Cubilin P1297L mutation associated with hereditary megaloblastic anemia 1 causes impaired recognition of intrinsic factor–vitamin B12 by cubilin

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 405-409 ◽  
Author(s):  
Mette Kristiansen ◽  
Maria Aminoff ◽  
Christian Jacobsen ◽  
Albert de la Chapelle ◽  
Ralf Krahe ◽  
...  
Keyword(s):  
Vitamin B12 ◽  
Megaloblastic Anemia ◽  
Intrinsic Factor ◽  
Autosomal Recessive Disorder ◽  
Site Directed Mutagenesis ◽  
Mutant Form ◽  
Wild Type ◽  
Surface Plasmon Resonance Analysis ◽  
Mammalian Expression ◽  
Iodine 125

Megaloblastic anemia 1 (MGA1) is an autosomal recessive disorder caused by the selective intestinal malabsorption of intrinsic factor (IF) and vitamin B12/cobalamin (Cbl) in complex. Most Finnish patients with MGA1 carry the disease-specific P1297L mutation (FM1) in the IF-B12 receptor, cubilin. By site-directed mutagenesis, mammalian expression, and functional comparison of the purified wild-type and FM1 mutant forms of the IF–Cbl-binding cubilin region (CUB domains 5-8, amino acid 928-1386), we have investigated the functional implications of the P1297L mutation. Surface plasmon resonance analysis revealed that the P1297L substitution specifically increases the Kd for IF–Cbl binding several-fold, largely by decreasing the association rate constant. In agreement with the binding data, the wild-type protein, but not the FM1 mutant protein, potently inhibits 37°C uptake of iodine 125–IF–Cbl in cubilin-expressing epithelial cells. In conclusion, the data presented show a substantial loss in affinity of the FM1 mutant form of the IF–Cbl binding region of cubilin. This now explains the malabsorption of Cbl and Cbl-dependent anemia in MGA1 patients with the FM1 mutation.

Cubilin P1297L mutation associated with hereditary megaloblastic anemia 1 causes impaired recognition of intrinsic factor–vitamin B12 by cubilin

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 405-409 ◽  
Author(s):  
Mette Kristiansen ◽  
Maria Aminoff ◽  
Christian Jacobsen ◽  
Albert de la Chapelle ◽  
Ralf Krahe ◽  
...  
Keyword(s):  
Vitamin B12 ◽  
Megaloblastic Anemia ◽  
Intrinsic Factor ◽  
Autosomal Recessive Disorder ◽  
Site Directed Mutagenesis ◽  
Mutant Form ◽  
Wild Type ◽  
Surface Plasmon Resonance Analysis ◽  
Mammalian Expression ◽  
Iodine 125

Abstract Megaloblastic anemia 1 (MGA1) is an autosomal recessive disorder caused by the selective intestinal malabsorption of intrinsic factor (IF) and vitamin B12/cobalamin (Cbl) in complex. Most Finnish patients with MGA1 carry the disease-specific P1297L mutation (FM1) in the IF-B12 receptor, cubilin. By site-directed mutagenesis, mammalian expression, and functional comparison of the purified wild-type and FM1 mutant forms of the IF–Cbl-binding cubilin region (CUB domains 5-8, amino acid 928-1386), we have investigated the functional implications of the P1297L mutation. Surface plasmon resonance analysis revealed that the P1297L substitution specifically increases the Kd for IF–Cbl binding several-fold, largely by decreasing the association rate constant. In agreement with the binding data, the wild-type protein, but not the FM1 mutant protein, potently inhibits 37°C uptake of iodine 125–IF–Cbl in cubilin-expressing epithelial cells. In conclusion, the data presented show a substantial loss in affinity of the FM1 mutant form of the IF–Cbl binding region of cubilin. This now explains the malabsorption of Cbl and Cbl-dependent anemia in MGA1 patients with the FM1 mutation.

The functional cobalamin (vitamin B12)–intrinsic factor receptor is a novel complex of cubilin and amnionless

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1573-1579 ◽  
Author(s):  
John C. Fyfe ◽  
Mette Madsen ◽  
Peter Højrup ◽  
Erik I. Christensen ◽  
Stephan M. Tanner ◽  
...  
Keyword(s):  
Vitamin B12 ◽  
Autosomal Recessive ◽  
Megaloblastic Anemia ◽  
Intrinsic Factor ◽  
Gel Filtration ◽  
Autosomal Recessive Disorder ◽  
Lysosomal Degradation ◽  
Amino Terminal ◽  
Genes Encoding ◽  
Vitamin B12 Malabsorption

Abstract Imerslund-Gräsbeck syndrome (I-GS, megaloblastic anemia 1) is an autosomal recessive disorder characterized by intestinal cobalamin (vitamin B12) malabsorption and proteinuria. I-GS–causing mutations are found in either of 2 genes encoding the epithelial proteins: cubilin and amnionless (AMN). Cubilin recognizes intrinsic factor (IF)–cobalamin and various other proteins to be endocytosed in the intestine and kidney, respectively, whereas the function of AMN is unknown. Here we show that cubilin and AMN colocalize in the endocytic apparatus of polarized epithelial cells and copurify as a tight complex during IF-cobalamin affinity and nondenaturing gel filtration chromatography. In transfected cells expressing either AMN or a truncated IF-cobalamin–binding cubilin construct, neither protein alone conferred ligand endocytosis. In cubilin transfectants, cubilin accumulated in early biosynthetic compartments. However, in cells cotransfected with AMN and the cubilin construct, cubilin trafficked to the cell surface and endosomes, and the cells exhibited IF-cobalamin endocytosis and lysosomal degradation of IF. These data indicate that cubilin and AMN are subunits of a novel cubilin/AMN (cubam) complex, where AMN binds to the amino-terminal third of cubilin and directs subcellular localization and endocytosis of cubilin with its ligand. Therefore, mutations affecting either of the 2 proteins may abrogate function of the cubam complex and cause IG-S.

scholarly journals The Human Intrinsic Factor-Vitamin B12 Receptor,Cubilin: Molecular Characterization and Chromosomal Mapping of the Gene to 10p Within the Autosomal Recessive Megaloblastic Anemia (MGA1) Region

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3593-3600 ◽  
Author(s):  
Renata Kozyraki ◽  
Mette Kristiansen ◽  
Asli Silahtaroglu ◽  
Claus Hansen ◽  
Christian Jacobsen ◽  
...  
Keyword(s):  
Vitamin B12 ◽  
Autosomal Recessive ◽  
Megaloblastic Anemia ◽  
Intrinsic Factor ◽  
Recessive Gene ◽  
Proteolytic Processing ◽  
Chromosomal Mapping ◽  
Surface Plasmon Resonance Analysis ◽  
Amino Terminal ◽  
Resonance Analysis

Abstract Uptake of vitamin B12 (cyanocobalamin) is facilitated by the cobalamin-binder gastric intrinsic factor (IF), which recognizes a 460-kD receptor, cubilin, present in the epithelium of intestine and kidney. Surface plasmon resonance analysis of ligand-affinity-purified human cubilin demonstrated a high-affinity calcium- and cobalamin-dependent binding of IF-cobalamin. Complete cDNA cloning of the human receptor showed a 3597 amino acid peripheral membrane protein with 69% identity to rat cubilin. Amino-terminal sequencing of the receptor indicates that the cDNA sequence encodes a precursor protein undergoing proteolytic processing due to cleavage at a recognition site (Arg7-Glu8-Lys9-Arg10) for the trans-Golgi proteinase furin. Using fluorescence in situ hybridization, radiation hybrid mapping, and screening of YAC clones, the human cubilin gene was mapped between the markers D10S1661 and WI-5445 on the short arm of chromosome 10. This is within the autosomal recessive megaloblastic anemia (MGA1) 6-cM region harboring the unknown recessive-gene locus of juvenile megaloblastic anemia caused by intestinal malabsorption of cobalamin (Imerslund-Gräsbeck's disease). In conclusion, the present molecular and genetic information on human cubilin now provides circumstantial evidence that an impaired synthesis, processing, or ligand binding of cubilin is the molecular background of this hereditary form of megaloblastic anemia.

scholarly journals The Human Intrinsic Factor-Vitamin B12 Receptor,Cubilin: Molecular Characterization and Chromosomal Mapping of the Gene to 10p Within the Autosomal Recessive Megaloblastic Anemia (MGA1) Region

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3593-3600 ◽  
Author(s):  
Renata Kozyraki ◽  
Mette Kristiansen ◽  
Asli Silahtaroglu ◽  
Claus Hansen ◽  
Christian Jacobsen ◽  
...  
Keyword(s):  
Vitamin B12 ◽  
Autosomal Recessive ◽  
Megaloblastic Anemia ◽  
Intrinsic Factor ◽  
Recessive Gene ◽  
Proteolytic Processing ◽  
Chromosomal Mapping ◽  
Surface Plasmon Resonance Analysis ◽  
Amino Terminal ◽  
Resonance Analysis

Uptake of vitamin B12 (cyanocobalamin) is facilitated by the cobalamin-binder gastric intrinsic factor (IF), which recognizes a 460-kD receptor, cubilin, present in the epithelium of intestine and kidney. Surface plasmon resonance analysis of ligand-affinity-purified human cubilin demonstrated a high-affinity calcium- and cobalamin-dependent binding of IF-cobalamin. Complete cDNA cloning of the human receptor showed a 3597 amino acid peripheral membrane protein with 69% identity to rat cubilin. Amino-terminal sequencing of the receptor indicates that the cDNA sequence encodes a precursor protein undergoing proteolytic processing due to cleavage at a recognition site (Arg7-Glu8-Lys9-Arg10) for the trans-Golgi proteinase furin. Using fluorescence in situ hybridization, radiation hybrid mapping, and screening of YAC clones, the human cubilin gene was mapped between the markers D10S1661 and WI-5445 on the short arm of chromosome 10. This is within the autosomal recessive megaloblastic anemia (MGA1) 6-cM region harboring the unknown recessive-gene locus of juvenile megaloblastic anemia caused by intestinal malabsorption of cobalamin (Imerslund-Gräsbeck's disease). In conclusion, the present molecular and genetic information on human cubilin now provides circumstantial evidence that an impaired synthesis, processing, or ligand binding of cubilin is the molecular background of this hereditary form of megaloblastic anemia.

scholarly journals 11 Study of Polynucleotide Kinase/Phosphatase (PNKP) Mutations Found in a Patient with Microcephaly, Seizures, and Developmental Delay (MCSZ) and Glioblastoma

2018 ◽  
Vol 45 (S3) ◽  
pp. S2-S2
Author(s):  
Bingcheng Jiang ◽  
Chibawanye I. Ene ◽  
Bonnie Cole ◽  
Jeff Ojemann ◽  
Sarah Leary ◽  
...  
Keyword(s):  
Human Cancer ◽  
Neurodevelopmental Disorder ◽  
Site Directed Mutagenesis ◽  
Expression Vectors ◽  
Wild Type ◽  
Double Mutation ◽  
Mutant Proteins ◽  
Human Cancer Cell Lines ◽  
Mammalian Expression ◽  
Polynucleotide Kinase

The enzyme polynucleotide kinase/phosphatase (PNKP) plays a key role in DNA repair by resolving the chemistry at DNA strand breaks. Mutations in PNKP (chromosome 19q13.4) are known to cause MCSZ, a serious neurodevelopmental disorder, but to date there has been no link to cancer initiation or progression. However, a child with MCSZ recently presented at Seattle Children's Hospital with a 3-cm glioblastoma. The child was shown to have two germline mutations in PNKP. To study the effects of the PNKP mutations found in this patient, we generated mutant PNKP cDNAs carrying either the individual mutations or the double mutation using site directed mutagenesis. These cDNAs were incorporated into bacterial and mammalian expression vectors. The bacterially expressed mutant proteins as well as the wild type have been purified and are undergoing testing for PNKP DNA kinase and phosphatase activity. The PNKP cDNAs, fused to GFP, were expressed in Hela and HCT116 human cancer cell lines. High-content analysis and micro-irradiation techniques are being used to determine PNKP localization within the cells and recruitment to damaged DNA. Our preliminary results indicate that the mutations alter the ratio of nuclear to cytoplasmic PNKP compared to the wild-type protein.

scholarly journals Imerslund-Grasbeck syndrome- a rare cause of megaloblastic anemia in an infant with homozygous mutation in CUBN gene: a case report and review of the literature

2021 ◽  
Vol 8 (7) ◽  
pp. 1281
Author(s):  
Mehak Agarwal ◽  
Sanmathi Suresh ◽  
Dhaarani Jayaraman ◽  
Sri Gayathri Shanmugam
Keyword(s):  
Vitamin B12 ◽  
Renal Tubule ◽  
Megaloblastic Anemia ◽  
Intrinsic Factor ◽  
Tubular Reabsorption ◽  
Megaloblastic Anaemia ◽  
Nutritional Deficiencies ◽  
Homozygous Mutation ◽  
Review Of The Literature ◽  
Common Causes

Megaloblastic anaemia is one of the important causes of pancytopenia in children and nutritional deficiencies of vitamin B12 and folate are the most common causes comprising 95% of these cases. Defects in absorption, transport and metabolism of vitamin B12 are well described, however, are very rare. We report a rare case of Imersland Grasbeck syndrome, in an infant who presented with pancytopenia, with defective absorption of B12-intrinsic factor complex at the ileum and defective tubular reabsorption of proteins in renal tubule due to same protein defect caused by mutations in two genes – CUBN (cubilin) and AMN (amnionless).

scholarly journals Imerslund-Grasbeck Syndrome: A Case Report

2011 ◽  
Vol 31 (3) ◽  
pp. 249-250
Author(s):  
TN Ghosh ◽  
K Nayek ◽  
A Banerjee
Keyword(s):  
Case Report ◽  
Early Detection ◽  
Vitamin B12 ◽  
Key Words ◽  
Genetic Counselling ◽  
Terminal Ileum ◽  
Autosomal Recessive ◽  
Intrinsic Factor ◽  
Autosomal Recessive Disorder ◽  
Megaloblastic Anaemia

Imerslund-Grasbeck syndrome is a rare autosomal recessive disorder due to selective malabsorption of Vitamin B12 at the level of cobalamin-intrinsic factor receptor mutation in the terminal ileum resulting in megaloblastic anaemia with proteinuria. Early detection of this rare disorder would enable screening and genetic counselling for asymptomatic family members. Key words: Imerslund Grasbeck Syndrome; Megaloblastic anaemia; Proteinuria DOI: http://dx.doi.org/10.3126/jnps.v31i3.5159 J Nep Paedtr Soc 2011;31(3): 249-250

Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiae

1991 ◽  
Vol 11 (6) ◽  
pp. 3105-3114
Author(s):  
J Schnier ◽  
H G Schwelberger ◽  
Z Smit-McBride ◽  
H A Kang ◽  
J W Hershey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Peptide Bond ◽  
Translation Initiation Factor ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
Initiation Factor ◽  
Mutant Form ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae

Translation intitiation factor eIF-5A (previously named eIF-4D) is a highly conserved protein that promotes formation of the first peptide bond. One of its lysine residues is modified by spermidine to form hypusine, a posttranslational modification unique to eIF-5A. To elucidate the function of eIF-5A and determine the role of its hypusine modification, the cDNA encoding human eIF-5A was used as a probe to identify and clone the corresponding genes from the yeast Saccharomyces cerevisiae. Two genes named TIF51A and TIF51B were cloned and sequenced. The two yeast proteins are closely related, sharing 90% sequence identity, and each is ca. 63% identical to the human protein. The purified protein expressed from the TIF51A gene substitutes for HeLa eIF-5A in the mammalian methionyl-puromycin synthesis assay. Strains lacking the A form of eIF-5A, constructed by disruption of TIF51A with LEU2, grow slowly, whereas strains lacking the B form, in which HIS3 was used to disrupt TIF51B, show no growth rate phenotype. However, strains with both TIF51A and TIF51B disrupted are not viable, indicating that eIF-5a is essential for cell growth in yeast cells. Northern (RNA) blot analysis shows two mRNA species, a larger mRNA (0.9 kb) transcribed from TIF51A and a smaller mRNA (0.8 kb) encoded by TIF51B. Under the aerobic growth conditions of this study, the 0.8-kb TIF51B transcript is not detected in the wild-type strain and is expressed only when TIF51A is disrupted. The TIF51A gene was altered by site-directed mutagenesis at the site of hypusination by changing the Lys codon to that for Arg, thereby producing a stable protein that retains the positive charge but is not modified to the hypusine derivative. The plasmid shuffle technique was used to replace the wild-type gene with the mutant form, resulting in failure of the yeast cells to grow. This result indicates that hypusine very likely is required for the vital in vivo function of eIF-5A and suggests a precise, essential role for the polyamine spermidine in cell metabolism.

scholarly journals Resistance to β-Lactamase Inhibitor Protein Does Not Parallel Resistance to Clavulanic Acid in TEM β-Lactamase Mutants

2002 ◽  
Vol 46 (11) ◽  
pp. 3568-3573 ◽  
Author(s):  
William A. Schroeder ◽  
Troy R. Locke ◽  
Susan E. Jensen
Keyword(s):  
Clavulanic Acid ◽  
Double Mutant ◽  
Acid Resistance ◽  
Site Directed Mutagenesis ◽  
Mutant Form ◽  
Inhibitor Protein ◽  
Wild Type ◽  
Defective Mutant ◽  
Mutant Forms ◽  
Lactamase Inhibitor

ABSTRACT In order to compare patterns of resistance to inhibition by clavulanic acid with patterns of resistance to inhibition by a β-lactamase inhibitor protein (BLIP), R164S, R244S, and R164S/R244S mutant forms of TEM β-lactamase were prepared by site-directed mutagenesis. When kinetic parameters were determined for these mutant and wild-type forms of TEM, the single mutants showed properties that were similar to those in the literature but the double mutant showed properties that were very different. The R164S/R244S double mutant form of TEM retained its resistance to inhibition by clavulanic acid (characteristic of the R244S mutation) but lost all its ability to hydrolyze ceftazidime (characteristic of the R164S mutation). While these characteristics are contrary to those previously reported for an R164S/R244S double mutant, this discrepancy resulted from the use of a defective mutant in the earlier study. Both the single and double mutant forms of TEM remained highly sensitive when tested for inhibition by BLIP, showing only slightly increased resistance compared to that of the wild type; this pattern of resistance is quite different from the pattern of clavulanic acid resistance. The slight increases in resistance to inhibition by BLIP seen in the mutants may have been related to the fact that all of the mutations effected changes in the net charge on the TEM protein that could impede interactions with BLIP.

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