Calcium-binding Protein Calbindin D9k Research Articles

Measurement of Very Fast Exchange Rates of Individual Amide Protons in Proteins by NMR Spectroscopy.

NMR spectroscopy is a pivotal technique to measure hydrogen exchange rates in proteins. However, currently available NMR methods to measure backbone exchange are limited to rates of up to a few per second. To raise this limit, we have developed an approach that is capable of measuring proton exchange rates up to approximately 104 s-1 . Our method relies on the detection of signal loss due to the decorrelation of antiphase operators 2Nx Hz by exchange events that occur during a series of pi pulses on the 15 N channel. In practice, signal attenuation was monitored in a series of 2D H(CACO)N spectra, recorded with varying pi-pulse spacing, and the exchange rate was obtained by numerical fitting to the evolution of the density matrix. The method was applied to the small calcium-binding protein Calbindin D9k , where exchange rates up to 600 s-1 were measured for amides, where no signal was detectable in 15 N-1 H HSQC spectra. A temperature variation study allowed us to determine apparent activation energies in the range 47-69 kJ mol-1 for these fast exchanging amide protons, consistent with hydroxide-catalyzed exchange.

New Insights into Mechanisms Of Vitamin D Action Revealed Through The Study Of Transgenic, Knockout, and Aging Mice

Vitamin D is an important regulator of calcium homeostasis. However, the role of vitamin D in the distal intestine, where 70% of calcium absorption occurs, has been unexplored. Hectorol, a vitamin D analog, which is less calcemic than the active form of vitamin D, 1,25(OH)2D3, has been suggested for the treatment of osteoporosis. We hypothesize that the distal segments of the intestine are as important as the proximal intestine in vitamin D mediated intestinal calcium absorption and that transgenic mice expressing the vitamin D receptor exclusively in the distal intestine express vitamin D target genes in the distal intestine at levels equivalent to wild type. We also hypothesize that hectorol is as effective as 1,25(OH)2D3 in the maintenance of calcium homeostasis. Intestine and kidney from WT mice, Tg, and VDR KO mice were harvested. Total RNA was isolated, and protein extracts were prepared. The expression of vitamin D target genes was analyzed by RT‐PCR. Protein expression was studied using Western blot. VDR mRNA was undetectable in all segments of the intestine in VDR KO mice and was 1.7 fold elevated in ileum, cecum and colon of Tg mice compared to WT mice. The calcium binding protein calbindin‐D9k mRNA was decreased in VDR KO mice and in Tg mice levels of calbindin‐D9k mRNA in the distal intestine were equivalent to WT. mRNA for CYP24A1, an enzyme regulated by 1,25(OH)2D3, was undetectable in intestine of VDR KO mice and was expressed at equivalent levels in the distal intestine of Tg and WT mice. Hectorol was as effective as 1,25‐D3 in inducing calbindin‐D9k mRNA in all regions of the intestine of vitamin D deficient mice.

Placental-specific Igf2 knockout mice exhibit hypocalcemia and adaptive changes in placental calcium transport

Evidence is emerging that the ability of the placenta to supply nutrients to the developing fetus adapts according to fetal demand. To examine this adaptation further, we tested the hypothesis that placental maternofetal transport of calcium adapts according to fetal calcium requirements. We used a mouse model of fetal growth restriction, the placental-specific Igf2 knockout (P0) mouse, shown previously to transiently adapt placental System-A amino acid transporter activity relative to fetal growth. Fetal and placental weights in P0 mice were reduced when compared with WT at both embryonic day 17 (E17) and E19. Ionized calcium concentration [Ca(2+)] was significantly lower in P0 fetal blood compared with both WT and maternal blood at E17 and E19, reflecting a reversal of the fetomaternal [Ca(2+)] gradient. Fetal calcium content was reduced in P0 mice at E17 but not at E19. Unidirectional maternofetal calcium clearance ((Ca) K (mf)) was not different between WT and P0 at E17 but increased in P0 at E19. Expression of the intracellular calcium-binding protein calbindin-D(9K), previously shown to be rate-limiting for calcium transport, was increased in P0 relative to WT placentas between E17 and E19. These data show an increased placental transport of calcium from E17 to E19 in P0 compared to WT. We suggest that this is an adaptation in response to the reduced fetal calcium accumulation earlier in gestation and speculate that the ability of the placenta to adapt its supply capacity according to fetal demand may stretch across other essential nutrients.

Active Intestinal Calcium Transport in the Absence of Transient Receptor Potential Vanilloid Type 6 and Calbindin-D9k

To study the role of the epithelial calcium channel transient receptor potential vanilloid type 6 (TRPV6) and the calcium-binding protein calbindin-D9k in intestinal calcium absorption, TRPV6 knockout (KO), calbindin-D9k KO, and TRPV6/calbindin-D(9k) double-KO (DKO) mice were generated. TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice have serum calcium levels similar to those of wild-type (WT) mice ( approximately 10 mg Ca2+/dl). In the TRPV6 KO and the DKO mice, however, there is a 1.8-fold increase in serum PTH levels (P < 0.05 compared with WT). Active intestinal calcium transport was measured using the everted gut sac method. Under low dietary calcium conditions there was a 4.1-, 2.9-, and 3.9-fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D9k KO mice, respectively (n = 8-22 per group; P > 0.1, WT vs. calbindin-D9k KO, and P < 0.05, WT vs. TRPV6 KO on the low-calcium diet). Duodenal calcium transport was increased 2.1-fold in the TRPV6/calbindin-D9k DKO mice fed the low-calcium diet (P < 0.05, WT vs. DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or KO mice. 1,25-Dihydroxyvitamin D3 administration to vitamin D-deficient null mutant and WT mice also resulted in a significant increase in duodenal calcium transport (1.4- to 2.0-fold, P < 0.05 compared with vitamin D-deficient mice). This study provides evidence for the first time using null mutant mice that significant active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D9k, thus challenging the dogma that TRPV6 and calbindin-D9k are essential for vitamin D-induced active intestinal calcium transport.

The effects of Vitamin D metabolites on expression of genes for calcium transporters in human duodenum

The role of Vitamin D in the regulation of calcium absorption in the intestine is well recognized but the mechanisms of the effects on human genes are surprisingly poorly understood. We have determined the expression of transcripts of the apical membrane calcium transporter TRPV6, the cytoplasmic calcium binding protein calbindin-D9k, the basolateral plasma membrane Ca2+-ATPase (PMCA1) and the Vitamin D receptor (VDR) in normal endoscopic duodenal mucosal biopsies using quantitative real-time RT-PCR and related baseline expression to Vitamin D metabolites. TRPV6 transcript levels have been shown to be significantly correlated with serum 1,25(OH)2D levels in men, but not overall in women, where negative effects of age predominate. TRPV6 and VDR expression were significantly related in both men and women, but were significantly lower in older women. Associations with bone mineral density and fractional calcium absorption were also studied. In a second series of subjects, duodenal biopsies were incubated in organ culture for 6h with Vitamin D metabolites. TRPV6 expression was significantly increased by 1,25(OH)2D3 (10−9mol/l) as was PMCA1 to a much smaller extent. TRPV6 expression also increased with 25(OH)D3. CYP27B1 expression was found in all samples, and CYP24 transcripts were detected after incubation with 1,25(OH)2D3 or 25(OH)D3.

Anti-progestogenic effect of flutamide on uterine expression of calbindin-D 9k mRNA and protein in immature mice

A calcium binding protein, calbindin-D 9k (CaBP-9k), is a cytosolic protein and regulated by steroid hormones in the reproductive tissues. Mouse CaBP-9k gene was predominantly regulated by progesterone (P4), whereas rat CaBP-9k was mainly regulated by 17beta-estradiol (E2) in the uterus. The induction of CaBP-9k can be employed as a biomarker for steroidal substrates as endocrine disruptors (EDs). Flutamide (FLU) is a non-steroidal anti-androgen or pro-drug that is rapidly metabolized to hydroxyflutamide, which may have both an anti-androgenic and anti-progestogenic activities. Thus, in the present study, we employed immature mice (14-day-old) subcutaneously injected with P4 (20 mg/kg/day) and/or FLU (5 mg/kg/day) for 3 consecutive days in the presence or absence of RU486, a pure PR antagonist (30 mg/kg/day), to analyze uterine CaBP-9k expression in this model. When immature mice were treated with P4, the expression levels of CaBP-9k mRNA and protein were significantly increased by P4. P4-induced expression levels of CaBP-9k mRNA and protein were abolished by FLU, in part, suggesting that FLU is a partial antagonist of P4 in the regulation of uterine CaBP-9k in immature mice. In addition, P4-induced CaBP-9k expression was completely reversed by RU486. Increased expression levels of CaBP-9k mRNA and protein were maintained for 24 h after final injection with P4 in a time-dependent manner. However, CaBP-9k mRNA rapidly disappeared after 48 h and its protein level is similar with its mRNA. Treatment with FLU suppressed partially P4-induced CaBP-9k mRNA and protein until 24 h. Taken together, these results indicate that FLU has an anti-progestogenic activity and plays a role as a partial antagonist of P4 in the regulation of uterine CaBP-9k in immature mouse model.

Probing Fragment Complementation by Rigid-Body Docking: in Silico Reconstitution of Calbindin D9k

Fragment complementation is gaining an increasing impact as a nonperturbing method to probe noncovalent interactions within protein supersecondary structures. In this study, the fast Fourier transform rigid-body docking algorithm ZDOCK has been employed for in silico reconstitution of the calcium binding protein calbindin D9k, from its two EF-hands subdomains, namely, EF1 (residues 1-43) and EF2 (residues 44-75). The EF1 fragment has been used both in its wild type and in nine mutant forms, in line with in vitro experiments. Consistent with in vitro data, ZDOCK reconstituted the proper fold of wild-type and mutated calbindin, locating the nativelike structures (i.e., holding a root-mean-square deviation < 1 A with respect to the X-ray structure) among the first 10 top-scored solutions out of 4000. Moreover, the three independent in silico reconstitutions of wild-type calbindin ranked a nativelike structure at the top of the output list, that is, the best scored one. The algorithm has been also successfully challenged in reconstituting the EF2 homodimer from two identical copies of the monomer. Furthermore, quantitative models consisting of linear correlations between thermodynamic data and ZDOCK scores were built, providing a tested tool for very fast in silico predictions of the free energy of association of protein-protein complexes solved at the atomic level and known to not undergo significant conformational changes upon binding.

The Lin12-Notch Repeats of Pregnancy-associated Plasma Protein-A Bind Calcium and Determine Its Proteolytic Specificity

The Lin12-Notch repeat (LNR) module of about 35 residues is a hallmark of the Notch receptor family. Three copies, arranged in tandem, are invariably present in the extracellular portion of the Notch receptors. Although their function is unknown, genetic and biochemical data indicate that the LNR modules participate in the regulation of ligand-induced proteolytic cleavage of the Notch receptor, a prerequisite to intramembrane cleavage and Notch signaling. Outside the Notch receptor family, the LNR module is present only in the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) and its homologue PAPP-A2, which also contain three copies. Curiously, LNR modules 1 and 2 are present within the proteolytic domain of PAPP-A/A2, but LNR3 is separated from LNR2 by more than 1000 amino acids. The growth factor antagonists insulin-like growth factor-binding protein (IGFBP)-4 and -5 are both substrates of PAPP-A. We provide here evidence that the PAPP-A LNR modules function together to determine the proteolytic specificity of PAPP-A. Analysis of C-terminally truncated PAPP-A mutants followed by the analysis of LNR deletion mutants demonstrated that each of the three PAPP-A LNR modules is strictly required for proteolytic activity against IGFBP-4 but not for proteolytic activity against IGFBP-5. Individual substitution of conserved LNR residues predicted to participate in calcium coordination caused elimination (D341A, D356A, D389A, D1484A, D1499A, and D1502A) or a significant reduction (D359A and E392A) of IGFBP-4 proteolysis, whereas IGFBP-5 proteolysis was unaffected. The activity of the latter mutants against IGFBP-4 could be partially rescued by calcium, and the addition of the calcium-binding protein calbindin D9k to wild-type PAPP-A eliminated activity against IGFBP-4 but not against IGFBP-5, demonstrating that the PAPP-A LNR modules bind calcium ions. We propose a model in which LNR3 is spatially localized in proximity to LNR1 and -2, forming a single functional unit.

Purification of recombinant calbindin D9k.

AbstractThe calcium-binding protein calbindin D9k is a relatively small member (8.5 kDa) of the EF-hand helix-loop-helix family of calcium-binding proteins. Unlike many other calcium-regulatory proteins in this family, binding of the two calcium ions to calbindin does not cause a large conformational change, which exposes a hydrophobic surface. Therefore, calbindin D9K probably acts as a calcium buffer, rather than a calcium-trigger protein. Because there is no calcium-dependent conformational change, hydrophobic interactions as a purification step for recombinant calbindin is not feasible. The purification scheme described here for recombinant calbindin is based on the same procedure as used for purification of calbindin D9k from intestine (1,2). The method starts with a heat shock step that takes advantage of calbindin’s unique stability. The second step is anion-exchange chromatography in the presence of ethylenediaminetetracetic acid (EDTA). The third step, gelfiltration, is used to remove small proteins, peptides, and other Escherichia coli components that do not elute from the ion exchange column with any distinct pattern. The fourth step is anion-exchange chromatography in the presence of calcium. Because calbindin changes its net charge upon binding calcium, it will elute at a different salt concentration in calcium compared to EDTA. Therefore, proteins that copurify in the EDTA step will be removed in the calcium step. The last anionexchange step will also remove any deamidated calbindin that may arise during the purification (3).KeywordsPump SpeedLarge Conformational ChangeBarbital BufferImidazole BufferCalcium LactateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Locating the metal ion in calcium-binding proteins by using cerium(III) as a probe.

The detection and assignment of NMR spectroscopic signals of carbon atoms from carbonyl and carboxylate groups in the loop hosting the Ce(III) ion was performed for the cerium-substituted calcium-binding protein calbindin D9k. This provided a tool to characterize in solution the first coordination sphere of the metal ion. Due to the well-documented possibility of replacing calcium with metal ions of the Ln(III) series, this approach turns out to be extremely efficient for characterizing in solution the coordination of calcium ions in proteins, independently of the availability of X-ray crystal structures. The present approach completes the structural characterization of lanthanide-substituted calcium-binding proteins, for which the role of long-range constraints arising from hyperfine interaction and self-orientation has already been assessed.

Thyroid Hormone Receptor Does Not Heterodimerize with the Vitamin D Receptor but Represses Vitamin D Receptor-Mediated Transactivation

The 9,000 Mr calcium-binding protein calbindin-D9k (CaBP9k) is markedly induced by 1,25-dihydroxyvitamin D3[ 1,25-(OH)2D3] in mammalian intestine. However, although a vitamin D response element (VDRE) has been reported in the promoter of the rat CaBP9k gene (at −490/−472), the CaBP9k promoter is weakly transactivated by 1,25-(OH)2D3. Previous studies indicated that when MCF-7 cells are transfected with the rat CaBP9k VDRE ligated to the thymidine kinase promoter and treated with both 1,25-(OH)2D3 and T3 there is an enhancement of the response observed with 1,25-(OH)2D3 alone, suggesting direct cross-talk between thyroid hormone and the vitamin D endocrine system and activation via the formation of vitamin D receptor (VDR)-thyroid hormone receptor (TR) heterodimers. To determine whether the weak response of the rat CaBP9k natural promoter to 1,25-(OH)2D3 could be enhanced by T3, CaBP9k promoter/reporter chloramphenicol acetyltransferase constructs were transfected in MCF-7 cells, and the cells were treated with the two hormones alone or in combination. No induction with T3 alone and no enhancement of reporter activity in the presence of both hormones was observed. To determine whether a lack of effect by T3 was specific for the CaBP9k promoter and to further examine the possibility of cross-talk between the TR- and VDR-signaling pathways, the 1,25-(OH)2D3-responsive rat 24 hydroxylase [24(OH)ase] promoter and the rat osteocalcin VDRE (−457/−430), both fused to reporter genes were similarly examined in MCF-7 cells. Again, no enhancement of the response to 1,25-(OH)2D3 was observed in the presence of T3. In addition, a similar lack of response to T3 but responsiveness to 1,25-(OH)2D3 was observed when UMR106–01 osteosarcoma cells [which, like MCF-7 cells, express VDR, TR, and the retinoid X receptor (RXR) endogenously] were transfected with a 1,25-(OH)2D3 responsive mouse osteopontin promoter reporter. In vitro DNA binding assays were carried out using purified human VDR, human RXRα, and chick T3Rα and 24(OH)ase, osteocalcin, osteopontin, and CaBP9k VDRE oligonucleotide probes. No VDR-TR heterodimer binding on any of these VDREs was observed, although, as expected, there was binding by the VDR-RXR complex and strong TR-RXR binding to a consensus thyroid hormone response element. Simultaneous gel retardation assays using similar and lower concentrations of TR with RXR showed strong binding of TR-RXR on a 32P-labeled thyroid response element. Studies using the yeast two-hybrid system also did not provide evidence for the formation of a VDR-TR protein-protein interaction. In addition, in vivo data showed that transfection of TR, in fact, repressed VDR-mediated transcription and that the repression could be reversed by the addition of RXR. Thus, in vitro and in vivo experiments do not support ligand-sensitive transactivation mediated by VDR-TR heterodimer formation but rather suggest that TR expression can repress 1,25-(OH)2D3-induced transcription predominantly by sequestering RXR.

Thyroid hormone receptor does not heterodimerize with the vitamin D receptor but represses vitamin D receptor-mediated transactivation.

The 9,000 Mr calcium-binding protein calbindin-D9k (CaBP9k) is markedly induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in mammalian intestine. However, although a vitamin D response element (VDRE) has been reported in the promoter of the rat CaBP9k gene (at -490/-472), the CaBP9k promoter is weakly transactivated by 1,25-(OH)2D3. Previous studies indicated that when MCF-7 cells are transfected with the rat CaBP9k VDRE ligated to the thymidine kinase promoter and treated with both 1,25-(OH)2D3 and T3 there is an enhancement of the response observed with 1,25-(OH)2D3 alone, suggesting direct cross-talk between thyroid hormone and the vitamin D endocrine system and activation via the formation of vitamin D receptor (VDR)-thyroid hormone receptor (TR) heterodimers. To determine whether the weak response of the rat CaBP9k natural promoter to 1,25-(OH)2D3 could be enhanced by T3, CaBP9k promoter/reporter chloramphenicol acetyltransferase constructs were transfected in MCF-7 cells, and the cells were treated with the two hormones alone or in combination. No induction with T3 alone and no enhancement of reporter activity in the presence of both hormones was observed. To determine whether a lack of effect by T3 was specific for the CaBP9k promoter and to further examine the possibility of cross-talk between the TR- and VDR-signaling pathways, the 1,25-(OH)2D3-responsive rat 24 hydroxylase [24(OH)ase] promoter and the rat osteocalcin VDRE (-457/-430), both fused to reporter genes were similarly examined in MCF-7 cells. Again, no enhancement of the response to 1,25-(OH)2D3 was observed in the presence of T3. In addition, a similar lack of response to T3 but responsiveness to 1,25-(OH)2D3 was observed when UMR106-01 osteosarcoma cells [which, like MCF-7 cells, express VDR, TR, and the retinoid X receptor (RXR) endogenously] were transfected with a 1,25-(OH)2D3 responsive mouse osteopontin promoter reporter. In vitro DNA binding assays were carried out using purified human VDR, human RXRalpha, and chick T3Ralpha and 24(OH)ase, osteocalcin, osteopontin, and CaBP9k VDRE oligonucleotide probes. No VDR-TR heterodimer binding on any of these VDREs was observed, although, as expected, there was binding by the VDR-RXR complex and strong TR-RXR binding to a consensus thyroid hormone response element. Simultaneous gel retardation assays using similar and lower concentrations of TR with RXR showed strong binding of TR-RXR on a 32P-labeled thyroid response element. Studies using the yeast two-hybrid system also did not provide evidence for the formation of a VDR-TR protein-protein interaction. In addition, in vivo data showed that transfection of TR, in fact, repressed VDR-mediated transcription and that the repression could be reversed by the addition of RXR. Thus, in vitro and in vivo experiments do not support ligand-sensitive transactivation mediated by VDR-TR heterodimer formation but rather suggest that TR expression can repress 1,25-(OH)2D3-induced transcription predominantly by sequestering RXR.

A Calbindin D9k Mutant with Reduced Calcium Affinity and Enhanced Cooperativity. Metal Ion Binding, Stability, and Structural Studies

In the native calcium-binding protein calbindin D9k (M(r) 8.700; 75aa; 2 EF-hands), the backbone carbonyl oxygen of Glu60 coordinates the Ca2+ ion in the C-terminal site (site II). The carboxylate group of the same residue forms a hydrogen bond to a water molecule that constitutes a Ca2+ ligand in the N-terminal site (site I). The mutant E60D, with the charge-conserving substitution Glu60-->Asp, has been prepared to study the role of Glu60 in subjoining the two Ca(2+)-binding sites and its role in the cooperative Ca2+ binding. Ca(2+)-binding studies of the mutant show that the overall affinity for calcium has decreased by a factor of 38 in comparison with wild-type calbindin D9k. The largest reduction is seen in the first macroscopic binding step. The Ca2+ affinities for both sites in the protein are reduced to a similar extent. In contrast, the mutation leads to a large increase in the cooperativity of calcium binding. Differential scanning calorimetry has been used to determine the thermal stability which is almost as high as in the wild-type protein. Cadmium binding has been assessed with 1H and 113Cd NMR. X-ray crystallographic studies of the E60D mutant in its calcium-bound form show very small structural changes relative to the wild-type protein. Almost all differences are within the error limits of the method. The largest crystallographic effects are seen in the crystal packing. Two E60D molecules with slightly different structure are found in the asymmetric unit in contrast to the single molecule in the wild-type crystal.(ABSTRACT TRUNCATED AT 250 WORDS)