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PubMed1985

Increased leucocyte alkaline phosphatase and transcobalamin III in chronic myeloid leukaemia associated with lithium therapy.

A 38-year-old woman developed chronic myeloid leukaemia after 2 years of lithium carbonate therapy. A peculiar feature of her leukaemia, as well as of the 5 patients previously reported in whom CML has developed in the course of lithium therapy, was the unusually high degree of granulocyte maturation manifested in normal leucocyte alkaline phosphatase (LAP) score and, in 1 case, selective increase of transcobalamin III. Although a cause and effect relation between lithium therapy and CML has not yet been established, in view of the stimulatory effect of lithium on granulocyte proliferation, such treatment should be avoided in patients with established myeloproliferative disorders, or in patients at high risk of developing leukaemia.

Adult↗
PubMed2006

Steroid-responsive functional B12 deficiency in association with transcobalamin II polymorphism 776C --> G.

We present a case of intracellular vitamin B12 deficiency presenting with confusion, subacute combined degeneration of the cord, megaloblastic anaemia and intrinsic factor antibodies in the serum. Diagnosis was delayed by a normal serum B12 level and was confirmed by a grossly elevated serum homocysteine. There was a dramatic response to steroids. The patient was heterozygous for the transcobalamin (TC) II polymorphism 776C --> G. This case demonstrates the importance of functional assessment of intracellular B12 activity (e.g. serum homocysteine) in excluding B12 deficiency, the role of steroids in pernicious anaemia and a possible clinical correlation of a TCII polymorphism.

Aged↗
PubMed2005

Mapping the functional domains of human transcobalamin using monoclonal antibodies.

Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA...TAAM(103-198) or the C-terminal peptide LEPA...LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS...QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA...HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment KRSN...RTVR(207-227), which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor.

Amino Acid Sequence↗
PubMed2006

Application of a fluorescent cobalamin analogue for analysis of the binding kinetics. A study employing recombinant human transcobalamin and intrinsic factor.

Fluorescent probe rhodamine was appended to 5' OH-ribose of cobalamin (Cbl). The prepared conjugate, CBC, bound to the transporting proteins, intrinsic factor (IF) and transcobalamin (TC), responsible for the uptake of Cbl in an organism. Pronounced increase in fluorescence upon CBC attachment facilitated detailed kinetic analysis of Cbl binding. We found that TC had the same affinity for CBC and Cbl (K(d) = 5 x 10(-15) m), whereas interaction of CBC with the highly specific protein IF was more complex. For instance, CBC behaved normally in the partial reactions CBC + IF(30) and CBC + IF(20) when binding to the isolated IF fragments (domains). The ligand could also assemble them into a stable complex IF(30)-CBC-IF(20) with higher fluorescent signal. However, dissociation of IF(30)-CBC-IF(20) and IF-CBC was accelerated by factors of 3 and 20, respectively, when compared to the corresponding Cbl complexes. We suggest that the correct domain-domain interactions are the most important factor during recognition and fixation of the ligands by IF. Dissociation of IF-CBC was biphasic, and existence of multiple protein-analogue complexes with normal and partially corrupted structure may explain this behaviour. The most stable component had K(d) = 1.5 x 10(-13) m, which guarantees the binding of CBC to IF under physiological conditions. The specific intestinal receptor cubilin bound both IF-CBC and IF-Cbl with equal affinity. In conclusion, the fluorescent analogue CBC can be used as a reporting agent in the kinetic studies, moreover, it seems to be applicable for imaging purposes in vivo.

Binding Sites↗
PubMed2004

Rat transcobalamin: cloning and regulation of mRNA expression.

Transcobalamin (TC) has been cloned and used for studying its gene expression in the rat. TC mRNA is distributed widely in adult rat tissues, but at different levels (kidney > liver > lung > yolk sac > intestine > heart > brain > spleen > muscle). TC mRNA levels were 4-fold higher in the jejunum and ileum compared to its levels in the duodenum. During postnatal development, TC mRNA levels in the ileum declined 4-fold from day 4 to day 12, but increased by 5-fold between days 12 and 24. In contrast, TC mRNA levels increased by 2.5-fold in the kidney from day 4 to day 12 and then declined by 2-fold by day 24. Adrenalectomy of adult rats resulted in a 4-fold decline in ileal levels of TC mRNA and a 50% decline in the ileal mucosal formation of the TC-[(57)Co] cobalamin (Cbl) complex following oral administration of [(57)Co]Cbl complexed to gastric intrinsic factor (IF). Cortisone treatment reversed these changes noted in the ileum. In contrast to ileum, kidney TC mRNA levels were not altered significantly in adrenalectomized rats before and after cortisone treatment. Taken together, this study has provided evidence for the regulation of TC gene expression in the rat kidney and intestine during their postnatal development, and cortisone selectively regulates ileal but not kidney TC mRNA levels.

Adrenalectomy↗
PubMed1995

Long-term follow up of patients with transcobalamin II deficiency.

Five cases of transcobalamin II deficiency presenting to our institution were reviewed. A delay in diagnosis often led to acute deterioration. Two patients have long term neurological sequelae. Minimal treatment in these patients may be dangerous. While haematological normality may be maintained, the adequate therapeutic dose of vitamin B-12 to allow normal neurological development and function is not easily determined and damage sustained early in life may be irreversible.

Drug Administration Schedule↗
PubMed1993

Correlations between holo-transcobalamin II, holo-haptocorrin, and total B12 in serum samples from healthy subjects and patients.

AIMS: To study the correlations between total vitamin B12(B12), holo-haptocorrin, and holo-transcobalamin II (holo-TCII) concentrations in human sera; the association between reduced holo-TCII concentrations and macrocytosis attributable to B12 deficiency. METHODS: Serum samples from 38 healthy volunteers, 113 patients with normal total serum B12 concentrations and 93 patients with low total serum B12 were studied. Holo-TCII was removed from whole serum by adsorption with amorphous precipitated silica, and both whole serum and adsorbed serum were assayed for B12 using the Becton Dickinson vitamin B12 [57Co] radioassay kit. RESULTS: In all three groups of subjects studied there were strong correlations between the logarithms of the total serum B12 and the holo-haptocorrin concentrations with regression coefficients between 0.884 and 0.967. By contrast, the correlations between the logarithms of the total serum B12 and holo-TCII concentrations were weaker, especially in the patients with normal or low total serum B12, for whom the regression coefficients were 0.491 and 0.391, respectively. Analysis of the clinical records of a proportion of the patients studied indicated that there were many more patients with low holo-TCII concentrations than with haematological disturbances related to B12 deficiency. CONCLUSIONS: The total serum B12 concentration is a relatively poor indicator of holo-TCII concentrations and, therefore, of the ability of serum to deliver B12 to tissues. Additional information regarding B12 values can therefore be gleaned from measuring holo-TCII concentrations in the serum. Low holo-TCII concentrations, however, are an early sign of negative B12 balance and are frequently unassociated with haematological abnormalities caused by B12 deficiency.

Adult↗
PubMed1996

Limited value of serum holo-transcobalamin II measurements in the differential diagnosis of macrocytosis.

AIM: To study the value of serum holo-transcobalamin II (holo-TCII) measurements in the differential diagnosis of macrocytosis. METHODS: Holo-TCII concentrations were measured in serum samples from 50 healthy non-vegetarian subjects and 30 patients with macrocytosis, using a technique based on the adsorption of holo-TCII with amorphous, precipitated silica. Deoxyuridine (dU) suppression tests were performed on the bone marrow cells of all the patients. Haematological diagnoses were made using standard criteria. RESULTS: The causes of macrocytosis were cobalamin (Cbl) deficiency due to pernicious anaemia or following partial gastrectomy (10 patients), dietary folate deficiency with/without Cb1 deficiency (four patients), chronic alcoholism (four patients), myelodysplastic syndrome (five patients), treatment with methotrexate or azathioprine (three patients), and congenital dyserythropoietic anaemia (CDA) (four patients). Undetectable or low holo-TCII concentrations were found in all patients with Cb1 deficiency and in some or all patients from each of the other diagnostic categories. There was also no correlation between the dU suppressed value and the holo-TCII concentration: all 15 patients with high dU suppressed values and nine of 15 with normal dU suppressed values, including four patients with CDA, had low holo-TCII concentrations. CONCLUSIONS: Measurements of serum holo-TCII concentrations by the silica adsorption method are not of value in the differential diagnosis of macrocytosis. The finding of low serum holo-TCII concentrations in patients with macrocytosis due to causes other than Cb1 deficiency may result not only from a state of negative Cb1 balance but also from other factors, such as increased utilisation of holo-TCII as a consequence of erythroid hyperplasia.

Biomarkers↗
PubMed2003

Function and stability of human transcobalamin II: role of intramolecular disulfide bonds C98-C291 and C147-C187.

The current studies have investigated the role of three disulfide bonds of human transcobalamin II (TC II), a plasma transporter of cobalamin (Cbl; vitamin B12), in its function and stability. When translated in vitro in the presence or absence of microsomal vesicles, TC II constructs with a single substitution, C3S or C249S, demonstrated synthesis of a stable functional protein. However, TC II synthesized in the presence of microsomal vesicles using constructs with a single (C98S, C147S, C187S, C291S), double (C3/147/S, C98/147/S) or triple (C3/98/147/S) substitution was unstable. In the absence of microsomal vesicles, the percentage of binding to Cbl-Sepharose matrix by TC II expressed by constructs C3S, C3/147/S, C98/147/S, or C3/98/147/S was 100, 49, 52, and 35%, respectively. Upon their reductive alkylation, the binding of TC II expressed by these constructs was reduced to approximately 25-30%. TC II constructs C3S or C249S, when expressed in TC II-deficient fibroblasts, produced a stable functional protein, but those expressed by constructs C147S, C187S, C291S, C3/147/S, C98/147/S, or C3/98/147/S were rapidly degraded. The intracellular degradation of TC II expressed by these constructs was inhibited by lactacystin or MG-132 but not by the lysosomal degradation inhibitors ammonium chloride or chloroquine. These studies suggest that optimal binding of Cbl by human TC II is supported by disulfide bonds C98-C291 and C147-C187 and that their disruption results in loss of Cbl binding and their rapid degradation by the proteasomal machinery.

Alkylation↗
PubMed1989

Endothelial cells from human umbilical vein secrete functional transcobalamin II.

Transcobalamin II (TCII) is a cobalamin (Cbl) binding protein in the plasma that mediates the cellular uptake of Cbl. Although the synthesis of TCII by a variety of cultured mammalian cells and by some isolated perfused organs has been reported, no single tissue has been identified as the source of TCII in vivo. In this study, we demonstrate that cultured human umbilical vein endothelial cells secrete a protein that binds CN[57Co]Cbl, elutes from a Sephacryl S-200 column in the same position as TCII, and precipitates with an antiserum to purified human TCII. The biosynthesis of TCII by these cells was confirmed by demonstrating the incorporation of [35S]methionine into a nascent protein that immunoprecipitated with anti-TCII and which, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) had an Mr of 43,000, the same as human TCII. This secreted protein also had the functional properties of TCII because it facilitated the uptake of CN[57Co]Cbl by the same endothelial cells that secreted it as well as other cell lines that express the membrane receptor for TCII. We also present evidence that the venous endothelium could be the source of TCII in vivo by showing that an intact umbilical vein in an isolated umbilical cord, when perfused with medium containing [35S]methionine, secretes a radiolabeled nascent protein with the same immunoreactive and electrophoretic properties as human TCII. These studies demonstrate that the endothelial cell, which has been shown to secrete a number of plasma proteins, also synthesizes and secretes TCII both in vitro and as an intact endothelium in situ, and therefore, could be the source of circulating TCII in vivo.

Cells, Cultured↗
PubMed1989

Cobalamin release from intrinsic factor and transfer to transcobalamin II within the rat enterocyte.

To ascertain the mechanism of release of cobalamin (Cbl) from intrinsic factor (IF) and subsequent formation of transcobalamin II (TC-II)-Cbl complex, we studied the intracellular distribution of 57Co-labeled Cbl after its uptake in suckling and adult rats. The amount of Cbl bound to IF, to the IF-Cbl receptor via IF, and to TC-II was determined by immunoprecipitation with monospecific antisera raised to these proteins. IF-Cbl receptor activity was found to be very low in suckling rats up to 12 days after birth. Oral administration of leupeptin in amounts known to alter protein turnover had no effect on the release of Cbl from IF nor did it inhibit the formation of the TC-II-Cbl complex in either adult or suckling animals. However, oral administration of chloroquine resulted in a transient increase in the intestinal concentration of Cbl in both adult and suckling rats and in total inhibition of Cbl released from IF in adults rats. Chloroquine prevented completely the transfer of Cbl to TC-II in adult rats and inhibited the transfer by 50% in suckling rats. These data demonstrate that in adult mucosa utilizing receptor-mediated endocytosis, Cbl is transferred from IF to TC-II. This transfer does not require the IF-Cbl receptor, as it occurs in suckling rats. Finally, transfer of Cbl to TC-II is decreased by a drug that alters vesicular pH. Because Cbl can be released at acid pH from IF, it is proposed that release of Cbl from IF and its transfer to TC-II occurs in an acidic vesicle.

Administration, Oral↗
PubMed1999

Transcobalamin II synthesized in the intestinal villi facilitates transfer of cobalamin to the portal blood.

This study was designed to identify the cellular component of the intestinal villus where transcobalamin II (TCII) is synthesized, because this protein provides an essential function in the intestinal absorption of vitamin B(12) (cobalamin, Cbl). When a segment of proximal or distal small intestine of the guinea pig is cultured in medium containing [(57)Co]Cbl, TCII-[(57)Co]Cbl appears within 15 min. Northern blot analysis of RNA from both proximal and distal small intestine identified the TCII transcript. In situ hybridization of the distal ileum with (35)S-labeled TCII antisense transcript localized grains predominantly in crypts and in the lower third and central core of the villi. Grains were also evident at the base of the enterocytes in close apposition with the vascular network, whereas few grains appeared in the apical region of the columnar cells. This study provides evidence that TCII is constitutively expressed in the intestinal villi where vascular endothelium is abundant. In the distal ileum, where the intrinsic factor (IF) receptor is expressed, after uptake of IF-Cbl and the subsequent binding of free Cbl to TCII synthesized in the villi, the TCII-Cbl complex enters the microcirculation and passes into the portal blood.

Animals↗
PubMed2002

Megalin is essential for renal proximal tubule reabsorption and accumulation of transcobalamin-B(12).

Megalin has previously been shown to bind and mediate endocytosis of transcobalamin (TC)-B(12). However, the physiological significance of this has not been established, and other TC-B(12) binding proteins have been suggested to mediate renal uptake of this vitamin complex. The present study demonstrates by the use of megalin-deficient mice that megalin is, in fact, essential for the normal renal reabsorption of TC-vitamin B(12) and for renal accumulation of this highly conserved vitamin. Megalin-deficient mice excrete increased amounts of TC and B(12) in the urine, revealing a defective renal tubular uptake of TC-B(12). The urinary B(12) excretion is increased approximately 4-fold, resulting in an approximately 28-fold higher renal B(12) clearance. This is associated with an approximately 4-fold decrease in B(12) content in megalin-deficient kidney cortex. Thus megalin is important to prevent urinary loss of vitamin B(12). In addition, light- and electron-microscopic immunocytochemistry demonstrate lysosomal accumulation of B(12) in rat and mouse proximal tubules. In rats this accumulation is correlated with vitamin intake. Thus renal lysosomal B(12) accumulation is dependent on vitamin status, indicating a possible reserve function of this organelle in the rat kidney.

Animals↗
PubMed2000

Production of gastric intrinsic factor, transcobalamin, and haptocorrin in opossum kidney cells.

Opossum kidney epithelial cells were shown previously to synthesize and secrete two cobalamin (Cbl)-binding proteins, presumed to be haptocorrin (Hc) and transcobalamin II (TCII). The present study examines the hypothesis that renal tubular cells also produce intrinsic factor (IF), and this production provides an explanation for the presence of IF in urine. By using antisera raised against human IF and against TCII, the presence of TCII was confirmed, and that of IF discovered in the media of opossum kidney (OK) cells in culture. The apparent molecular weight of IF and TCII was 68 and 43 kDa, respectively. Immunoreactivity on Western blot of the putative IF protein was blocked by recombinant human IF. When proteins secreted into the media were separated electrophoretically under nondenaturing conditions after binding with [(57)Co]Cbl, a broad major band migrated at a relative front independently of recombinant IF or TCII, and probably represents Hc, as the Cbl binding is blocked by cobinamide. Small amounts of bound [(57)Co]Cbl migrated in the position of both IF and TCII, when cobinamide was present. The presence of IF and TCII in OK cells was confirmed by immunohistology. Specific reactivity for IF (blocked by recombinant IF) was found in proximal tubules of opossum kidney, but not in other portions of the nephron, confirming the ability of anti-human IF antiserum to detect opossum IF. A 732-bp fragment of IF, nearly identical in sequence to rat IF, was isolated by RT-PCR from opossum kidney mRNA, and Western blot confirmed the presence of IF protein. The presence of IF was also documented in rat kidney by isolation of an RT-PCR fragment, immunocytochemistry, and Western blot. IF should be added to the list of renal (proximal) tubular antigens that are shared by other epithelia.

Animals↗
PubMed2000

Transcytosis and coenzymatic conversion of [(57)Co]cobalamin bound to either endogenous transcobalamin II or exogenous intrinsic factor in caco-2 cells.

We have examined the intracellular route, coenzyme conversion and transcytosis rate of [(57) Co]-labeled cobalamin (Cbl) in function of its presentation to the apical side of Caco-2 cells, either free or bound to intrinsic factor (IF). The free-presented Cbl was progressively bound to endogenous transcobalamin II (TCII) which may stem, in part, from a basolateral to apical passage. Its transcytosis was TCII-mediated as it was abolished when antibodies to TCII were added to the apical medium. The apparent permeability coefficient (P(app)) was estimated at 20.8+/-3.6, 103.5+/-17.7, 0.9+/-0.3 x 10(-5) cm/h for TCII-Cbl, IF-Cbl and haptocorrin-Cbl, respectively. Chloroquine inhibited the transcytosis rate of both TCII and IF-bound Cbl in a dose-dependent manner. Approximately 80% of apical Cbl, bound to either exogenous IF or endogenous TCII, was transported to the basolateral side as intact cyano[(57)Co]Cbl whereas the remainder was converted into Ado-Cbl and CH(3)-Cbl within the cells, as shown by HPLC analyses of a 1,000-g pellet and a 12,000-g supernatant. Coenzymatic conversion was virtually abolished by chloroquine. In conclusion, we suggest that apically presented free Cbl is internalized via TCII-dependent transport. The apically internalized CN-Cbl, bound to either IF or TCII, is processed via an acidic vesicle and part of it is converted to coenzymes, whereas bulk of CN-Cbl is transcytosed intact.

Biological Transport↗
PubMed1988

Transcobalamin II deficiency: long-term follow-up of two cases.

Two Spanish siblings, a boy and girl, with transcobalamin II (TCII) deficiency are described. Both have grown and developed mentally normally after 10 years of vitamin B12 therapy. Two other siblings died soon after birth and it is almost certain that they also suffered from TCII deficiency.

Anemia, Megaloblastic↗
PubMed1983

Prognostic significance of changes in serum transcobalamin levels in cancer patients with chemotherapy-induced granulocytopenia.

Serum transcobalamin (TC) levels were determined daily in 14 adults suffering from advanced nonhematological malignancies and hospitalized because of chemotherapy-induced leukopenia and fever. Even during the nadir leukocyte count, TCI and TCIII serum levels were normal or only slightly decreased indicating that bone marrow activity was not completely suppressed. A significant increase in serum TCII level was observed in all patients, with peak values occurring an average of 4.5 days from admission at a time when the median leukocyte count was 2,300/mm 3. Full white cell count recovery followed this peak TCII elevation within a mean of 5.5 days in all patients, coinciding with a fall in TCII levels to basal values. The rise in serum TCII level appears to be an early indicator for imminent bone marrow recovery in myelosuppressed patients.

Agranulocytosis↗
PubMed1975

Transcobalamins I and II as natural transport proteins of vitamin B12.

There are two conflicting theories of how plasma vitamin B12 (B12) is transported in man: (a) by two distinct transport proteins, transcobalamins I and II (TC I and II), each having a specific role and time of function; and (b) by three active transport proteins, TC I, II, and III, that take up B12 randomly in proportion to the unsaturated amounts of each. To test these theories a man was given 1.12 mug, 229 muCi, of [57Co]B12 mixed with food. Blood samples were taken several times on the 1st day and at lengthening intervals up to day 51. The amount of TC II-B12 was measured in each sample by: gel filtration and by precipitation with (NH4)2SO4. Total serum R-B12 was then separated into TC I and TC III by: (a) a single step anion exchange system and (b) isoelectric focusing (IEF). As the B12 was being absorbed, 92-95% of that in venous blood was carried by TC II. Absolute and percentage transport by TC II declined sharply during the first 24 h; between days 7 and 51 20-33% of the label was on TC II, and the rest was carried by R-type binders. Absolute transport by TC I did not reach a maximum until after day 1 and before day 3. Transport by an alpha2 R-type binder, TC III, could not be demonstrated. TC I was isoelectrically heterogenous, with the components focusing between pH 2.9 and 3.35. It was concluded that (a) TC II is the dominant carrier of B12 immediately after absorption; (b) maximum transport by TC I requires the passage of time after absorption; (c) after the absorbed B12 reaches equilibrium with the total body B12, about one fourth of the plasma B12 is carried by TC II and three fourth by TC I; and (d) TC I and TC II are the only functional transport proteins of plasma B12.

Biological Transport↗