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.     

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.     

Biosynthesis of transcobalamin II by adult rat liver parenchymal cells in culture.

The biosynthesis of transcobalamin II was investigated in primary cultures of adult rat liver parenchymal cells maintained in serum-free media. The data indicate that these hepatocytes secrete a vitamin B₁₂-binding substance into the culture medium which is identical to rat serum transcobalamin II as judged by the following criteria: (i) gel filtration on columns of Sephadex G-200; (ii) ion-exchange chromatography on columns of diethyl aminoethyl cellulose and carboxymethyl cellulose; (iii) polyacrylamide-gel electrophoresis at pH 9.5; and (iv) the ability to facilitate cellular vitamin B₁₂ uptake by HeLa cells and mouse L-929 fibroblasts in culture. The secretion of transcobalamin II by the liver parenchymal cells was blocked by cycloheximide, puromycin, and p-fluorophenylalanine. The inhibition by cycloheximide, but not that of the other inhibitors, was partially reversed upon removal of the drug. The liver parenchymal cells incorporated radioactive amino acids into transcobalamin II which was absorbed from the growth medium using affinity chromatography on Sepharose containing covalently linked B₁₂. Collectively, these data indicate that rat liver parenchymal cells, in culture, are capable of the biosynthesis de novo of transcobalamin II and the subsequent secretion of this protein into the culture media.     

Isolation of vitamin B-12-binding proteins by combined immuno and affinity chromatography. Comparative studies on the isolated and unisolated proteins

Intrinsic factor or cobalophilin were removed by incubating human gastric juice and pig pyloric extract with purified anti-intrinsic factor and anti-cobalophilin immunoglobulin-G, respectively, covalently coupled to Sepharose. Cobalophilin (transcobalamin I) was also removed from pig serum either by using anti-cobalophilin immunoglobulin-G Sepharose or by Sephadex G-200 chromatography. The one remaining semipurified vitamin B-12-binding protein (intrinsic factor, cobalophilin or transcobalamin II) was then isolated by vitamin B-12-Sepharose affinity chromatography. Intrinsic factors, cobalophilins and transcobalamin II isolated by this two-step procedure were compared by double isotope techniques with the corresponding protein not subjected to affinity chromatography and found to be identical in reaction to antiserum, gel filtration and electrofocusing. The avidity of the isolated and unisolated intrinsic factors for the ileal intrinsic factor receptor was also the same.     

Receptors for transferrin and transcobalamin II display segregated distribution on microvilli of leukemia L1210 cells

Simultaneous addition of uniform latex particles derivatized with transferrin (0.532 micron) and transcobalamin II (0.345 micron) to leukemia L1210 cells resulted in segregated binding to individual microvilli as demonstrated by scanning electron microscopy. This segregated distribution suggests that individual microvilli are endowed either transferrin or transcobalamin II receptors but not both. Intracellular sorting and segregation of newly synthesized or recycling receptors probably occur prior to expression on the plasmalemma microvilli.     

Porcine serum cobalophilin and transcobalamin. Identification, isolation and properties including electrofocusing patterns

Pooled porcine serum was found to contain cobalophilin (also called transcobalamin I) and transcobalamin (also called transcobalamin II). The two proteins were harvested by batchwise absorption with vitamin B-12 covalently coupled to Sepharose, and then separated from each other either by gel filtration or using an immunoadsorbent. Both proteins were finally isolated as single proteins using a second vitamin B-12-Sepharose chromatography step. Cobalophilin and transcobalamin complexed with vitamin B-12 had molecular weights by gel filtration of 135 000 and 38 000 and by the formula of Svedberg 104 000 and 44 000, Stokes radii 4.97 nm and 2.65 nm, and sedimentation coefficients 5.39 S and 3.75 S, respectively. Electrofocusing resolved the cobalophilin complex into three main isoproteins isoelectric at pH 3.23, 3.42 and 3.69, and transcobalamin into only the main component isoelectric at a value as low as pH 3.47. Neither protein was capable of binding to the ileal intrinsic factor receptor.     

Transcobalamin II--cobalamin binding sites are present on rabbit germ cells.

Specific binding sites for rabbit transcobalamin II have been found on isolated adult rabbit germ cells. Scatchard analysis revealed a single class of binding sites for [57Co]cyanocobalamin-transcobalamin II with an association constant (Ka) of 1.3 x 10(10) M-1 and 700 sites per cell. Binding was reversible, saturable and calcium dependent. Electron microscope radioautography following incubation with iodinated transcobalamin II at 4 degrees C led to a detectable labeling mainly restricted to the plasma membrane.     

Evidence for intestinal origin of transcobalamin II during vitamin B12 absorption.

The plasma binding of newly absorbed, radioactively labelled vitamin B12 was studied during a urinary excretion (Schilling) test. Vitamin B12, after being absorbed from the gut, enters blood attached to transcobalamin II, which seems to be derived from the ileal enterocyte. The absorbed B12 re-enters the blood stream after the transcobalamin II-B12 complex is cleared by the liver and it is then excreted into the urine during the Schilling test.     

Human plasma R-type vitamin B12-binding proteins. II. The role of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B12-binding protein in the plasma transport of vitamin B12.

The normal human granulocyte vitamin B12-binding protein, transcobalamin I, and transcobalamin III, have been labeled with 125I-labeled N-succinimidyl 3-(4-hydroxyphenyl)propionate and utilized for plasma clearance studies performed with rabbits. Both moieties of 125I-labeled granulocyte vitamin B12-binding protein-[57Co]vitamin B12 were cleared rapidly from the plasma (is less than 90% by 5 min) by the liver. After 30 min, the bulk of the 125I reappeared in the plasma in small molecular weight (less than 1000) form and was rapidly excreted in the urine. After 60 min the bulk of the [57Co]vitamin B12 reappeared in the plasma bound to rabbit transcobalamin II and was subsequently taken up by a variety of tissues. Approximately 15% of the 125I-labeled granulocyte vitamin B12-binding protein-[57Co-a1vitamin B12 was excreted intact into the bile during the period from 10 to 80 min after injection. The hepatic uptake of the protein-vitamin B12 complex was blocked by the prior injection of desialyzed fetuin but not by native fetuin. Similar results were obtained with 125I-labeled transcobalamin III-[57Co]vitamin B12. Approximately 90% of both moieties of 125I-labeled transcobalamin I-[57Co]vitamin B12 had prolonged plasma survivals similar to that of 125I-labeled bovine serum albumin. After treatment with neuraminadase, both moieties of the 125I-labeled transcobalamin I-[57Co]vitamin B12 complex were cleared rapidly from the plasma by the liver in a manner that was indistinguishable from that observed in the case of untreated granulocyte vitamin B12-binding protein and transcobalamin III. These observations indicate that desialyzed transcobalamin I and the native forms of the granulocyte vitamin B12-binding protein and transcobalamin III are cleared from plasma by the mechanism elucidated by Ashwell and Morell (Ashwell, G., and Morell A. G. (1974) Adv. Enzymol. 41, 99-128) that is capable of clearing a wide variety of asialoglycoproteins. These observations have implications concerning the function of the human R-type vitamin B12-binding proteins, the nature of the enterohepatic circulation of vitamin B12, the biological significance of the mechanism described by Ashwell and Morell, and the etiology of the increased plasma concentration of human R-type protein that occurs frequently in chronic myelogenous leukemia and occasionally in hepatocellular carcinoma and other solid tumors.     

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.     

Low holo-transcobalamin levels are prevalent in vegetarians and is associated with coronary artery disease in Indian population

Coronary artery disease (CAD) has been increasing alarmingly in India. We had earlier shown that vitamin B₁₂ deficiency is associated with CAD in Indian population. However, only about a quarter of the total vitamin B₁₂ is internalised in the cells by the proteins transcobalamin II. Vitamin B₁₂-bound transcobalamin II (holotranscobalamin, holoTC) is thus referred to as biologically active B₁₂. In this study, we ascertained the levels of holoTC in 501 CAD cases and 1253 healthy controls and for the first time show that holoTC levels are significantly lower (p?=?2.57E-4) in CAD (26.81?pmol/l) cases as compared to controls (29.97?pmol/l).     

Purification of human transcobalamin II-cyanocobalamin by affinity chromatography using thermolabile immobilization of cyanocobalamin.

Transcobalamin II-cyanocobalamin was isolated from Cohn fraction III of pooled human plasma by affinity chromatography on cyanocobalamin-Sepharose and some conventional separation methods. The affinity ligand cyanocobalamin was coupled to AH-Sepharose by a thermolabile linkage. The unsaturated binding protein was absorbed at 4 degrees C and eluted from the column at 37 degrees C as transcobalamin II-cyanocobalamin complex. The final preparation had a specific cyanocobalamin-binding capacity of 0.98 mol cyanocobalamin/mol transcobalamin II, the yield was 55% and the purification index amounted to 1.1 . 10(6). In dodecyl sulphate polyacrylamide gel electrophoresis one major protein band was observed at a molecular weight of 37 000 and a faint band at a molecular weight of 29 000. In polyacrylamide gel isolectric focusing the pure preparation turned out to be heterogeneous with isoelectric points ranging from pH 6.2 to 6.8, possibly by the occurrence of isoproteins.     

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.     

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.     

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.     

Human plasma R-type vitamin B12-binding proteins. I. Isolation and characterization of transcobalamin I. TRANSCOBALAMIN III. and the normal granulocyte vitamin B12-binding protein.

Transcobalamin I and transcobalamin III have been purified approximately 6,000,000- and 3,000,000-fold, respectively, from normal human plasma using a purification scheme consisting of immunoadsorption, dialysis against 7.5 M guanidine HCl to remove endogenous vitamin B12, and affinity chromatography on vitamin B12-Sepharose. The two proteins were separated from each other subsequently by chromatography on DEAE-cellulose. The vitamin B12-binding protein present in granulocytes obtained from normal subjects has been purified approximately 5000-fold using affinity chromatography on vitamin B12-Sepharose as the sole purification technique. The final preparations of all three proteins were homogeneous based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transcobalamin I and transcobalamin III belong to the R-typed class of vitamin B12-binding proteins and are indistinguishable from each other, and from the human granulocyte, milk, and saliva R-type vitamin B12-binding proteins, when studied by immunodiffusion with rabbit anti-human milk vitamin B12-binding protein sera. The carbohydrate compositions, expressed as moles of carbohydrate per mole of vitamin B12, of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B12-binding protein, respectively, are: sialic acid, 18, 11, 11; fucose, 9, 20, 24; galactose, 41, 51, 46; mannose, 24, 22, 20; galactosamine, 2, 2, 2; and glucosamine, 46, 54, 46. The high sialic acid content of transcobalamin I appears to account for the fact that this protein elutes after transcobalamin III and the normal granulocyte vitamin B12-binding protein during chromatography on DEAE-cellulose. This observation provides support for the hypothesis that differences among the R-type vitamin B12-binding proteins are due to differences in carbohydrate content. The similarities in carbohydrate composition and other properties of transcobalamin III and the granulocyte vitamin B12-binding protein provide support for the hypothesis that human plasma transcobalamin III is derived from granulocytes. The differences observed between transcobalamin I and the normal granulocyte vitamin B12-binding protein suggest that transcobalamin I may not be derived from granulocytes.     

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)     

An improved method for large scale purification of human holo-transcobalamin II

25 mg of human holo-transcobalamin II with a specific cobalamin-binding capacity of 0.95 mol cobalamin/mol TC II was purified from 122 kg Cohn fraction III with a yield of 73% and a purification factor of 9.34 · 10⁵. Consecutive purification steps comprised CM-Sephadex batchwise ion-exchange chromatography, affinity chromatography, using cyanocobalamin as a ligand, thermolabilly attached to 3.3′-diaminodipropylamine-substituted CH-Sepharose, and gel filtration. The high yield of the purification procedure was achieved by improving the stability of apo-transcobalamin II in the eluate of the CM-Sephadex, and by a few other modifications of a former procedure. In the latter, rapid denaturation of apo-transcobalamin II prohibited the use of long term affinity chromatography, which is obligatory for processing large amounts of Cohn fraction. In addition, subfractionation of transcobalamin II into smaller fragments which occurred in SDS-polyacrylamide gel electrophoresis in previous studies, was now reduced, indicating that proteolysis in the CM-Sephadex eluate had been prevented effectively.     

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.