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. (+info)
Sequence, S-S bridges, and spectra of bovine transcobalamin expressed in Pichia pastoris.
Transcobalamin (TC) -encoding cDNA was isolated from a bovine mammary gland cDNA library. Hybridization of the cloned bovine TC-cDNA to RNA samples from bovine tissues showed that the most intensive synthesis of a TC positive 1.9-kilobase mRNA occurred in kidney, lymphatic nodes, and liver. Bovine TC was expressed in yeast Pichia pastoris, and the isolated recombinant protein showed cobalamin (Cbl) and receptor binding properties similar to TCs from other sources. Alignment of the related Cbl carriers (haptocorrins and intrinsic factors from other species) with bovine TC (414 residues) revealed four conservative clusters in the sequence (85-98, 137-147, 178-190, and 268-288), which may be responsible for Cbl binding. Three S-S bonds connected Cys residues 3-252, 98-294, and 147-190. Treatment with an S-S reducing agent caused liberation of Cbl from TC-Cbl. A significant change was observed in the TC-Cbl absorbance spectrum upon substitution of Co(2+)-coordinated H(2)O by azide. The reaction developed several orders of magnitude slower, and the spectral distortions were much stronger than those in free Cbl. This may be caused by significant deformation of the Cbl molecule and/or by its shielding when bound to TC. (+info)
Cellular import of cobalamin (Vitamin B-12).
Recent studies have isolated and characterized human gastric intrinsic factor (IF) and transcobalamin II (TC II) genes, whose products mediate the import of cobalamin (Cbl; Vitamin B-12) across cellular plasma membranes. Analyses of cDNA and genomic clones of IF and TC II have provided some important insights into their sites of expression, structure and function. IF and TC II genes contain the same number, size and position of exons, and four of their eight intron-exon boundaries are identical. In addition, they share high homology in certain regions that are localized to different exons, indicating that IF and TC II may have evolved from a common ancestral gene. Both IF and TC II mediate transmembrane transport of Cbl via their respective receptors that function as oligomers in the plasma membrane. IF-mediated import of Cbl is limited to the apical membranes of epithelial cells; it occurs via a multipurpose receptor recently termed "cubilin," and the imported Cbl is usually exported out of these cells bound to endogenous TC II. On the other hand, TC II-mediated Cbl import occurs in all cells, including epithelial cells via a specific receptor, and the Cbl imported is usually retained, converted to its coenzyme forms, methyl-Cbl and 5'-deoxyadenosyl-Cbl, and utilized. (+info)
Conformational changes of transcobalamin induced by aquocobalamin binding. Mechanism of substitution of the cobalt-coordinated group in the bound ligand.
Binding of aquo-, cyano-, or azidocobalamin (Cbl.OH(2), Cbl.CN, and Cbl.N(3), respectively) to the recombinant human transcobalamin (TC) and haptocorrin from human plasma was investigated via stopped-flow spectroscopy. Association of cobalamins with haptocorrin always proceeded in one step. TC, however, displayed a certain selectivity for the ligands: Cbl.CN or Cbl.N(3) bound in one step with k(+1) = 1 x 10(8) M(-1) s(-1) (20 degrees C), whereas binding of Cbl.OH(2) under the same conditions occurred in two steps with k(+1) = 3 x 10( 7) M(-1) s(-1) (E(a) = 30 kJ/mol) and k(+2) = 0.02 s(-1) (E(a) = 120 kJ/mol). The second step of Cbl.OH(2) binding was interpreted as a transformation of the initial "open" intermediate TC.Cbl.OH(2) to the "closed" conformation TC(Cbl) with displaced water. The backward transition from the closed to the open conformation was the reason for the identical rate-limiting steps during substitution of H(2)O in TC.Cbl.OH(2) for cyanide or azide according to the reaction TC(Cbl) --> TC.Cbl.OH(2) + CN(-)/N(3)(-). The cyano and azido forms of holo-TC which were produced behaved as the open proteins. Different conformations of holo-TC, determined by the nature of the active group in the bound Cbl, may direct transportation of cobalamins in the organism. (+info)
Extremely low activity of methionine synthase in vitamin B-12-deficient rats may be related to effects on coenzyme stabilization rather than to changes in coenzyme induction.
Severely vitamin B-12 (B-12)-deficient rats were produced by feeding a B-12-deficient diet. The status of B-12 deficiency was confirmed by an increase in urinary methylmalonate excretion and decreases in liver B-12 concentrations and cobalamin-dependent methionine synthase activity. Rat liver methionine synthase existed almost exclusively as the holoenzyme. In B-12-deficient rats, the level of methionine synthase protein was lower, although the mRNA level was not significantly different from that of control rats. When methylcobalamin, the coenzyme for methionine synthase, was administered to the B-12-deficient rats, growth, liver B-12 concentrations and urinary excretion of methylmalonate were reversed although not always to control (B-12-sufficient) levels in a short period. During this recovery process, methionine synthase activity and its protein level increased, whereas the mRNA level was unaffected. We reported previously that rat apomethionine synthase is very unstable and is stabilized by forming a complex with methylcobalamin. Thus, the extremely low activity of methionine synthase in B-12-deficient rats may be related to effects on "coenzyme stabilization" (stabilization of the enzyme by cobalamin binding) rather than to changes in "coenzyme induction." (+info)
Measurement of transcobalamin by ELISA.
BACKGROUND: Transcobalamin is essential for the cellular internalization of cobalamin. Methods to quantify the unsaturated protein are available, but few attempts have been made to develop methods to quantify the sum of unsaturated and cobalamin saturated transcobalamin. METHODS: gamma-Globulins from two polyclonal rabbit antibodies against recombinant human transcobalamin were used as capture and detection antibodies, and recombinant human transcobalamin was used as calibrator in an ELISA design. RESULTS: The ELISA is specific for transcobalamin and has a detection limit of <1.6 pmol/L. The imprecision (CV) is 4-6% for mean concentrations of 13-70 pmol/L. The central 95% interval for serum from healthy blood donors (n = 77) was approximately 600-1500 pmol/L and showed limited variation with age and sex. No correlation was observed between the marker of acute phase reaction, C-reactive protein, and transcobalamin in plasma. CONCLUSIONS: The ELISA measures total transcobalamin in serum and thus can be used for measurement of transcobalamin in patients treated with cobalamin. (+info)
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. (+info)
Transcobalamin codon 259 polymorphism in HT-29 and Caco-2 cells and in Caucasians: relation to transcobalamin and homocysteine concentration in blood.
Transcobalamin (TC) is the plasma transporter that delivers vitamin B(12) to cells. We have already reported that HT-29 and Caco-2 cells secrete different TC variants. HT-29 secretes 2 TC isoproteins (codon 259-Pro/Arg [259-P/R]), exhibiting unequal concentrations (TC 259-P > TC 259-R), and Caco-2 cells only secrete the phenotype 259-R. We investigated the relation between phenotypic and genetic TC polymorphism in HT-29 cells transfected with Caco-2 TC complementary DNA and in 159 healthy Caucasians. We found that codon 259-R is buried and, thus, the genetic polymorphism provides no explanation why the TCs from HT-29 and Caco-2 cells have different isoelectric points in nondenaturing isoelectric focusing (IEF). The newly translated TC in HT-29 cells from the Caco-2 complementary DNA recombinant plasmid had the same isoelectric point as the TC constitutively expressed in HT-29 cells, suggesting that TC phenotypic variability involves a specific cell folding of the protein. The codon 259 polymorphism was found to have a biallelic distribution: homozygotes P = 34.6%, heterozygotes R/P = 47.8%, and homozygotes R = 17.6%. In heterozygous samples, the IEF showed that the TC 259-P/TC 259-R ratio = 1.6. The blood apo-TC concentration of 259-P homozygous Caucasians was significantly higher than that of homozygous 259-R (P <.0001) and heterozygous (P <.0006) Caucasians. The heterozygotes 259-R/P had homocysteine concentration significantly higher than the homozygotes 259-R and 259-P (P =.02 and P =.01, respectively). In conclusion, TC codon-259 polymorphism affects TC plasma concentration and may interfere in vitamin B(12) cellular availability and homocysteine metabolism. (+info)