrome, IGS). Here, we present the crystal structure of AMN in complex with the amino-terminal region of cubilin, revealing a sophisticated assembly of three cubilin subunits combining into a single intertwined β-helix domain that docks to a corresponding three-faced β-helix domain in AMN. This β-helix-β-helix association thereby anchors three ligand-binding cubilin subunits to the transmembrane AMN. Electron microscopy of full-length cubam reveals a 700–800 Å long tree-like structure with the potential of dimerization into an even larger complex. Furthermore, effects of known human mutations causing IGS are explained by the structural information.

Advances of the experimental models of idiopathic membranous nephropathy (Review)

“…These studies confirm that the membrane attack complex (MAC; c5b-9) formed by the activation of the immune complex is the main inflammatory mediator of Heymann nephritis and is closely related to the production of pathological proteinuria (32,33). Furthermore, although megalin is expressed in human podocytes (34,35), it is not detected in the glomerular subepithelial immune complex and no circulating anti-megalin antibodies are found in patients with iMn (36). in addition, the complement pathway and subclass of igG in this model are still unknown (37)(38)(39).…”

Human C-terminal CUBN variants associate with chronic proteinuria and normal renal function

“…With regard to the protein structures, megalin and amnionless are type I transmembrane proteins, whereas cubilin is a peripheral protein that requires amnionless for anchoring to the membrane. Anchoring occurs via β-helix-β-helix association between amnionless and the N-terminal hydrophobic stretches of 3 cubilin subunits (17). Each cubilin protomer has 8 EGF domains and 27 CUB (complement C1r/C1s, UEGF [EGF-related sea urchin protein] and bone morphogenic protein 1) domains, some of which are involved in Ca 2+ -dependent ligand binding (17,18).…”

“…Anchoring occurs via β-helix-β-helix association between amnionless and the N-terminal hydrophobic stretches of 3 cubilin subunits (17). Each cubilin protomer has 8 EGF domains and 27 CUB (complement C1r/C1s, UEGF [EGF-related sea urchin protein] and bone morphogenic protein 1) domains, some of which are involved in Ca 2+ -dependent ligand binding (17,18). Most IGS mutations of CUBN are in the N-terminal half of cubilin, either Table 4).…”

“…affecting the interactions with amnionless or the vitamin B12/ intrinsic factor-binding (IF-binding) CUB domains 5-8 (CUB5-8) (17). Interestingly, 1 individual with a homozygous deletion of exon 53 harboring CUB20 was shown to have proteinuria without vitamin B12 malabsorption (19).…”

Suspected Metformin-induced Cobalamin Deficiency Mimicking Thrombotic Thrombocytopenic Purpura

“…The proposed theories have included inhibition of cobalamin absorption, changes in intrinsic factor levels, normal flora or gastrointestinal (GI) motility [5]. The cubulin endocytic receptor formed by the 460-kDa protein cubilin and the 45-kDa transmembrane protein amnionless, is responsible for the absorption of cobalamin-intrinsic factor complex in the intestine [6]. Metformin has been found to disrupt the calcium-dependent membrane activity of this receptor, and hence decreases the absorption of cobalamin by the body [5].…”