Search

Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo.

Mice were supplemented with low and high doses of natural and synthetic vitamin E, T cells from the spleen isolated and stimulated with plate-bound anti-CD3 and soluble anti-CD28, and gene expression changes assessed by gene array experiments. The data obtained indicate significant qualitative and quantitative differences between the two vitamin forms in regulating gene expression in response to T-cell stimulation. Marker genes have been found whose expression can be considered significant in establishing the level of, and response to vitamin E for both natural and synthetic vitamin E supplementation; unique markers for synthetic vitamin E supplementation and unique markers for natural vitamin E supplementation have been identified.

Vitamin D-binding protein (DBP), a multi-functional serum glycoprotein, has a triple-domain modular structure. Mutation of Trp145 (in Domain I) to Ser decreased 25-OH-D(3)-binding by 80%. Furthermore, recombinant Domain I (1-203) and Domain I + II (1-330) showed specific and strong binding for 25-OH-D(3), but Domain III (375-427) did not, suggesting that only Domains I and II might be required for vitamin D sterol-binding. Past studies have suggested that Domain III is independently capable of binding G-actin. We exploited this apparently independent ligand-binding property of DBP to purify DBP-actin complex from human serum and rabbit muscle actin by 25-OH-D(3) affinity chromatography. Competitive (3)H-25-OH-D(3) binding curves for native DBP and DBP-actin complex were almost identical, further suggesting that vitamin D sterol- and actin-binding activities by DBP might be largely independent of each other. Trypsin treatment of DBP produced a prominent 25 kDa band (Domain I, minus 5 amino acids in N-terminus), while actin was completely fragmented by such treatment. In contrast, tryptic digestion of purified DBP-actin complex showed two prominent bands, 52 (DBP, minus 5 amino acids in the N-terminus) and 34 kDa (actin, starting with amino acid position 69) indicating that DBP, particularly its Domains II and III were protected from trypsin cleavage upon actin-binding. Similarly, actin, except its N-terminus, was also protected from tryptic digestion when complexed with DBP. These results provided the basis for our studies to crystallize DBP-actin complex, which produced a 2.5 A crystal, primitive orthorhombic with unit cell dimensions a=80.2A, b=87.3A, and c=159.6A, P2(1)2(1)2(1) space group, V(m)=2.9. Soaking of crystals of actin-DBP in crystallization buffer containing various concentrations of 25-OH-D(3) resulted in cracking of the crystal, which was probably a reflection of a ligand-induced conformational change in the complex, disrupting crystal contacts. In conclusion, we have provided data to suggest that although binding of 25-OH-D(3) to DBP might result in discrete conformational changes in the holo-protein to influence actin-binding, these binding processes are largely independent of each other in solution.

The multiple physiological properties of vitamin D-binding protein (DBP) include organ-specific transportation of vitamin D(3) and its metabolites, manifested by its ability to bind vitamin D sterols with high affinity. In the present investigation we probed the vitamin D sterol-binding pocket of human DBP with affinity labeling analogs of 25-hydroxyvitamin D(3) ¿25-OH-D(3) and 1, 25-dihydroxyvitamin D(3) ¿1,25(OH)(2)D(3) containing bromoacetate alkylating probe at C-3 (A-ring), C-6 (triene), C-11 (C-ring), and C-19 (exocyclic methylene) of the parent sterol. Competitive binding assays with DBP showed approximately 22-, 68-, and 2000-fold decrease in the binding of 1,25(OH)(2)-D(3)-11-BE, 25-OH-D(3)-3-BE, and 25-OH-D(3)-6-BE, respectively, compared to that seen with 25-OH-D(3), while there was no significant difference in the DBP-binding affinity of 25-OH-D(3)-19-BE and 25-OH-D(3). Surprisingly, ¿(14)C25-OH-D(3)-11-BE and ¿(14)C1, 25(OH)(2)-D(3)-19-BE failed to label DBP despite high-affinity DBP-binding, indicating the absence of any nucleophilic amino acid in the vicinity of their bromoacetate moiety to form a covalent bond, while these analogs are inside the binding pocket. In contrast, ¿(14)C25-OH-D(3)-6-BE and ¿(14)C25-OH-D(3)-3-BE specifically labeled DBP. BNPS-skatole digestion of DBP labeled with ¿(14)C25-OH-D(3)-3-BE or ¿(14)C25-OH-D(3)-6-BE produced two peptides (M(r) 17,400 and 33,840), with radioactivity associated with the N- and C-terminal peptides, respectively, raising the possibility that either different areas of the same vitamin D sterol-binding pocket, or different domains of DBP might be labeled by these analogs. Successful affinity labeling of recombinant domain I (1-203) of DBP with both reagents indicated that different areas of the same vitamin D-binding pocket (domain I) were labeled. These affinity analogs are potentially useful for "mapping" the vitamin D sterol-binding pocket and developing a functional model.

Vitamin D binding protein (DBP) plays an essential role in the vitamin D hormone endocrine system in sequestering vitamin D3 and its metabolites with high affinity, and transporting them to various target organs and tissues. In the present investigation, 25-hydroxyvitamin D3-3 beta-(1,2-epoxypropyl)ether (25-OH-D3-epoxide) and 25-hydroxyvitamin D3-3 beta-bromoacetate (25-OH-D3-BE), synthetic analogs of 25-hydroxyvitamin D3 (25-OH-D3), were developed as affinity alkylating reagents for the covalent modification of the 25-OH-D3-binding site in rat vitamin D binding protein (rDBP). Competitive radioligand binding assays of 25-OH-D3-BE and 25-OH-D3-epoxide with affinity-purified rDBP demonstrated that these analogs displaced 25-hydroxy[26(27)-3H]vitamin D3 (3H-25-OH-D3), specifically bound to rDBP, in a dose-dependent fashion. Incubation of rDBP samples with radiolabeled versions of these analogs, i.e., 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE, resulted in the covalent labeling of rDBP. This labeling was largely prevented when incubations were carried out in the presence of an excess of 25-OH-D3, the natural ligand for rDBP. Labeling-specificity by these analogs was further demonstrated by the covalent labeling, inhibited by coincubation with a large excess of 25-OH-D3, of a single protein band, upon incubating rat serum Cohn IV fraction with 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE. Collectively, these results strongly suggested that 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE covalently modified the 25-OH-D3-binding site in rDBP. The reagents described in this report could be important in mapping the 25-OH-D3-binding pocket in rDBP.

The effective treatment of the rachitic symptoms of pseudo-vitamin D-deficiency rickets, type I (PVDRI) by massive doses of vitamin D3 was examined. For this purpose, the affinities and the maximum binding capacities (Bmax) of the plasma vitamin D-binding protein (DBP) and of the intestinal 1,25-dihydroxyvitamin D3 (1,25- (OH)2D3) receptor for vitamin D3, 25-hydroxyvitamin D3 (25-OHD3) and 1,25-(OH)2D3, were investigated in normal piglets and in rachitic piglets that suffered from PVDRI. The piglets were 5 to 10 weeks old and of both sexes. The Bmax of plasma DBP for 25-OHD3 was 6.77 ± 0.45 μ m for PVDRI piglets and 7.30 ± 0.41 μ m for control piglets and showed no differences between the two groups. Equilibrium association constants (Ka) of DBP for 25-OHD3 were 4.3 × 108 m −1 for PVDRI piglets and 4.0 × 108 m −1 for controls and showed also no differences between the two groups. Similarly the Ka of DBP for 1,25-(OH)2D3 was also the same for rachitic and control piglets (1.45 × 107 and 1.54 × 107 m −1, respectively). Due to the lower circulating concentration of 1,25-(OH)2D3 in the plasma of rachitic piglets compared to that of controls its free metabolite index was significantly lower in rachitic (0.42 ± 0.05 × 10−5) than in control piglets (3.63 ± 0.30 × 10−5). The Kd and Bmax of the intestinal nuclear receptor for 1,25-(OH)2D3 of rachitic and control piglets were 0.31 ± 0.05 and 0.33 ± 0.05 n m and 674 ± 103 and 719 ± 123 fmol/mg protein, respectively, and were also not different between the two groups of piglets. It was concluded from these observations that the rachitic symptoms of PVDRI piglets resulted solely from the lower free 1,25-(OH)2D3 concentration in plasma compared to that of normal piglets. The relative affinities of the intestinal 1,25-(OH)2D3 receptor for vitamin D3 and 25-OHD3 were also measured. It was found that 50% displacement of 1,25-(OH)2D3 from the intestinal receptor of PVDRI and control piglets required a 220,000- and 245,000-fold excess of the free concentration of vitamin D3, respectively, and a 20- to 42- and 23- to 71-fold excess of the free concentration of 25-OHD3, respectively. Treatment of PVDRI piglets with an effective dose of 7.5 × 104 IU of vitamin D3 led to a transient 1073-fold excess of the free concentration of vitamin D3 and a 59-fold excess of the free concentration of 25-OHD3 over free 1,25-(OH)2D3 in plasma. Since a 59-fold excess of free 25-OHD3 in plasma of treated piglets could cause at least 50% displacement of 1,25-(OH)2D3 from the intestinal 1,25-(OH)2D3 receptor it is suggested that after administration of pharmacological amounts of vitamin D3 to PVDRI piglets both 25-OHD3 and 1,25-(OH)2D3 acted upon the intestinal 1,25-(OH)2D3 receptor. The two metabolites probably exerted a coordinate effect in healing the rachitic symptoms.

Cancer procoagulant (CP) and tissue factor (TF; only in complex with Factor VIIa (FVIIa)) can activate FX to FXa. Controversy still exists whether or not CP is an entity different from TF, or whether CP activity is due to contamination of CP preparations with TF/FVIIa complex. We therefore looked for proteins in CP preparations that were detected by anti-TF antibodies and then sequenced these proteins. One- and two-dimensional gels of CP and TF were used to identify proteins immunoreactive to monoclonal anti-CP and anti-TF antibodies (Mabs). Those proteins in the CP preparation recognized by anti-TF antibodies were sequenced. Angiotensinogen precursor, alpha-1-antitrypsin precursor, and vitamin D-binding protein were identified along with one so far unidentified sequence; however, no TF-sequences were identified. Also, no proteins with the correct molecular weight for TF were identified using anti-TF antibodies. It seems possible that CP preparations contain proteins that have some epitopes similar to the epitopes recognized in TF by anti-TF Mab. However, these proteins do neither have the molecular weight nor the amino acid sequence of TF.

Vitamin D-binding protein (DBP) is primarily involved in the binding and transportation of vitamin D3 and its various metabolites to target organs and tissues. This is manifested by the ability of DBP to bind vitamin D3 and its metabolites with high affinity. In the present study we developed 25-hydroxyvitamin D3-3 beta-(1,2-epoxypropyl)ether (25-OH-D3-epoxide) as an affinity labeling reagent of human DBP (hDBP). Competitive radioligand binding assays of 25-OH-D3-epoxide with hDBP demonstrated that the binding affinity of this analog was similar to that of 25-hydroxyvitamin D3 (25-OH-D3). Incubation of 25-hydroxy[26(27)-3H]-vitamin D3-3 beta-(1,2-epoxypropyl)ether [[3H]25-OH-D3-epoxide] with hDBP covalently labeled the protein. When the incubation was carried out in the presence of a large excess of 25-OH-D3, labeling was removed completely. When human Cohn IV fraction, containing hDBP, was incubated with [3H]25-OH-D3-epoxide a single protein band, corresponding to hDBP, was labeled. Labeling was completely obliterated in the presence of a large amount of 25-OH-D3. However, an equivalent amount of 7-dehydrocholesterol had no effect on labeling. These results demonstrated that [3H]25-OH-D3-epoxide most probably labeled the vitamin D sterol-binding domain of hDBP.

Biochemical analyses revealed that a thyroxine (T4)-binding protein (TBP) in the blood of some turtles exhibits a high degree of structural homology to mammalian vitamin D-binding protein (DBP), instead of mammalian T4-binding protein (TBG). Like DBP, TBP is rich in Cys (ca. 28/molecule). The 31 NH2-terminal amino acids of TBP show 68% identity to the corresponding region of mammalian (rat, mouse, and human) DBP and one peptide from Lys-C proteolysis shows similar homology to residues 35-49 of DBP (the predicted sterol binding domain). Another peptide shows a 75% identity to a sequence corresponding to residues 134-154 of DBP, but seven others showed no homology to any proteins in GenBank. Digestion with N-glycosidase F reduced the M(r) of TBP by only 4.5K compared with 14K for TBG; T4-binding activity of TBP was unaffected. TBP (and DBP) also behaves as a much smaller molecule (approximately 57 kDa) than TBG (> 70 kDa) by size-exclusion chromatography, despite similarities in their estimated sizes from sodium dodecyl sulfate electrophoresis (approximately 60 kDa). Binding studies confirm that the turtle T4-binding protein likely also represents the major blood DPB. Thus, in the turtle, a single binding protein resembling DBP performs two major transport functions which are normally served by proteins representing two different multigene families in mammals.

The vitamin D-binding protein in human serum (the group-specific component) is an α 2 -globulin which is genetically polymorphic in all populations studied. Previous work ( J. Svasti and B. H. Bowman (1978) J. Biol. Chem. 253 , 5188–5194 , and J. Svasti, A. Kurosky, A. Bennett, and B. H. Bowman (1979) Biochemistry 18 , 1611–1617 ) has shown that the electrophoretic variations of the proteins controlled by two allelic genes, Gc 1 and Gc 2 , are due to at least three amino acid substitutions between Gc1 and Gc2 ( Svasti et al. (1979) ) and to heterogeneity in the Gc1 phenotype arising from carbohydrate dissimilarities. Gc1 migrates electrophoretically as two protein bands, while Gc2 migrates cathodally as a single band. This study demonstrates a post-translational glycosylation difference occurring in a single area of the Gc1 sequence which accounts for the heterogeneity observed previously. The glycosylation site, a threonine residue, appears to be in a sequence which differs between Gc1 and Gc2. The O -glycosidic bond, which is typical of mucins, is rare in plasma proteins. The cyanogen bromide fragment containing the galactosamine-containing carbohydrate in Gc1 was partially sequenced through 20 residues from the amino terminus. No detectable galactosamine could be found in the homologous cyanogen bromide fragment in Gc2. A new purification procedure for the vitamin D-binding protein in human plasma has been developed. Three chromatographic steps provide purified protein.

A protein has been detected in chick brain which is immunologically identical to the vitamin D-induced calcium-binding protein found in intestinal tissue. It is present in highest concentration in the cerebellum and at lower levels in the remainder of the brain. Brain calcium-binding protein has the same molecular weight and electrophoretic properties as intestinal calcium-binding protein. Although the synthesis of intestinal calcium-binding protein is totally dependent upon a source of vitamin D, this has not yet been shown for brain calcium-binding protein. The total calcium-binding protein content of the cerebellum of chicks fed a vitamin D-free diet continued to increase during growth from 1 to 5 weeks, and is not responsive to exogenous vitamin D or 1,25-dihydroxyvitamin D.

The binding of the natural and unnatural diastereoisomers 25-hydroxyvitamin D 3 -26, 23-lactone and 1,25 dihydroxyvitamin D 3 -26,23-lactone to the vitamin D-binding protein (DBP) and 1,25 dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] chick intestinal receptor have been investigated. Also, the biological activities, under in vivo conditions, of these compounds, in terms of intestinal calcium absorption (ICA) and bone calcium mobilization (BCM), in the chick are reported. The presence of the lactone ring in the C23-C26 position of the seco-steroid side chain increased two to three times the ability of both 25(OH)D 3 and 1,25(OH) 2 D 3 to displace 25(OH)[ 3 H]D 3 from the D-binding protein; however, the DBP could not distinguish between the various diastereoisomers. In contrast, the unnatural form (23 R ,25 S ) of the 25-hydroxy-lactone was found to be 10-fold more potent than the natural form, and the unnatural (23 R ,25 S )1,25(OH) 2 D 3 -26,23-lactone three times more potent than the natural 1,25-dihydroxy-lactone in displacing 1,25(OH) 2 [ 3 H]D 3 from its intestinal receptor. While studying the biological activity of these lactone compounds, it was found that the natural form of the 25-hydroxy-lactone increased the intestinal calcium absorption 48 h after injection (16.25 nmol), while bone calcium mobilization was decreased by the same dose of the 25-hydroxy-lactone. The 1,25-dihydroxyvitamin D 3 -26,23-lactone in both its natural and unnatural forms was found to be active in stimulating ICA and BCM. These results suggest that the 25-hydroxy-lactone has some biological activity in the chick and that 1,25(OH) 2 D 3 -26,23-lactone can mediate ICA and BCM biological responses, probably through an interaction with 1,25-(OH) 2 D 3 specific receptors in these target tissues.

A new fluoro analog of 1,25-dihydroxyvitamin D 3 , i.e., 26,26,26,27,27,27-hexafluoro-1, 25-dihydroxyvitamin D 3 , has been compared with the native hormone, 1,25-dihydroxyvitamin D 3 , in its biological potency, duration of action, and binding to the vitamin D transport protein and intestinal receptor protein. The fluoro analog is about 5 times more active than the native hormone in healing rickets and elevating serum inorganic phosphorus levels of rachitic rats. It is about 10 times more active than 1,25-dihydroxyvitamin D 3 in increasing intestinal calcium transport and bone calcium mobilization of vitamin D-deficient rats fed a low-calcium diet. Furthermore, the higher biopotency is manifested in animals after oral dosing. Of great importance is that the action of the fluoro analog is longer lasting than that of 1,25-dihydroxyvitamin D 3 . This is especially apparent in the elevation of serum phosphorus and bone mineralization responses. The fluoro analog is only slightly less competent than 1,25-dihydroxyvitamin D 3 in binding to the vitamin D transport protein in rat blood, and is one-third as competent as 1,25-dihydroxyvitamin D 3 in binding to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D 3 . These results suggest that the basis for increased potency of this analog is likely the result of less rapid metabolism.

An interaction among serum protein, materials containing intrinsic factor, and vitamin B12 has been described. Not all serum proteins combine more vitamin B12 in the presence of materials containing intrinsic factor than in the absence of such materials, but only those that migrate in an electric field as α2- or β-globulin. The increased combining activity of serum protein in the presence of materials containing intrinsic factor is many times greater than was observed if the binding capacities of serum and materials containing intrinsic factor were determined separately and added together. The data indicate that a factor that is present in serum of normal individuals and, also to a lesser extent in serum from patients with pernicious anemia, is responsible for the B12 combining reaction.

The production of the plasma proteins, prothrombin and factor VII, which are essential in the blood clotting process, is dependent on the action of vitamin K. In intact vitamin K-deficient rats, administration of actinomycin D did not block the vitamin K-induced increase in plasma prothrombin. An isolated perfused rat liver preparation that released factor VII activity to the perfusate in response to vitamin K administration was developed. The factor VII production in this system was inhibited by puromycin but not by actinomycin D. These data indicate that the site of action of the vitamin is at some in step protein biosynthesis subsequent to the formation of a specific messenger RNA and prior to release of the complete protein from the liver.

Vitamin B 12 in normal rat serum is found to exist largely in the bound form and almost entirely associated with α 2 -globulin. With increasing in vitro additions of the vitamin to serum, up to 1.5 mμg./ml., the vitamin is chiefly recovered in the α 2 - and β-globulin fractions, the bound vitamin being confined to the α 2 -fraction. Any extra vitamin B 12 added migrates in the free state with the γ-globulin fraction.

Calcitroic acid was recently identified as a major metabolite of 1,25-dihydroxyvitamin D 3 (Esvelt, Schnoes, and DeLuca, Biochemistry 18 , 3977, 1979). The metabolite was found to have little, although significant, activity in healing rickets, and causing bone mineral mobilization but elicited no significant elevation in intestinal calcium transport. The compound showed little affinity for either the serum 25-hydroxyvitamin D binding protein or the intestinal cytosol receptor for 1,25-dihydroxyvitamin D 3 . Various tissues of the rat were examined for the presence of calcitroic acid following a 120-ng dose of 1,25-dihydroxy-[3α- 3 H]vitamin D 3 . The metabolite was detected in liver, intestinal mucosa, kidneys, and blood with livers and mucosa containing the highest concentrations. In each of these tissues the calcitroic acid content increased during the period between 4 and 12 h after the dose. The presence of calcitroic acid in femurs was indicated but could not be confirmed. Bile duct cannulation reduced but did not abolish the intestinal calcitroic acid content. In addition to calcitroic acid, other polar metabolites of 1,25-dihydroxyvitamin D 3 were detected in these experiments.

The tissue protein which tightly binds the human serum binding protein for vitamin D and its metabolites (HDBP) was studied in soluble extracts of human skeletal muscle. A tissue protein-HDBP complex was effected in vitro by the addition of human serum Cohn IV to high-speed supernatant from muscle, and the complex was partially purified by ion-exchange chromatography, gel filtration, and affinity chromatography. The faster-sedimenting complex was retained longer than HDBP on DEAE-Sephacel columns, and was estimated to have a size of 100,000 daltons by gel filtration. The complex displayed inhibitory activity to deoxyribonuclease I (DNase I), whereas HDBP alone did not. When the complex was applied to affinity chromatography columns, immunoassayable HDBP was retained by DNase I-agarose and two dominant proteins of ~58,000 and 45,000 Mr were retained by the IgG fraction of anti-HDBP serum covalently bonded to amino-agarose, as revealed by sodium dodecyl sulfate-polyacrylamide electrophoresis. Pure HDBP does not bind to nor inhibit DNase I, but an actin-HDBP complex does. These data suggested that the tissue component with high affinity for HDBP was actin. Incubation of equimolar amounts of polymerized actin and pure HDBP in its apo form resulted in the depolymerization of the actin. This depolymerizing activity was also observed with HDBP saturated with cholecalciferol, 25-hydroxycholecalciferol, 24R, 25-dihydroxycholecalciferol, or 1,25-dihydroxycholecalciferol.