Human Vitamin D-binding Protein Research Articles

Genome-wide association study (GWAS) of circulating vitamin D outcomes among individuals of African ancestry

BackgroundVitamin D deficiency is more common among African-ancestry individuals and may be associated with adverse health outcomes. Vitamin D binding protein (VDBP) regulates concentrations of biologically active vitamin D. ObjectiveWe conducted genome-wide association study (GWAS) of VDBP and 25-hydroxyvitamin D among African-ancestry individuals. MethodsData were collected from 2,602 African American adults from the Southern Community Cohort Study (SCCS) and 6,934 African- or Caribbean-ancestry adults from the UK Biobank. Serum VDBP concentrations were available only in the SCCS and were measured by using the Polyclonal Human VDBP ELISA kit. Serum 25-hydroxyvitamin D concentrations for both study samples were measured by using Diasorin Liason, a chemiluminescent immunoassay. Participants were genotyped for single nucleotide polymorphisms (SNPs) with genome-wide coverage by using Illumina or Affymetrix platforms. Fine-mapping analysis was performed by using forward stepwise linear regression models including all variants with P value < 5 × 10−8 and within 250 kbps of a lead SNP. ResultsWe identified 4 loci notably associated with VDBP concentrations in the SCCS population: rs7041 (per allele β = 0.61 μg/mL, SE = 0.05, P = 1.4 × 10−48) and rs842998 (per allele β = 0.39 μg/mL, SE = 0.03, P = 4.0 × 10−31) in GC, rs8427873 (per allele β = 0.31 μg/mL, SE = 0.04, P = 3.0 × 10−14) near GC and rs11731496 (per allele β = 0.21 μg/mL, SE = 0.03, P = 3.6 × 10−11) in between GC and NPFFR2. In conditional analyses, which included the above-mentioned SNPs, only rs7041 remained notable (P = 4.1 × 10−21). SNP rs4588 in GC was the only GWAS-identified SNP associated with 25-hydroxyvitamin D concentration. Among UK Biobank participants: per allele β = −0.11 μg/mL, SE = 0.01, P = 1.5 × 10−13; in the SCCS: per allele β = −0.12 μg/mL, SE = 0.06, P = 2.8 × 10−02). rs7041 and rs4588 are functional SNPs that influence the binding affinity of VDBP to 25-hydroxyvitamin D. ConclusionsOur results were in line with previous studies conducted in European-ancestry populations, showing that GC, the gene that directly encodes for VDBP, would be important for VDBP and 25-hydroxyvitamin D concentrations. The current study extends our knowledge of the genetics of vitamin D in diverse populations.

Fermentation of hemp seed proteins leads to formation of peptides that share sequence similarity with human vitamin D-binding protein

Fermentation of hemp seed proteins leads to formation of peptides that share sequence similarity with human vitamin D-binding protein

Proteomic analysis on the alteration of protein expression in the early-stage placental villous tissue of electromagnetic fields associated with cell phone exposure.

To explore the possible adverse effects and search for cell phone electromagnetic field (EMF)-responsive proteins in human early reproduction, a proteomics approach was employed to investigate the changes in protein expression profile induced by cell phone EMF in human chorionic tissues of early pregnancy in vivo. Volunteer women about 50 days pregnant were exposed to EMF at the average absorption rate of 1.6 to 8.8 W/kg for 1 hour with the irradiation device placed 10 cm away from the umbilicus at the midline of the abdomen. The changes in protein profile were examined using 2-dimensional electrophoresis (2-DE). Up to 15 spots have yielded significant change at least 2- to 2.5-folds up or down compared to sham-exposed group. Twelve proteins were identified- procollagen-proline, eukaryotic translation elongation factor 1 delta, chain D crystal structure of human vitamin D-binding protein, thioredoxin-like 3, capping protein, isocitrate dehydrogenase 3 alpha, calumenin, Catechol-O-methyltransferase protein, proteinase inhibitor 6 (PI-6; SerpinB6) protein, 3,2-trans-enoyl-CoA isomerase protein, chain B human erythrocyte 2,3-bisphosphoglycerate mutase, and nucleoprotein. Cell phone EMF might alter the protein profile of chorionic tissue of early pregnancy, during the most sensitive stage of the embryos. The exposure to EMF may cause adverse effects on cell proliferation and development of nervous system in early embryos. Furthermore, 2-DE coupled with mass spectrometry is a promising approach to elucidate the effects and search for new biomarkers for environmental toxic effects.

Abstract 670: Cigarette smoking, race, and genetic ancestry in association with plasma vitamin D-binding protein (VDBP) in healthy African Americans (AA) and Caucasian Americans (CA)

Abstract Vitamin D and VDBP affect several pathways involved in inflammation, tumor growth, and immune surveillance relevant to carcinogenesis. In a previous pilot study (Bortner et al., 2010), using a proteomics approach, VDBP was down-regulated in the plasma of chronic cigarette smokers by as much as 3-fold. Therefore, using an existing study of healthy AA and CA participants, we conducted an investigation of the differences in VDBP between 39 current smokers and 82 never smokers, frequency matched on age, sex and race/ethnicity. VDBP plasma concentrations were determined in duplicate using the Quantikine® Human VDBP ELISA (CV%, 5.6%). Genotyping was conducted for a set of 112 previously validated Ancestry Informative Markers for the percent West African and European genetic ancestry, and VDBP coding region changes via rs7041 (Asp416Glu, T/G) and rs4588 (Thr420Lys, C/A). Vitamin D metabolites (25OHD3 and 24,25(OH)2D3) were determined using LC-MS/MS (CV% 2.7 and 6.3, respectively). T-test, Chi-Square, Fisher's Exact, and Spearman's correlation coefficients were used to determine statistically significant bivariate differences. Multiple logistic regression was used to adjust for multiple factors using the log-normal transformation of VDBP concentration with the Cochran-Armitage test for trend for VDBP genotype. Multiplicative interaction terms were tested along with their main-effects, adjusted for co-variates. There were no significant differences between current smokers and never smokers by age, VDBP genotype, body mass index, vitamin D metabolites, or genetic ancestry. VDBP concentrations were significantly negatively correlated with body mass index (BMI, r=−0.19, p=0.033), West African ancestry (r=−0.66, p&amp;lt;0.001), vitamin D metabolites (p-values&amp;lt;0.001), VDBP rs4588-A and rs7041-T alleles (VDBP genotypes were 100% correlated). VDBP genotypes coding Asp416Asp and Lys420Lys were at least 3.3-fold lower, compared with Glu416Glu and Thr420Thr, respectively, and this was regardless of smoking status (p-trend&amp;lt;0.001). There was no association between VDBP concentration and age or smoking status. In multiple regression models, serum vitamin D metabolites, self-reported race/ethnicity, West African ancestry and GC genotypes remained significantly associated with VDBP concentration. The magnitude of difference in these variables was similar among current and never smokers, although BMI remained significantly negatively associated with VDBP concentrations among never smokers (p-interaction =0.028). Smoking status was not highly correlated with VDBP plasma concentration, although may have some interactive effect with BMI. Future studies will need to account for the strong correlations with VDBP genotype and genetic ancestry. Support: 1K22CA120092-01A2 and 3K22CA120092-02S1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 670. doi:1538-7445.AM2012-670

An intronic locus control region plays an essential role in the establishment of an autonomous hepatic chromatin domain for the human vitamin D-binding protein gene.

The human vitamin D-binding protein (hDBP) gene exists in a cluster of four liver-expressed genes. A minimal hDBP transgene, containing a defined set of liver-specific DNase I hypersensitive sites (HSs), is robustly expressed in mouse liver in a copy-number-dependent manner. Here we evaluate these HSs for function. Deletion of HSI, located 5' to the promoter (kb -2.1) had no significant effect on hDBP expression. In contrast, deletion of HSIV and HSV from intron 1 repressed hDBP expression and eliminated copy number dependency without a loss of liver specificity. Chromatin immunoprecipitation analysis revealed peaks of histone H3 and H4 acetylation coincident with HSIV in the intact hDBP locus. This region contains a conserved array of binding sites for the liver-enriched transcription factor C/EBP. In vitro studies revealed selective binding of C/EBPalpha to HSIV. In vivo occupancy of C/EBPalpha at HSIV was demonstrated in hepatic chromatin, and depletion of C/EBPalpha in a hepatic cell line decreased hDBP expression. A nonredundant role for C/EBPalpha was confirmed in vivo by demonstrating a reduction of hDBP expression in C/EBPalpha-null mice. Parallel studies revealed in vivo occupancy of the liver-enriched factor HNF1alpha at HSIII (at kb 0.13) within the hDBP promoter. These data demonstrate a critical role for elements within intron 1 in the establishment of an autonomous and productive hDBP chromatin locus and suggest that this function is dependent upon C/EBPalpha. Cooperative interactions between these intronic complexes and liver-restricted complexes within the target promoter are likely to underlie the consistency and liver specificity of the hDBP activation.

The human vitamin D-binding protein gene contains locus control determinants sufficient for autonomous activation in hepatic chromatin.

The human vitamin D-binding protein (hDBP) gene is a member of a cluster that includes albumin, α-fetoprotein and α-albumin genes. The common origin, physical linkage and hepatic expression of these four genes predict shared regulatory element(s). However, separation of hDBP from the other three genes by 1.5 Mb argues that hDBP may be under autonomous control. To test for hDBP autonomy, mouse lines were generated with a transgene containing the hDBP gene along with extensive flanking sequences. Expression of this transgene was hepatic, robust and proportional to transgene copy number. DNase I hypersensitive site (HS) mapping revealed five liver-specific HS at the hDBP locus: HSI and HSIII at −2.1 kb and −0.13 kb upstream of the transcription initiation site, HSIV and HSV within intron 1 and HSVII located 3′ to the poly(A) site. A second transgene with minimal flanking sequences confirmed the sufficiency of these gene-proximal determinants for hepatic activation. The hepatic-specific HS aligned with segments of phylogenetically conserved non-coding sequences. These data demonstrate the autonomy of the hDBP locus and suggest that this control is mediated by chromatin-based locus control determinants in close proximity to, and within the transcription unit.

Crystal structure of the complex between actin and human vitamin D-binding protein at 2.5 A resolution.

A high-affinity complex formed between G-actin and plasma vitamin D-binding protein (DBP) is believed to form part of a scavenging system in the plasma for removing actin released from damaged cells. In the study presented here, we describe the crystal structure of the complex between actin and human vitamin D-binding protein at 2.5 A resolution. The complex contains one molecule of each protein bound together by extensive ionic, polar, and hydrophobic interactions. It includes an ATP and a calcium ion bound to actin, but no evidence of vitamin D metabolites bound to the DBP. Both actin and DBP are multidomain molecules, two major domains in actin and three in DBP. All of these domains contribute to the interaction between the molecules. DBP enfolds the end of the actin molecule, principally in actin subdomain 3 but with additional interactions in actin subdomain 1. This orientation is similar to the binding of profilin to actin, as predicted from previous studies. The more extensive interactions of DBP give an affinity for actin some 3 orders of magnitude higher than that for profilin. The larger "footprint" of DBP on actin also leads to an overlap with the actin-binding site of gelsolin domain I.

Biochemical and preliminary crystallographic characterization of the vitamin D sterol- and actin-binding by human vitamin D-binding protein

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 Å crystal, primitive orthorhombic with unit cell dimensions a=80.2 Å, b=87.3 Å, and c=159.6 Å, P2 12 12 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.

Errata: A structural basis for the unique binding features of the human vitamin D-binding protein

Nature Structural Biology 9, 131–136 (2002). Two mistakes were introduced during production of this paper. On page 131, in the first paragraph of the introduction, a sentence “The multifunctional DBP also binds monomeric actin (G-actin) and slowly depolymerizes filamentous actin.” was inadvertently inserted.

A structural basis for the unique binding features of the human vitamin D-binding protein

The human serum vitamin D-binding protein (DBP) has many physiologically important functions, ranging from transporting vitamin D3 metabolites, binding and sequestering globular actin and binding fatty acids to functioning in the immune system. Here we report the 2.3 A crystal structure of DBP in complex with 25-hydroxyvitamin D3, a vitamin D3 metabolite, which reveals the vitamin D-binding site in the N-terminal part of domain I. To more explicitly explore this, we also studied the structure of DBP in complex with a vitamin D3 analog. Comparisons with the structure of human serum albumin, another family member, reveal a similar topology but also significant differences in overall, as well as local, folding. These observed structural differences explain the unique vitamin D3-binding property of DBP.

C-6 functionalized analogs of 25-hydroxyvitamin D 3 and 1α,25-dihydroxyvitamin D 3: synthesis and binding analysis with vitamin D-binding protein and vitamin D receptor

In this article, we describe the development of a general synthetic strategy to functionalize the C-6 position of vitamin D 3 and its biologically important metabolites, i.e. 25-hydroxyvitamin D 3 (25-OH-D 3) and 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3]. We employed Mazur’s cyclovitamin D method to synthesize vitamin D 3 analogs with several functionalities at the C-6 position. In addition, we synthesized 6-(3-hydroxypropyl) and 6-[(2-bromoacetoxy)propyl] derivatives of 25-OH-D 3 15 and 16, respectively, and 6-(3-hydroxypropyl) derivative of 1,25(OH) 2D 3 17. Competitive binding assays of 15– 17 with human serum vitamin D-binding protein showed that all these analogs specifically bound to this protein, although with significantly lower affinity than the 25-OH-D 3, the strongest natural binder, but with comparable affinity with 1,25(OH) 2D 3, the hormone. On the other hand, 6-[3-hydroxypropyl], 1α,25-dihydroxyvitamin D 3 17 did not show any specific binding for recombinant nuclear vitamin D receptor. These results indicated that the region containing the C-6 position of the parent seco-steroid [1,25(OH) 2D 3] may be an important recognition marker towards vitamin D receptor binding. Information, delineated in this article, will be important for evaluating structure-activity relationship in synthetic analogs of vitamin D and its metabolites.

Synthesis and Binding-analysis of 5E-[19-(2-bromoacetoxy)methyl]25-hydroxyvitamin D 3 and 5E-25-hydroxyvitamin D 3-19-methyl[(4-azido-2-nitro)phenyl]glycinate: Novel C 19-modified Affinity and Photoaffinity Analogs of 25-hydroxyvitamin D 3

Synthesis of novel C 19-modified affinity and photoaffinity analogs of vitamin D 3 and 25-hydroxyvitamin D 3(25-OH-D 3) is described. A key step in the synthesis is a Horner–Emmons reaction between C 19-nor-cyclovitamin D 3-C 19-ketone or C 19-nor-25-hydroxy-cyclovitamin D 3-C 19-ketone and diethyl cyanomethylphosphonate. Competitive radioligand binding assays with human serum vitamin D-binding protein (DBP) and 5E-[19-(2-bromoacetoxy)methyl]25-hydroxyvitamin D 3 and 5E-25-hydroxyvitamin D 3-19-methyl[(4-azido-2-nitro)phenyl]glycinate, 25-OH-D 3-analogs containing affinity and photoaffinity probes at C 19-position, demonstrated that these compounds displaced radiolabeled 25-OH-D 3 from the binding pocket of DBP in a dose-dependent manner. Thus, these affinity and photoaffinity analogs are potentially useful in determining the ligand binding site topographies of DBP and possibly the vitamin D receptor.

97/04768 Conceptual considerations on PV systems composed of AC modules: Kurokawa, K. et al. Solar Energy Materials and Solar Cells, 1997, 47, (1–4), 243–250

The structure and organization of the human vitamin D-binding protein (DBP) gene has been determined. The gene is composed of 13 exons and 12 intervening sequences. With the help of the polymerase chain reaction (PCR) introns were amplified using exon-specific oligonucleotide primers, and were sequenced after subcloning; the exon/intron borders were determined. The introns 2, 5, 7, 9 and 10 were sequenced completely; the introns 1, 3, 4, 6, 8, 11 and 12 were sequenced in part. We designed intron-specific primers for the amplification of each exon by the PCR-method. This permits the analysis of mutational and function-related sites. By comparison with the genes for human albumin and α-fetoprotein the gene for DBP/GC is confirmed as a member of this multigene family. The location of the introns in the coding region of the human DBP-gene is identical with the position of the introns in the rat DBP-gene.

Bacterial Expression of Human Vitamin D-Binding Protein (Gc2) in Functional Form

In this report, we report the first expression of human vitamin D-binding protein (hDBP), a serum protein with several functions and a multidomained structure, inEscherichia coli.The recombinant protein (reDBP) was expressed as a fusion partner of glutathioneS-transferase in order to facilitate proper folding of the reDBP;E. coli-expressed DBP was found to be fully functional with respect to vitamin D sterol binding, interaction with actin, and cross-reactivity with anti-DBP antibody. Furthermore, both natural DBP and reDBP were affinity-labeled with 25-hydroxyvitamin D3-3-bromo[1-14C]acetate in a similar fashion. Availability of an expression system for hDBP in functional form provides opportunity to develop mutants and truncated DBPs to study multiple ligand-binding properties of this protein in relationship with its structure.

Affinity Purification of Human Plasma Vitamin-D-Binding Protein

During the course of our studies to probe the vitamin D ligand-binding domains of vitamin D-binding protein and vitamin D receptor, we developed a synthetic procedure to modify the 3β-hydroxyl group of vitamin D 3 and its 25-hydroxy- and 1,25-dihydroxy metabolites with a 3′-aminopropylether group, In the present study we have coupled 25-hydroxyvitamin D 3-3β-3′-aminopropylether to an activated Sepharose matrix, Using this stable and reusable affinity matrix we have purified human vitamin D-binding protein from human plasma to homogeneity.

A novel sequence polymorphism in exon 8 of the human vitamin D-binding protein (GC) gene in an African population

A novel sequence polymorphism due to a T to C transition at the third nucleotide of the codon for Cys283 of the vitamin D-binding protein (GC) gene assigned to chromosome 4q13-4q21.1 was revealed by sequence analysis. Population studies by single strand conformation polymorphism (SSCP) analysis showed this GC-283.3 site was polymorphic in a Black African population but monomorphic in a European population.

Trp-145 Is Essential for the Binding of 25-Hydroxyvitamin D3 to Human Serum Vitamin D-binding Protein

Chemical modification of specific amino acid residues in a protein has been a valuable tool in identifying amino acid residues that are responsible for ligand binding of a protein. In the present investigation, we targeted Trp and His residues in human serum vitamin D-binding protein (hDBP) by modifying them with specific chemical modifiers. We also evaluated the results of these modifications in the binding of 25-hydroxy[26(27)-3H]vitamin D3 ([3H]25-OH-D3) to hDBP. We observed a dose-dependent loss of binding activity by N-bromosuccinimide (specific for Trp). Similar results were observed with diethylpyrocarbonate (specific for His). Furthermore, loss of [3H]25-OH-D3-binding was protected by preincubation of hDBP samples with an excess of 25-hydroxyvitamin D3. These results strongly emphasized the importance of Trp (single residue at position 145) and 1 His residue (out of a total of 6) in the vitamin D sterol-binding by vitamin D-binding protein.

Sequence and organization of the human vitamin D-binding protein gene

The structure and organization of the human vitamin D-binding protein (DBP) gene has been determined. The gene is composed of 13 exons and 12 intervening sequences. With the help of the polymerase chain reaction (PCR) introns were amplified using exon-specific oligonucleotide primers, and were sequenced after subcloning; the exon/intron borders were determined. The introns 2, 5, 7, 9 and 10 were sequenced completely; the introns 1, 3, 4, 6, 8, 11 and 12 were sequenced in part. We designed intron-specific primers for the amplification of each exon by the PCR-method. This permits the analysis of mutational and function-related sites. By comparison with the genes for human albumin and α-fetoprotein the gene for DBP/GC is confirmed as a member of this multigene family. The location of the introns in the coding region of the human DBP-gene is identical with the position of the introns in the rat DBP-gene.

A novel sequence polymorphism in exon 1 of the human vitamin D-binding protein (GC) gene.

Journal Article A novel sequence polymorphism in exon 1 of the human vitamin D-binding protein (GC) gene Get access A. Braun, A. Braun Institute of Anthropology and Human Genetics, University of MunichRichard-Wagner Str 10/1, D-80333 München 2, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar A. Kofler, A. Kofler Institute of Anthropology and Human Genetics, University of MunichRichard-Wagner Str 10/1, D-80333 München 2, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar R. Bichlmaier, R. Bichlmaier Institute of Anthropology and Human Genetics, University of MunichRichard-Wagner Str 10/1, D-80333 München 2, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar S. Kammerer, S. Kammerer Institute of Anthropology and Human Genetics, University of MunichRichard-Wagner Str 10/1, D-80333 München 2, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar H. Cleve H. Cleve * Institute of Anthropology and Human Genetics, University of MunichRichard-Wagner Str 10/1, D-80333 München 2, Germany *To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Human Molecular Genetics, Volume 2, Issue 10, October 1993, Page 1750, https://doi.org/10.1093/hmg/2.10.1750-a Published: 01 October 1993

A novel sequence polymorphism in exon 1 of the human vitamin D-binding protein (GC) gene

A novel sequence polymorphism in exon 1 of the human vitamin D-binding protein (GC) gene