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PubMed2006

Characterization of a monoclonal antibody with specificity for holo-transcobalamin.

BACKGROUND: Holotranscobalamin, cobalamin-saturated transcobalamin, is the minor fraction of circulating cobalamin (vitamin B12), which is available for cellular uptake and hence is physiologically relevant. Currently, no method allows simple, direct quantification of holotranscobalamin. We now report on the identification and characterization of a monoclonal antibody with a unique specificity for holotranscobalamin. METHODS: The specificity and affinity of the monoclonal antibodies were determined using surface plasmon resonance and recombinant transcobalamin as well as by immobilizing the antibodies on magnetic microspheres and using native transcobalamin in serum. The epitope of the holotranscobalamin specific antibody was identified using phage display and comparison to a de novo generated three-dimensional model of transcobalamin using the program Rosetta. A direct assay for holotrnscobalamin in the ELISA format was developed using the specific antibody and compared to the commercial assay HoloTC RIA. RESULTS: An antibody exhibiting >100-fold specificity for holotranscobalamin over apotranscobalamin was identified. The affinity but not the specificity varied inversely with ionic strength and pH, indicating importance of electrostatic interactions. The epitope was discontinuous and epitope mapping of the antibody by phage display identified two similar motifs with no direct sequence similarity to transcobalamin. A comparison of the motifs with a de novo generated three-dimensional model of transcobalamin identified two structures in the N-terminal part of transcobalamin that resembled the motif. Using this antibody an ELISA based prototype assay was developed and compared to the only available commercial assay for measuring holotranscobalamin, HoloTC RIA. CONCLUSION: The identified antibody possesses a unique specificity for holotranscobalamin and can be used to develop a direct assay for the quantification of holotranscobalamin.

Journal Article↗
PubMed1977

Purification and characterization of rabbit transcobalamin II.

Rabbit transcobalamin II has been purified by labile ligand affinity chromatography and G-200 Sephadex gel filtration. Structural studies indicate Stokes' radii of 2.7 nm and 3.0 nm for transocobalamin II saturated and unsaturated with cobalamin. The amino acid content of the protein is very similar to that of human transcobalamin II (Allen, R. H. (1975) Prog. Hematol. 9, 59-84). The aminoterminal sequence for transcobalamin II is reported for the first time: Glu-Ile-Cys-Gly-Val-Pro-Lys-Val-Asp-Ser-Glu-Leu-Val-Glu-Lys-Leu-Gly-Gln-Arg-Leu-Leu-Pro-(Trp)-Met-Thr). The ultraviolet and circular dichroic spectra of aquo-, hydroxo-, azido- and cyanocobalamin bound to rabbit transcobalamin II are described. On complex formation the molar absorption of the cobalamins increases and the major bands shift to longer wavelengths. The spectra are little affected by change in the fifth ligand, which indicates that the electron density around the cobalt atom is kept fairly constant by the transcobalamin II molecule. This is in contrast to the observations for the same cobalamins attached to human intrinsic factor and human transcobalamin I (Nexo, E. and Olesen, H. (1976) Biochim. Biophys. Acta 446, 143-150).

Amino Acid Sequence↗
PubMed1985

Transcobalamin II, a serum protein reflecting autoimmune disease activity, its plasma dynamics, and the relationship to established serum parameters in systemic lupus erythematosus.

Earlier investigations have shown that the activity of autoimmune diseases appears to correlate with increased levels of the vitamin B12 (cobalamin)-binding serum protein, transcobalamin II (M. Fráter-Schröder et al., Schweìz. Med. Wochensch 110, 1441, 1980; M. Fráter-Schröde et al., Lancet 2, 238, 1978). These preliminary findings were confirmed and extended with regard to SLE in the present prospective study. The correlation of serum levels with the degree of disease activity (determined by clinical scoring of 44 patients with systemic lupus erythematosus (SLE), fulfilling four or more of the American Rheumatoid Association criteria) was shown to be most reliable for transcobalamin II (P less than 0.001), when compared to other serological parameters. An answer to the question "what induces increased levels of transcobalamin II in active SLE?" was sought by injecting 15 SLE patients with cyanocobalamin which influences plasma dynamics of transcobalamin II. Results indicate that transcobalamin II-cobalamin "clearance" is probably unchanged in SLE, and that increased production or stimulation of transcobalamin II secretion may be the cause of elevated plasma levels in active SLE.

Antibodies, Antinuclear↗
PubMed1976

Increased circulating levels of transcobalamin ii in gaucher's disease.

The presence of several serum protein abnormalities in Gaucher's disease prompted a study of vitamin B12 binding proteins, in which 14 of 15 consecutive patients displayed increased circulating transcobalamin II unassociated with elevations of serum vitamin B12 or other vitamin B12 binders. Transcobalamin II levels were most significantly increased in nine patients with disease severe enough to require splenectomy (P less than 0.01), but were not correlated with liver size or levels of any other laboratory feature of Gaucher's disease studied. Splenectomy, per se, did not alter circulating transcobalamin II. Chracterization of the binder in Gaucher's disease revealed identity with normal serum transcobalamin II in acid inhibition of vitamin B12 binding, chromatographic behavior, immunologic specificity and functional integrity in vitamin B12 delivery. This observation suggests a relation between reticuloendothelial-cell activity and transcobalamin II metabolism. Elevated transcobalamin II levels may provide an additional means for diagnosis and assessment of Gaucher's disease.

Adolescent↗
PubMed1976

Changes in the ultraviolet and circular dichroism spectra of aquo-, hydroxy-, azido-, and cyanocobalamin when bound to human intrinsic factor or human transcobalamin I.

The ultraviolet and the circular dichroic spectra of aquo-, hydroxy-, azido-, and cyanocobalamin free or bound to purified human intrinsic factor or purified human transcobalamin I were recorded. Except for azidocobalamin-transcobalamin I, the molar absorption for the gamma1-band in the ultraviolet spectra increased with a factor between 1.2 and 1.4 when the cobalamins were bound to protein. The ultraviolet spectrum of azidocobalamin-transcobalamin I changed so that the gamma2-band became the most intense. Minor changes were observed in the other ultraviolet spectra. The most prominent change in the CD-spectra of protein bound cobalamins was the increase in negative elipticity above 400 nm. This increase was most intense for cyano- and azidocobalamin-transcobalamin I. The study emphasizes that the mechanism by which human intrinsic factor and human transcobalamin I bind cobalamins differ.

Binding Sites↗
PubMed1978

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.

Animals↗
PubMed2006

Polymorphism C776G in the transcobalamin II gene and homocysteine, folate and vitamin B12 concentrations. Association with MTHFR C677T and A1298C and MTRR A66G polymorphisms in healthy children.

One of the etiologies of hyperhomocysteinemia is decreased vitamin B(12). Genetic variation in the transcobalamin II gene, the transporter of vitamin B(12) to the cells, may produce altered homocysteine levels. We determined transcobalamin II C776G polymorphism, homocysteine, folate and vitamin B(12) levels and analyzed the interactive effect with the methylenetetrahydrofolate reductase C677T and A1298C and methionine synthase reductase A66G polymorphisms in 207 healthy Brazilian children. The prevalence of GG genotype of transcobalamin II C776G polymorphism in this Brazilian population, a highly miscigeneous population was 12.5% and the statistical analysis showed that this population is in Hardy-Weinberg equilibrium, it could be considered representative of the general population. We observed a significant increase in homocysteine in the 776GG vs. 776CC genotype, corroborating the influence of age as a determinant of homocysteine in relation to this polymorphism. When we analyzed vitamin B(12) and its relationship with the C776G polymorphism, we found no significant differences. Only 776CG/66AA or 776GG/66AG genotypes presented a significant increase in homocysteine when compared with other groups. In the multivariate analysis, transcobalamin II C776G (CC/CG vs. GG), methylenetetrahydrofolate reductase C677T (CC/CT vs. TT), folate, gender and age presented statistical significance in relation to the homocysteine. These can be considered independent risk factors for hyperhomocysteinemia in this children group. Our results, if confirmed in other populations, highlight the necessity for investigation of the transcobalamin II C776G polymorphism in the research for hyperhomocysteinemia risk factors.

Brazil↗
PubMed1986

Indication against genetic localisation of the human transcobalamin II gene (TC2) on chromosome 16.

The genetic locus of human transcobalamin II (TC2) is not yet known. The mouse transcobalamin II gene has been assigned to mouse chromosome 11, linked to hemoglobin A. This fact suggested a similar linkage of transcobalamin II in man, assigning it thus to human chromosome 16. Our linkage investigation in a family material of more than 600 individuals demonstrated absence of linkage between transcobalamin II and phosphoglycolate phosphatase, which is very closely linked to hemoglobin A on chromosome 16. Additionally we confirmed absence of linkage with the chromosome 16 gene marker system haptoglobin. These two gene marker systems are located far from each other, and the total length of chromosome 16 is estimated only about 100 cM. Together with recent results of investigations in somatic mouse-man cell hybrids, we conclude that TC2 is not located on chromosome 16. Additionally we found absence of linkage between transcobalamin II and 6-phosphogluconate dehydrogenase, rhesus blood group (both on chromosome 1), GC (chromosome 4), Esterase D (chromosome 13) and AG; absence of close linkage with "debrisoquin polymorphism".

Animals↗
PubMed1984

Transcobalamin II in human seminal plasma.

Study of cobalamin-binding proteins revealed seminal plasma to be the most concentrated site of transcobalamin II in man. The next richest normal fluid, blood, has approximately one-tenth its concentration. Normal seminal unsaturated cobalamin-binding capacity averaged 15,030 +/- 7,290 pg/ml, of which 11,550 +/- 6,660 pg/ml was transcobalamin II. Transcobalamin II levels were markedly diminished in subjects lacking seminal vesicles (1520-1660 pg/ml), but not after vasectomy. This suggests that seminal vesicles are the chief source of this protein in semen. R binder concentration was increased in postvasectomy subjects (9,970 +/- 4,900 pg/ml vs. 2,980 +/- 1,370 pg/ml in normals) and varied in other patients. The endogenous cobalamin content of semen was only 88-699 pg/ml, and was carried largely by R binder rather than by transcobalamin II. The function of the unusually large seminal transcobalamin II pool in reproduction is unknown, but seems unlikely to be related solely to cobalamin transport needs, at least within the male reproductive tract itself.

Humans↗
PubMed1975

[Transcobalamins in megaloblastic anemias].

Transcobalamins are proteins which carry vitamin B12 and which are normally partly unsaturated. This study of transcobalamins was carried out in 17 subjects with megaloblastic anemia (12 true cases of pernicious anemia and 5 cases of folate deficiency). Among the latter, the transcobalamins were studied in 4 cases of pernicious anemia, before and after treatment with vitamin B12. The distribution of endogenous B12 was determined in four normal controls and two cases of pernicious anemia. This vitamin is normally distributed roughly equally between the three transcobalamins, whereas in B12 deficiency, T.C.2 is very unsaturated together with T.C.1 to a lesser degree. The latent fixation capacity of the serum is increased together with the latent fixation capacity of T.C. I and, above all, T.C. II but that of T.C. III is reduced in patients with pernicious anemia. In folate deficiency, only the latent fixation capacity of T.C. II is increased. When vitamin B12 is administered in physiological dosage, T.C. I becomes gradually saturated. In pharmacological dosage, total fixation capacity together with that of T.C. I and T.C. II become gradually reduced, but in spite of high levels of circulating B12, these proteins remain partially unsaturated. Various theories are suggested to explain the variations of these three transcobalamins in megaloblastic anemia but the problem is still unclear.

Anemia, Macrocytic↗
PubMed1986

Plasma transcobalamins in haematological disorders.

Plasma UBBC-B12 and transcobalamins were measured in 112 patients suffering from different haematological disorders. The data showed different patterns of changes in plasma transcobalamin profile in different haematological disorders. Plasma UBBC-B12 and transcobalamins were significantly higher than normal in untreated chronic myeloid leukaemia, acute promyelocytic leukaemia, nutritional megaloblastic anaemia and in refractory anaemias with hypercellular marrow. Normal levels of these proteins were noted in chronic lymphatic leukaemias, in primary and secondary hypereosinophilic states and in multiple myeloma. Subnormal levels of these proteins were observed in hypoplastic anaemia and acute lymphoblastic leukaemia. Chronic myeloid leukaemia patients during blast crisis and acute myeloid leukaemia patients except those suffering from acute promyelocytic leukaemia showed varying pattern of plasma transcobalamins depending on type of blast crisis or FAB subtype of AML. The significance of these changes in plasma transcobalamins have been discussed along with the experience of other workers in this field.

Anemia↗
PubMed1980

Bone marrow participates in the biosynthesis of human transcobalamin II.

Studies concerning the site of synthesis of the vitamin B12 binding serum protein, transcobalamin II, have been done in various mammalian animals. The actual site of biosynthesis in man has not yet been defined. The finding that bone marrow derived cells release apo-transcobalamin II in the mouse led us to examine the genetic patterns of transcobalamin II in man, both before and after marrow transplantation. A gradual but incomplete transformation of the recipient's transcobalamin II type into donor's type, corresponding to 75% or less of the total activity, was registered in 4 cases. Surprisingly, persistent host-type TC II, in spite of different donor type, was observed in 4 further marrow recipients. We conclude that hematopoietic cells transferred with the transplanted marrow participate in the biosynthesis of human transcobalamin II.

Anemia, Aplastic↗
PubMed1997

Transcobalamin II receptor imaging via radiolabeled diethylene-triaminepentaacetate cobalamin analogs.

UNLABELLED: Rapidly dividing cells up-regulate the number of transcobalamin II receptors during DNA replication. We have developed diethylene-triaminepentaacetate (DTPA) cobalamin analogs for the purpose of imaging transcobalamin II receptors in malignant and nonmalignant tissue. METHODS: Methyl-, adenosyl- and cyanocobalamin-b-(4-aminobutyl)-amide-DTPA analogs were synthesized. In vitro binding of the analogs to the transcobalamin proteins was assessed by the unsaturated vitamin B12 binding capacity assay and compared to DTPA and cyanocobalamin. The biodistribution of the 111In-DTPA cobalamin analogs was measured at 24 hr after injection into sarcoma-bearing mice and non-tumor-bearing mice and pigs. RESULTS: Methyl-, adenosyl- and cyanocobalamin-b-(4-aminobutyl)-amide-DTPA analogs and DTPA were 94.0%, 90.4%, 66.4%, and 3.6%, respectively, as efficient in binding to the transcobalamin proteins when compared to cyanocobalamin. At 24 hr after administration, the cobalamin analogs had 5-17 times and 20-29 times, respectively, the amount of uptake within the resected tissue samples and transplanted sarcomas when compared to 111In-DTPA. CONCLUSION: The radiolabeled DTPA cobalamin analogs are biologically active. Preliminary animal studies suggest that the analogs could be effective in vivo transcobalamin II receptor imaging agents.

Animals↗
PubMed1975

Extreme elevation of serum transcobalamin I in patients with metastatic cancer.

Elevation of transcobalamin I and serum vitamin B12 levels has usually been associated with increased granulocytic proliferation, such as occurs in chronic myelogenous leukemia. Two patients with metastatic cancer had extremely high serum vitamin B12 and transcobalamin I levels--greater than those seen in even the most intense granulocytic proliferation--that were not explainable by leukocytosis. The subjects' serum vitamin B12 levels were 18,750 and 21,221 pg per milliliter (normal, 471 plus or minus 174 pg per milliliter, mean plus or minus S.D.) and unsaturated vitamin B12 binding capacity 158,750 and 5,400 pg per milliliter (normal, 1153 plus or minus 313 pg per milliliter) respectively. The abnormally elevated serum binder was shown to be identical with transcobalamin balamin I in every respect. Levels of transcobalamin II and serum third binder were normal. The cause of the binder abnormality is unknown, but factors other than granulocyte proliferation may control or contribute to the production or accumulation of transcobalamin I.

Adenocarcinoma↗
PubMed1983

Transcobalamin II deficiency associated with unusual bone marrow findings and chromosomal abnormalities.

A female infant presented at seven weeks of age with failure to thrive, progressively severe pancytopenia, hypogammaglobulinemia and, mucosal ulceration. Bone marrow morphology showed severed megaloblastic changes in the myeloid series with a shift to the left and an increased number of blasts with abnormal morphology. Erythroid precursors and megakaryocytes were markedly decreased. Cytogenetic studies showed marked aneuploidy and increased chromosomal breakage. Treatment with high doses of vitamin B12 resulted in a dramatic clinical response with hematological values becoming normal. The patient's serum showed absence of transcobalamin II, and very little TC I and TC III binding. The patient's parents had only half the lower limits of normal transcobalamin II. QUSO G-32 was used for separation of transcobalamins, and the results were confirmed by Sephacryl S-300. This case illustrates the usefulness of QUSO in the rapid diagnosis of transcobalamin II deficiency.

Blood Cell Count↗
PubMed1982

Allelic forms of mouse transcobalamin 2.

Transcobalamin 2 is the only vitamin B12-binding protein found in mouse serum. Two allelic forms of mouse transcobalamin 2 are described. The two forms differ in their mobilities on polyacrylamide gel electrophoresis. The slowly migrating form has been found in serum from 25 inbred mouse strains. The more rapidly migrating form was detected in 3 inbred mouse strains (NZB, ST/bJ, and CPB-WV). Both parental variants were expressed in F1 progeny of appropriate interstrain crosses, showing codominant expression of the transcobalamin 2 alleles. In backcrosses between F1 and parental individuals, the two electrophoretic variants were inherited as single Mendelian traits. The strain distribution pattern of the two variants in recombinant inbred lines likewise suggested a single-gene mode of inheritance and indicated a lack of close linkage with a number of genetic loci on chromosomes 1, 2, 4, 5, 6, 7, 9, 12, 14, 15, and 17. We propose the symbol Tcn-2 for the polymorphic gene locus coding for transcobalamin 2 in the mouse and Tcn-2s and Tcn-2f for the two alleles.

Alleles↗
PubMed1983

Application of a simple immunoadsorption assay for the measurement of saturated and unsaturated transcobalamin II and R-binders.

In this paper an immunoadsorption method for the selective measurement of the concentrations of saturated and unsaturated transcobalamin II and R-binders in human plasma is presented. With this assay the concentrations of saturated transcobalamin II found in the plasma of 70 normal individuals ranged from 20-220 pmol per litre, with a mean of 70 pmol/l. The concentration of R-binders, carrying cobalamin, ranged from 87-491 pmol/l (mean 195 pmol/l). The 33-203 pmol/l (mean 111 pmol/l) of cobalamin analogues were found to be almost exclusively bound to the R-binder fraction. The level of saturated binding proteins is not, or only marginally, influenced by the absorption of ingested cobalamin, even with an oral dose of 15 micrograms. The concentration of transcobalamin II-bound cobalamin is apparently not determined by the availability of unsaturated binding protein. On the contrary, a positive correlation was found between the R-binder-cobalamin concentration and total R-binder level. These observations suggest that the concentration of transcobalamin II-bound cobalamin is primarily determined by the concentration of cobalamin in the tissues.

Humans↗
PubMed1981

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 . 10(5). Consecutive purification steps comprised CM-Sephadex batchwise ion-exchange chromatography, affinity chromatography, using cyanocobalamin as a ligand, thermolability 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 Crohn 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.

Animals↗