Uptake and degradation of vitamin D binding protein and vitamin D binding protein-actin complex in vivo in the rat.

We have labelled the rat vitamin D binding protein (DBP), DBP-actin and rat albumin with 125I-tyramine-cellobiose (125I-TC). In contrast with traditional 125I-labelling techniques where degraded radioactive metabolites are released into plasma, the 125I-TC moiety is trapped intracellularly in the tissues, where the degradation of the labelled proteins takes place. By using this labelling method, the catabolism of proteins can be studied in vivo. In this study we have used this labelling technique to compare the tissue uptake and degradation of DBP, DBP-actin and albumin in the rat. DBP-actin was cleared from plasma at a considerably faster rate than DBP. After intravenous injection of labelled DBP-actin complex, 48% of the radioactive dose was recovered in the liver after 30 min, compared with 14% when labelled DBP was administered. Only small amounts of DBP-actin complex were recovered in the kidneys. In contrast with the results obtained with DBP-actin complex, liver and kidneys contributed about equally in the uptake and degradation of DBP determined 24 h after the injection. When labelled DBP was compared with labelled albumin, the amount of radioactivity taken up by the liver and kidneys by 24 h after the injection was 2 and 5 times higher respectively. In conclusion, liver and kidneys are the major organs for catabolism of DBP in the rat. Furthermore, binding of actin to DBP enhances the clearance of DBP from circulation as well as its uptake by the liver.     

Species differences in the binding kinetics of 25-hydroxyvitamin D3 to vitamin D binding protein

The specific binding of 25-hydroxyvitamin D3 to its binding protein was studied in serum of the human, rhesus monkey, cow, horse, and rat. The free fraction of 25-hydroxyvitamin D3 in the rat was 0.34 +/- 0.15 pmol free/nmol total (+/- SD) and this was lower than in any of the other species (p less than 0.01). In the human, the free fraction was 1.5 +/- 0.32 pmol free/nmol total, which was higher than in any of the other species (p less than 0.001). The differences in the free fraction were mainly due to differences in dissociation constant. The relative levels of free 25-hydroxyvitamin D should be taken into account when extrapolating findings about vitamin D metabolism in animals to the human. A technical outcome of this study is that of the species tested, vitamin D binding protein from rat serum is the most suitable as a reagent component for methods used to measure total 25-hydroxyvitamin D by competitive protein binding assay.     

Relations between vitamin D and fatty acid binding properties of vitamin D-binding protein

The interaction of fatty acids with bovine vitamin D-binding protein (DBP) was studied using a partition equilibrium method. This protein has one high affinity site for binding of fatty acids with an association constant Ka = 7 x 10(5) M-1 for palmitic acid and Ka = 6 x 10(5) M-1 for arachidonic acid. Competition experiments showed that palmitic acid hardly competes with 25-hydroxycholecalciferol for binding to DBP. However, arachidonic acid showed comparatively a stronger competition for binding to this protein. The great difference in competition of palmitic and arachidonic acids with 25-hydroxycholecalciferol may be related to changes in DBP conformation promoted by the binding of different ligands.     

Glycosylation status of vitamin D binding protein in cancer patients

On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase‐derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O‐linked trisaccharide glycosylation of serum‐derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization‐based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages.     

Immunoperoxidase localization of vitamin D dependent calcium binding protein.

Calcium binding protein (CaBP) was localized by the indirect peroxidase-labeled antibody method in chick duodenum 72 hr after administering 32.5 nmol of cholecalciferol to vitamin D-deficient chicks. CaBP was observed in cytoplasm and nuclei of absorptive cells but was absent from goblet cells. Our results are consistent with the suggested functional role for CaBP in the prevention of intracellular accumulation of calcium by preventing mitochondrial accumulation of calcium, enhancing removal of calcium from absorptive cells, and/or preventing the "leaking" of calcium into cells through the lateral borders. They are not consistent with an extracellular functional role for CaBP.     

Distribution of vitamin D binding protein expressing neurons in the rat hypothalamus

We observed immunostaining for vitamin D binding protein (DBP) in rat hypothalamus. Part of the supraoptic and of the paraventricular neurons showed DBP immunoreactivity, in part colocalized with Arg-vasopressin. DBP was also observed in widespread axonal projections throughout the lateral hypothalamus, the median eminence and the posterior pituitary lobe. A portion of ependymal cells, the choroids plexus epithelium and some of the endocrine cells in the anterior pituitary lobe contained DBP immunoreactivity. In situ hybridization of semithin sections with a synthetic oligonucleotide probe to DBP mRNA resulted in staining of magnocellular hypothalamic neurons, but not of ependymal cells or anterior lobe cells. Our observations indicate an intrinsic expression of DBP in the rat hypothalamus. DBP may be synthesized and transported along with the classical neurohypophyseal hormones. The multiple locations of DBP-expressing neurons indicate multiple functional properties: DBP may be released from in the posterior lobe, it may act as a hypophyseotropic factor and as a central neuroactive substance.     

Population distribution of the human vitamin D binding protein: anthropological considerations

The polymorphism of the serum vitamin D binding protein (DBP) in humans is based on the existence of three common alleles, Gc1F, Gc1S, and Gc2, and 84 rare alleles. The geographical distribution of Gc1F, Gc1S, and Gc2 alleles shows north to south clines, together with a balanced equilibrium between the Gc1F or Gc1S allele frequency and the Gc2 frequency. The distribution of the FST values shows high variability within a geographical area. For European and North Asiatic groups, the FST values are the lowest observed, and the reason may be a long process of homogenization. Aboriginal populations from Australia and New Guinea and groups from both North Africa and South America show the greatest heterogeneity of their allele frequencies. Systematic factors such as genetic drift and selection may account for this distribution. In contrast with the three main DBP alleles, the distribution of the rare alleles corresponds to patterns of human migrations that occurred during prehistoric and historic periods. Thus, the rare mutants are of particular relevance to anthropological and genetical investigations.     

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.     

Effect of ligand binding on the conformation of human plasma vitamin D binding protein (group-specific component).

Several techniques have been used to demonstrate that the binding of specific ligands to human plasma vitamin D binding protein induces a change in protein conformation. Apoprotein and holoprotein show circular dichroism spectra of similar form in the peptide region with double minima at 207 and 218 nm. The minimum mean residue ellipticity of apoprotein (20.6 X 10(3) degrees.cm2.dmol-1) is decreased by about 8% after vitamin D3 binding, suggesting a small change in the backbone conformation. Spectrofluorimetric studies showed that 25-hydroxycholecalciferol causes a saturable enhancement of intrinsic fluorescence of human vitamin D binding protein and alters the pH profile of protein fluorescence, suggesting that there are alterations in the local environment of tryptophan residue(s) after ligand binding. Furthermore, in the presence of 25-hydroxycholecalciferol, the rate of chemical modification of the amino groups in human vitamin D binding protein is decreased and the susceptibility of intact vitamin D binding protein to proteolytic degradation is reduced, suggesting that some surface sites in the vitamin D binding protein molecule are less accessible to external agents. In addition, although the absorbance of vitamin made if difficult to interpret the ultraviolet spectra of holoprotein and apoprotein, the presence of vitamin D binding protein appears to stabilize the vitamin in an aqueous environment, a phenomenon that may be of physiological importance.     

Genetic polymorphism of the vitamin D binding protein and another post-albumin protein in horse serum

Horizontal polyacrylamide gel electrophoresis, on 10% separation gel, of horse serum revealed polymorphism of the vitamin D binding protein (Gc protein) and another post-albumin protein (Pa). Family data supported the hypothesis that Gc and Pa types were controlled by autosomal codominant alleles. For both Gc and Pa proteins, the homozygous types showed a single fraction while the heterozygous type had two fractions. Pa types were found to be identical to the post-albumin types reported earlier by starch gel electrophoresis. Two Gc alleles, Gc F and Gc S , and three Pa alleles, Pa D, Pa F and Pa S , were observed in samples from Swedish (four breeds), Lipizzaner and Arab horses. The frequency of the more common allele at the two loci, i.e. Gc F and Pa F , ranged from 0.72–0.93 and from 0.58–0.99, respectively, in the different breeds studied. Plasma samples showed an extra protein fraction near the Gc S fraction and thus were found unsuitable for Gc typing.     

A sensitive competitive protein binding assay for vitamin D in plasma

A sensitive protein binding assay for vitamin D is described. The vitamin D3 was extracted from plasma with diethyl ether and methylene chloride. The lipid extract was purified in Sephadex LH-20 followed by Lipidex 5000 and finally by high pressure liquid chromatography on a Zorbax Sil column (0.79 x 25 cm) developed in 0.25:99.75 isopropanol: methylene chloride. The vitamin D fraction was collected and quantitated by competitive protein binding assay with a 1/50,000 dilution of sheep plasma in 0.05 M potassium phosphate buffer (pH 7.5) containing 0.01% gelatin. [H3]-25-Hydroxyvitamin D3 was used as a radioactive tracer in the assay. We found that under these conditions, sheep plasma had equal affinity for vitamin D2 and vitamin D3 and could detect as little as 0.1 ng of vitamin D. When rat, cow, or human plasma was substituted for the sheep plasma, the decline in sensitivity to vitamin D2 was fivefold to tenfold. With this assay, we found excellent agreement (r = 0.98) between the results obtained by competitive protein binding analysis and direct U.V. absorbance analysis by high pressure liquid chromatography.     

Gc (vitamin D binding protein) subtypes in rheumatoid arthritis

Summary Two hundred and six patients with rheumatoid arthritis were examined for their association with the subtypes of Gc (vitamin D binding protein). In patient groups there is 11% excess of individuals with Gc^*2 allele compared to the control group, giving a relative risk of 1.55. This risk increases with the humoral status of the individual. A possible physiological basis between the association of vitamin D binding protein and rheumatoid arthritis is discussed.     

Determination of the affinity of vitamin D metabolites to serum vitamin D binding protein using assay employing lipid-coated polystyrene beads.

We have developed an assay to measure the affinity of serum vitamin D binding protein for 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, and vitamin D3, using uniform diameter (6.4 microns) polystyrene beads coated with phosphatidylcholine and vitamin D metabolites as the vitamin D donor. The lipid metabolite coated beads have a solid core, and thus all of the vitamin D metabolites are on the bead surface from which transfer to protein occurs. After incubating these beads in neutral buffer for 3 h, essentially no 3H-labeled vitamin D metabolites desorb from this surface. Phosphatidylcholine/vitamin D metabolite-coated beads (1 microM vitamin D metabolite) were incubated with varying concentrations of serum vitamin D binding protein under conditions in which the bead surfaces were saturated with protein, but most of the protein was free in solution. After incubation, beads were rapidly centrifuged without disturbing the equilibrium of binding and vitamin D metabolite bound to sDBP in solution was assayed in the supernatant. All three vitamin D metabolites became bound to serum vitamin D binding protein, and after 10 min of incubation the transfer of the metabolites to serum vitamin D binding protein was time independent. The transfer followed a Langmuir isotherm, and the Kd for each metabolite binding to serum vitamin D binding protein was derived by nonlinear least-squares fit analysis. From this analysis the following values for the Kd were obtained: 5.59 x 10(-6) M, 25-hydroxyvitamin D; 9.45 x 10(-6) M, 1,25-dihydroxyvitamin D; and 9.17 x 10(-5) M, vitamin D. In conclusion, we have developed a method which avoids problems encountered in previous assays and allows the precise and convenient determination of binding affinities of vitamin D metabolites and serum vitamin D binding protein.     

Genetic polymorphism of the vitamin D binding protein and another post-albumin protein in horse serum.

Horizontal polyacrylamide gel electrophoreses, on 10% separation gel, of horse serum revealed polymorphism of the vitamin D binding protein (Gc protein) and another post-albumin protein (Pa). Family data supported the hypothesis that Gc and Pa types were controlled by autosomal codominant alleles. For both Gc and Pa proteins, the homozygous types showed a single fraction while the heterozygous type had two fractions. Pa types were found to be identical to the post-albumin types reported earlier by starch gel electrophoresis. Two Gc alleles, GcF and GcS, and three Pa alleles, Pa D, Pa F and Pa S, were observed in samples from Swedish (four breeds), Lipizzaner and Arab horses. The frequency of the more common allele at the two loci, i.e. GcF and PaF, ranged from 0.72-0.93 and from 0.58-0.99, respectively, in the different breeds studied. Plasma samples showed an extra protein fraction near the GcS fraction and thus were found unsuitable for Gc typing.     

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.