Uptake of transcobalamin II-bound cobalamin by HL-60 cells: effects of differentiation induction

Binding and uptake of transcobalamin II-bound cobalamin by HL-60 promyelocytic leukemia cells proceed through receptor-mediated endocytosis. The affinity constant of the receptor for transcobalamin II-cobalamin was found to be 6.1 liter/nmol and the maximal rate of uptake 12 pmol/10(9) cells/h. This uptake is mediated by about 3000 receptor sites per cell. Evidence is presented that the receptor recirculates from the cell surface to the lysosomes and vice versa. Upon differentiation induction of the cells by either DMSO in granulocytic direction or by 1,25-dihydroxy-vitamin D3 in monocytic direction a rapid decline in cellular uptake and cell surface binding of the protein-bound vitamin ensues. In particular the internalization of the complex decreases faster than all other observed signs of the ongoing differentiation process, such as reduction in the OKT9-reactive transferrin receptor, increase in lineage-specific surface markers, and decrease in [3H]thymidine incorporation and actual cell proliferation. The transcobalamin II receptor on the cell surface appears to be a proliferation-associated membrane component in human leukemic cells.     

The uptake of R-type cobalamin-binding protein by isolated rat liver cells

The uptake of R-type cobalamin-binding protein from human granulocytes and plasma by isolated parenchymal rat liver cells has been studied. When [⁵⁷Co]cyanocobalamin-saturated granulocyte-binding protein or transcobalamin III was incubated with the liver cells in a concentration of 500 pM, more than 80% of the vitamin was taken up in 1 h. Vitamin B-12 bound to plasma transcobalamin I, however, was not taken up unless the protein was desialylated by neuraminidase from Vibrio cholerae. The uptake of iodinated pure granulocyte-binding protein, saturated with cobalamin, reached 100% and was accompanied by increasing intracellular proteolytic degradation of the binding protein. EGTA and asialo-orosomucoid completely inhibited this process of uptake and degradation, whereas partial inhibition was caused by chloroquine and colchicine. These observations provide evidence that these (asialo)-R-type cobalamin-binding proteins are taken up by the cell through the plasma membrane receptor for asialoglycoproteins by means of endocytosis followed by proteolysis of the binding protein in the lysosomes.     

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.     

High affinity binding of the transcobalamin II-cobalamin complex and mRNA expression of haptocorrin by human mammary epithelial cells

Little is known about the acquisition of cobalamin by the mammary gland and its secretion into milk. Human milk and plasma contain at least two types of cobalamin binding proteins: transcobalamin II (TC) and haptocorrin (HC). In plasma, TC is responsible for the transport of cobalamin to tissues and cells; however, cobalamin in milk is present exclusively bound to HC. We show that human mammary epithelial cells (HMEC) exhibit high affinity for TC; Scatchard analysis revealed a single class of binding sites for the TC-[(57)Co]cyanocobalamin complex with a dissociation constant (K(d)) of 4.9 x 10(-11) M. Uptake of the TC-[(57)Co]cyanocobalamin complex at 37 degrees C was saturable by 24 h. Binding of free [(57)Co]cyanocobalamin to HMEC was not saturable and very limited binding of the HC-[(57)Co]cyanocobalamin complex was observed. Expression of the haptocorrin gene by HMEC was confirmed by Northern blot and PCR analysis. Thus, a specific cell surface receptor for the TC-cobalamin complex exists in the mammary gland and once cobalamin is internalized, it may be transferred to HC and subsequently secreted into milk as a HC-cobalamin complex.     

How does cobalamin (vitamin B12) enter and traverse mammalian cells?

This overview is intended to present recent information available on the work done in understanding the mode of entry of cobalamin into and out of mammalian cells. I have focused primarily on the uptake of cobalamin bound to 3 proteins: intrinsic factor, transcobalamin II and R-proteins. Even though the nature of these cobalamin binding proteins, and the receptors involved in the internalization of these proteins is known, a total picture on the intracellular trafficking of cobalamin and the binding proteins is far from complete.     

Complete disulfide bond assignment of a recombinant immunoglobulin G4 monoclonal antibody

Surface plasmon resonance biosensor analysis was used to evaluate the thermodynamics and binding kinetics of naturally occurring and synthetic cobalamins interacting with vitamin B(12) binding proteins. Cyanocobalamin-b-(5-aminopentylamide) was immobilized on a biosensor chip surface to determine the affinity of different cobalamins for transcobalamin, intrinsic factor, and nonintrinsic factor. A solution competition binding assay, in which a surface immobilized cobalamin analog competes with analyte cobalamin for B(12) protein binding, shows that only recombinant human transcobalamin is sensitive to modification of the corrin ring b-propionamide of cyanocobalamin. A direct binding assay, where recombinant human transcobalamin is conjugated to a biosensor chip, allows kinetic analysis of cobalamin binding. Response data for cyanocobalamin binding to the transcobalamin protein surface were globally fitted to a bimolecular interaction model that includes a term for mass transport. This model yields association and dissociation rate constants of k(a) = 3 x 10(7) M(-1) s(-1) and k(d) = 6 x 10(-4) s(-1), respectively, with an overall dissociation constant of K(D) = 20 pM at 30 degrees C. Transcobalamin binds cyanocobalamin-b-(5-aminopentylamide) with association and dissociation rates that are twofold slower and threefold faster, respectively, than transcobalamin binding to cyanocobalamin. The affinities determined for protein-ligand interaction, using the solution competition and direct binding assays, are comparable, demonstrating that surface plasmon resonance provides a versatile way to study the molecular recognition properties of vitamin B(12) binding proteins.     

High affinity binding of the transcobalamin II–cobalamin complex and mRNA expression of haptocorrin by human mammary epithelial cells

Little is known about the acquisition of cobalamin by the mammary gland and its secretion into milk. Human milk and plasma contain at least two types of cobalamin binding proteins: transcobalamin II (TC) and haptocorrin (HC). In plasma, TC is responsible for the transport of cobalamin to tissues and cells; however, cobalamin in milk is present exclusively bound to HC. We show that human mammary epithelial cells (HMEC) exhibit high affinity for TC; Scatchard analysis revealed a single class of binding sites for the TC–[⁵⁷Co]cyanocobalamin complex with a dissociation constant (K_d) of 4.9×10⁻¹¹ M. Uptake of the TC–[⁵⁷Co]cyanocobalamin complex at 37°C was saturable by 24 h. Binding of free [⁵⁷Co]cyanocobalamin to HMEC was not saturable and very limited binding of the HC–[⁵⁷Co]cyanocobalamin complex was observed. Expression of the haptocorrin gene by HMEC was confirmed by Northern blot and PCR analysis. Thus, a specific cell surface receptor for the TC–cobalamin complex exists in the mammary gland and once cobalamin is internalized, it may be transferred to HC and subsequently secreted into milk as a HC–cobalamin complex.     

The binding properties of the human receptor for the cellular uptake of vitamin B12

Cellular uptake of vitamin B(12) (cobalamin, Cbl) is mediated by a receptor expressed on the plasma membrane that binds transcobalamin (TC) saturated with Cbl and internalizes the TC-Cbl by endocytosis. A few reports have described the characterization of the receptor protein. However, many discrepancies have emerged in the functional and structural properties of the receptor and therefore, the identity and primary structure of this protein remains unconfirmed. In this report, we provide evidence of a 58 kDa monomeric protein as the likely receptor for the uptake of TC-Cbl and that the functional activity is not associated with a 72/144 kDa monomer/dimer with immunoglobulin Fc structural domain that has been purported to be the receptor in a number of publications.     

Transcobalamin II Receptor Interacts with Megalin in the Renal Apical Brush Border Membrane

Purified human transcobalamin II receptor (TC II-R) binds to megalin, a 600 kDa endocytic receptor with an association constant, K(a), of 66 n M and bound(max) of 1.1 mole of TC II-R/mole of megalin both in the presence and absence of its ligand, transcobalamin II (TC II). Immunoprecipitation followed by immunoblotting of Triton X-100 extracts of the apical brush border membrane (BBM) from rabbit renal cortex revealed association of these two proteins. (35)[S]-TC II complexed with cobalamin (Cbl; Vitamin B(12)) bound to Sepharose-megalin affinity matrix and the binding was enhanced 5-fold when TC II-R was prebound to megalin. Megalin antiserum inhibited both the TC II-R-dependent and -independent binding of (35)[S]-TC II-Cbl to megalin, while TC II-R antiserum inhibited only the TC II-R-dependent binding. In rabbits with circulating antiserum to megalin, renal apical BBM megalin was present as an immune complex, but its levels were not altered. However, the protein levels of both TC II-R and the cation-independent mannose 6-phosphate receptor (CIMPR) were drastically reduced and the urinary excretion of TC II, albumin, and other low-molecular weight proteins was significantly increased. These results suggest that megalin contains a distinct single high-affinity binding site for TC II-R and their association in the native renal BBM is important for tubular reabsorption of many proteins, including TC II.     

Comparison of recombinant human haptocorrin expressed in human embryonic kidney cells and native haptocorrin

Haptocorrin (HC) is a circulating corrinoid binding protein with unclear function. In contrast to transcobalamin, the other transport protein in blood, HC is heavily glycosylated and binds a variety of cobalamin (Cbl) analogues. HC is present not only in blood but also in various secretions like milk, tears and saliva. No recombinant form of HC has been described so far. We report the expression of recombinant human HC (rhHC) in human embryonic kidney cells. We purified the protein with a yield of 6 mg (90 nmol) per litre of cell culture supernatant. The isolated rhHC behaved as native HC concerning its spectral properties and ability to recognize both Cbl and its baseless analogue cobinamide. Similar to native HC isolated from blood, rhHC bound to the asialoglycoprotein receptor only after removal of terminal sialic acid residues by treatment with neuraminidase. Interestingly, rhHC, that compared to native HC contains four excessive amino acids (…LVPR) at the C-terminus, showed subtle changes in the binding kinetics of Cbl, cobinamide and the fluorescent Cbl conjugate CBC. The recombinant protein has properties very similar to native HC and although showing slightly different ligand binding kinetics, rhHC is valuable for further biochemical and structural studies.     

The cDNA sequence and the deduced amino acid sequence of human transcobalamin II show homology with rat intrinsic factor and human transcobalamin I.

The cellular uptake of cobalamin (Cbl, vitamin B12) is mediated by transcobalamin II (TCII), a plasma protein that binds Cbl and is secreted by human umbilical vein endothelial (HUVE) cells. These cells synthesize and secrete TCII and, therefore, served as the source of the complementary DNA (cDNA) library from which the TCII cDNA was isolated. This full-length cDNA consists of 1866 nucleotides that code for a leader peptide of 18 amino acids, a secreted protein of 409 amino acids, a 5'-untranslated segment of 37 nucleotides, and a 3'-untranslated region of 548 nucleotides. A single 1.9-kilobase species of mRNA corresponding to the size of the cDNA was identified by Northern blot analysis of the RNA isolated from HUVE cells. TCII has 20% amino acid homology and greater than 50% nucleotide homology with human transcobalamin I (TCI) and with rat intrinsic factor (R-IF). TCII has no homology with the amino-terminal region of R-IF that has been reported to have significant primary as well as secondary structural homology with the nucleotide-binding domain of NAD-dependent oxidoreductases. The regions of homology that are common to all three proteins are located in seven domains of the amino acid sequence. One or more of these conserved domains is likely to be involved in Cbl binding, a function that is common to all three proteins. However, the difference in the affinity of TCII, TCI, and R-IF for Cbl and Cbl analogues indicates, a priori, that structural differences in the ligand-binding site of these proteins exist and these probably resulted from divergence of a common ancestral gene.     

Effect of lectins on the cobalamin-protein binding reactions: implications for the tissue uptake of cobalamin

Plant lectins have been thought to impair nutrient absorption, both by specific and nonspecific interference in the absorptive process. The main objective of this investigation was to study the effect of lectins on the various binding reactions involving cobalamin (cbl)-protein complexes and their receptors, and to identify the rate-limiting step important in maintaining tissue levels of cobalamin. Among the lectins tested in vivo, only concanavalin A (ConA) was able to inhibit the transport of cobalamin to the tissues and caused a 70% to 75% inhibition of [(57)Co] cobalamin transported to the liver and kidney. The inhibition of transport to the tissues was independent of route of administration of cobalamin, whether intragastric or systemic, and was not due to decreased gastrointestinal uptake. When tested in vitro, concanavalin A inhibited the binding of transcobalamin II-cbl to its receptor, but not the binding of cobalamin to intrinsic factor or intrinsic factor-cobalamin complex to the ileal receptor. These results suggest that late events during transcellular transport of cobalamin through the enterocytes is the rate-limiting step determining tissue levels of cobalamin and that ConA inhibits these latter events.     

Glial cells as a model for the role of cobalamin in the nervous system: impaired synthesis of cobalamin coenzymes in cultured human astrocytes following short-term cobalamin-deprivation.

The conversion of cyanocobalamin to adenosyl- and methylcobalamin is impaired in cobalamin-deficient cultured human glial cells. In contrast cultured human skin fibroblasts retained their ability to synthesize coenzyme forms when grown in cobalamin-deficient medium. Cells were pre-conditioned by growing in cobalamin-deficient media for six weeks and then subcultured in medium containing either free or transcobalamin II-bound 57Co-cyanocobalamin. Although both coenzyme levels were low in cobalamin-deficient glial cells, the decrease in methylcobalamin was more marked than that of adenosylcobalamin. Methionine synthase and Cb1 reductase activities were markedly decreased in cobalamin-deficient glial cells but were unchanged in fibroblasts cultured in cobalamin-deficient medium. Our data suggest that in glial cells, cobalamin coenzyme synthesis and function is exquisitely sensitive to short-term cobalamin deprivation. Glial cells apparently synthesize and secrete transcobalamin II since antibodies directed against the transport protein inhibit the uptake of free cobalamin.     

Structural study on ligand specificity of human vitamin B12 transporters

Studies comparing the binding of genuine cobalamin (vitamin B12) to that of its natural or synthetic analogues have long established increasing ligand specificity in the order haptocorrin, transcobalamin and intrinsic factor, the high-affinity binding proteins involved in cobalamin transport in mammals. In the present study, ligand specificity was investigated from a structural point of view, for which comparative models of intrinsic factor and haptocorrin are produced based on the crystal structure of the homologous transcobalamin and validated by results of published binding assays. Many interactions between cobalamin and its binding site in the interface of the two domains are conserved among the transporters. A structural comparison suggests that the determinant of specificity regarding cobalamin ligands with modified nucleotide moiety resides in the beta-hairpin motif beta3-turn-beta4 of the smaller C-terminal domain. In haptocorrin, it provides hydrophobic contacts to the benzimidazole moiety through the apolar regions of Arg357, Trp359 and Tyr362. Together, these large side chains may compensate for the missing nucleotide upon cobinamide binding. Intrinsic factor possesses only the tryptophan residue and transcobalamin only the tyrosine residue, consistent with their low affinity for cobinamide. Relative affinity constants for other analogues are rationalized similarly by analysis of steric and electrostatic interactions with the three transporters. The structures also indicate that the C-terminal domain is the first site of cobalamin-binding since part of the beta-hairpin motif is trapped between the nucleotide moiety and the N-terminal domain in the final holo-proteins.     

Synthesis and secretion of the human vitamin B12-binding protein, transcobalamin II, by cultured skin fibroblasts and by bone marrow cells

Human skin fibroblasts and bone marrow cells were tested for their ability to synthesize the cobalamin-binding protein transcobalamin II. Cobalamin binders secreted in the media of cultured fibroblasts and of dextran-sedimented bone marrow cells in liquid culture could be identified as transcobalamin II on the basis of immunological, electrophoretical and chromatographical identity with serum transcobalamin II. The net secretion of transcobalamin II increased linearly with time of culture, up to 30 days after confluence. The reversible inhibition of transcobalamin II secretion by cycloheximide demonstrated that human fibroblasts are capable of de novo transcobalamin II synthesis. Addition of cyanocobalamin to the fibroblast culture medium induced a reduction of transcobalamin II net secretion, most likely due to preferred uptake of transcobalamin II saturated with cobalamin, as opposed to unsaturated protein. Addition of lysozymal enzyme inhibitors, ammonium chloride and chloroquine, resulted in a markedly increased secretion of transcobalamin II. In the culture medium of fibroblasts, obtained from two transcobalamin II-deficient patients, functionally deficient transcobalamin II was demonstrated on the basis of strongly reduced secretion of immunoreactive transcobalamin II, and the absence of apotranscobalamin II. Individual phenotypes in the culture media of the fibroblasts and bone marrow cells were identical to the corresponding serum transcobalamin II types.     

Cobalamin transport proteins and their cell-surface receptors

The primary function of cobalamin (Cbl; vitamin B12) is the formation of red blood cells and the maintenance of a healthy nervous system. Before cells can utilise dietary Cbl, the vitamin must undergo cellular transport using two distinct receptor-mediated events. First, dietary Cbl bound to gastric intrinsic factor (IF) is taken up from the apical pole of ileal epithelial cells via a 460 kDa receptor, cubilin, and is transported across the cell bound to another Cbl-binding protein, transcobalamin II (TC II). Second, plasma TC II-Cbl is taken up by cells that need Cbl via the TC II receptor (TC II-R), a 62 kDa protein that is expressed as a functional dimer in cellular plasma membranes. Human Cbl deficiency can develop as a result of acquired or inherited dysfunction in either of these two transmembrane transport events. This review focuses on the biochemical, cellular and molecular aspects of IF and TC II and their cell-surface receptors.     

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.     

Genetic patterns of transcobalamin II and the relationships with congenital defects

The vitamin B12-binding protein, transcobalamin II, is a trace component of plasma with a rapid turnover. This protein is essential for absorption, transport, cellular uptake and for recycling of vitamin B12 (cobalamin). Congenital transcobalamin II deficiency, an inborn error of metabolism is inherited as a recessive trait. The homozygous form of the deficiency is accompanied by severe clinical, hematological and immunological disturbances in the first months of life. Analytical, genetic, biochemical and clinical aspects of transcobalamin II in man and in vertebrates have been reviewed here. A genetic polymorphism for the protein has been found in man, rabbits and mice. Family studies revealed that the genetic patterns in man are determined by four polymorphic and several rare alleles. This genetic variability has been applied in paternity testing and in population studies. Transcobalamin II typing in families of patients with the inherited functional deficiency has led to identification of various deficient alleles in heterozygous carriers of the defects. Applying transcobalamin II typing after bone marrow transplantation demonstrated that this protein originates partly in the bone marrow. Subsequent investigations in cell culture have shown that human skin fibroblasts and cultured bone marrow synthesize and secret isotypes of a transport protein corresponding to the genetic isotypes observed in plasma. Comparison of transcobalamin II types in umbilical cord serum with the maternal types, has proven that the transcobalamin II activity in the cord serum is derived from the fetus. This finding will be of crucial importance in the early diagnosis of the deficiency syndrome.     

Cell signaling in interstitial cystitis/painful bladder syndrome

Evidence for several types of cell signaling abnormalities has been presented for patients with interstitial cystitis/painful bladder syndrome (IC/PBS), a poorly understood chronic painful bladder disorder for which currently there is no reliable effective therapy. Increases or decreases in various urine cytokines and growth factors have been found in patient specimens, along with abnormal expression of epithelial differentiation markers, growth factors, cell membrane proteins, neurotransmitters, and other cytokines in tissue biopsies and/or explanted bladder cells from IC/PBS patients. Some of the abnormalities found in bladder epithelial cells from IC/PBS patients have been shown to be induced in normal cells by an antiproliferative factor from IC/PBS bladder epithelial cells that binds to a functional cell membrane receptor (CKAP4/p63). Greater understanding of cell signaling events associated with this debilitating disorder may lead to the development of more effective therapies.