Vitamin D-Binding Research Articles

Enhancing vitamin D3 bioaccessibility: Unveiling hydrophobic interactions in soybean protein isolate and vitamin D3 binding via an infant in vitro digestion model

In the domain of infant nutrition, optimizing the absorption of crucial nutrients such as vitamin D3 (VD3) is paramount. This study harnessed dynamic-high-pressure microfluidization (DHPM) on soybean protein isolate (SPI) to engineer SPI-VD3 nanoparticles for fortifying yogurt. Characterized by notable binding affinity (Ka = 0.166 × 105 L·mol−1) at 80 MPa and significant surface hydrophobicity (H0 = 3494), these nanoparticles demonstrated promising attributes through molecular simulations. During simulated infant digestion, the 80 MPa DHPM-treated nanoparticles showcased an impressive 74.4% VD3 bioaccessibility, delineating the pivotal roles of hydrophobicity, bioaccessibility, and micellization dynamics. Noteworthy was their traversal through the gastrointestinal tract, illuminating bile salts' crucial function in facilitating VD3 re-encapsulation, thereby mitigating crystallization and augmenting absorption. Moreover, DHPM treatment imparted enhancements in nanoparticle integrity and hydrophobic properties, consequently amplifying VD3 bioavailability. This investigation underscores the potential of SPI-VD3 nanoparticles in bolstering VD3 absorption, thereby furnishing invaluable insights for tailored infant nutrition formulations.

Vitamin D(3) auto-/paracrine system in rat brain relating to vitamin D(3) status in experimental type 2 diabetes mellitus

Growing evidence suggests that vitamin D3 (D3, cholecalciferol) deficiency and impaired signaling of the hormonally active form of D3, 1α,25(OH)2D3 (1,25D3), through its cellular receptor (VDR) can be significant risk factors for the development of numerous multifactorial diseases, including diabetes. Our investigation was aimed at researching the D3 status in relation to the state of the D3 auto-/paracrine system in the brain and clarifying the effectiveness of the therapeutic use of D3 as a neuroprotective agent in experimental type 2 diabetes mellitus (T2DM). T2DM was induced in male Wistar rats by a combination of a high fat diet and a low dose of streptozotocin (25 mg/kg BW). Diabetic animals were treated with or without cholecalciferol (1,000 IU/kg BW, 30 days). The content of 25-hydroxyvitamin D3 (25D3) in blood serum and brain tissue was determined by ELISA. Analysis of mRNA expression of CYP24A1 and CYP27B1 genes was performed by RT-PCR. Protein levels of VDR, vitamin D3 binding protein (VDBP), CYP27B1 and CYP24A1 were investigated by Western blotting. A significant T2DM-associated decrease in the content of 25D3 in the blood serum was revealed, which correlated with a reduced content of this metabolite in the brain tissue. Impaired D3 status in animals with T2DM was accompanied by an increase in the levels of mRNA and protein of both 25D3 lα-hydroxylase (CYP27B1) and 1,25-hydroxyvitamin D3-24-hydroxylase (CYP24A1), which, respectively, provide local formation and degradation in the nervous tissue of the hormonally active form of D3 – 1,25D3. At the same time, a significant T2DM-induced down-regulation of the brain content of VDBP was shown. In addition, diabetes caused a slight increase in the protein expression of the VDR, through which the auto-/paracrine effects of 1,25D3 are realized in the brain. We have established a complete or partial corrective effect of cholecalciferol on D3 status, its bioavailability in the CNS and the level of protein expression of CYP27B1 and CYP24A1 in the brain of rats with T2DM. Abnormal D3 status in animals with T2DM was accompanied by compensatory changes in the expression of key components of the auto-/paracrine vitamin D3 system. Cholecalciferol was demonstrated to be partially effective in counteracting the impairments caused by T2DM. Keywords: 25-hydroxyvitamin D3, brain, type 2 diabetes, vitamin D3, vitamin D3 auto-/paracrine system

Analysis of the Biological Properties of Blood Plasma Protein with GcMAF Functional Activity.

The main problem related to the studies focusing on group-specific component protein-derived macrophage-activating factor (GcMAF) is the lack of clarity about changes occurring in different types of macrophages and related changes in their properties under the effect of GcMAF in various clinical conditions. We analyzed the antitumor therapeutic properties of GcMAF in a Lewis carcinoma model in two clinical conditions: untreated tumor lesion and tumor resorption after exposure to Karanahan therapy. GcMAF is formed during site-specific deglycosylation of vitamin D3 binding protein (DBP). DBP was obtained from the blood of healthy donors using affinity chromatography on a column with covalently bound actin. GcMAF-related factor (GcMAF-RF) was converted in a mixture with induced lymphocytes through the cellular enzymatic pathway. The obtained GcMAF-RF activates murine peritoneal macrophages (p < 0.05), induces functional properties of dendritic cells (p < 0.05) and promotes in vitro polarization of human M0 macrophages to M1 macrophages (p < 0.01). Treatment of whole blood cells with GcMAF-RF results in active production of both pro- and anti-inflammatory cytokines. It is shown that macrophage activation by GcMAF-RF is inhibited by tumor-secreted factors. In order to identify the specific antitumor effect of GcMAF-RF-activated macrophages, an approach to primary reduction of humoral suppressor activity of the tumor using the Karanahan therapy followed by macrophage activation in the tumor-associated stroma (TAS) was proposed. A prominent additive effect of GcMAF-RF, which enhances the primary immune response activation by the Karanahan therapy, was shown in the model of murine Lewis carcinoma. Inhibition of the suppressive effect of TAS is the main condition required for the manifestation of the antitumor effect of GcMAF-RF. When properly applied in combination with any chemotherapy, significantly reducing the humoral immune response at the advanced tumor site, GcMAF-RF is a promising antitumor therapeutic agent that additively destroys the pro-tumor properties of macrophages of the tumor stroma.

Vitamin C and Vitamin D3 show strong binding with the amyloidogenic region of G555F mutant of Fibrinogen A alpha-chain associated with renal amyloidosis: proposed possible therapeutic intervention.

G555F mutant of Fibrinogen A alpha-chain (FGA) is reported to be associated with kidney amyloidosis. In the current study, we have modelled the G555F mutant and examined the mutation's effect on the structural and functional level. We have also docked Vitamin C and D3 on the mutant's amyloidogenic region to identify if these vitamins can bind amyloidogenic regions. Further, we analyzed if they could prevent or modulate amyloid formation by stopping critical interactions in amyloidogenic regions in FGA. We used the wild type FGA model protein as a control. Our docking and molecular dynamics simulation results indicate stronger Vitamin D3 binding than Vitamin C to the amyloidogenic region of the mutant protein. The RMSD, radius of gyration, and RMSF values were higher for the G555F mutant than the FGA wild type protein. Overall, the results support these vitamins' potential as a therapeutic and anti-amyloidogenic agent for FGA renal amyloidosis.

Tear-Derived Exosome Proteins Are Increased in Patients with Thyroid Eye Disease.

Exosomes contain proteins, lipids, RNA, and DNA that mediate intercellular signaling. Exosomes can contribute to the pathological processes of various diseases, although their roles in ocular diseases are unclear. We aimed to isolate exosomes from tear fluids (TF) of patients with Thyroid eye disease (TED) and analyze the exosomal proteins. TFs were collected from eight patients with TED and eight control subjects. The number of TF exosomes were measured using nanoparticle-tracking analysis. The expression of specific proteins in the purified exosome pellets were analyzed using a Proteome Profiler Array Kit. Cultured normal orbital fibroblasts were incubated with TF exosomes from patients with TED and control subjects, and changes in inflammatory cytokine levels were compared. TF exosomes from TED patients showed more exosomes than the control subjects. The expression levels of exosomal proteins vitamin D-binding (VDB) protein, C-reactive protein (CRP), chitinase 3-like 1 (CHI3L1), matrix metalloproteinase-9 (MMP-9), and vascular adhesion molecule-1 (VCAM-1) were significantly increased in patients with TED, compared to those of controls. Orbital fibroblasts exposed to TF exosomes from patients with TED showed significantly higher levels of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) production than those treated with control TF exosomes. Specific proteins showed higher expression in exosomes from TED patients, implying that they may play keys roles in TED pathogenesis.

Label-Free Fluorescent Aptasensor for Small Targets via Displacement of Groove Bound Curcumin Molecules.

Signal transduction based on fluorescence is one of the most common optical aptasensors for small molecules. Sensors with a number of unique features including high sensitivity, low cost, and simple operation can be constructed easily. However, the label-free fluorescent approach is limited to synthetic dyes that bind strongly to the aptamer sequence and result in a diminished sensor operation with high detection limits. In this study, we report the use of curcumin as a fluorescent probe to signal aptamer/small target binding events. A substantial enhancement in curcumin’s fluorescent emission was observed when bound into the grooves of vitamin D3 (VTD3) binding aptamer, as an example. However, the introduction of the target molecule causes the aptamer to undergo a conformational change that favors complexing the target molecule over binding the curcumin dye. The sensor was able to detect VTD3 down to 1 fM concentration in buffer solutions and extracted blood samples, operate at a wide dynamic range, and discriminate against potential biological interfering molecules including VTD2. The operation of the curcumin based fluorescent sensor is at least six orders of magnitude more sensitive than a VTD3 sensor constructed with the synthetic dye SYBR Green I. The generality of the reported label-free approach was applied with a previously isolated 75-mer bisphenol-A (BPA) aptamer, confirming that the reported sensing strategy is not confined on a particular aptamer sequence. Our work not only reports a novel sensor format for the detection of small molecules, but also serves fluorescent sensor’s most pressing need being novel fluorophores for multiplex targets detection.

A thorny pathway of macrophage activating factor (GcMAF): from bench to bedside

Vitamin D3 Binding Protein (DBP) is a multifunctional glycoprotein whose main role is to transport vitamin D3 and its metabolites, but it also is the precursor of the macrophage activating factor (GcMAF). DBP is converted to GcMAF as a result of site-specific selective deglycosylation under the action of β-galactosidase and sialidase, localized on activated B and T cells, respectively. GcMAF exerts its biological activity primarily as the capability of activating macrophages by enhancing their phagocytic function and producing ROS. Activation results in elevated expression of the specific macrophageal surface receptors involved in the recognition of tumor-associated antigens, as well as in the implementation of direct anticancer activity by inducing the apoptosis or necrosis of tumor cells. Increased interest in GcMAF is associated with its potential to be used in the clinic as a new antitumor drug. Besides its anti-tumor activity, GcMAF exerts a potential against a number of viral and neurodegenerative diseases associated with increased activity of N-acetylgalactosaminidase (nagalase) in the blood serum of patients. Nagalase is an enzyme that completely (rather than selectively) deglycosylates DBP so it cannot be converted to GcMAF, leading to immunodeficiency. Circulating DBP is composed of unmodified and O-glycosylated molecules with the glycosylation degree being dependent on the allelic variants of the gene encoding DBP. The role of DBP in the resistance of organism against a number of diseases is supported by the increased risk of a variety of severe illnesses (amyotrophic lateral sclerosis, colorectal cancer etc.) in patients deficient for GcMAF due to homozygosity for defective DBP alleles. In this review, we also will examine in detail the current data i) on the structure and functions of DBP, as the main precursor of GcMAF, ii) on the main mechanisms of GcMAF anticancer effect, iii) on the tumor strategy for neutralizing GcMAF activity, iv) on the results of GcMAF clinical trials in various cancers; and will discuss the available controversies regarding the positioning of GcMAF as an effective antitumor drug.

Improved Visual Acuity and Retinal Integrity with Resveratrol Based Supplementation in Patients with Macular Degeneration

These case reports evaluate oral supplementation with a resveratrol based red wine supplement, containing vitamin D3 and labile iron binding inositol hexaphosphate. We appraised function Visual Acuity (VA) and Structure Domain Optical Coherence Tomography (SDOCT) in 3 geriatric patients with atrophic AMD, without clinical alternatives.

The role of receptor topology in the vitamin D3 uptake and Ca2+ response systems

The steroid hormone, vitamin D3, regulates gene transcription via at least two receptors and initiates putative rapid response systems at the plasma membrane. The vitamin D receptor (VDR) binds vitamin D3 and a second receptor, importin-4, imports the VDR-vitamin D3 complex into the nucleus via nuclear pores. Here we present evidence that the Homo sapiens VDR homodimer contains two transmembrane (TM) helices (327E – D342), two TM “half-helix” (264K N276), one or more large channels, and 16 cholesterol binding (CRAC/CARC) domains. The importin-4 monomer exhibits 3 pore-lining regions (226E – L251; 768V – G783; 876S – A891) and 16 CRAC/CARC domains. The MEMSAT algorithm indicates that VDR and importin-4 may not be restricted to cytoplasm and nucleus. VDR homodimer TM helix-topology predicts insertion into the plasma membrane, with two 84 residue C-terminal regions being extracellular. Similarly, MEMSAT predicts importin-4 insertion into the plasma membrane with 226 residue extracellular N-terminal regions and 96 residue C-terminal extracellular loops; with the pore-lining regions contributing gated Ca2+ channels. The PoreWalker algorithm indicates that, of the 427 residues in each VDR monomer, 91 line the largest channel, including two vitamin D3 binding sites and residues from both the TM helix and “half-helix”. Cholesterol-binding domains also extend into the channel within the ligand binding region. Programmed changes in bound cholesterol may regulate both membrane Ca2+ response systems and vitamin D3 uptake as well as receptor internalization by the endomembrane system culminating in uptake of the vitamin D3-VDR-importin-4 complex into the nucleus.

Alpha-lactalbumin: A new carrier for vitamin D3 food enrichment

Vitamin D is a fat-soluble regulatory vitamin maintaining blood calcium and phosphorus concentrations within a narrow physiological range. Binding to an appropriate delivery system can enhance vitamin D3 solubility, simplify its transport, protect it from degradation and increase its bioavailability. Alpha-lactalbumin as a milk protein is a good candidate for a vitamin encapsulation. Binding properties and conformational change of bovine apo alpha-lactalbumin upon interaction with vitamin D3 were investigated by calorimetry, spectroscopy and by molecular docking. Tryptophan fluorescence quenching indicates that the protein conformation changes in the presence of vitamin D3. However, according to far UV CD results, the secondary structure of the protein was altered in the presence of vitamin D3. The molecular modeling showed that Van der Waals interactions, hydrogen bond and hydrophobic interactions play a major role in the binding of vitamin D3 to the alpha-lactalbumin hydrophobic pocket. The particle size of alpha-lactalbumin and vitamin D3 complex is much larger than the native protein. Surprisingly, in the presence of vitamin D3, the thermal stability of the protein decreases. The binding constant and standard Gibbs free energy change (ΔG°) of binding vitamin D3 to the protein obtained from ITC are 3.66 × 105 M−1 and −7.6 kcal mol−1, respectively, what agrees with results obtained by measurement of fluorescence and by molecular docking. The formed complex is a suitable candidate in order to enrich the low-fat food and non-alcohol drinks. According to the results, alpha-lactalbumin can be introduced suitable carrier for vitamin D3.

Effect of vitamin d receptor polymorphisms on the development and progression of malignant melanoma

Malignant melanoma is one of the most aggressive cutaneous tumors in men and women. The risk of developing a malignant melanoma depends on several external factors along with deregulation of mutual interaction of genotype and phenotype. Nowadays, growing attention is focused on the study of the interactions of the active form of vitamin D3 with its receptor and inhibitory effect of vitamin D3 receptor polymorphisms on multiple signaling pathways involved in proliferative and metastatic processes. This review article addresses the relationship between factors involved in the development of malignant melanoma through Hedgehog signaling pathway (HH). It summarizes current knowledge of malignant melanoma in regard to the role of the active form of vitamin D3 binding to vitamin D3 receptor (VDR), as well as it describes the influence of polymorphisms of VDR on the inhibition of HH. Understanding of these mechanisms and critical assessment of available data is beneficial to both primary and secondary prevention of malignant melanoma particularly by means of chemo -preventive substances.

Very important pharmacogene summary for VDR

The vitamin D receptor (VDR) binds the active form of vitamin D (1,25-dihydroxyvitamin D3). It belongs to the family of trans-acting transcriptional regulatory factors and shows sequence similarity to the steroid and thyroid hormone receptors. The gene was cloned by Baker and colleagues in 1988 and maps to chromosome 12q13.11 [1]. It consists of nine exons with at least six isoforms of exon 1, spans 63.5 kb, and encodes a 427 amino acid protein [2]. Alternative splicing results in multiple transcript variants encoding the VDR protein of different lengths [3,4]. The interaction of 1,25-dihydroxyvitamin D3 with VDR modulates many biological activities of the neural, immune, and endocrine systems, including calcium and phosphorous homeostasis, apoptosis, and cell differentiation (reviewed by Hewison and colleagues [5-7]). Its pleiotropic property is reflected by the findings that VDR is expressed in at least 37 tissues, which can roughly be grouped into seven biological systems (calcium homeostasis, immune, pancreatic β cells, muscle, cardiovascular, brain, and lung) [8-10]. The most characterized mechanism is the binding of vitamin D3 to nuclear VDR, which activates the receptor to form a heterodimer with the retinoid-X receptor and interacts with a specific DNA sequence on the gene promoter regions called the ‘vitamin D response element’ (VDRE). Transcription repressors occupying the VDRE are then replaced by transcription activators to initiate transcription of targeted genes. Microarray analyses using different human cell lines have identified over 100 genes with VDREs in the promoter regions, all potential targets of the 1,25-dihydroxyvitamin D3–VDR complex [11-13]. In human oral squamous carcinoma SCC25 cells, genes with characterized VDREs and of which demonstrated greater than 10-fold increase after treatment with 1,25-dihydroxy-vitamin D3 include 24-hydroxylase (CYP24), 17β-hydroxy-steroid dehydrogenase (HSD17B2), CD14 (CD14), type XIII collagen (COL13A1), and 5-lipoxygenase (ALOX5) [13]. The effect of 1,25-dihydroxyvitamin D3 by binding to VDR in the nucleus is often described as the slow-acting genomic effect; the effect will take days or hours to be noticed. Alternatively, 1,25-dihydroxyvitamin D3 can bind to VDR located in the caveolae in the plasma membrane to exert rapid responses through production of second messengers [14]. Pathways that have been shown to be activated by the 1,25-dihydroxyvitamin D3–VDR[memb] complex include mitogen-activated protein kinase-dependent [15], cAMP-dependent [16], phospholipase A2-dependent [17], phospholipase C-dependent [18], phosphatidylinositol 3-kinase-dependent [19], and protein kinase C-dependent [20] pathways. Examples of such rapid response include rapid Ca2+ absorption in the duodenum [21,22], opening of Cl− channels and secretion in osteoblasts, insulin secretion from pancreatic β-cells [23,24], and vascular smooth muscle cell migration [19].

Abstract 82: A comprehensive characterization of cholecalciferol supplementation induced changes in the vitamin D3 metabolic pathway in metastatic colorectal cancer patients

Abstract Epidemiological data suggest that vitamin D3 deficiency plays a role in the pathogenesis and progression of human colorectal adenomas and adenocarcinomas. We have characterized the changes in plasma vitamin D3 metabolites (25-D3, 24,25-D3 and 1,25-D3), PTH and vitamin D3 binding protein (VDBP) levels, and peripheral blood mononuclear cells (PBMC) CYP24A1 activity in metastatic colorectal cancer (CRC) patients receiving 2000 IU of oral cholecalciferol daily over a 12 month period. Plasma samples were collected on day 0, 14, 30, 60, 90, 180, 270 and 360. Serum 25-D3 and 24,25-D3 were measured by LC/MS/MS method; plasma PTH and 1,25-D3 levels were determined by RIA. Plasma VDBP levels were measured by ELISA. PBMC CYP24A1activity was determined by HPLC method. A total of 50 CRC patients completed the study. Baseline 25-D3 were significantly influenced by season (p= 0.0145) . Baseline plasma 25-D3 levels were &amp;lt;30ng/mL in 66% of patients. Baseline plasma 24,25-D3, 1,25-D3 and VDBP levels were within the normal range. (The normal ranges are 0- 5ng/mL for 24,25-D3, 16 −80pg/mL for 1,25-D3 and 200 - 550μg/mL for VDBP). Mean baseline plasma PTH levels in patients with plasma 25-D3 &amp;lt;20ng/mL and &amp;gt;30ng/mL were 74.82 ± 27.02 and 56.46 ± 18.10pg/mL, respectively (p&amp;gt;0.05). Baseline PBMC CYP24A1activity was not affected by baseline 25-D3 levels. The increase in serum 25-D3 and 24,25-D3 levels during cholecalciferol treatment was characterized by an initial phase of rapid increase (between day 1 and day 90) followed by a phase of stable plasma 25-D3 and 24,25-D3 levels. A linear relation between plasma 25-D3 and 24,25-D3 was observed at baseline and during cholecalciferol supplementation (r2 = 0.55). No consistent changes in PBMC CYP24A1 activity was observed after 90 days of cholecalciferol treatment. The plasma 24,25-D3/25-D3 ratio at day 60 and 90 of cholecalciferol treatment was weakly correlated (r2=0.03 and 0.06, respectively) to changes in plasma 25-D3 levels. No CRC patient subsets with disproportional increase in plasma 24,25-D3 levels relative to 25-D3 levels during cholecalciferol treatment were identified suggesting that that variable 25-D3 responses cannot be explained by increased 24-hydroxylation. The increase in 1,25-D3 was linear, remained below 100pg/mL and did not correlate with plasma 25-D3 levels. A decrease in plasma PTH levels from 62.83 ± 24.84 to 41.61 ± 18.42 pg/mL was observed after 2 months of cholecalciferol supplementation (p&amp;lt;0.0001). Plasma VDBP protein levels were inconsistent across the study duration; no specific patterns or correlations with 25-D3 or 1,25-D3 could be identified. In summary, vitamin D3 deficiency, defined as 25-D3 &amp;lt;30 ng/mL, is prevalent in metastatic CRC patients. Furthermore, CYP24A1-mediated catabolism of 25-D3 to 24,25-D3 does not appear to be a major determinant of systemic 25-D3 levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 82.

Rational Design for Crystallization of β-Lactoglobulin and Vitamin D3 Complex: Revealing a Secondary Binding Site

β-Lactoglobulin (LG) is a major milk whey protein containing primarily a calyx for vitamin D3 binding, although the existence of another site beyond the calyx is controversial. Using fluorescence spectral analyses in the previous study, we showed the binding stoichiometry for vitamin D3 to LG to be 2:1 and a stoichiometry of 1:1 when the calyx was “disrupted” by manipulating the pH and temperature, suggesting that a secondary vitamin D binding site existed. To help localize this secondary site using X-ray crystallography in the present study, we used bioinformatic programs (Insight II, Q-SiteFinder, and GEMDOCK) to identify the potential location of this site. We then optimized the occupancy and enhanced the electron density of vitamin D3 in the complex by altering the pH and initial ratios of vitamin D3/LG in the cocrystal preparation. We conclude that GEMDOCK can aid in searching for an extra density map around potential vitamin D binding sites. Both pH (8) and initial ratio of vitamin D3/LG (3:1) are c...

Inactivation of the Human Vitamin D Receptor by Caspase-3

Calcitriol actions are mediated by the vitamin D receptor (VDR), a nuclear transcription factor of the steroid-retinoid-thyroid nuclear receptor gene superfamily. Calcitriol inhibits the growth of many cells including cancer cells by inducing cell cycle arrest. In some cancer cell lines, calcitriol also induces apoptosis. In the LNCaP prostate cancer cell line, induction of apoptosis and caspase-3/7 activities by staurosporine (STS) abolished [(3)H]1,25-dihydroxy vitamin D(3) binding and VDR protein, suggesting that the VDR may be targeted for inactivation by caspases during apoptosis. A potential caspase-3 site (D(195)MMD(198)S) was identified in the human VDR ligand-binding domain. Mutations D195A, D198A, and S199A were generated in the putative capase-3 cleavage site. In transfected COS-7 cells, STS treatment resulted in the cleavage of the wild-type (WT) VDR and S199A mutant VDR but not the D195A or D198A mutants. In in vitro assays, the WT VDR and S199A mutant VDR were cleaved by caspase-3, although the D195A and D198A mutants were resistant to caspase-3. In vitro, the WT VDR was also cleaved by caspase-6 and caspase-7 and in extracts of STS-treated LNCaP cells. In STS-treated LNCaP cells and human skin fibroblasts, the proteasome inhibitor MG-132 protected the VDR caspase cleavage fragment from further degradation by the 26S proteasome. The rat VDR that does not contain the caspase-3 cleavage site was not cleaved in STS-treated COS-7 cells. In conclusion, our results demonstrate that the human VDR is a target of caspase-3 and suggest that activation of caspase-3 may limit VDR activity.

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Deglycosylation of Serum Vitamin D3-Binding Protein by α-N-Acetylgalactosaminidase Detected in the Plasma of Patients with Systemic Lupus Erythematosus

A serum glycoprotein, Gc protein (vitamin D3-binding protein), can be converted by β-galactosidase of B cells and sialidase of T cells to a potent macrophage-activating factor (MAF), a protein withN-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor for MAF. Treatment of Gc protein with immobilized β-galactosidase and sialidase generates a remarkably high titered macrophage-activating factor (GcMAF). When peripheral blood monocytes/macrophages (designated macrophages) of 33 systemic lupus erythematosus patients were incubated with GcMAF (100 pg/ml), the macrophages of all patients were activated as determined by superoxide generation. However, the precursor activity of patient plasma Gc protein was lost or reduced in these patients. Loss of the precursor activity was the result of deglycosylation of plasma Gc protein by α-N-acetylgalactosaminidase activity found in the patient plasma. Levels of plasma α-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Deglycosylated Gc protein cannot be converted to macrophage-activating factor. The resulting defect in macrophage activation may lead to an inability to clear pathogenic immune complexes. Thus, elevated plasma α-N-acetylgalactosaminidase activity resulting in the loss of MAF precursor activity and reduced macrophage activity may play a role in the pathogenesis of systemic lupus erythematosus.

Vitamin D3-Binding Protein as a Precursor for Macrophage Activating Factor in the Inflammation-Primed Macrophage Activation Cascade in Rats

When rat peritoneal nonadherent cells were treated with inflammatory lipid metabolites and cultured with adherent cells in 1% fetal calf serum (FCS) supplemented medium RPMI 1640 (FCS medium) for 3 hr, markedly enhanced phagocytic and superoxide generating capacities of macrophages were observed. Stepwise preparation of conditioned medium of lysophosphatidylcholine (lyso-Pc)-treated B cells and untreated T cells in FCS medium generated a potent macrophage activating factor whereas cultivation of lyso-Pc-treated B cells alone in a 1% adult rat serum supplemented medium efficiently generated the macrophage activating factor. Generation of macrophage activating factor requires a precursor protein, serum vitamin D3-binding protein (DBP), as well as participation of lymphocyte glycosidases. The lyso-Pc-inducible β-galactosidase of B lymphocytes and theNeu-1sialidase of T lymphocytes modified bovine DBP (bDBP) to yield the macrophage activating factor, a protein withN-acetylgalactosamine as the remaining sugar. In contrast, lyso-Pc-inducible β-galactosidase of B cells alone modified rat DBP (rDBP) to yield the macrophage activating factor, a protein withN-acetylgalactosamine as the remaining sugar. Thus, we conclude that bDBP carries a trisaccharide composed ofN-acetylgalactosamine, galactose, and sialic acid while rDBP carries a disaccharide composed ofN-acetylgalactosamine and galactose.

All‐trans retinoic acid inhibits binding of 1,25‐dihydroxy‐vitamin D3 to the vitamin D receptor in cultured human keratinocytes

Psoriasis is characterized by hyperproliferation and impared differentiation of epidermal keratinocytes (KCs). Psoriasis can be treated with derivatives of retinoic acid (RA) and vitamin D3. Analogues of vitamin D3 are able to inhibit proliferation and stimulate differentiation of KCs. In contrast, RA inhibits terminal differentiation of KCs. Interactions are known to occur between RA and vitamin D3 signalling pathways. The purpose of the present study was to determine the effect of all-trans RA on the binding of 1,25-dihydroxy-vitamin D3 (1,25 (OH)2D3) to the vitamin D3 receptor (VDR) of cultured human KCs. Cultured KCs from normal adults were incubated with or without RA (10-9-10-7M) for 4-24 h. Cells were then harvested, homogenized and ultrasonicated. The extracted protein was incubated with 3H-1,25 (OH)2D3 (0.015-1.0 nM) with or without 250-fold excess nonradioactive 1,25 (OH)2D3 for 24 h and specific binding was determined by use of the dextran coated charcoal binding assay. Western blot analysis utilizing the monoclonal antibody 9A7 gamma to VDR was performed on protein extracted from the KCs. The bands resulting from Western blot analysis were visualized by enhanced chemiluminescence. From Scatchard analysis it was found that KCs bind 1,25 (OH)2D3 with high affinity (Kd = 0.175 nM). This binding was dose and time dependently inhibited by RA (60% inhibition at 10-7 M after 24 h of incubation). By Western blot analysis, RA had no effect on the amount of protein extracted from KCs at any of the RA concentrations tested. In conclusion, these results show that binding of vitamin D3 to its receptor of human KCs can be inhibited markedly by RA without effecting the amount of protein. These results are in contrast to results with other cell types in which RA upregulates binding of 1,25 (OH)2D3 to the VDR. Because interaction between retinoids and vitamin D3 may occur at different levels during signal transduction, it is not possible to predict from our results whether RA will inhibit the effects of vitamin D3 in vivo.

Alpha-fetoprotein signal sequences: a proposed mechanism for subcellular localization and organelle targeting

Alpha-fetoprotein (AFP) is an oncofetal protein, classified in an “albuminoid” superfamily (with albumin and Vitamin-D binding (Gc) protein) comprising molecules with three characteristic globular domains. The cellular uptake and internalization of AFP and its subcellular compartmentalization has previously been reported in a multitude of cell types. Studies have also emerged which have detected and characterized binding proteins complexed to AFP in various cell membranes and intracellular compartments. However, the literature is devoid of any attempts to relate these binding proteins to possible intracellular trafficking interactions of AFP. Recombinant DNA mutation/deletion technology has provided a means to pinpoint the amino acid sequence location of organelle localization signals on various transcription factors and/or receptors. Several subdomain regions on AFP have been reported to mimic heptad dimerization regions of the steroid/thyroid nuclear receptors. In light of these transcription factor-like docking motifs reported for AFP, the present report purposes various subdomain regions which might constitute basic amino acid sequences resembling recognition signals for binding (dimerizing) proteins. AFP appears to possess multiple prototypic amino acid sequence cassettes on each domain which consist of (i) classical short, compact sequences found on both steroid and thyroid receptors; (ii) proto-signals resembling a steroid receptor type; and (iii) degenerative signal sequence similar to the thyroid/retinoic acid receptor type. The concepts identifying binding or escort proteins for AFP together with organelle signal sequences on AFP have been reconciled in a composite hypothesis to formulate a mechanism which could account for some of the growth-regulating properties described for AFP during the last decade.