Amnionless function is required for cubilin brush-border expression and intrinsic factor-cobalamin (vitamin B12) absorption in vivo

Abstract

IntroductionAdult-onset gastrointestinal malabsorption of the essential micronutrient, cobalamin (vitamin B 12 ), leads to potentially lethal manifestations as diverse as megaloblastic anemia and neutropenia, degeneration of spinal cord nerve tracts, and dementia. 1 In the very young, signs of cobalamin malabsorption may also include growth retardation or loss of developmental milestones or both. ImerslundGräsbeck syndrome (I-GS; also called megaloblastic anemia 1, OMIM no. 261100) is an autosomal recessive disorder characterized by selective malabsorption of cobalamin in the intestine and, most often, of specific low-molecular-weight proteins in renal proximal tubules. 2,3 Patients with I-GS typically present at 2 to 4 years of age with signs of cobalamin deficiency and proteinuria. More than 200 human cases and familial clusters have been reported in Finland, Norway, the Middle East, and Northern Africa. 1 Various null and missense mutations of the cubilin (CUBN) 4 and amnionless (AMN) genes 5 have been implicated in I-GS kindreds, but both loci have been excluded in some. 6 In addition, a number of patients suffering from gastric intrinsic factor (IF) deficiency have been misdiagnosed as having I-GS. 7 Cubilin is a 460-kDa multiligand receptor protein that mediates endocytosis of the IF-cobalamin complex from distal small intestinal chyme and of several proteins from glomerular filtrate in renal proximal tubules. 8 AMN is an approximately 48-kDa apical membrane protein also expressed in intestinal and proximal tubule epithelia but whose function is poorly defined. 5,9 Recent studies suggested that cubilin and AMN function as subunits of a receptor complex, now called "cubam." 10 However, no studies have directly assessed the effect of CUBN or AMN mutations on cubam expression in patients with I-GS because these proteins are expressed in inaccessible tissues, and the clinical disease is easily treated. Unfortunately, the AMN knockout mouse exhibits an embryonic lethal phenotype. 9 Canine I-GS was derived originally from purebred giant schnauzers (GSs) as a naturally occurring animal model of the human disorder [11][12][13] and has contributed significantly to understanding of its molecular biologic basis as well as aspects of cubilin function. 14-17 CUBN was excluded from the GS disease locus, 18 and the disorder was recently mapped to an approximately 4-Mb interval predicted to harbor AMN. 19 To define the molecular basis of the canine I-GS model, we sought AMN mutations segregating in the GS kindred and an unrelated kindred of Australian shepherd (AS) dogs and investigated in vivo AMN and cubilin expression in affected dogs. Additionally, comparison of these results to expression of mutant canine AMN in a heterologous mammalian-cell transfection system validates the cell-culture system for ex vivo Materials and methods AnimalsDogs were handled according to principles and protocols approved by the MSU All University Committee for Animal Use and Care. MSU University Laboratory Animal Resources housed dogs of...

Luminal expression of cubilin is impaired in Imerslund-Grasbeck syndrome with compound AMN mutations in intron 3 and exon 7

“…This interdependency of cubilin and amnionless in cubilin brushborder expression has also been shown in a spontaneous Imerslund-Gräsbeck canine model with a mutated AMN homolog. 23 Our cases with a compound mutation in AMN and no mutation in CUBN provide the first evidence in humans that cubilin depends on AMN for its correct expression in the epithelium of the proximal tubule. An inverse situation was recently reported where the altered interdependency of both proteins was due to a CUBN mutation in exon 23 and no mutation in AMN.…”

Degenerative Liver Disease in Young Beagles with Hereditary Cobalamin Malabsorption Because of a Mutation in the Cubilin Gene

“…This is a rare autosomal recessive disorder which, if left untreated, results in failure to thrive, megaloblastic anemia, proteinuria, and neurological damage 2. In dogs, primary cobalamin malabsorption, which is analogous to IGS in humans, has been reported in young Australian Shepherds,3 a Beagle,4 Border Collies,5, 6, 7 and Giant Schnauzers 8. The genetic defects in affected Border Collies and Beagles recently have been identified as 2 independent mutations in the CUBN gene 9, 10.…”

Inherited metabolic disease in companion animals: Searching for nature’s mistakes

“…As previously reported by Fordyce et al (2000), this patient was successfully treated by parenteral vitamin B12 administration every 2-4 weeks. Cobalamin malabsorption has also been described in giant Schnauzers (Fyfe et al, 1991), Border collies (Battersby et al, 2005), and Australian shepherd dogs (He et al, 2005). Investigations of affected Giant schnauzers and Australian shepherd dogs at the molecular level (Fyfe et al, 2004;He et al, 2005) have confirmed that this disease is a true homologue of congenital megaloblastic anaemia due to vitamin B12 deficiency because of a defect in the vitamin B12/intrinsic factor receptor (Imerslund-Gräsbeck syndrome) in humans.…”

“…Cobalamin malabsorption has also been described in giant Schnauzers (Fyfe et al, 1991), Border collies (Battersby et al, 2005), and Australian shepherd dogs (He et al, 2005). Investigations of affected Giant schnauzers and Australian shepherd dogs at the molecular level (Fyfe et al, 2004;He et al, 2005) have confirmed that this disease is a true homologue of congenital megaloblastic anaemia due to vitamin B12 deficiency because of a defect in the vitamin B12/intrinsic factor receptor (Imerslund-Gräsbeck syndrome) in humans. This disease is caused by mutations in either the gene encoding cubilin (Aminoff et al, 1999) or amnionless (Tanner et al, 2003).…”