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. 2005 Aug 15;106(4):1447-53.
doi: 10.1182/blood-2005-03-1197. Epub 2005 Apr 21.

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

Qianchuan He  1 , Mette Madsen, Adam Kilkenney, Brittany Gregory, Erik I Christensen, Henrik Vorum, Peter Højrup, Alejandro A Schäffer, Ewen F Kirkness, Stephan M Tanner, Albert de la Chapelle, Urs Giger, Søren K Moestrup, John C Fyfe
Affiliations

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

Qianchuan He et al. Blood. .

Abstract

Amnionless (AMN) and cubilin gene products appear to be essential functional subunits of an endocytic receptor called cubam. Mutation of either gene causes autosomal recessive Imerslund-Gräsbeck syndrome (I-GS, OMIM no. 261100) in humans, a disorder characterized by selective intestinal malabsorption of cobalamin (vitamin B12) and urinary loss of several specific low-molecular-weight proteins. Vital insight into the molecular pathology of I-GS has been obtained from studies of dogs with a similar syndrome. In this work, we show that I-GS segregates in a large canine kindred due to an in-frame deletion of 33 nucleotides in exon 10 of AMN. In a second, unrelated I-GS kindred, affected dogs exhibit a homozygous substitution in the AMN translation initiation codon VSports手机版. Studies in vivo demonstrated that both mutations abrogate AMN expression and block cubilin processing and targeting to the apical membrane. The essential features of AMN dysfunction observed in vivo are recapitulated in a heterologous cell-transfection system, thus validating the system for analysis of AMN-cubilin interactions. Characterization of canine AMN mutations that cause I-GS establishes the canine model as an ortholog of the human disorder well suited to studies of AMN function and coevolution with cubilin. .

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"V体育2025版" Figures

Figure 1.
Figure 1.
Tissue expression AMN and CUBN cDNA. Total RNA isolated from the designated tissues was amplified by RT-PCR. AMN primers were designed to amplify the full-length coding sequence (product size 1.4 kbp). CUBN primers amplified exons 8-12 (product size 0.5 kbp). PPID primers were used as a positive control for success of the RT reaction in each RNA sample (product size 0.6 kbp). Lanes: 1, kidney; 2, muscle; 3, spleen; 4, thymus; 5, liver; 6, heart; 7, lung; 8, pituitary; 9, testis; 10, cerebellum; 11, cerebrum; 12, placenta; 13, duodenum; 14, jejunum; 15, ileum; 16, colon; 17, pancreas.
Figure 2.
Figure 2.
Immunodetection and characterization of AMN in canine kidney. AMN was enriched from membrane (lanes 1-4) or supernatant (lane 5) fractions of homogenates of normal dog kidney cortex by IF-cobalamin affinity column chromatography. Eluted proteins were separated by SDS-PAGE (lanes 1-3) or by 2-dimensional gel electrophoresis (blots 4 and 5). Proteins in lanes 1 and 2 were subjected to mock and PNGase F digestion, respectively, prior to electrophoresis and blotting. The additional, lightly stained band in lane 2 migrates at the expected position of PNGase F. Antiserum against the extracellular domain peptide A, as shown in the schematic, was used for immunodetection in 1, 2, 4, and 5, and antiserum against the cytoplasmic domain peptide B was used in lane 3. The schematic of the mature, full-length AMN protein indicates the relative positions of the site of N-linked oligosaccharide addition (NXT/S), the cysteine-rich domain (CRD), and peptides A (amino acid residues 340-353) and B (residues 388-400) against which antisera were raised. The excess positive charge of the intracellular domain protein sequence including peptide B is + 3. Two vertical lines indicate the single transmembrane domain.
Figure 3.
Figure 3.
AMN mutations in canine I-GS. (A) Normal canine AMN cDNA sequence in exon 10 (above) and the corresponding c.1113_1145del mutation sequence of the GS kindred disease allele (below) are shown. Two copies of a near-perfect 24-bp repeat (underlined) apparently predisposed this locus to deletion by unequal crossover. (B) Normal canine AMN cDNA sequence in exon 1 (above) and the corresponding c.3G>A mutation sequence (arrowhead) of the AS kindred disease allele (below) are shown. The putative translation initiation site of the normal sequence is underlined and conforms to the Kozak consensus. (C) Representative mutation screening tests performed with gDNA templates are shown.
Figure 4.
Figure 4.
I-GS segregating in an Australian shepherd dog (AS) pedigree. Squares are males and circles are females, and the arrow indicates the proposita. Open and partially filled symbols indicate dogs that were clinically healthy as determined by physical examination, if genotyped, or by family history, if not. Symbols filled in the upper left quadrant indicate dogs that were genotyped at the KNS2 locus, and symbols filled in the upper right quadrant indicate dogs genotyped for the AMN c.3G>A mutation. Symbols filled in the lower right quadrant indicate dogs determined by genotyping to be heterozygous (G/A) at the mutation site. The 3 clinically affected dogs, indicated by symbols filled in all quadrants, were genotyped at both loci and determined to be homozygous A/A at the mutation site. The 2 symbols filled in the lower right but not the upper right quadrant indicate dogs that were inferred from family data to be heterozygous at the mutation site. The female dog labeled A is an ancestor common to all dogs in the kindred determined to be carriers of the c.3G>A AMN allele.
Figure 5.
Figure 5.
Cubilin and AMN expression in normal and I-GS affected dog kidney in vivo. (A) Membrane fractions prepared in bulk from normal (N) and GS kindred affected (A) dog kidney cortex were detergent solubilized and subjected to IF-cobalamin affinity chromatography. Eluted proteins were concentrated, separated by SDS-PAGE on an 8% to 16% gradient gel, and silver stained. Two bands missing from the affected dog preparation migrated at approximately 48 and 40 kDa (arrowheads) and comigated with anti-AMN immunoreactive bands on Western blots of duplicate gels. They were identified by LC/MS/MS peptide mapping as full-length and C-terminally truncated AMN, respectively. (B) Anti-AMN Western blots of proteins isolated by large-scale IF-cobalamin affinity chromatography from normal dog kidney (lane 1) or by small-scale IF-cobalamin pull-down from kidney cortex homogenates of a normal dog (lane 2), a GS kindred affected dog (lane 3), and an AS kindred affected dog (lane 4). (C) Immunoperoxidase staining of cubilin in renal cortex of an unrelated normal dog (left) and I-GS affected dogs of the GS (middle) and AS (right) kindreds expressing AMN mutations c.1113-1145del and c.3G>A, respectively. Total original magnification, 200 ×.
Figure 6.
Figure 6.
Cubilin expression in CHO cells transfected with wild-type or c.1113-1145del AMN cDNA. (A) CHO cell lines expressing a “mini-cubilin” construct of rat origin were additionally transfected with wild-type (WT) or c.1113-1145del (mut) canine AMN cDNA constructs, and stable double transfectants expressing cubilin and AMN were selected. Nonpermeabilized cells were stained for confocal fluorescence microscopy by incubation at 4°C with anticubilin and subsequently with labeled anti-rabbit IgG. Abundant surface staining of cubilin was observed in cells expressing wild-type, but not mutant, AMN. (B) Proteins isolated by IF-cobalamin pull-down from lysates of double-transfectant cell lines were subjected to endo H or mock digestion followed by SDS-PAGE and Western blot with anticubilin serum. Golgi maturation of cubilin N-linked oligosaccharides to endo H-resistant forms was observed in cells expressing wild-type AMN, but not in cells expressing the mutant AMN. (C) Lysates of double-transfectant cell lines were subjected to Western blotting with anti-myc, confirming expression of wild-type and mutant AMN.
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V体育安卓版 - References

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    1. Gräsbeck R, Gordin R, Kantero I, Kuhlback B. Selective vitamin B12 malabsorption and proteinuria in young people. Acta Med Scand. 1960;167: 289-296. - PubMed
    1. Aminoff M, Carter JE, Chadwick RB, et al. Mutations in CUBN, encoding the intrinsic factor-vitamin B12 receptor, cubilin, cause hereditary megaloblastic anaemia 1. Nat Genet. 1999;21: 309-313. - PubMed
    1. Tanner SM, Aminoff M, Wright FA, et al. Amnionless, essential for mouse gastrulation, is mutated in recessive hereditary megaloblastic anemia. Nat Genet. 2003;33: 426-429. - PubMed

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