Variant-specific differences in human unsaturated transcobalamin II

Electrophoresis and subsequent autoradiography of ⁵⁷Co-cobalamin (⁵⁷Co-Cbl)-labeled serum show intensity differences between the genetic variants of human transcobalamin II (TC2), suggesting differences in the unsaturated (apo-) TC2 concentration. In order to distinguish between variant-specific differences in the Cbl binding affinity and those in the total-TC2 concentration, techniques were developed to determine total, apo-, and holo-TC2. Prolonged incubation at 37° C with a 20-fold excess of ⁵⁷Co-Cbl resulted in an almost complete exchange of endogenously bound Cbl, which allowed determination of the total TC2. The holo-TC2 concentration of both gene products in TC2 heterozygotes could be estimated by comparison of the labeling levels of apo- and total TC2, using densitometric quantification of the autoradiographs. By means of ion-exchange chromatography, TC2 could be separated from other Cbl-binding proteins, permitting a simple quantitative assay of apo- and total TC2, the results of which correlate fairly well with those measured by an immunoadsorption assay. The results obtained in the present investigation indicate that the variant-specific variation in the apo-TC2 concentration is caused by differences in the total-TC2 concentration rather than in the Cbl binding affinity.     

The function of cellular transcobalamin II in cultured human cells

The known function of human transcobalamin II (TC II) is to transport cobalamin (Cbl) in the circulation to tissue receptors for TC II-Cbl. Several types of human cells synthesize apo (unsaturated) TC II and the present study was conducted in order to evaluate possible functions of this endogenous TC II. The approach consisted of a correlation between the abilities of cultured cells to produce apo TC II and to internalized Cbl when presented in the free form. The amount of apo TC II produced by six lines of cultured human cells ranged from abundant to nil. The amount of free Cbl internalized by these cells correlated directly with the capacity to produce apo TC II. The interactions between endogenous TC II and free Cbl took place either at the cell surface or in the medium surrounding the cell. It was also shown that cells in culture contain free Cbl and release free Cbl into the surrounding medium. Thus it was concluded that the apo TC II produced by human cells remains intact to interact with free Cbl and to participate in the cellular metabolism of Cbl.     

Methionine dependency of cultured human lymphocytes

Human peripheral blood lymphocytes stimulated with phytohemagglutinin and a lymphocyte model consisting of the RPMI 6410 cell, a human virus-transformed B cell, required added methionine (Met) for growth of the cultures. This failure to meet all needs for Met via endogenous synthesis, which is characteristic of oncogenic transformation, occurred even in the presence of adequate homocysteine, methylfolate (5-CH3-H4PteGlu) and cobalamin (Cbl)-dependent methionine synthetase activity. Folinic acid (5-CHO-H4PteGlu), which provides available folate independently of Cbl, improved growth only slightly in the absence of Met. Free Cbl at 222 nM, an amount great enough to alter other intracellular events, failed to increase growth in the absence of Met, but 0.22 nM Cbl bound to transcobalamin II did, however, enhance growth.     

Isolation of the complementary DNA for human transcobalamin II

A complementary DNA (cDNA) clone coding for transcobalamin II (TCII) has been isolated from a human umbilical vein endothelial cell cDNA library. The cDNA is 1.9 Kb and includes the nucleotide sequence which encodes the NH2-terminal 19 amino acids of human TCII. The size of the cDNA is sufficient to code for the entire protein and also contains the nucleotide sequence coding for a 24 amino acid leader peptide and a long untranslated 3' region. The availability of this cDNA will provide the opportunity to characterize genetic disorders of TCII.     

Synthesis of transcobalamin II by cultured human hepatocytes

Cultured HepG2 cells, derived from a human hepatoma synthesized and released unsaturated, immunoreactive transcobalamin II. Synthesis was confirmed by the blocking with inhibitors of protein synthesis and by incorporation of tritiated leucine into transcobalamin II.     

Purification and molecular characterization of human transcobalamin II

Transcobalamin II (TCII) has been purified from Cohn fraction III of human plasma by batchwise binding to and then elution from carboxymethyl-Sephadex, affinity chromatography using photo-labile aminopropyl cobalamin coupled to activated Sephacryl S-200, and finally chromatography through carboxymethyl cellulose. The yield was approximately 80%. The addition of protease inhibitors in all steps of the purification procedure and extensive washing of the carboxymethyl-Sephadex prior to eluting the TCII minimized degradation of the protein and the final preparation of holo-TCII contained 1 mol of cobalamin/mol of protein. A single polypeptide of 43,000 daltons was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal 19 amino acids have been determined for human TCII. 12 of the amino acids are homologous with rabbit TCII and six are homologous with human R-binder, but there is no homology with human intrinsic factor.     

Monoclonal antibodies to different sites on human transcobalamin II

Two IgG1K monoclonal antibodies to human transcobalamin II (TC II) were generated. These antibodies, 16.1 and 16.6, did not cross-react with the other two types of human cobalamin-binding proteins, intrinsic factor and R binder (TC I). Both antibodies cross-reacted with orangutan and simiang TC II but not with TC II from cynomolgus and howler monkeys, who are less closely related to humans. This finding suggests close structural similarity of human to ape TC II. The antibodies also did not react with TC II of lower mammals which included the horse, dog, guinea pig, and mouse; in particular, reaction did not occur with rabbit TC II, which has been considered structurally close to human TC II. Neither of the two antibodies was directed at the cobalamin-binding site of TC II. However, antibody 16.6 hindered TC II binding to cell receptor. This reactivity with the receptor-binding site should prove particularly useful in studies of that region of the TC II molecule.     

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.     

Species differences in the properties of mammalian transcobalamin II

Striking differences in physical and immunologic properties of transcobalamin II (TC II) in six mammalian species were noted. Polyacrylamide gel electrophoresis of TC II suggested the presence of isoproteins in several species. A microfine precipitate of silica (Quso), adsorbed TC II directly only from human and canine plasma. TC II in some species appears to be associated with a high molecular weight constituent of plasma, resulting in the TC II being unavailable to bind to Quso. Quso should therefore not be used to assay TC II in the plasma of all species without prior validation.     

Solid-phase immunoassay for the vitamin B12-binding protein transcobalamin II in human serum

A solid-phase radio immunoassay was developed for total immunoreactive transcobalamin II (TC II). Rabbit antihuman TC II antiserum (which recognizes both apo- and holo-TC II), was immobilized by covalent binding to acrylamide-acrylic acid copolymer beads. A normal mean and SD for immunoreactive TC II in serum was determined in 130 healthy adult individuals and found to be 1150 ± 250 ng/liter cobalamin equivalent. Mean holo-TC II (N = 30), estimated by substraction of apo-TC II from total TC II, was 137 ng/liter bound cobalamin (or 12% of total TC II). Three patients with lack of functional TC II had immunoreactive TC II levels between 22 and 39% of normal mean, which demonstrated that the solid-phase bound antiserum recognized deficient TC II molecules, whereas the same antiserum in its soluble form did not. Eight out of nine individuals, recognized as heterozygous for TC II deficiency, had TC II levels below the normal range, on the order of 50% of the normal mean. The stability of immunoreactive TC II was strongly enhanced by the presence of an unknown serum factor not corresponding to serum albumin.     

Mouse transcobalamin II metabolism: The effects of antibiotics on the clearance of vitamin B12 from the serum transcobalamin II-vitamin B12 complex and the reappearance of the free serum transcobalamin II in the mouse

The mechanism of the clearance of vitamin B₁₂ from the serum transcobalamin II-vitamin B₁₂ (Tc-II-B₁₂) complex and the reappearance of free Tc-II in mouse have been studied. When a saturating dose of vitamin B₁₂ is given parenterally to normal mice, a portion of the Tc-II-bound vitamin B₁₂ is rapidly cleared and free Tc-II promptly reappears until it reaches a constant level in 6–8 h. The remaining vitamin B₁₂ is cleared slowly from the rest of the Tc-II-B₁₂ complex. In cycloheximide or puromycin-treated mice, free Tc-II fails to reappear and the bound Tc-IIdecreases. Treatment with actinomycin D has no effect on the reappearance of free Tc-II. The probable mechanism of this inhibition is discussed. The results suggest that mouse serum Tc-II has a stable messenger RNA template and a fast turnover. The free Tc-II which reappears in the serum after Tc-II has been saturated with vitamin B₁₂, appears to be newly synthesized.     

Inheritance and genetic linkage of transcobalamin II

Summary The genetic polymorphism of the vitamin B₁₂ transport protein transcobalamin II (TC II) was studied in a Caucasian population and in families. There are five codominent alleles of TC II which show a Mendelian mode of inheritance. No genetic linkage of TC II was found with gene loci for ADA, GLOI, Pi, HLA, AB0 and AK₁. TC II like proteints could be detected on autoradiograph of PAGE in two patients with congenital homozygosity for functional TC II deficiency. These vitamin B₁₂ binding proteins in the patients' serum were shown not to be normal R-proteins.Supported in part by grants from U.S. Public Health Service, NCI CA-22507, CA-19267, CA-08748, NIAID AI-07073. A portion of this work was conducted through the Clinical Research Center Facility of the University of Washington (RR-37)     

The role of the transferrin receptor for the activation of human lymphocytes

The proliferative response of peripheral blood mononuclear cells (PBMC) in synthetic serum-free media depends on the presence of sufficient amounts of transferrin (Tf). In the present communication we show that the reduction of Tf concentration in culture media results in a decreased proliferation, whereas lymphokine production and the expression of activation markers (IL-2 receptor; transferrin receptor, (TfR); HLA class II) remain unchanged. To examine whether this effect is due to iron depletion we added iron chelates (ferric citrate, FeCi; ferric nitrilotriacetic acid, FeNTA) which can be internalized by cells without the requirement for Tf. The iron chelates could fully restore the proliferative response even in complete absence of Tf, suggesting that the observed inhibitory effect was indeed caused by iron depletion. Addition of a monoclonal TfR antibody, J 64, also caused a marked inhibition of proliferation of PBMC in regular serum-containing medium as well as in Tf-free synthetic medium; this effect could not be overcome by any of the tested iron chelates. Therefore, growth inhibition caused by J 64 cannot simply be attributed to iron starvation. These data suggest that J 64 may interfere with processes others than iron uptake and that the TfR might confer a necessary promoting signal for lymphocyte proliferation.