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.     

Immunocytochemical localization of rat intestinal vitamin D-dependent calcium-binding protein

Vitamin D-dependent calcium-binding protein (CaBP) was localized in intestinal tissue sections obtained from rats raised under three different nutritional conditions: a normal vitamin D-replete diet, a vitamin D-free diet followed by supplementation with vitamin D3, or a vitamin D-free diet without additional supplementation. An indirect immunoperoxidase technique, with immunocontrols, was used to visualize the specific sites of CaBP. CaBP was visualized only in the cytoplasm of absorptive cells. In the duodenum of animals raised on a normal diet, CaBP was present in absorptive cells from the upper crypt region to the villus tips. In the jejunum, many fewer absorptive cells contained CaBP, while in the ileum only random absorptive cells near the villus tips contained CaBP. In rats raised on a vitamin D-deficient diet then supplemented with vitamin D3, CaBP was present in cells at the full depth of the crypts and in absorptive cells along the total villus length in the duodenum. Rats raised on the same deficient diet but without supplementation with additional vitamin D exhibited no CaBP in crypt cells nor in absorptive cells more than half way up the villi. Absorptive cells higher on the villi contained immunoreactive CaBP but the intensity of immunostaining and number of CaBP-containing cells was markedly reduced compared to the vitamin D-supplemented group.     

Intestinal and placental calcium-binding proteins in vitamin D-deprived or -supplemented rats

In rats, at day 20 of pregnancy, the placenta and the fetal intestine contain calcium-binding proteins (CaBPs) which closely resemble the vitamin D-dependent CaBP of the adult rat duodenal mucosa. A significant and specific increase of the dam intestinal CaBP likely synthesized as a result of pregnancy, is observed. A 5 week-vitamin D-depletion promoted a decrease of the CaBP content of the dam intestine and of its calcemia. No changes were detected in the non-pregnant animals. Likewise, neither fetal calcemia nor CaBP contents of the feto-placental unit were affected. Such findings suggest i) that pregnancy elicits the vitamin D-sensitivity of rats and ii) that a slight vitamin D-deficiency acts only on the maternal compartment. Although the vitamin D-dependence of placental and fetal CaBPs remains to be demonstrated, our results suggest that these proteins act in concert with the maternal CaBP, to favour a mother to fetus transfer of calcium in order to satisfy the needs of the mineralizing fetal skeleton.     

Calmodulin and rat vitamin D-dependent calcium-binding proteins: Biochemical and immunochemical comparison

Purified vitamin D-dependent rat intestinal (M_r 10,000) and rat renal (M_r 28,000) calcium-binding proteins (CaBPs) have been compared to vertebrate calmodulin, and the vitamin D-dependent CaBPs have been found to be distinct from calmodulin by biochemical and immunochemical criteria. Rat renal and rat intestinal CaBPs do not stimulate 3′,5′-cyclic nucleotide phosphodiesterase, do not compete with iodinated calmodulin for binding to phenothiazine-Sepharose conjugates, do not cross-react immunochemically, and do not contain N^?-trimethyllysine. In addition, although calmodulin exhibits a characteristic calcium-dependent mobility shift on polyacrylamide gels in the presence of sodium dodecyl sulfate, a similar mobility shift is not observed for the vitamin D-dependent CaBPs. Immunocytochemically, calmodulin has a widespread localization in the kidney, whereas CaBP is present specifically in the distal tubules of the kidney. These localizations suggest a specialized role for CaBP in the kidney. Thus, although the vitamin D-dependent CaBPs and calmodulin are similar in that they are small, acidic, calcium-binding proteins, these two classes of proteins are biochemically and immunochemically distinct.     

Specific in vitro activation of Ca,Mg-ATPase by vitamin D-dependent rat renal calcium binding protein (calbindin D28K)

The vitamin D-dependent, calcium-binding protein from rat kidney, calbindin D28k (renal CaBP) specifically stimulates Ca,Mg-ATPase activity of human erythrocyte plasma membranes in a dose-dependent, calcium-sensitive manner. This stimulation was about two-fold compared to a three-fold stimulation by calmodulin. The effect was specific since other calcium-binding proteins and low molecular weight proteins did not stimulate Ca,Mg-ATPase activity. Renal CaBP did not stimulate cyclic nucleotide phosphodiesterase at concentrations greater than those which stimulated Ca,Mg-ATPase activity. This is the first report of a specific in vitro effect of renal CaBP on an enzyme system.     

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.     

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.     

Vitamin D-dependent calcium-binding protein of mouse yolk sac. Biochemical and immunochemical properties and responses to 1,25-dihydroxycholecalciferol

The present studies were performed to further characterize a mouse yolk sac protein which is similar or identical to the vitamin D-dependent intestinal calcium-binding protein (CaBP). Yolk sac protein and purified rat intestinal CaBP displayed full identity upon immunodiffusion (Ouchterlony) using antiserum to the rat intestinal CaBP. Immunoreactive CaBP in yolk sac homogenates eluted from gel permeation columns with the low molecular weight peak of 45Ca2+ binding (Chelex assay), and the electrophoretic mobility of the protein was markedly increased by EDTA. On days 11-13 of gestation, the concentrations of immunoreactive CaBP in yolk sac were 4-5-fold higher than in placenta; by days 16-17, the concentrations in yolk sac and placenta were similar. Incubation of yolk sac with [3H]leucine demonstrated synthesis of immunoprecipitable [3H]CaBP. A single band of 3H-labeled protein was seen on sodium dodecyl sulfate gel electrophoresis of the immunoprecipitate. This protein co-migrated with radioactive placental CaBP with an apparent Mr of 10,050. Addition of 1,25-dihydroxycholecalciferol (calcitriol) to organ culture media with or without serum increased the amount and concentration of CaBP in yolk sac (p less than 0.001) at 48 h. CaBP synthesis in yolk sac appeared to be independent of calcitriol concentrations in the maternal circulation since injection of the hormone into the maternal compartment produced no change in yolk sac CaBP despite increases of maternal intestinal and renal CaBP. These studies demonstrate that yolk sac immunoreactive CaBP is synthesized in yolk sac and has an apparent molecular size and calcium-binding properties characteristic of mammalian vitamin D-dependent calcium-binding proteins. The in vitro response of yolk sac CaBP to calcitriol is the first evidence of a vitamin D effect on the fetal membranes and suggests one function for calcitriol receptors in these tissues.     

Biochemical characterization of mouse vitamin D-dependent calcium-binding protein. Evidence for its presence in embryonic life.

We compared immunochemical and biochemical properties of the vitamin D-dependent Ca2+-binding protein (CaBP) from rat and mouse intestine. The two intestinal CaBP species were extensively purified by gel filtration and successive anion-exchange chromatographies. Both had a similar mol.wt. of 9000. Their pI values differed markedly, being 8.0 and 4.9 in rat and mouse CaBP respectively. Accordingly, mouse CaBP displayed more anodal migration in electrophoresis under non-denaturing conditions. Both mouse and rat CaBP only exhibited partial immunochemical similarities, but their amino acid compositions were very similar. Chromatofocusing was also found to be a good method of detecting calcium-dependent changes in their pI. We developed a sensitive radioimmunoassay for mouse CaBP enabling us to detect substantial amounts of CaBP in uterus, yolk sac and chorio-allantoic placenta. During normal mouse gestation, CaBP appeared on day 12 in the chorio-allantoic placenta but was already present on day 9 in the yolk sac, where its level rose sharply between days 9.5 and 10. CaBP may therefore be considered as a new marker for mouse yolk-sac differentiation.     

Vitamin D-dependent calcium-binding protein. Immunochemical studies and synthesis by placental tissue in vitro

A protein similar to rat intestinal calcium-binding protein (CaBP) has been identified in both mouse placenta and mouse small intestine. The mouse protein had a molecular weight of approximately 10,000, exhibited cation-binding properties, and demonstrated immunologic identity with vitamin D-dependent rat CaBP. Under normal dietary conditions, the concentrations of CaBP in mouse placenta and intestine increased 6- and 3-fold, respectively, during the third trimester of pregnancy in parallel with the fetal demands for skeletal mineral. Studies of in vitro protein synthesis indicated that CaBP was synthesized by placental tissue. Slices of mouse or rat placental tissue (12-18-day gestation) were incubated with [3H]leucine and the biosynthesis of placental CaBP was quantified by an immunoprecipitation method using rabbit antiserum to rat intestinal CaBP. Sodium dodecyl sulfate gel electrophoresis of the radioactive immune complex revealed a single 3H-labeled peak corresponding to the molecular weight of rat and mouse CaBP (10,050). The amount of CaBP synthesized by mouse placental tissue was dependent upon gestational age of the placenta and reflected the in vivo changes in placental CaBP content observed during gestation. These data indicate that CaBP is synthesized by placenta and provide an in vitro model for studying the developmental control of placental CaBP synthesis.     

Multiple forms of vitamin D-dependent calcium-binding protein in rat kidney

The technique of two-dimensional electrophoresis was used in combination with a highly sensitive silver stain to study vitamin D-dependent calcium-binding protein (CaBP) in rat kidney. Rat renal CaBP was shown to co-migrate almost exactly with CaBP purified from chick intestine suggesting evolutionary conservation of this protein. In some cases rat renal CaBP appeared not as a single polypeptide, but rather as a cluster of 4 polypeptides. Formation of the satellite cluster of CaBP in response to high doses of 1,25-dihydroxyvitamin D3 occurred in young rats which had been maintained on a vitamin D-deficient diet for 2 weeks, as well as in older rats which had been maintained on the same diet for 5 months. The 4 forms of CaBP were not the result of various states of Ca2+ binding, but rather the result of an enzymatic reaction. This was shown by 3 experiments. 1) Adding excess EGTA to samples containing the 4 satellite forms did not change the two-dimensional electrophoretogram. 2) Incubation of purified chick intestinal CaBP with kidney cytosols from D-deficient rats brought about the formation of the satellite CaBP forms from the chick protein. However, purified chick CaBP was unchanged by incubation in buffer alone for up to 2 h at 37 degrees C. 3) Placing rat kidney cytosols in a boiling water bath for 10 min inactivated the factor which generated the satellite forms as would be expected for an enzyme. The physiological significance of these forms of CaBP is as yet unknown.     

Tooth formation and the 28,000-dalton vitamin D-dependent calcium-binding protein: an immunocytochemical study

Antiserum to the 28-kilodalton vitamin D-dependent calcium-binding protein (CaBP) was used to localize CaBP in histologic sections of the continuously erupting incisor in mandibles obtained from normal rats. With the peroxidase--anti-peroxidase technique, no CaBP was detected in undifferentiated ameloblasts or in those which had become columnar and were facing pulp. Calcium-binding protein was first noted in the cytoplasm of random ameloblasts facing dentin in the presecretion zone. As the ameloblasts became more mature in the zone of enamel secretion, CaBP was uniformly present in their cytoplasm. Ameloblasts with Tome's processes clearly contained CaBP in these processes as well as in the cell-body cytoplasm. Near the later developmental stages of the zone of enamel secretion, some of the adjacent underlying cells of the stratum intermedium also contained CaBP in their cytoplasm. In some stratum intermedium cells and papillary cells, CaBP extended into the zone of enamel maturation, but not to the end of that zone. Cytoplasmic CaBP continued to be present in ameloblasts as they progressed through the zone of enamel maturation to the final, shortened cells at the gingival margin of the erupting incisor. No CaBP was detected in odontoblasts, pulpal cells, the stellate reticulum, or the outer dental epithelium.     

Intestinal calcium-binding protein

Summary The vitamin D-dependent calcium-binding protein (CaBP) was studied in relation to the age of the cell, in isolated epithelial cell populations removed from rat duodenum. Alkaline phosphatase and thymidine kinase activities were used as markers to characterize differentiated villus cells and undifferentiated (mitotically active) crypt cells, respectively. CaBP distribution along the length of the villus, as established by radioimmunoassay, appears as a gradient increasing from the crypt to the tip of the villus. CaBP concentration in cells is shown to be (i) negatively correlated with the thymidine kinase activity of cells, and (ii) positively correlated with the alkaline phosphatase activity of cells. This indicates that CaBP is absent in crypt cells and appears in differentiated cells with the development of the brush border. Thus CaBP, like alkaline phosphatase, can be considered as an indicator of enterocyte maturation. These data were also confirmed by studying the cellular localization of the protein. In addition both indirect immunofluorescence and immunoperoxidase staining methods reveal that antibody against CaBP decorates the terminal web, but not the microvilli of the brush border of mature absorptive cells. The results suggest that CaBP may act as a modulator of some Ca²⁺-mediated biochemical processes at the level of the enterocyte brush border.Portions of this work were presented at the Fourth International Workshop on Calcified Tissues, Israel (March 1980)     

Diabetes and renal calcium binding protein in the rat

Renal calcium binding protein (CaBP), a vitamin D-dependent protein of 28,000 Mr, may be involved in calcium transport by cells of the renal tubule. The streptozotocin-diabetic rat is hypercalciuric and shows markedly decreased concentration of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] in serum and of CaBP in small intestine. To examine the relationship of renal CaBP in diabetes to 1,25-(OH)2D3 and urinary calcium excretion, renal CaBP, serum 1,25-(OH)2D3, and urinary calcium were measured in control, diabetic, and insulin-treated diabetic rats. Treatment of the diabetic rat with insulin decreased urinary calcium excretion and elevated 1,25-(OH)2D3 toward normal. Renal CaBP was found to be the same in controls and diabetics despite a tenfold difference in concentration of 1,25-(OH)2D3 in serum, and to be unaffected by insulin treatment, which elevated 1,25-(OH)2D3 by a factor of 7 above untreated diabetics. It is concluded that in the diabetic rat either (1) the threshold concentration of 1,25-(OH)2D3 for inducing synthesis of renal CaBP is set at a much lower level than that for intestinal CaBP, or (2) since both 1,25-(OH)2D3 and renal CaBP are produced in the kidney, 1,25-(OH)2D3 exerts a paracrine effect on renal CaBP production because of its high local concentration. The increased urinary calcium excretion in the untreated streptozotocin-diabetic rat is not secondary to an alteration in renal CaBP.     

Mechanisms of intestinal calcium absorption

Calcium is absorbed in the mammalian small intestine by two general mechanisms: a transcellular active transport process, located largely in the duodenum and upper jejunum; and a paracellular, passive process that functions throughout the length of the intestine. The transcellular process involves three major steps: entry across the brush border, mediated by a molecular structure termed CaT1, intracellular diffusion, mediated largely by the cytosolic calcium-binding protein (calbindinD(9k) or CaBP); and extrusion, mediated largely by the CaATPase. Chyme travels down the intestinal lumen in approximately 3 h, spending only minutes in the duodenum, but over 2 h in the distal half of the small intestine. When calcium intake is low, transcellular calcium transport accounts for a substantial fraction of the absorbed calcium. When calcium intake is high, transcellular transport accounts for only a minor portion of the absorbed calcium, because of the short sojourn time and because CaT1 and CaBP, both rate-limiting, are downregulated when calcium intake is high. Biosynthesis of CaBP is fully and CaT1 function is approximately 90% vitamin D-dependent. At high calcium intakes CaT1 and CaBP are downregulated because 1,25(OH)(2)D(3), the active vitamin D metabolite, is downregulated.     

Anti-clotting activity of endothelial cell cultures and heparan sulfate proteoglycans

A photoaffinity probe for the vitamin D-dependent chick intestinal calcium binding protein (CaBP) has been prepared by conjugation of methyl-4-azidobenzoimidate (MABI) to lactoperoxidase-¹²⁵I-iodinated CaBP to yield ¹²⁵I-CaBP-MABI: [3 moles MABI per mole CaBP]. After incubation $\text{in}\text{vitro}$ of ¹²⁵I-CaBP-MABI (28,000 daltons) in model systems with bovine intestinal alkaline phosphatase (AP) (67,000 daltons), a UV light-dependent crosslinking occurred to yield a conjugate with a molecular weight of 95,000 (by SDS-gel electrophoresis); no crosslinking occurred with $\text{E.}\text{coli}$ alkaline phosphatase. The formation of the ¹²⁵I-CaBP-MABI-AP was found to occur only in the presence of calcium.     

Cloning and analysis of calbindin-D28K cDNA and its expression in the central nervous system

The vitamin D-dependent calcium-binding protein (CaBP), calbindin-D28K (CaBP28K), is present in the central nervous system (CNS), the sensory system, and kidneys of mammals and birds. Recent studies have indicated that several other CaBPs of very similar Mrs are also present in the CNS. This study was carried out to establish the relationship between CaBP28K and other CaBP, particularly spot 35, to provide a basis for further studies on the tissue-specific regulation and distribution of CaBP28K. A cloned pC28 cDNA was isolated from a rat brain expression library using synthetic oligodeoxyribonucleotides (oligos) complementary to rat spot-35 mRNA. This pC28 cDNA had an open reading frame (ORF) of 783 nucleotides (nt) coding for a 261-aa, 30-kDa protein. There was 100% homology between the pC28 sequence and that of the CaBP28K isolated from rat brain cDNA library using a chicken intestinal CaBP28K probe (Hunziker and Schrickel, 1988). Thus the aa and nt sequences of rat CaBP28K and spot 35 are identical. Primer extension studies and Northern analyses show that the major species of CaBP28K mRNA contains a 5'-untranslated region of 132 nt, a coding region of 261 codons and a 3'-untranslated region of 804 nt without the poly(A) tail. The rat CaBP28K probe hybridizes to one major RNA species (1.9 kb) and two minor ones (2.8 and 3.2 kb) in the cerebellum, hippocampus, retina and kidney. This distribution correlates well with the distribution of CaBP28K itself in these organs. Comparison of the genomic organization of the CaBP28K gene with that of other members of the 'EF-hand' CaBP family emphasizes that the CaBP28K gene diverged from the others at the first duplication of the gene encoding one CaBP domain. All the members of the 'EF-hand' gene CaBP family evolved by exon shuffling and specific genomic rearrangements.