Evidence for calcium mediated conformational changes in calbindin-D28K (the vitamin D-induced calcium binding protein) interactions with chick intestinal brush border membrane alkaline phosphatase as studied via photoaffinity labeling techniques

The role of the vitamin D-induced calcium binding protein termed calbindin-D (CaBP) in the biological response to 1,25-dihydroxyvitamin D₃ was assessed by photoaffinity labeling techniques. The heterobifunctional cross-linking reagent methyl-4-azidobenzoimidate was employed for studies with the 28 KD chick intestinal calbindin-D_28K. Calcium-dependent interactions were evident with purified chick intestinal CaBP-immunoglobulins and bovine intestinal alkaline phosphatase; in the absence of Ca²⁺ there was a greatly diminished crosslinking process. There were also at least two membrane components of chick intestinal brush border membranes, with M_R = 60,000 and 130,000, which were photoaffinity cross-linked with CaBP in a calcium-dependent manner. Similar interactions were demonstrated following incubations of CaBP with phosphatidylinositol-specific phospholipase C (PI-PLC)-treated supernatant fractions from chick intestinal brush borders. PI-PLC was shown to release 14% of the alkaline phosphatase from chick intestinal brush borders compared to greater than 80% for rabbit and chick kidney BBM preparations. Specific interactions between CaBP and brush border membrane proteins could also be demonstrated in the absence of photoaffinity labeling by Sephadex G-150 chromatography of Triton X-100 solubilized incubations between calbindin-D_28K and chick intestinal BBMS, with 17% of the radiolabelled CaBP comigrating with alkaline phosphatase activity. These studies collectively demonstrate that calbindin-D_28K undergoes calcium-dependent conformational changes which alter its subsequent interactions with cellular proteins in a way consistent with other calcium-binding proteins such as calmodulin or troponin C.     

Binding Proteins from Animals with Possible Transport Function

Several proteins from various animal tissues with possible transport function have been briefly described, with emphasis given to a vitamin D-induced calcium-binding protein (CaBP) implicated in calcium translocation across epithelial membranes. The latter protein was shown to be present in the small intestine, colon, kidney, and the uterus (shell gland) of the chicken. CaBP was also found in the small intestine of the rat, dog, bovine, and monkey. This protein has been isolated in high purity from chick intestinal mucosa and some of its properties determined. Its molecular weight is about 28,000, its formation constant, about 2.6 x 10⁵ M^-1, and its binding capacity, 1 calcium atom per protein molecule. Correlative studies have shown that CaBP concentration in intestinal mucosa varies with the calcium absorptive capacity of the gut, thereby suggesting that CaBP is intimately involved in the process of calcium absorption. CaBP has been localized in the brush border region of the intestinal absorptive cell and within goblet cells. Among other proteins mentioned were the intrinsic factor required for vitamin B₁₂ absorption and the protein(s) associated with iron translocation.     

1,25-Dihydroxyvitamin D3-induced calcium-binding protein: localization in organ-cultured embryonic chick duodenum

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) induces de novo biosynthesis of a specific calcium-binding protein (CaBP) in embryonic chick duodenum in organ culture. Using a highly sensitive and specific, peroxidase-antiperoxidase immunocytochemical procedure, 1,25(OH)2D3-induced CaBP in the organ-cultured duodenum was found only in the cytoplasm of absorptive cells, corresponding to its localization in rachitic chick duodenal cells after a single injection of 1,25(OH)2D3 in vivo. This observation, along with evidence correlating CaBP with calcium transport, strongly supports the use of the embryonic chick duodenal organ culture system as a physiologically relevant model of the vitamin D-dependent calcium absorptive mechanism.     

Embryonic chick intestine in organ culture. A unique system for the study of the intestinal calcium absorptive mechanism

Duodena from 20-day-old chick embryos can be maintained in large scale organ culture on specially designed stainless-steel grids in contact with serum-free medium for 48 h with excellent preservation of mucosal structure at both the light and electron microscope levels. Although mitotic rate was subnormal, several other factors attest to the essential viability of the cultured intestine: L-leucine incorporation into protein, as well as the synthesis of a specific vitamin D₃-induced calcium-binding protein (CaBP), increased over a 48-h culture period, and the electropotential gradient across the intestine was maintained throughout the culture period as was a concentration gradient for calcium. The tissue responded to vitamin D₃ in the medium by synthesizing the calcium-binding protein within 6 h and by exhibiting enhanced ⁴⁵Ca uptake within 12–24 h. Concentrations of vitamin D₃, or its 25-hydroxylated derivative, higher than necessary for CaBP induction, also increased the activity of alkaline phosphatase. The 1,25-dihydroxylated derivative of vitamin D₃, at a level extremely potent in CaBP induction, did not stimulate alkaline phosphatase. Mucosal to serosal transport of ⁴⁵Ca could also be measured in everted duodenal sacs, subsequent to culture under similar conditions, and was also increased by vitamin D₃ in the medium. Other embryonic organs, esophagus, stomach, liver, pancreas, lung, skin, and muscle, did not produce CaBP in response to vitamin D₃ in the culture medium. However, CaBP-synthesizing capacity was present in the entire intestinal tract, exclusive of the rectum. ⁵⁹Fe and ³²P uptake by cultured duodenum were also stimulated by vitamin D₃. The system has proven quite useful in the study of the vitamin D-mediated calcium absorptive mechanism but should be applicable to the study of the absorption of other nutrients, drugs, hormones, etc., as well as other studies of intestinal function.     

Vitamin D-dependent calcium-binding protein synthesis by chick kidney and duodenal polysomes

The hormonally active form of vitamin D, 1,25-dihydroxy vitamin D₃, is known to induce in the intestine and kidney of chicks the synthesis of a calcium-binding protein (CaBP). Here we report a correlation between the tissue levels of CaBP and the levels of apparent messenger RNA in total polysomes as determined by the vitamin D and dietary calcium status. Polysomes from pooled duodenal mucosa and kidney were prepared by the Mg²⁺ precipitation method. After translation in a heterologous, rabbit nuclease-treated reticulocyte system, the immunoprecipitated pellet of CaBP was dissolved and the proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels. When 13 nmol of D₃ was given to 4-week-old rachitic chicks which were sacrificed 48 h later, it was found that the duodenum had eightfold more apparent mRNA for CaBP in the polysomes than the kidney. This was also reflected in the values of CaBP/mg protein in these tissues (duodenum, 7 μg/mg vs kidney, 0.9 μ/mg). Also, after giving D₃, there was a twofold increase in both apparent mRNA levels in the polysomes and in CaBP levels in the duodena of chicks which were raised on low-calcium diets versus chicks raised on high-calcium diets. While apparent mRNA for CaBP was present in polysomes from rachitic chick kidney, it was not detectable in the duodenum. From these studies it appears that the induction of CaBP by 1,25(OH)₂D₃ in both the intestine and kidney is determined by similar control mechanisms.     

Pancreatic vitamin D-dependent calcium binding protein: biochemical properties and response to vitamin D

The biochemical properties of a chick pancreatic calcium binding protein (CaBP) and its response to vitamin D status and dietary calcium and phosphorus levels were studied and compared with the known vitamin D-dependent CaBPs present in the chick intestine and kidney. Pancreatic CaBP is homologous to the intestinal CaBP on the basis of immunological cross-reactivity, molecular size (28,200 Da), and charge properties (chromatographic mobility on DEAE-Sephadex in the presence of either EDTA or Ca2+). Pancreatic levels of CaBP respond to changes in vitamin D status and dietary Ca and P level in a fashion similar to the intestinal CaBP. Thus, in the absence of dietary vitamin D, both pancreatic and intestinal CaBPs were essentially undetectable, while in the presence of dietary vitamin D, a low dietary P (0.05%) elevated the pancreatic and intestinal CaBP 1.5X and 1.6X, respectively, compared to the CaBP levels present with normal dietary Ca and P (1.0%, 1.0%). The tissue levels of pancreatic CaBP (6-10 ng/mg protein) are about 0.2% of the intestine (5000 ng/mg protein) and 1% of the kidney CaBP (700 ng/mg protein). However, when corrections are made for the CaBP distribution in the tissues and expressed as CaBP concentration per CaBP-containing cells, the pancreatic CaBP level was 30% of the intestine and 10% of the kidney. Collectively, these results suggest that the chick pancreatic vitamin D-dependent CaBP is a homologous protein to the intestinal CaBP, both with regards to its relative cellular concentration as well as in its response to changing dietary levels of Ca and P.     

Determination of the rates of synthesis and degradation of vitamin D-dependent chick intestinal and renal calcium-binding proteins

Vitamin D₃ and its biologically active metabolite 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] are shown to induce in the chick intestine and kidney the biosynthesis of a calcium binding protein (CaBP). In vitamin D₃-replete chickens raised under adequate dietary calcium (Ca) and phosphorus (P) conditions, the steady-state level of intestinal CaBP (30–50 g/mg protein) is 5- to 20-fold greater than that of renal CaBP. Whereas dietary phosphorus restriction is known to elevate both intestinal and renal CaBP levels, dietary calcium restriction elevates only intestinal CaBP. The present study reports the rates of biosynthesis in vivo and in vitro, and of biodegradation in vivo, of both intestinal and renal CaBP after administration of vitamin D₃ or 1,25(OH)₂D₃ to rachitic chicks. The apparent rate constant of degradation for intestinal CaBP was 0.024 h^?1 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 29}\text{h}$) and that for renal CaBP was 0.019 h^?1 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 36}\text{h}$) while total cellular soluble protein in the intestine and kidney had half-lives of 43 and 70 h, respectively. The time course of induction of the synthesis of CaBP was determined in intestine and kidney after administration of a physiological dose of 1,25(OH)₂D₃ to rachitic chicks. Intestinal CaBP synthesis was detectable by 3 hours, reached a maximal rate by 10 hours, and sharply decayed by 16–20 hours. The time course of induction of renal CaBP synthesis was very similar, although the rate of renal CaBP synthesis was readily detectable at the initial time of administration of 1,25(OH)₂D₃. The relative rates of synthesis of CaBP in the intestine and kidney under a variety of dietary Ca and P conditions in the vitamin D₃-replete chick exactly paralleled the steady-state level of CaBP in these two tissues. These results are consistent with a model in which the steady-state levels of intestinal and renal CaBP are solely determined by their respective rates of biosynthesis; the CaBP biosynthetic capability, in turn, is regulated by the availability of 1,25(OH)₂D₃ to each target organ.     

Lead-binding properties of intestinal calcium-binding proteins

The bovine and chick vitamin D-induced intestinal calcium-binding proteins (CaBP) bind lead. Bovine CaBP binds 2 atoms of lead/molecule, and chick CaBP binds 4 atoms of lead per molecule and these values are identical to those for calcium binding. 45Calcium-displacement studies indicate significantly higher affinities for lead than for calcium for both proteins. All evidence indicates that lead is bound to the 4 high affinity calcium-binding sites on chick CaBP and to the corresponding 2 high affinity sites on bovine CaBP, and that binding of lead to sulfhydryl groups is, relatively, not significant. Calmodulin, troponin C, and oncomodulin also bind lead with high affinities and in preference to calcium, indicating that lead binding is a general property of proteins belonging to the troponin C superfamily of calcium-binding proteins.     

Calcium transport and related functions in the chorioallantoic membrane of cultured shell-less chick embryos

In order to investigate the influence of the egg shell on the process of shell calcium mobilization by the chorioallantoic membrane (CAM), chick embryos were maintained in long-term cultures in vitro without the shells. The shell-less embryos were severely calcium deficient and showed signs of retarded development and anomalous skeletal calcification. Throughout development, calcium transport and calcium-binding protein (CaBP) activities were diminished in the CAM of shell-less embryos as compared to those of control embryos which developed in ovo. The levels of developmentally expressed carbonic anhydrase activity remained, however, similar. By means of a single radial immunodiffusion assay of CaBP using a specific anti-CaBP antiserum, the level of immunoreactive CaBP was found to be significantly increased in the CAM of the shell-less embryos. These studies indicate that the CAM of chick embryos cultured under shell-less conditions is defective in calcium transport, probably as a result of the expression of an inactive form of the CaBP.     

Influence of cycloheximide and 1,25-dihydroxyvitamin D3 on mitochondrial and vesicle mineralization in the intestine

1,25(OH)₂D₃ increases cell permeability to calcium. This increase is not mediated by proteins sensitive to cycloheximide or actinomycin D inhibition. We propose that CaBP may associate with intracellular membranes and organelles to prevent intracellular calcium accumulation and the potential cytotoxic effects of such accumulation. In support of this hypothesis, the amount of mitochondrial mineralization in chick intestinal cells was markedly increased by 1,25(OH)₂D₃ treatment when CaBP synthesis was simultaneously blocked by cycloheximide treatment. Mineral in membrane vesicles was increased by 1,25(OH)₂D₃ treatment, but was blocked by simultaneous treatment with cycloheximide.     

Evidence that sodium deprivation influences vitamin D dependent rat renal calcium binding protein

In order to provide some insight concerning the role of renal calcium binding protein (CaBP) in the functioning of the mammalian kidney, the response of renal CaBP to dietary alterations was examined. Three week old rats were fed diets deficient in calcium, phosphorous or sodium supplemented with vitamin D for a four week period. The specific activity of renal CaBP (as measured by the chelex resin assay; Ca²⁺ bound protein/Ca²⁺ bound resin per mg protein) in the 28,000 M_r region was found to increase four fold in rats fed the low phosphorus diet and two fold rats fed the low calcium diet when compared to rats fed the control diet. Renal CaBP/mg protein from rats fed the low sodium diet decreased 50% from the control values. Changes in renal CaBP were confirmed by polyacrylamide gel analysis of the 28,000 M_r fraction by densitometric tracing using a purified CaBP marker. The greater response to dietary phosphorus restriction suggests that renal CaBP may be regulated by a mechanism different from that of intestinal CaBP. The decrease in renal CaBP in rats fed the low sodium diet suggests for the first time that sodium is required for vitamin D dependent distal tubular calcium transport processes.     

Intestinal vitamin D-induced calcium-binding protein: time-course of immunocytological localization following 1,25-dihydroxyvitamin D3

The vitamin D-induced calcium-binding protein (CaBP) was localized in histological sections of chick duodenum using the peroxidase-antiperoxidase immunocytochemical technique. The time-course of appearance of CaBP in rachitic chicks was investigated from 0 to 120 hr after stimulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). CaBP was not routinely detected at 0 hr after 1,25(OH)2D3 administration. CaBP was first noted in some, but not all, of the samples taken 2 hr following 1,25(OH)2D3 and was detected in all 2 1/2 hr samples. The number of CaBP-containing absorptive cells and the apparent CaBP concentration both increased to a maximum at about 16-24 hr. At later times, as CaBP free cells migrated up the villi, the CaBP-containing cells decreased in number, but even at 120 hr post 1,25(OH)2D3 dose there were significant numbers of CaBP-containing cells present. The relationships between time-course of CaBP location on intestinal villi, enterocyte migration rates, and the time-course of 1,25(OH)2D3 stimulated intestinal calcium transport are discussed.     

Functional analysis of placental 57-kDa Ca(2+)-binding protein: overexpression and downregulation in a trophoblastic cell line.

The placental trophoblastic epithelium functions to transport nutrients needed by the fetus, including calcium, which is required in the greatest amounts during the last third of pregnancy when the majority of fetal skeletal mineralization occurs. The mechanism of placental calcium transport and the developmental changes in the trophoblast that facilitate this process are currently incompletely understood. We have previously identified a 57-kDa, Ca(2+)-binding protein (CaBP) functionally implicated in placental calcium transport and trophoblast differentiation. In this study we have directly examined the role of CaBP in these processes by (1) recombinantly overexpressing CaBP in an inducible manner and (2) downregulating CaBP expression using antisense technology, using the rat choriocarcinoma cell line Rcho-1 as a trophoblastic cell model system. Our results show that overexpression of CaBP stimulates both cellular calcium uptake and vectorial calcium transport activities in Rcho-1 cells. Those cells stably expressing CaBP also exhibit higher levels of steady-state intracellular calcium and enhanced calcium-buffering ability. In addition, prolonged overexpression of CaBP in Rcho-1 cultures promotes trophoblast differentiation. Conversely, downregulation of CaBP expression had a negative effect on calcium uptake, calcium transport, and trophoblast differentiation in Rcho-1 cells. These data indicate that CaBP plays a direct role in placental calcium transport, functioning both as an intracellular calcium buffer and as a shuttle. These results also support a more direct role for CaBP in the trophoblast differentiation pathway.     

The effect of parathyroid hormone and vitamin D3 metabolites on CaBP synthesis by embryonic duodenum, in vitro

Embryonic chick duodena were incubated, in vitro, for two days in the presence of vitamin D analogues and metabolites and the ability of PTH to potentiate the vitamin-D-dependent synthesis of CaBP measured. PTH potentiated CaBP synthesis in the presence of vitamin D3, 25-OH-D3, 1 alpha-OH-D3, DHT, and 1,25(OH)2D3. The synthesis of CaBP was directly related to the log of the meddium PTH concentration over the range 1 x 10(-8) M to 5 x 10(-7) M.     

Vitamin D, calcium-binding protein and calcium absorption in the intestines

The author presents his own and literature data concerning the action of vitamin D and its analogs on the biosynthesis of calcium-binding protein (CaBP) in the chick intestinal mucosa. The mechanism of involvement of this protein in calcium transport across the intestinal epithelium is discussed. CaCP is considered as a specific test-substance for vitamin D. A close correlation between the CaCP content in the intestinal mucosa and the vitamin antirachitic effect is demonstrated. The quantitative estimates of the biological activity of various forms of vitamin D, their metabolites and synthetic analogs with respect to the CaCP content are given.     

Calcium-binding protein of the chick chorioallantoic membrane. II. Vitamin K-dependent expression

A simple method was devised for the maintenance of the chorioallantoic membrane (CAM) of chick embryos in organ culture. Explants of CAM survived for up to 5 days in this system and retained the characteristic three-layered morphology (ectoderm, mesoderm, and endoderm). Induction of the CAM calcium-binding protein (CaBP) by effectors of calcium metabolism was studied in these organ cultures. Vitamin K was found to elicit a seven- to eightfold increase in CaBP, whereas no increase in CaBP activity occurred on supplementation with vitamin A, parathyroid hormone, an analogue of vitamin D, vitamin D and its hydroxylated metabolites, or with elevated calcium levels. The vitamin K-mediated induction of CaBP was dose-dependent, inhibited by the vitamin K antagonists warfarin and dicoumarol, selective for vitamin K5, and maximal at the developmental stage (13-15 days of incubation) corresponding to the onset of calcium transport by the CAM in vivo. CaBP levels increased after 60-70 h in cultures of 13-15 day CAM supplemented with vitamin K and reached maximal levels around 80-90 h of culture. The CAM ectoderm underwent extensive proliferation and often assumed a villuslike morphology in the vitamin K cultures.     

Calcium-dependent translocation of calbindin-D28k from intestine to blood

Calbindin-D (vitamin D-induced calcium-binding protein; CaBP) is known to be present in blood at concentrations which vary directly with levels in the intestinal mucosa. Employing a sensitive radioimmunoassay and sampling mesentery venous blood, the present experiments demonstrated a direct relationship between intestinal calcium absorption and serum CaBP. Solutions containing 150 mM NaCl and 45Ca-labeled calcium chloride (5 or 20 mM) were placed in the lumen of ligated duodenal preparations in situ and mesentery venous blood sampled with time. The concentration of absorbed 45Ca in serum was maximal at 5 min, followed by a significant increase in mesentery CaBP maximizing at 15-20 min. Elevation of serum CaBP was not observed when calcium in the dosing solution was omitted or replaced by either glucose or glycine. The possible transfer of absorbed calcium from the enterocyte to the circulation as a CaBP complex was ruled out by calculations revealing that considerably more calcium was transferred than could be accounted for by the low and high affinity binding sites on the protein. It is proposed that vitamin D-dependent enhanced transcellular calcium transport constitutes a stimulus for the increased release of intestinal CaBP into the circulation.     

Immunocytochemical localization of the vitamin D-dependent calcium binding protein in chick duodenum

The vitamin D-dependent calcium binding protein (CaBP) of chick duodenum has been localized by immunocytochemistry and by radioimmunoassay. Light microscopically, CaBP was seen to be present in the absorptive cells of the villi while in other cell types of the villi and the crypts, including goblet cells and endocrine cells, no CaBP was seen. At the electron microscopic level, CaBP was shown to be localized in the cytosol and the euchromatin of the nucleus but not in membrane-bounded cytoplasmic compartments. Quantitative evaluation of the immunocytochemical protein A-gold label showed that the terminal web and the cytosol of basal cellular regions were most highly labeled while the brush border was weakly labeled. The radioimmunoassay evaluation of intestinal subcellular fractions indicated that 96% of the homogenate CaBP is in the cytosol high-speed supernatant fraction. Collectively, these results support the hypothesis that the vitamin D- dependent intestinal CaBP may play a role in either regulation of intracellular calcium concentration or movement of calcium across the brush border membrane from the gut lumen.