Cbl-binding Proteins Research Articles

Organometallic DNA-B12 Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin-Transport Proteins.

The synthesis and structural characterization of Co-(dN)25 -Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN)39 -Cbl, which are organometallic DNA-B12 conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA-Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA-Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA-Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN)25 -Cbl, or radiolabeled vitamin B12 (57 Co-CNCbl) and Co-(dN)25 -Cbl or Co-(dN)39 -Cbl. Binding of the new DNA-Cbl conjugates was fast and tight with TC, but poorer with HC and IF, which extends a similar original finding with the simpler DNA-Cbl, Co-(dN)18 -Cbl. The contrasting affinities of TC versus IF and HC for the DNA-Cbl conjugates are rationalized herein by a stepwise mechanism of Cbl binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl-binding proteins to the DNA-Cbl, which is first bound to the respective β domains. This transition is fast with TC, but slow with IF and HC, with which weaker binding results. The invariably tight interaction of the DNA-Cbl conjugates with TC makes the Cbl moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells.

Binding of aquocobalamin to bovine casein and its peptides via coordination to histidine residues

Vitamin B12 (cobalamin, Cbl) is an essential nutrient of bovine milk, bound to the casein fraction and the Cbl-specific protein transcobalamin at a ratio of 50:50. This study aimed to elucidate the mechanism of interaction between Cbl and the caseins. It was found that the isolated caseins bind aquocobalamin (HOCbl) via histidine-[Co3+]Cbl coordination. The casein–Cbl complex slowly dissociated in the presence of KCN due to formation of CNCbl. The “active” His groups (5.7 mm measured in 41.6 mg mL−1 casein) accumulate ≤ 3 mm HOCbl at a high rate (t½ ≈ 10 min, 20 °C) and with a high affinity (Kd = 0.1 mm). Low pH hinders the binding, but does not accelerate a very slow dissociation (t½ ≈ 8 h). Increased temperature and/or the presence of the specific Cbl-binding proteins accelerate the dissociation. The consequences of casein–Cbl interactions for the intestinal uptake of Cbl remain unclear.

Mouse Transcobalamin Has Features Resembling both Human Transcobalamin and Haptocorrin

In humans, the cobalamin (Cbl) -binding protein transcobalamin (TC) transports Cbl from the intestine and into all the cells of the body, whereas the glycoprotein haptocorrin (HC), which is present in both blood and exocrine secretions, is able to bind also corrinoids other than Cbl. The aim of this study is to explore the expression of the Cbl-binding protein HC as well as TC in mice. BLAST analysis showed no homologous gene coding for HC in mice. Submaxillary glands and serum displayed one protein capable of binding Cbl. This Cbl-binding protein was purified from 300 submaxillary glands by affinity chromatography. Subsequent sequencing identified the protein as TC. Further characterization in terms of glycosylation status and binding specificity to the Cbl-analogue cobinamide revealed that mouse TC does not bind Concanavalin A sepharose (like human TC), but is capable of binding cobinamide (like human HC). Antibodies raised against mouse TC identified the protein in secretory cells of the submaxillary gland and in the ducts of the mammary gland, i.e. at locations where HC is also found in humans. Analysis of the TC-mRNA level showed a high TC transcript level in these glands and also in the kidney. By precipitation to insolubilised antibodies against mouse TC, we also showed that >97% of the Cbl-binding capacity and >98% of the Cbl were precipitated in serum. This indicates that TC is the only Cbl-binding protein in the mouse circulation. Our data show that TC but not HC is present in the mouse. Mouse TC is observed in tissues where humans express TC and/or HC. Mouse TC has features in common with both human TC and HC. Our results suggest that the Cbl-binding proteins present in the circulation and exocrine glands may vary amongst species.

High Measured Cobalamin (Vitamin B12) Concentration Attributable to an Analytical Problem in Testing Serum from a Patient with Pernicious Anemia

Vitamin B12 assays that depend on the competition of endogenous cobalamin (Cbl) in a sample with labeled Cbl for binding to intrinsic factor (IF) must effectively denature or inactivate any Cbl-binding proteins and autoantibodies to IF that are present in the sample. Blocking antibodies specific for IF are present in more than one-half of all patients with pernicious anemia. The alkaline hydrolysis denaturation step, coupled with 37 °C incubation, has been shown to inactivate …

Cobalamin (vitamin B 12) binding, phylogeny, and synteny of human transcobalamin

Selected residues in a highly conserved 15-residue region, 174SVDTAAMAGLAFTC L 188 of human transcobalamin (TC), a cobalamin (Cbl: vitamin B 12) binding protein, were subjected to site-directed mutagenesis. The mutant constructs were expressed in TC-deficient fibroblasts or in vitro to assess the effect of these mutations on Cbl binding. Phylogenetic analyses and protein parsimony indicated that TC evolved earlier than other mammalian Cbl-binding proteins, intrinsic factor and haptocorrins, and divergence occurred between mouse/rat and human dispersing TC gene to different chromosomes. These studies show that (a) two of the three polar residues, S174, T177, or D176 and two of the three conserved alanine residues, A179 and A184 present in the 15-residue evolutionary conserved region are essential for Cbl-binding by human TC, and (b) TC gene is transferred in a syntenic manner to different chromosomes, at least before the divergence of mouse/rat and human.

Analysis of the transcriptional regulation of mouse prostacyclin synthase gene

Human transcobalamin II (TC II) gene was isolated and partially sequenced. The gene is composed of nine exons and eight introns spanning ∼20 kb. Multiple potential transcription start sites were revealed by primer extension analysis. The 5′-flanking region of the gene contained no TATA-like motif, but a binding motif for HIP1, which is suggested to be important in the transcription of TATA-less housekeeping genes, was identified in a region very close to the initiator methionine codon. In addition, potential binding sites for a variety of transcription factors such as SP1, AP2, CF1, NF-IL6, Ets-l, Myb and E2A were also observed. Comparison of the genomic structure of TC II to other Cbl-binding proteins, human gastric intrinsic factor (IF) and transcobalamin I (TC I) revealed similar intron-exon organizations with respect to the number, position and size of exons. These results suggest that TC II, TC I and IF genes have originated by gene duplications of an ancestral gene and TC II, unlike the other two Cbl-binding proteins, is the product of a "housekeeping" gene.

Assimilation of [57Co]-labeled cobalamin in human fetal gastrointestinal xenografts into nude mice.

Cobalamin (Cbl) and its Cbl-binding proteins are present in amniotic fluid. Because amniotic fluid is swallowed by the embryo-fetus, we studied the ability of Cbl to be transported and metabolized across the embryo-fetal digestive tract. Human embryonic stomachs and intestines were transplanted into nude mice. The basal secretion of Cbl-binding proteins was studied by gel filtration of the graft juices. Intrinsic factor (IF) was looked for in gastric mucosa by immunohistochemistry. The uptake of [57Co]-labeled Cbl by the intestinal graft was studied by Schilling tests and HPLC. IF, haptocorrin, and a transcobalamin-like protein were detected in gastric juice, with concentration ranges of 5.0-26.4, 1.9-27.1, and 5.2-12.6 pmol/mL, respectively. The IF [57Co]Cbl complex had a single isoprotein with a pI at 5.6, which was maintained after incubation with neuraminidase. Urine excretion percentages (Schilling tests) ranged from 5.5 to 21.2% and from 0.3 to 1.6% when cyano-[57Co]Cbl-IF or cyano-[57Co]Cbl, respectively, was instilled in intestinal grafts. Chloroquine reduced significantly the percentage of excreted [57Co]Cbl. The [57Co]Cbl was mainly excreted as cyano-[57Co]Cbl in urines, showing a low coenzyme conversion. In conclusion, IF is secreted by the nonstimulated embryonic stomach and lacks sialic acid. Cbl binds to it and is subsequently transported across the xenografted embryo-fetal intestine. This suggests that amniotic fluid may contribute to Cbl delivery to the embryo-fetus.

Transport and endogenous release of vitamin B 12 in the dually perfused human placenta

The kinetics for vitamin B 12, cobalamin (Cbl), transfer across the human placenta, and the retention of the endogenous Cbl and its release into the maternal and fetal compartments were investigated in a dually perfused human term placenta in vitro. After 4 hours of perfusion following a single bolus injection (peak maternal perfusate 94 fmol/ml) of 57Co-Cbl into the maternal reservoir, the maternal [ 57Co-Cbl] rapidly decreased whereas the fetal [ 57Co-Cbl] was only 9% of the final maternal [ 57Co-Cbl]. Even though there was a limited transfer of 57Co-Cbl to the fetus, the placenta rapidly accumulated Cbl. At 4 hours, 18% of the initial 57Co-Cbl dose was in the placenta; only 3% of the initial dose was in the fetal perfusate. Also after 4 hours, [ 57Co-Cbl] was bound >95% to transcobalamin (TC)I/III-like proteins in the maternal perfusate, whereas Cbl was bound to TCI/III- and TCII-like proteins with some free in the fetal perfusate. In the cytosol, >95% of the [ 57Co-Cbl] was bound (80% to TCII-like and 19% to TCI/III-like proteins). When no exogenous Cbl was added, total endogenous [Cbl] in the maternal circulation increased with time during 8 hours of perfusion at a rate of 0.25 ± 0.12 pmol/gm per hour. Only 2% of this Cbl was free, whereas 98% was bound to specific binding proteins. Neither plateau values nor equilibration with the fetal side were noted. In the fetal circulation, there was a release of Cbl at a rate of 0.015 ± 0.003 pmol/gm per hour, which was 99.99% bound. Thus the human placenta rapidly concentrates Cbl in the perfused lobule with little distribution of Cbl to nonperfused areas. Therefore, the human placenta modulates the asymmetric transfer of vitamin B 12 on the basis of release of specific Cbl-binding proteins (TCI/III- and TCII-like) into the maternal and fetal perfusates. (J Pediatr 1998;132:S35-S42.)

Transcobalamin from cow milk: isolation and physico-chemical properties

The concentration of endogenous cobalamin (Cbl) in cow milk was 3.3 nM while the Cbl-binding capacity was 0.05 nM. Both endogenous and newly added Cbl showed similar quantitative distribution between a 280 kDa protein complex (45%) and a 43 kDa Cbl-binder (55%). Long time incubation, as well as urea treatment, was accompanied by a slow release of the 43 kDa Cbl-binder from the 280 kDa fraction. No other Cbl-binding proteins appeared after these procedures. The 43 kDa binder from cow milk, depleted of the ligand by urea treatment, reacted with Cbl even in the presence of a B 12 analogue cobinamide (Cbi) at the ratio Cbl:Cbi = 1:40. The Stokes radius of the binder changed from 2.7 nm for the Cbl-free protein to 2.5 nm for the Cbl-saturated form and the Cbl-saturated binder was able to displaze human transcobalamin (TC) from the TC-receptor. The interaction between the protein and Cbl was significantly suppressed at pH 2.0. The N-terminal sequence of the purified 43 kDa Cbl-binder revealed homology with TC from human and rabbit plasma. In conclusion we have shown that TC is the main Cbl-binding protein in cow milk. This is surprising, since previous studies on human and rat milk have shown another Cbl-binder, apo-haptocorrin, to be the dominating Cbl-binding protein.

Genomic Structure of Human Transcobalamin II: Comparison to Human Intrinsic Factor and Transcobalamin I

Human transcobalamin II (TC II) gene was isolated and partially sequenced. The gene is composed of nine exons and eight introns spanning ∼20 kb. Multiple potential transcription start sites were revealed by primer extension analysis. The 5′-flanking region of the gene contained no TATA-like motif, but a binding motif for HIP1, which is suggested to be important in the transcription of TATA-less housekeeping genes, was identified in a region very close to the initiator methionine codon. In addition, potential binding sites for a variety of transcription factors such as SP1, AP2, CF1, NF-IL6, Ets-l, Myb and E2A were also observed. Comparison of the genomic structure of TC II to other Cbl-binding proteins, human gastric intrinsic factor (IF) and transcobalamin I (TC I) revealed similar intron-exon organizations with respect to the number, position and size of exons. These results suggest that TC II, TC I and IF genes have originated by gene duplications of an ancestral gene and TC II, unlike the other two Cbl-binding proteins, is the product of a "housekeeping" gene.

Secretion of acid, gastrin, and cobalamin-binding proteins by the fetal pig stomach: developmental regulation by cortisol.

The role of cortisol in the development of gastric function was investigated in the fetal pig. Pregnant sows (term = 114 +/- 2 days, n = 10) were anaesthetized (pentobarbitone) and osmotic minipumps containing either saline or cortisol inserted subcutaneously into fetuses at 82-84 (n = 11) or 91-96 (n = 14) days gestation. Six days later the infused pigs were removed by Caesarean section and the stomach and its contents collected for analysis. Samples were also obtained from unoperated (control) fetal pigs (n = 51) removed under anaesthesia at intervals from 70 days gestation until term. The concentrations of acid in gastric fluid, gastrin in plasma and antral tissue (bioactive as well as precursor forms), and cobalamin(CBL)-binding proteins in fundic tissue were low in control pigs until 100 days gestation. During the last 2 weeks before term, large increases occurred in gastric fluid acidity (from pH 6-7 to pH 3 at birth), plasma amidated gastrin (from 10 to 90 pM) and CBL-binding protein concentration (from 10 to 80 pmol (g fundus)-1). These changes occurred in parallel with a surge in plasma cortisol (from 20 to 200 ng ml-1). Immature fetuses infused with cortisol had significantly higher gastric acidity, plasma amidated gastrin, and concentration of CBL-binding proteins than the corresponding control fetuses (P < 0.05). Across age and treatment groups, significant correlations were found between log10 plasma cortisol values and the above parameters (r = 0.59-0.76, P < 0.01). The results suggest that endogenous cortisol secretion stimulates the secretion of gastric acid, gastrin and CBL-binding proteins in the prenatal period of the pig.

Isolation and sequence analysis of variant forms of human transcobalamin II

Two cDNA clones (1.9 kb and 1.5 kb, respectively) encoding full length human TC II have been isolated from a human endothelial cell cDNA library and sequenced. The differences between the two clones are the length of the 5′ end and the 3′ end non-coding regions and the codon at position 198 and 219. Both the clones differ from the recently isolated (human endothelial cell) cDNA for TC II (Platica, O., Janecko, R., Quadros, E.V., Regee, A., Romain, R. and Rothenberg, S.P. (1991) J. Biol. Chem. 266, 7860–7863) in codon 259 and 376 and in their calculated pI values. In vitro transcription followed by translation in a reticulocyte lysate system and SDS-PAGE revealed that the isolated cDNA clones encode a protein of 43 kDa. Upon treatment with canine pancreatic microsomes, the molecular mass of the in vitro translated product was reduced to 41.5 kDa, indicating the presence of an ≈ 1.5 kDa signal peptide. This translation product was immunoprecipitated with rabbit anti-serum to human TC II and was able to bind to Cbl-Sepharose beads. The amino acid sequence alignment of TC II with that of other Cbl binding proteins (rat intrinsic factor, human transcobalamin I and porcine haptocorrin) revealed only 33% overall homology. However, there were four regions of greater than 80% homology and two regions of about 60% homology. These regions encompass the majority of the hydrophobic areas of the Cbl-binders. Based on these studies, we suggest that structural basis for the expression of different polymorphic forms of TC II may be due to single point mutations and that TC II, like other mammalian Cbl-binders, have evolved from a common ancestral gene. Furthermore, the Cbl-binding functional domain most probably resides in a hydrophobic pocket which is formed by all or some of the six regions of high homology.

Synthesis and secretion of Cobalamin binding proteins by opossum kidney cells

Opossum kidney epithelial cells synthesize and secrete two Cobalamin (Cbl) binding proteins of Mr 66,000 and 43,000. When grown on culture inserts, the apical medium contained both these proteins while the basolateral medium contained only the 43 kDa Cbl binder. Colchicine, a microtubule disruptive drug, increased two fold the apical but not the basolateral secretion of the Cbl binding proteins. Although the opossum Cbl binders did not cross react with antiserum raised to Cbl binders from other species, the identity based on Cbl binding and size suggest that the 66 kDa and 43 kDa proteins are haptocorrin and transcobalamin II.

Purification and some properties of cytosolic cobalamin-binding protein in Euglena gracilis.

In Euglena gracilis SM-ZK, a bleached mutant of E. gracilis z, the cobalamin- (Cbl-)binding activity was distributed in cytosol (49.2%), mitochondria (20.3%) and microsomal fraction (20.4%). The cytosolic Cbl-binding activity gave two major peaks in isoelectric focusing. The Cbl-binding protein with pI 3.2 was purified 6500-fold in a yield of 19.9%, and that with pI 4.7 5800-fold in a yield of 11.9%. The monomeric Mr values of both Cbl-binding proteins were about 66,000. The Cbl-binding activity of both proteins showed a very low pH-dependency, and thiol groups and metal ions were not concerned with the Cbl-binding activities. The Ks values of the Cbl-binding proteins with pI 3.8 and 4.7 for CN-Cbl were 1.0 and 2.0 nM respectively. The Cbl-binding protein with pI 3.8 was shown to be immunologically identical with the protein with pI 4.7 by double-immunodiffusion experiments against antibody to the protein with pI 3.8. The two cytosolic Cbl-binding proteins did not show the activities of Cbl-dependent enzymes in E. gracilis, N5-methyltetrahydrofolate:homocysteine methyltransferase, methylmalonyl-CoA mutase and ribonucleotide reductase, suggesting that the two cytosolic Cbl-binding proteins play a physiological role as intracellular Cbl carriers.

Variant-specific differences in human unsaturated transcobalamin II.

Electrophoresis and subsequent autoradiography of 57Co-cobalamin (57 Co-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 degrees 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.

3] Isolation of native and proteolytically derived ileal receptor for intrinsic factor—cobalamin

This chapter describes the assay method, purification, and isolation of native and proteolytically derived ileal receptor for intrinsic factor–cobalamin. Receptor activity during the purification was assessed by measuring the binding of the hog IF-[ 57 Co]Cbl complex made with purified hog intrinsic factor (IF). Hog IF can be replaced by human IF. However the important consideration is that IF preparations should be free of any possible contamination of other Cbl-binding proteins (nonIF). The supernatant fraction containing unbound IF-[ 57 Co]Cbl and the pellet containing receptor bound IF-[ 57 Co]Cbl are assayed for radioactivity. Receptor specific binding is calculated as the difference between the binding observed in the presence of CaCl 2 and in the absence of calcium with EDTA. The most important step in the purification is the immunoaffinity chromatography using rabbit anti-hog intrinsic factor bound to Sepharose 4B. For the purification of the dog receptor attempts to use either intrinsic factor alone or the intrinsic factor-cobalamin complex as an affinity absorbent resulted in very poor binding of the receptor compared to rabbit anti-hog intrinsic factor antiserum. The advantage of this method is the recycling of both the intrinsic factor-cobalamin complex and the affinity ligand. After elution of the receptor the column can be washed with 0.2 M glycine-HCl buffer, pH 3.0, to remove the intrinsic factor-cobalamin complex which was bound to the antibody. The eluate is neutralized to pH 7 and can be reused after assessing the amount of complex present.

4] Purification of B12-binding proteins using a photodissociative affinity matrix

Publisher Summary This chapter outlines a general method for the affinity purification of Cbl-binding proteins from body fluids or crude extracts utilizing a photodissociative Cbl matrix. B 12 -binding proteins I play essential roles in the absorption, distribution, and retention of vitamin B 12 (cyanocobalamin; CN-Cbl) and its coenzyme forms, adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl).The unique organometallic carbon–cobalt bond of alkylcorrinoids homolyzes when exposed to light in the UV-visible range. In most alkylcorrinoids this bond is located in the β or upper-axial ligand position. Analogs containing aminoalkyl groups in the β position readily react with activated solid-phase matrices to give immobilized corrinoids that retain their high avidity for apoCbl-binding proteins. When exposed to visible light, the organometallic bond photodissociates and releases an aquacorrinoid-protein complex to solution. The chapter discusses the preparation of photodissociative corrinoid matrices. The affinity purification of Cbl-binding proteins involves: the preparation of body fluids and tissue extracts, adsorption of apoCbl-binding proteins by S-APCbl, and photodissociation of Cbl-binding proteins from S-APCbl.

Presence of cobalamin analogues in animal tissues.

Cobalamin (Cbl, vitamin B-12) has been extracted and isolated from a number of animal tissues by using (i) reverse-affinity chromatography on R protein-Sepharose followed by adsorption to and elution from charcoal-coated agarose and (ii) paper chromatography. Radioisotope dilution assays showed that only 75-97% of the Cbl chromatographed in the position of crystalline Cbl. The remaining 3-25% was present in a number of slower and faster moving fractions. This suggested that Cbl analogues are present in animal tissues because appropriate controls ruled out the possibility that this material was artifactually derived from Cbl during the extraction and purification procedures. With a large-scale isolation from rabbit kidney, the material in five such fractions contained cobalt and had absorption spectra that were similar to but different from the spectrum of Cbl, indicating that they were Cbl analogues. Compared to Cbl, these Cbl analogues had decreased but definite affinities for Cbl-binding proteins with the following order of strength of binding: R protein > transcobalamin II > intrinsic factor. Compared to Cbl, they also had decreased but definite growth-promoting activity for two microorganisms, Euglena gracilis and Lactobacillus leichmannii, which require Cbl for growth. These Cbl analogues differed from each other and from 18 synthetic Cbl analogues, including the most common Cbl analogues synthesized by microorganisms, in at least one of the above features. These studies indicate that animal tissues contain a number of Cbl analogues whose origins, structures, and biologic activities remain to be determined.