Uptake Of Cobalamin Research Articles

4-Ethylphenyl-Cobalamin Impairs Tissue Uptake of Vitamin B12 and Causes Vitamin B12 Deficiency in Mice

Coβ-4-ethylphenyl-cob(III) alamin (EtPhCbl) is an organometallic analogue of vitamin B12 (CNCbl) which binds to transcobalamin (TC), a plasma protein that facilitates the cellular uptake of cobalamin (Cbl). In vitro assays with key enzymes do not convert EtPhCbl to the active coenzyme forms of Cbl suggesting that administration of EtPhCbl may cause cellular Cbl deficiency. Here, we investigate the in vivo effect of EtPhCbl in mice and its ability, if any, to induce Cbl deficiency. We show that EtPhCbl binds to mouse TC and we examined mice that received 3.5 nmol/24h EtPhCbl (n=6), 3.5 nmol/24h CNCbl (n=7) or NaCl (control group) (n=5) through osmotic mini-pumps for four weeks. We analyzed plasma, urine, liver, spleen, submaxillary glands and spinal cord for Cbl and markers of Cbl deficiency including methylmalonic acid (MMA) and homocysteine (tHcy). Plasma MMA (mean±SEM) was elevated in animals treated with EtPhCbl (1.01±0.12 µmol/L) compared to controls (0.30±0.02 µmol/L) and CNCbl (0.29±0.01 µmol/L) treated animals. The same pattern was observed for tHcy. Plasma total Cbl concentration was higher in animals treated with EtPhCbl (128.82±1.87 nmol/L) than in CNCbl treated animals (87.64±0.93 nmol/L). However, the organ levels of total Cbl were significantly lower in animals treated with EtPhCbl compared to CNCbl treated animals or controls, notably in the liver (157.07±8.56 pmol/g vs. 603.85±20.02 pmol/g, and 443.09±12.32 pmol/g, respectively). Differences between the three groups was analysed using one-way ANOVA and, Bonferroni post-hoc test. EtPhCbl was present in all tissues, except the spinal cord, accounting for 35-90% of total Cbl. In conclusion, treatment with EtPhCbl induces biochemical evidence of Cbl deficiency. This may in part be caused by a compromised tissue accumulation of Cbl.

An exon 53 frameshift mutation in CUBN abrogates cubam function and causes Imerslund-Gräsbeck syndrome in dogs

Cobalamin malabsorption accompanied by selective proteinuria is an autosomal recessive disorder known as Imerslund-Gräsbeck syndrome in humans and was previously described in dogs due to amnionless (AMN) mutations. The resultant vitamin B12 deficiency causes dyshematopoiesis, lethargy, failure to thrive, and life-threatening metabolic disruption in the juvenile period. We studied 3 kindreds of border collies with cobalamin malabsorption and mapped the disease locus in affected dogs to a 2.9Mb region of homozygosity on canine chromosome 2. The region included CUBN, the locus encoding cubilin, a peripheral membrane protein that in concert with AMN forms the functional intrinsic factor-cobalamin receptor expressed in ileum and a multi-ligand receptor in renal proximal tubules. Cobalamin malabsorption and proteinuria comprising CUBN ligands were demonstrated by radiolabeled cobalamin uptake studies and SDS-PAGE, respectively. CUBN mRNA and protein expression were reduced ~10 fold and ~20 fold, respectively, in both ileum and kidney of affected dogs. DNA sequencing demonstrated a single base deletion in exon 53 predicting a translational frameshift and early termination codon likely triggering nonsense mediated mRNA decay. The mutant allele segregated with the disease in the border collie kindred. The border collie disorder indicates that a CUBN mutation far C-terminal from the intrinsic factor-cobalamin binding site can abrogate receptor expression and cause Imerslund-Gräsbeck syndrome.

A Frameshift Mutation in the Cubilin Gene (CUBN) in Border Collies with Imerslund-Gräsbeck Syndrome (Selective Cobalamin Malabsorption)

Imerslund-Gräsbeck syndrome (IGS) or selective cobalamin malabsorption has been described in humans and dogs. IGS occurs in Border Collies and is inherited as a monogenic autosomal recessive trait in this breed. Using 7 IGS cases and 7 non-affected controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 3.53 Mb interval on chromosome 2. We re-sequenced the genome of one affected dog at ∼10× coverage and detected 17 non-synonymous variants in the critical interval. Two of these non-synonymous variants were in the cubilin gene (CUBN), which is known to play an essential role in cobalamin uptake from the ileum. We tested these two CUBN variants for association with IGS in larger cohorts of dogs and found that only one of them was perfectly associated with the phenotype. This variant, a single base pair deletion (c.8392delC), is predicted to cause a frameshift and premature stop codon in the CUBN gene. The resulting mutant open reading frame is 821 codons shorter than the wildtype open reading frame (p.Q2798Rfs*3). Interestingly, we observed an additional nonsense mutation in the MRC1 gene encoding the mannose receptor, C type 1, which was in perfect linkage disequilibrium with the CUBN frameshift mutation. Based on our genetic data and the known role of CUBN for cobalamin uptake we conclude that the identified CUBN frameshift mutation is most likely causative for IGS in Border Collies.

Abstract 3537: Serum cobalamin level and clinical outcome in patients with acute myeloid leukemia.

Abstract Introduction: An elevated concentration of serum cobalamin was found in one third of patients with acute myeloid leukemia; however, the clinical implication of elevated level of cobalamin in disease prognosis is not clear. Procedures: We performed a retrospective analysis of the patients who were diagnosed to have de novo acute myeloid leukemia at China Medical University Hospital from January 2003 to December 2011. Two hundred and six patients were diagnosed as de novo acute myeloid leukemia, and thirty-four patients (16.5%) had been checked the level of serum cobalamin at diagnosis. Patients were divided into two groups, one with serum cobalamin level less than 400 pg/mL (n=10), the other with serum cobalamin level 400 pg/mL or higher (n=24). The clinical and cytogenetic characteristics between each group were compared. Findings: There was no difference in gender, age and cytogenetic risk between the two groups. Nine of ten patients in lower serum cobalamin group and eighteen of twenty-four patients in higher serum cobalamin group received intensive chemotherapy for acute myeloid leukemia (p=0.315). The patients with lower serum cobalamin had a trend to obtain complete remission (80.0 % vs. 45.8 %, p=0.072). For patients who reached complete remission after chemotherapy, those in the higher cobalamin group had a trend to have more disease relapse (81.8% vs. 37.5%, p=0.067). Interestingly, the relapse-free survival time after complete remission was 42.5 m and 9.2 m for patients with lower and higher cobalamin, respectively (p=0.010). Furthermore, serum cobalamin level remained significant in the multivariate analysis of risk factors for relapse-free survival (p=0.028). The median overall survival time was 18.3 m and 7.2 m for patients with lower and higher cobalamin, respectively. However, the difference in overall survival between the two groups did not reach statistical significance (p=0.140). In multivariate analysis, intensive chemotherapy for AML was the most important factor for overall survival. Conclusions: AML patients with higher serum level of cobalamin have a trend of worse response to chemotherapy, higher relapse rate and significantly shorter relapse-free survival time. Targeted therapy to interfere the cobalamin uptake or related pathways is worth of further investigation in treating patient with acute myeloid leukemia. Citation Format: Li-Yuan Bai, Tzu-Ting Chen, Long-Yuan Li, Chang-Fang Chiu. Serum cobalamin level and clinical outcome in patients with acute myeloid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3537. doi:10.1158/1538-7445.AM2013-3537

Cellular uptake of cobalamin: transcobalamin and the TCblR/CD320 receptor.

Cellular uptake of cobalamin is facilitated by a receptor-mediated endocytosis process involving transcobalamin, a plasma protein that binds cobalamin and a cell surface receptor that specifically binds transcobalamin saturated with cobalamin. Intracellular Cbl concentration is maintained by modulating the expression of the receptor, which is cell cycle associated with highest expression in actively proliferating cells and an efflux system that shunts the excess cobalamin out of the cells for mobilization to other tissues where it is most needed. This review describes the process, proteins involved and genes encoding these proteins.

Saporin Conjugated Monoclonal Antibody to the Transcobalamin Receptor TCblR/CD320 Is Effective in Targeting and Destroying Cancer Cells

Cobalamin uptake into cells is mediated by the CD320 receptor for transcobalamin-bound cobalamin. Optimum receptor expression is associated with proliferating cells and therefore, in many cancers this receptor expression is up regulated. Delivering drugs or toxins via this receptor provides increased targeting to cancer cells while minimizing toxicity to the normal tissues. Saporin conjugated monoclonal antibodies to the extracellular domain of TCblR were effectively internalized to deliver a toxic dose of Saporin to some cancer cell lines propagating in culture. Antibody concentration of 2.5 nM was effective in producing optimum inhibition of cell proliferation. The cytotoxic effect of mAb-Saporin appears to be dictated primarily by the level of receptor expression and therefore normal primary cells expressing low levels of CD320 were spared while tumor cell lines with higher CD320 expression were destroyed. Targeting the pathway for cellular uptake of vitamin B12 via the CD320 receptor with toxin-antibody conjugates appears to be a viable treatment strategy for certain cancers that over expresses this receptor.

A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor. In conclusion, only one soluble cobalamin-binding protein was identified in the rainbow trout, a protein that structurally behaves like an intermediate between the three human cobalamin-binding proteins.

Influence of cobalamin scarcity on diatom molecular physiology and identification of a cobalamin acquisition protein

Diatoms are responsible for ~40% of marine primary production and are key players in global carbon cycling. There is mounting evidence that diatom growth is influenced by cobalamin (vitamin B(12)) availability. This cobalt-containing micronutrient is only produced by some bacteria and archaea but is required by many diatoms and other eukaryotic phytoplankton. Despite its potential importance, little is known about mechanisms of cobalamin acquisition in diatoms or the impact of cobalamin scarcity on diatom molecular physiology. Proteomic profiling and RNA-sequencing transcriptomic analysis of the cultured diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana revealed three distinct strategies used by diatoms to cope with low cobalamin: increased cobalamin acquisition machinery, decreased cobalamin demand, and management of reduced methionine synthase activity through changes in folate and S-adenosyl methionine metabolism. One previously uncharacterized protein, cobalamin acquisition protein 1 (CBA1), was up to 160-fold more abundant under low cobalamin availability in both diatoms. Autologous overexpression of CBA1 revealed association with the outside of the cell and likely endoplasmic reticulum localization. Cobalamin uptake rates were elevated in strains overexpressing CBA1, directly linking this protein to cobalamin acquisition. CBA1 is unlike characterized cobalamin acquisition proteins and is the only currently identified algal protein known to be implicated in cobalamin uptake. The abundance and widespread distribution of transcripts encoding CBA1 in environmental samples suggests that cobalamin is an important nutritional factor for phytoplankton. Future study of CBA1 and other molecular signatures of cobalamin scarcity identified here will yield insight into the evolution of cobalamin utilization and facilitate monitoring of cobalamin starvation in oceanic diatom communities.

The Cobalamin-Binding Protein in Zebrafish Is an Intermediate between the Three Cobalamin-Binding Proteins in Human

In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio) and summarize current knowledge concerning the phylogenetic evolution of kindred proteins. We identified a cobalamin binding capacity in zebrafish protein extracts (8.2 pmol/fish) and ambient water (13.5 pmol/fish) associated with a single protein. The protein showed resistance toward degradation by trypsin and chymotrypsin (like human IF, but unlike human HC and TC). The cobalamin analogue, cobinamide, bound weaker to the zebrafish cobalamin binder than to human HC, but stronger than to human TC and IF. Affinity for another analogue, adenosyl-pseudo-cobalamin was low compared with human HC and TC, but high compared with human IF. The absorbance spectrum of the purified protein in complex with hydroxo-cobalamin resembled those of human HC and IF, but not TC. We searched available databases to further explore the phylogenies of the three cobalamin-binding proteins in higher vertebrates. Apparently, TC-like proteins are the oldest evolutionary derivatives followed by IF and HC (the latter being present only in reptiles and most but not all mammals). Our findings suggest that the only cobalamin-binding protein in zebrafish is an intermediate between the three human cobalamin binders. These findings support the hypothesis about a common ancestral gene for all cobalamin-binding proteins in higher vertebrates.

Transcobalamin Polymorphism 67A->G, but Not 776C->G, Affects Serum Holotranscobalamin in a Cohort of Healthy Middle-Aged Men and Women

Two polymorphic variants in the gene coding for transcobalamin II (TCN2),TCN2776C- > G andTCN267A- > G, may alter serum holotranscobalamin (holoTC), which in turn may affect cellular uptake of cobalamin (Cbl) and thereby Cbl status indicators. We studied the effects ofTCN2776C- > G andTCN267A- > G on blood concentrations of holoTC, Cbl, methylmalonic acid (MMA), and total homocysteine (tHcy) in 2411 individuals (50–64 y) that had been selected on the basis of theseTCN2genotypes from 10601 Norwegian inhabitants. The serum holoTC concentration was lower inTCN267AG (55 ± 0.75 pmol/L) and 67GG (48 ± 2.14 pmol/L) than in 67AA (62 ± 0.67 pmol/L) (P< 0.001) but did not differ amongTCN2776C- > G genotypes. The polymorphisms interacted as serum holoTC determinants (P= 0.001) and the presence ofTCN267AG and GG in strata of 776CC and CG, but not 776GG, increased the risk of having serum holoTC < 45.6 pmol/L [tertile 1 vs. tertiles 2 and 3: OR = 2.5 (95% CI 1.8–3.5) for 67AG; OR = 5.7 (95% CI 3.5–9.1) for 67GG in 776CC; OR = 2.1 (95% CI 1.6–2.9) for 67AG; and OR = 4.5 (95% CI 2.4–8.2) for 67GG in 776CG; all P< 0.001]. Plasma MMA, tHcy, and Cbl were not affected by either polymorphism. In summary, serum holoTC, but not plasma Cbl, MMA, or tHcy, varied according toTCN267A- > G genotypes. It remains to be determined whether this polymorphic effect on serum holoTC alters its diagnostic utility as Cbl status indicator.

Down-regulation of transcobalamin receptor TCblR/ CD320 by siRNA inhibits cobalamin uptake and proliferation of cells in culture

The clinical phenotype of cobalamin (Cbl) deficiency is dictated by the essential role of this vitamin in two key enzymatic reactions. Multiple proteins and receptors participate in the absorption, transport and delivery of this vitamin to tissue cells. Cellular uptake of Cbl is mediated by transcobalamin (TC), a plasma protein and a transmembrane receptor (TCblR) with high affinity for TC saturated with Cbl. Knockdown of TCblR with siRNA results in decreased TC–Cbl uptake. The ensuing Cbl deficiency leads to an increase in doubling time and decreased proliferation of these cells. The study confirms the seminal role of this receptor in the cellular uptake of Cbl and its down-regulation as a potential strategy to inhibit proliferation of cancer cells.

The prevalence of hypocobalaminaemia in cats with spontaneous hyperthyroidism

To determine the prevalence of hypocobalaminaemia in cats with moderate to severe hyperthyroidism and to investigate the relationship between cobalamin status and selected haematologic parameters. Serum cobalamin concentrations were measured in 76 spontaneously hyperthyroid cats [serum thyroxine (T(4) ) concentration ≥100 nmol/L] and 100 geriatric euthyroid cats. Erythrocyte and neutrophil counts in hyperthyroid cats with hypocobalaminaemia were compared with those in hyperthyroid cats with adequate serum cobalamin concentrations (≥290 ng/L). The median cobalamin concentration in hyperthyroid cats was lower than the control group (409 versus 672 ng/L; P=0·0040). In addition, 40·8% of hyperthyroid cats had subnormal serum cobalamin concentrations compared with 25% of controls (P=0·0336). Weak negative correlation (coefficient: -0·3281) was demonstrated between serum cobalamin and T(4) concentrations in the hyperthyroid population, and the median cobalamin concentration was lower in cats with T(4) above the median of 153 nmol/L compared with cats with T(4) below this value (P=0·0281). Hypocobalaminaemia was not associated with neutropenia or anaemia in hyperthyroid cats. This study indicates that a substantial proportion of cats with T(4) ≥100 nmol/L are hypocobalaminaemic and suggests that hyperthyroidism directly or indirectly affects cobalamin uptake, excretion or utilisation in this species.

Uptake of cobalamin and markers of cobalamin status: a longitudinal study of healthy pregnant women

Currently, it is unknown whether the decline in plasma cobalamin observed during pregnancy is caused by malabsorption of the vitamin. This study examined cobalamin absorption and markers of cobalamin status during normal pregnancy. Twenty-seven pregnant Danish women were examined at gestation weeks 13, 24 and 36. The absorption test CobaSorb was performed in all women implying measurement of holotranscobalamin or cyanocobalamin bound to transcobalamin before and after 2 days intake of 3 × 9 μg cobalamin. Serum cobalamin and the two cobalamin binding proteins transcobalamin and haptocorrin, including haptocorrin saturated with cobalamin or analogues, were measured, and so was plasma methylmalonic acid and homocysteine. No change in the uptake of cobalamin was observed throughout pregnancy. Serum cobalamin displayed a gradual decline during pregnancy (p<0.0001), while holotranscobalamin remained unchanged, despite an increase in total transcobalamin (p<0.0001). In accord with these results, total haptocorrin showed a decline from the 1st to 3rd trimester (p=0.007) and cobalamin bound to haptocorrin declined (p<0.0001). Interestingly, the amount of cobalamin analogues attached to haptocorrin remained unchanged. Methylmalonic acid (p=0.002) and homocysteine (p<0.0001) increased during pregnancy. Cobalamin absorption remains unchanged during normal pregnancy, as judged by the CobaSorb test. No change was observed in the biological active holotranscobalamin during pregnancy. Thus, the pregnancy-related decline in cobalamin is caused by alternations in haptocorrin-bound cobalamin. Surprisingly, no pregnancy-related change was observed in the amount of analogues attached to haptocorrin.

Targeted Delivery of Saporin Toxin by Monoclonal Antibody to the Transcobalamin Receptor, TCblR/CD320

Cellular uptake of cobalamin (Cbl) occurs by endocytosis of transcobalamin saturated with Cbl by the transcobalamin receptor (TCblR/CD320). The cell cycle-associated overexpression of this receptor in many cancer cells provides a suitable target for delivering chemotherapeutic drugs and cytotoxic molecules to these cells while minimizing the effect on the normal cell population. We have used monoclonal antibodies to the extracellular domain of TCblR to deliver saporin-conjugated secondary antibody to various cell lines propagating in culture. A molar ratio of 2.5:10 nmol/L of primary:secondary antibody concentration was identified as the lowest concentration needed to produce the optimum cytotoxic effect. The effect was more pronounced when cells were seeded at lower density, suggesting lack of cell division in a fraction of the cells at higher density as the likely explanation. Cells in suspension culture, such as K562 and U266 cells, were more severely affected than adherent cultures, such as SW48 and KB cells. This differential effect of the anti-TCblR-saporin antibody conjugate and the ability of an anti-TCblR antibody to target proliferating cells were further evident by the virtual lack of any effect on primary skin fibroblasts and minimal effect on bone marrow cells. These results indicate that preferential targeting of some cancer cells could be accomplished through the TCblR.

Characterizing monoclonal antibodies to antigenic domains of TCblR/CD320, the receptor for cellular uptake of transcobalamin-bound cobalamin

Monoclonal antibodies (mAbs) were generated to the extracellular domain of transcobalamin receptor (TCblR) and used to identify the regions of the receptor protein involved in antibody binding. Based on the effect of transcobalamin bound cobalamin (TC-Cbl) on antibody binding, this study identified both blocking and binding antibodies. Both types of antibodies bind apo as well as holo receptors, whereas the blocking antibody when bound to the apo receptor prevents the binding and cellular uptake of TC-Cbl. Binding of these antibodies to truncated receptor constructs has identified the peptide domains of the receptor involved in antibody binding. These antibodies have potential utility in blocking cellular uptake of Cbl and delivery of drugs via TCblR, which is over-expressed in many cancers.

Characterization of the promoter region of TCblR/CD320 gene, the receptor for cellular uptake of transcobalamin-bound cobalamin

Cellular uptake of cobalamin (Cbl) is mediated by the transcobalamin receptor (TCblR) that binds and internalizes transcobalamin (TC) saturated with Cbl. These receptors are expressed in actively proliferating cells and are down-regulated in quiescent cells. The 5’ region of TCblR gene was analyzed for promoter activity to determine transcriptional regulation of TCblR expression. The region −668 to −455 appears to regulate TCblR expression. We have identified transcription factors MZF-1 (myeloid zinc finger 1)/RREB-1 (Ras-responsive element binding protein 1), C/EBP (CCAAT/enhancer binding protein)/HNF-3β (hepatocyte nuclear factor 3) and AP-1(activator protein 1) as proteins likely to be involved in this regulation with the former region primarily involved in up regulation and the latter two regions involved in suppression of TCblR expression. These transcription factors are involved in cell proliferation and differentiation. Thus the cell cycle associated expression of TCblR appears to be tightly regulated in synchrony with the proliferative phase of the cell cycle.

Transcobalamin II receptor polymorphisms are associated with increased risk for neural tube defects

ObjectiveWomen who have low cobalamin (vitamin B12) levels are at increased risk for having children with neural tube defects (NTDs). The transcobalamin II receptor (TCblR) mediates uptake of cobalamin into...

Vitamin B12 deficiency in a 15-year old boy due to mutations in the intrinsic factor gene, GIF

The discoveries of new genes involved in cobalamin assimilation and metabolism has increased our understanding of the rare inborn errors of cobalamin metabolism (Quadros 2009). The following case documents a compound heterozygote mutation in the gastric intrinsic factor (GIF) gene, with a previously described mutation (c.79+1G>A) and a novel mutation (c.290T>C; M97T) leading to a megaloblastic anaemia in an adolescent.

Cobalamin uptake and reactivation occurs through specific protein interactions in the methionine synthase–methionine synthase reductase complex

Human methionine synthase reductase (MSR), a diflavin enzyme, restores the activity of human methionine synthase through reductive methylation of methionine synthase (MS)-bound cob(II)alamin. Recently, it was also reported that MSR enhances uptake of cobalamin by apo-MS, a role associated with the MSR-catalysed reduction of exogenous aquacob(III)alamin to cob(II)alamin [Yamada K, Gravel RA, TorayaT & Matthews RG (2006) Proc Natl Acad Sci USA103, 9476-9481]. Here, we report the expression and purification of human methionine synthase from Pichia pastoris. This has enabled us to assess the ability of human MSR and two other structurally related diflavin reductase enzymes (cytochrome P450 reductase and the reductase domain of neuronal nitric oxide synthase) to: (a) stimulate formation of holo-MS from aquacob(III)alamin and the apo-form of MS; and (b) reactivate the inert cob(II)alamin form of MS that accumulates during enzyme catalysis. Of the three diflavin reductases studied, cytochrome P450 reductase had the highest turnover rate (55.5 s(-1)) for aquacob(III)alamin reduction, and the reductase domain of neuronal nitric oxide synthase elicited the highest specificity (k(cat)/K(m) of 1.5 x 10(5) m(-1) s(-1)) and MSR had the lowest K(m) (6.6 microm) for the cofactor. Despite the ability of all three enzymes to reduce aquacob(III)alamin, only MSR (the full-length form or the isolated FMN domain) enhanced the uptake of cobalamin by apo-MS. MSR was also the only diflavin reductase to reactivate the inert cob(II)alamin form of purified human MS (K(act) of 107 nm) isolated from Pichia pastoris. Our work shows that reactivation of cob(II)alamin MS and incorporation of cobalamin into apo-MS is enhanced through specific protein-protein interactions between the MSR FMN domain and MS.

The protein and the gene encoding the receptor for the cellular uptake of transcobalamin-bound cobalamin

AbstractThe transcobalamin (TC, TCII) receptor (TCblR) on the plasma membrane binds TC- cobalamin (Cbl) and internalizes the complex by endocytosis. This receptor was purified from human placental membranes by affinity chromatography. Tryptic digest of the protein extracted from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and subjected to liquid chromatography/mass spectrometry identified 4 peptides that matched with a membrane protein in the data bank. TCblR belongs to the low-density lipoprotein receptor family, with 2 low-density lipoprotein receptor type A domains separated by a complement-like cysteine-rich region. The 282-amino acid sequence includes a signal peptide of 31 residues, extracellular domain of 198 residues, a transmembrane region of 21 residues, and a cytoplasmic domain of 32 residues. The binding of TC-Cbl does not require the cytoplasmic domain or its orientation in the plasma membrane because the recombinant extracellular domain binds TC-Cbl with high affinity and specificity. The protein is heavily glycosylated and accounts for the 58-kDa size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The human gene first identified as 8D6A and more recently as CD 320 encoding TCblR is located at p13.2 on the short arm of chromosome 19, spans a length of 6.224 kb, and is composed of 5 exons and 4 introns.