Multiplex LC-MS/MS for simultaneous determination of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D3, albumin, and vitamin D-binding protein with its isoforms: One-step estimation of bioavailable vitamin D and vitamin D metabolite ratio.

Bioavailable vitamin D and vitamin D metabolite ratio (VMR) have emerged as potential novel vitamin D markers. We developed a multiplex liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine all elements necessary for the calculation of bioavailable vitamin D and VMR, including 25-hydroxyvitamin D [25-(OH)D] and 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3], VDBP and its isoforms, and albumin. Following separate reactions of hexane extraction and trypsin digestion, serum samples were analyzed using LC-MS/MS to measure 25-(OH)D3, 25-(OH)D2, 24,25-(OH)2D3, VDBP and its isoforms, and albumin. Analytical performances were assessed. Korean (n = 229), Arab (n = 98), White (n = 99) and Black American (n = 99) samples were analyzed. Bioavailable vitamin D and VMR were calculated. All target molecules were clearly separated and accurately quantified by LC-MS/MS. Analytical performances, including imprecision, accuracy, ion suppression, limit of quantification, linearity, and comparison with existing methods were within acceptable levels. The allele frequencies of VDBP isoforms in various races resulted similar to previously known values. The levels of bioavailable vitamin D were highest in White Americans and lowest in Black Americans. We have successfully developed a multiplex LC-MS/MS-based assay method that can simultaneously perform the measurement of all parameters needed to calculate bioavailable vitamin D and VMR. Our devised method was robust and reliable in terms of analytical performances and could be applied to routine clinical samples in the future to more accurately assess vitamin D status.

Improved preparation of group-specific component (Gc) protein to derive macrophage activating factor.

Cancer immunotherapy has recently attracted attention as an approach for cancer treatment through the activation of the immune system. Group-specific component (Gc) protein is a precursor for macrophage activating factor (GcMAF), which has a promising immunomodulatory effect on the suppression of tumor growth and angiogenesis. In this study, we successfully purified Gc protein from human serum using anion-exchange chromatography combined with affinity chromatography using a 25-OH-D3-immobilized column. The purity of Gc protein reached 95.0% after anion-exchange chromatography. The known allelic variants of Gc protein are classified into three subtypes-Gc1F, Gc1S and Gc2. The fragment sequence of residues 412-424 determined according to their MS/MS spectra is available to evaluate the subtypes of Gc protein. The data showed that the Gc protein purified in this study consisted of the Gc1F and Gc2 subtypes. Our method improved the purity of Gc protein, which was not affected by the treatment to convert it into GcMAF using ß-galactosidase- or neuraminidase-immobilized resin, and will be useful for biological studies and/or advanced clinical uses of GcMAF, such as cancer immunotherapy.

Comparative Proteomics of Human and Macaque Milk Reveals Species-Specific Nutrition during Postnatal Development.

Milk has been well established as the optimal nutrition source for infants, yet there is still much to be understood about its molecular composition. Therefore, our objective was to develop and compare comprehensive milk proteomes for human and rhesus macaques to highlight differences in neonatal nutrition. We developed a milk proteomics technique that overcomes previous technical barriers including pervasive post-translational modifications and limited sample volume. We identified 1606 and 518 proteins in human and macaque milk, respectively. During analysis of detected protein orthologs, we identified 88 differentially abundant proteins. Of these, 93% exhibited increased abundance in human milk relative to macaque and include lactoferrin, polymeric immunoglobulin receptor, alpha-1 antichymotrypsin, vitamin D-binding protein, and haptocorrin. Furthermore, proteins more abundant in human milk compared with macaque are associated with development of the gastrointestinal tract, the immune system, and the brain. Overall, our novel proteomics method reveals the first comprehensive macaque milk proteome and 524 newly identified human milk proteins. The differentially abundant proteins observed are consistent with the perspective that human infants, compared with nonhuman primates, are born at a slightly earlier stage of somatic development and require additional support through higher quantities of specific proteins to nurture human infant maturation.

[Identification of asthenozoospermia-associated proteins in human seminal plasma by shotgun proteomic strategy].

OBJECTIVE: To identify asthenozoospermia-associated proteins in seminal plasma by the shotgun proteomic strategy. METHODS: Six seminal plasma samples were collected by Percoll respectively from healthy fertile and asthenozoospermia volunteers, balanced, mixed, and then the mixture was separated by SDS-PAGE. The proteins in the gel were enzymolyzed, extracted and identified by the shotgun proteomic strategy. The identified proteins with the unique peptide count > or =2 or the unique peptide count=1 but the total count > or =4 were compared between the two groups. RESULTS: A total of 172 differential proteins were identified, of which, 89 were exclusively from the asthenozoospermia and 83 exclusively from the healthy fertile men. According to the molecular function, these differential proteins were mainly the types of signal transduction and catalytic activity. CONCLUSION: Functionally, 10 of the proteins are particularly important, which include annexin VI isoform 2, isoform 1 of interleukin-6 receptor subunit beta precursor, Mr 400,000 protein, cytosolic dynein heavy chain, alpha-actinin-4, receptor-type tyrosine-protein phosphatase eta precursor, vitamin D-binding protein precursor, protein S100-A11, protein S100-A9 and ANXA4.

Protein chemical characterization of Gc globulin (vitamin D-binding protein) isoforms; Gc-1f, Gc-1s and Gc-2.

Gc globulin, also called vitamin D-binding protein, is a plasma protein involved in the extracellular actin-scavenger system, vitamin D transport and possibly also other biological activities. Low levels of Gc globulin have been found to correlate with multiple organ failure and non-survival of patients with fulminant hepatic failure and trauma. Here, we characterize the dominant isoforms of plasma-derived Gc globulin from Cohn fraction IV paste with respect to amino acid sequence and posttranslational modifications. Gc globulin was purified in large scale and the isoforms separated by ion exchange chromatography. The separated isoforms and several commercial preparations of individual isoforms were characterized by mass spectrometry. This revealed that the major isoforms were non-glycosylated. Compared to the Gc-1f isoform the other dominating isoforms represented an Asp/Glu substitution (Gc-1s) and a Thr/Lys substitution (Gc-2) in agreement with DNA sequencing studies. The commercial preparations were found to represent mainly one or two isoforms. An O-linked glycan with a mass of 656 Da and terminating with a sialic acid residue was detected on a minor proportion of Gc globulin molecules.

Upregulation of vitamin D binding protein (Gc-globulin) binding sites during neutrophil activation from a latent reservoir in azurophil granules.

Vitamin D binding protein (DBP) is a multifunctional plasma transport protein that is also found on the surface of many cell types. Cell surface DBP significantly enhances chemotactic activity of complement (C) peptides C5a and C5a des Arg. However, both DBP binding and C5a chemotaxis enhancement can vary among neutrophil donors. To test if activation during cell purification is responsible for this variability, neutrophils were isolated using both standard and lipopolysaccharide (LPS)-free protocols. Cells isolated by the LPS-free method had no DBP-enhanced chemotaxis to C5a or DBP binding to plasma membranes. Moreover, neutrophils treated with LPS bound more avidity to immobilized DBP than sham-treated cells. Subcellular fractionation of neutrophils (standard protocol) revealed a heavy plasma membrane (HM) band that contained components of light plasma membranes and all three granules. The HM band possessed most of the DBP binding activity (58%), and activation of cells with ionomycin greatly increased DBP binding to HM. Azurophil granules contained 33% of the total DBP binding sites and there was a highly significant positive correlation (r=0.988) between release of the granule marker myeloperoxidase and DBP binding. These results indicate that fusion of granules with the plasma membrane forms HM that contains DBP binding sites.

Large-scale purification and characterization of non-glycosylated Gc globulin (vitamin D-binding protein) from plasma fraction IV.

Gc globulin, also called vitamin D-binding protein, is a plasma protein involved in the extracellular actin-scavenger system. Low levels of Gc globulin have been found to correlate with multiple organ failure and nonsurvival of patients with fulminant hepatic failure and trauma. Therefore substitution therapy with Gc globulin might be beneficial for such patients, increasing their chance of survival. In the present study, we describe a large-scale purification process for the production of a virus-safe human plasma-derived Gc globulin from Cohn fraction IV paste. The process includes three ion-exchange-chromatography steps, followed by a gel filtration, and two virus-reduction steps are implemented. The Gc globulin product was characterized with respect to purity, functional activity, glycosylation and, finally, with respect to content of endotoxin. From the results, it can be concluded that human Gc globulin purified from Cohn fraction IV is non-glycosylated. The purified Gc globulin is able to mask the presence of endotoxin by 20%.

Association of the macrophage activating factor (MAF) precursor activity with polymorphism in vitamin D-binding protein.

BACKGROUND: Serum vitamin D-binding protein (Gc protein or DBP) is a highly expressed polymorphic protein, which is a precursor of the inflammation-primed macrophage activating factor, GcMAF, by a cascade of carbohydrate processing reactions. In order to elucidate the relationship between Gc polymorphism and GcMAF precursor activity, we estimated the phagocytic ability of three homotypes of Gc protein, Gc1F-1F, Gc1S-1S and Gc2-2, through processing of their carbohydrate moiety. MATERIALS AND METHODS: We performed Gc typing of human serum samples by isoelectric focusing (IEF). Gc protein from human serum was purified by affinity chromatography with 25-hydroxyvitamin D3-sepharose. A phagocytosis assay of Gc proteins, modified using beta-glycosidase and sialidase, was carried out. RESULTS: The Gc1F-1F phenotype was revealed to possess Galbeta1-4GalNAc linkage by the analysis of GcMAF precursor activity using beta1-4 linkage-specific galactosidase from jack bean. The GcMAF precursor activity of the Gc1F-1F phenotype was highest among three Gc homotypes. CONCLUSION: The Gc polymorphism and carbohydrate diversity of Gc protein are significant for its pleiotropic effects.

Preparation of Gc protein-derived macrophage activating factor (GcMAF) and its structural characterization and biological activities.

BACKGROUND: Gc protein has been reported to be a precursor of Gc protein-derived macrophage activation factor (GcMAF) in the inflammation-primed macrophage activation cascade. An inducible beta-galactosidase of B cells and neuraminidase of T cells convert Gc protein to GcMAF. MATERIALS AND METHODS: Gc protein from human serum was purified using 25(OH)D3 affinity column chromatography and modified to GcMAF using immobilized glycosidases (beta-galactosidase and neuraminidase) The sugar moiety structure of GcMAF was characterized by lectin blotting by Helix pomatia agglutinin. The biological activities of GcMAF were evaluated by a superoxide generation assay and a phagocytosis assay. RESULTS: We successfully purified Gc protein from human serum. GcMAF was detected by lectin blotting and showed a high biological activity. CONCLUSION: Our results support the importance of the terminal N-acetylgalactosamine moiety in the GcMAF-mediated macrophage activation cascade, and the existence of constitutive GcMAF in human serum. These preliminary data are important for designing small molecular GcMAF mimics.

Purification, crystallization and preliminary X-ray investigation of the complex of human vitamin D binding protein and rabbit muscle actin.

The vitamin D binding protein binds globular actin with high affinity and is involved in the clearance of actin from the blood circulation. A complex of the human vitamin D binding protein and rabbit muscle actin was subjected to purification steps. The pure complex was crystallized using the hanging-drop vapour-diffusion procedure. The best obtained crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 74.44, b = 74.90, c = 88.02 A, beta = 110.19 degrees. A complete data set to 2.4 A was collected from a single crystal using synchrotron radiation at DESY, Hamburg, Germany.

Characterization of 25-hydroxyvitamin D binding protein from intestinal cells.

We have previously purified a cytosolic vitamin D metabolite binding protein (cDBP) from rat enterocytes, which has characteristics distinct from other vitamin D binding proteins. In these studies, we demonstrate that cDBP in a semi-purified fraction from human intestinal cells (Caco-2 cells) binds 25-hydroxyvitamin D (25OHD) with at least a 1000-fold greater affinity than 1, 25-dihydroxyvitamin D (1,25(OH)(2)D) or 24,25-dihydroxyvitamin D. Treatment of cells with 1,25(OH)(2)D reduced 25OHD binding to approximately one third that of the untreated cells (0.42 CPM/mg total protein vs 1.34 CPM/mg total protein, respectively). Finally, the cDBP is not immunoreactive to antibodies prepared against the C-terminus of the nuclear vitamin D receptor (VDR). In summary, cDBP bound 25OHD with greater affinity than either 1,25(OH)(2)D or 24,25 dihydroxyvitamin D, the cytosolic binding activity was down-regulated by 1,25(OH)(2)D and cBDP is distinct from the nuclear VDR.

Functional characterization and purification of an intracellular vitamin D-binding protein in vitamin D-resistant new world primate cells. Amino acid sequence homology with proteins in the hsp-70 family.

Most genera of New World primates exhibit resistance to vitamin D. These monkeys harbor high circulating concentrations of the prohormone 25-hydroxyvitamin D and the active vitamin D hormone 1, 25-dihydroxyvitamin D. Previous work from this laboratory indicated that resistance is associated with the overexpression of a 60-65-kDa intracellular protein that binds vitamin D metabolites competitively. In the current studies 25-[3H]hydroxyvitamin D3 (25-OHD3) was used as a competitive ligand to investigate the ability of a number of small lipid molecules to interact with this intracellular vitamin D-binding protein (IDBP) in post-nuclear extracts of a prototypical lymphoblast cell line from the common marmoset, a vitamin D-resistant New World primate. Only those vitamin D metabolites with a hydroxyl moiety in the C-25 position were bound by IDBP. Disruption of the C-25 hydroxyl obviated binding, whereas more proximal alterations in the vitamin D side chain did not. Modifications in the A-ring of 25-hydroxylated vitamin D metabolites, most specifically hydroxylation of C-1, diminished but did not abolish ligand binding. Of more than two dozen other small lipid molecules examined, only the C-19 17-hydroxysteroids, 17beta-estradiol and testosterone, and the C-21 steroid progesterone were found to be capable of binding specifically to IDBP. Using a combination of physical and serial chromatographic techniques, we enriched IDBP 25-OHD3 binding activity 17,588-fold in extracts of B95-8 cells. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this purified fraction demonstrated a predominant 65-kDa molecular species with a pI approximately 4.5. Seven different peptide fragments were isolated from the 65-kDa protein, each possessing sequence similarity to the hsp-70 family of proteins. Ligand binding analyses confirmed that human inducibly expressed hsp-70-bound 25-OHD3 with approximately similar affinity ( approximately 10(-7) M) as did purified IDBP. In summary, these results suggest a novel action for the hsp-70 family of proteins as intracellular vitamin D- and gonadal steroid hormone-binding molecules.

Structural definition of a potent macrophage activating factor derived from vitamin D3-binding protein with adjuvant activity for antibody production.

Incubation of human vitamin D3-binding protein (Gc protein), with a mixture of immobilized beta-galactosidase and sialidase, efficiently generated a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase, and isolation of the intermediates with immobilized lectins, revealed that either sequence of hydrolysis of Gc glycoprotein by these glycosidases yields the macrophage-activating factor, implying that Gc protein carries a trisaccharide composed of N-acetylgalactosamine and dibranched galactose and sialic acid termini. A 3 hr incubation of mouse peritoneal macrophages with picomolar amounts of the enzymatically generated macrophage-activating factor (GcMAF) resulted in a greatly enhanced phagocytic activity. Administration of a minute amount (10-50 pg/mouse) of GcMAF resulted in a seven- to nine-fold enhanced phagocytic activity of macrophages. Injection of sheep red blood cells (SRBC) along with GcMAF into mice produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days.

Electrophoretic mobility shift assay identifies vitamin D binding protein (Gc-globulin) in human, rat, and mouse sera.

Serum vitamin D binding protein (DBP, also known as Gc-globulin) is a multifunctional protein capable of binding both vitamin D metabolites and actin. DBP can be visualized when analyzed by polyacrylamide gel electrophoresis followed by staining. Confirmation of its identity had previously required immunoprecipitation with specific anti-DBP antisera or occupancy of the protein with radioactive vitamin D sterols. We present studies showing that preincubation of G-actin with mammalian sera produced a discernible DBP protein band shift on native gel electrophoresis. Addition of DNaseI, a 33-kDa intracellular protein with an avid actin-binding site, to the incubations resulted in a supershift of DBP-actin complexes to an even more cathodal region of the gels. Following incubations with human, rat, and murine sera the same actin shift occurred as did the actin plus DNaseI supershift. The migrations of each complex were correlated with purified DBP migrations under identical conditions. It was confirmed that the supershifted bands contained DBP by Western blotting and detection of DBP by binding of 25-OH[3H]D3. After intravenous G-actin injections into living mice, a serum DBP-actin complex could be detected on native gels as the uncomplexed DBP band decreased in intensity. This simple, direct-staining technique appears to be suitable for identifying DBP/Gc phenotypes in human populations as well as for semiquantitatively monitoring the plasma actin-scavenger system in vivo in animal models or in human diseases.

Binding of GC (VDBP) to membranes of human B lymphocytes following stripping of extant protein.

The presence of Gc (vitamin D binding protein) has been consistently demonstrated on the membrane of B lymphocytes. This protein appears to be spatially associated with surface immunoglobulins. The origin of this surface protein has not yet been determined and the purpose of the present paper was to investigate if Gc may bind to human lymphocytes after immunoglobulin (Ig) capping. For this purpose the presence of Gc on B lymphocytes was examined by three different approaches. First, when cells were examined by immunofluorescence and quantified by flow cytometry, membrane Ig capping was followed by a dramatic decrease in positivity for Gc when compared to native cells. In addition, incubation of capped cells with purified Gc was followed by a significant increase in fluorescence, indicating that this protein had been able to bind again. Second, analysis of solubilized lymphocytes by Western blotting showed that native lymphocytes and capped cells incubated with purified Gc contained a large quantity of a 56kDa protein which was immunoreactive with anti Gc antibodies. This protein band was much weaker on blots from capped cells not treated with Gc. Third, radiobinding assays indicated that, following capping, cells were able to bind Gc in saturable fashion. These results suggest that membrane Gc could play a role in the entry of vitamin D metabolites into lymphocytes.

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 3 beta-hydroxyl group of vitamin D3 and its 25-hydroxy- and 1,25-dihydroxy metabolites with a 3'-aminopropylether group. In the present study we have coupled 25-hydroxyvitamin D3-3 beta-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.

The relation of the dual thyroxine/vitamin D-binding protein (TBP/DBP) of emydid turtles to vitamin D-binding proteins of other vertebrates.

The relation of a dual binding protein, involved in the transport of both thyroxine (T4) and vitamin D3, in the blood of emydid turtles to blood proteins in other vertebrates was examined immunologically. Binding studies with 25-OH-[3H]cholecalciferol (D3) confirmed the presence of a D3-binding protein (DBP) in the plasma of a wide variety of chelonian species representing both major suborders, as well as other species of reptiles, amphibians, birds, and mammals. Analysis by polyacrylamide gel electrophoresis (PAGE) showed that these binding proteins exhibited variable electrophoretic mobilities and in some species multiple DBPs were observed. Western blot analysis of these gels and additional tests with SDS-PAGE showed that an antiserum against the dual T4/D3-binding protein (TBP/DBP) from the turtle Trachemys scripta cross-reacted with the DBPs of diverse turtles, one crocodilian (Alligator), and birds; however, there was little or no cross-reaction with plasma from squamate reptiles (three snakes, four lizards), one crocodilian (Osteolaemus), amphibians (two anurans, one urodele), or mammals (six eutherians, one metatherian). The cross-reacting proteins (DBPs) all exhibit a similar M(r) (approximately 60 K). Adsorption of plasma by TBP/DBP-affinity chromatography confirmed this phylogenetic pattern of cross-reactions between the anti-TBP/DBP serum and functional DBPs in chicken, turtle, and alligator blood (affinity adsorption effectively eliminated D3 binding); there was only weak cross-reaction with DBP in a lizard and the second crocodilian (50% reduction in D3 binding) and none with DBP of snakes or mammals (D3 binding was unaffected). Results suggest that the plasma protein (TBP/DBP) that exhibits dual, high-affinity binding of T4 and D3 in one turtle family evolved from the more "primitive" vitamin D-binding protein of stem reptiles; thus, the high-affinity T4-binding site on this molecule is probably a derived characteristic of DBP in the Emydidae.

Regulation of inflammation-primed activation of macrophages by two serum factors, vitamin D3-binding protein and albumin.

A very small amount (0.0005 to 0.001%) of an ammonium sulfate [50% saturated (NH4)2SO4]-precipitable protein fraction of alpha 2-globulin efficiently supported inflammation-primed activation of macrophages. This fraction contains vitamin D3-binding protein essential for macrophage activation. Comparative macrophage activation studies with fetal calf serum, alpha 2-globulin fraction, 50% (NH4)2SO4 precipitate, and purified bovine vitamin D3-binding protein revealed that fetal calf serum and alpha 2-globulin fraction appear to contain an inhibitor for macrophage activation while ammonium sulfate precipitate contains no inhibitor. This inhibitor was found to be serum albumin. When bovine serum albumin (25 micrograms/ml) was added to a medium supplemented with 0.0005 to 0.05% (NH4)2SO4 precipitate or 1 to 10 ng of vitamin D3-binding protein per ml, activation of macrophages was inhibited.