Behind the scenes of vitamin D binding protein: more than vitamin D binding.

Although being discovered in 1959, the number of published papers in recent years reveals that vitamin D binding protein (DBP), a member of the albuminoid superfamily, is a hot research topic. Besides the three major phenotypes (DBP1F, DBP1S and DBP2), more than 120 unique variants have been described of this polymorphic protein. The presence of DBP has been demonstrated in different body fluids (serum, urine, breast milk, ascitic fluid, cerebrospinal fluid, saliva and seminal fluid) and organs (brain, heart, lungs, kidneys, placenta, spleen, testes and uterus). Although the major function is binding, solubilization and transport of vitamin D and its metabolites, the name of this glycoprotein hides numerous other important biological functions. In this review, we will focus on the analytical aspects of the determination of DBP and discuss in detail the multifunctional capacity [actin scavenging, binding of fatty acids, chemotaxis, binding of endotoxins, influence on T cell response and influence of vitamin D binding protein-macrophage activating factor (DBP-MAF) on bone metabolism and cancer] of this abundant plasma protein.

Molecular cloning and function characterization of a new macrophage-activating protein from Tremella fuciformis.

Silver ear mushroom ( Tremella fuciformis ) is an edible fungus with health benefits. In this study, we purified a new T. fuciformis protein (TFP) and demonstrated its ability to activate primary murine macrophages. The isolation procedure involved ammonium sulfate fractionation and ion exchange chromatography. TFP naturally formed a 24 kDa homodimeric protein and did not contain glycan residues. The TFP gene was cloned using the rapid amplification of cDNA ends method, and the cDNA sequence of TFP was composed of 408 nucleotides with a 336 nucleotide open reading frame encoding a 112 amino acid protein. TFP was capable of stimulating TNF-α, IL-1ß, IL-1ra, and IL-12 production in addition to CD86/MHC class II expression, mRNA expression of M1-type chemokines, and nuclear NF-κB accumulation in murine peritoneal macrophage cells. Furthermore, TFP failed to stimulate TLR4-neutralized and TLR4-knockout macrophages, suggesting that TLR4 is a required receptor for TFP signaling on macrophages. Taken together, these results indicate that TFP may be an important bioactive compound from T. fuciformis that induces M1-polarized activation through a TLR4-dependent NF-κB signaling pathway.

A novel role for a major component of the vitamin D axis: vitamin D binding protein-derived macrophage activating factor induces human breast cancer cell apoptosis through stimulation of macrophages.

The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH)(2)D3), its two binding proteins that are the vitamin D receptor (VDR) and the vitamin D-binding protein-derived macrophage activating factor (GcMAF). In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This allows 1,25(OH)(2)D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects.

Effects of body weight reduction on blood adipokines and subcutaneous adipose tissue adipokine mRNA expression profiles in obese ponies.

Fifteen obese ponies were used in a body weight (BW) reduction programme (BWRP, daily energy intake: 7.0-8.4 MJ/100 kg BW). A frequently sampled intravenous glucose tolerance test was used to assess insulin sensitivity. Subcutaneous adipose tissue biopsies of the tail head were obtained for mRNA gene expression profiles of adiponectin, retinol-binding protein 4 (RBP4), interleukin 6 (IL-6) and macrophage activation marker (CD68) before and after BWRP. Blood samples were analysed for serum leptin, serum RBP4 and plasma adiponectin. Significant BW losses occurred with 7 MJ DE/100 kg BW. Serum leptin and RBP4 were initially similar between insulin-resistant (IR) and insulin-sensitive (IS) ponies, and both significantly decreased during BWRP. Compared with IS ponies, IR ponies initially had significantly lower plasma adiponectin levels. At the beginning of BWRP, mRNA expression of RBP4, adiponectin, IL-6 and CD68 was similar between IR and IS ponies. Plasma adiponectin was strongly related to IR, whereas serum leptin and RBP4 were closely linked to adiposity, independent of insulin sensitivity. Adipose tissue mRNA expression profiles did not clearly reflect these differences. However, the role of subcutaneous adipose tissue in IR remains open.

The glycosylation and characterization of the candidate Gc macrophage activating factor.

The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF candidate was tested for its effect on cytokine release from macrophages in human whole blood.

Characterization of oncogene dysregulation in multiple myeloma by combined FISH and DNA microarray analyses.

Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus and various partner loci frequently are associated with multiple myeloma (MM). We investigated the expression profiles of the FGFR3/MMSET, CCND1, CCND3, MAF, and MAFB genes, which are involved in t(4;14)(p16.3;q32), t(11;14)(q13;q32), t(6;14)(p21;q32), t(14;16)(q32;q23), and t(14;20)(q32;q12), respectively, in purified plasma cell populations from 39 MMs and six plasma cell leukemias (PCL) by DNA microarray analysis and compared the results with the presence of translocations as assessed by dual-color FISH or RT-PCR. A t(4;14) was found in 6 MMs, t(11;14) in 9 MMs and 1 PCL, t(6;14) in 1 MM, t(14;16) in 2 MMs and 1 PCL, and t(14;20) in 1 PCL. In all cases, the translocations were associated with the spiked expression of target genes. Furthermore, gene expression profiling enabled the identification of putative translocations causing dysregulation of CCND1 (1 MM and 1 PCL) and MAFB (1 MM and 1 PCL) without any apparent involvement of immunoglobulin loci. Notably, all of the translocations were mutually exclusive. Markedly increased MMSET expression was found in 1 MM showing associated FGFR3 and MMSET signals on an unidentified chromosome. Our data suggest the importance of using combined molecular cytogenetic and gene expression approaches to detect genetic aberrations in MM.

A gene expression signature for oxidant stress/reactive metabolites in rat liver.

Formation of free radicals and other reactive molecules is responsible for the adverse effects produced by a number of hepatotoxic compounds. cDNA microarray technology was used to compare transcriptional profiles elicited by training and testing sets of 15 oxidant stressors/reactive metabolite treatments to those produced by approximately 85 other paradigm compounds (mostly hepatotoxicants) to determine a shared signature profile for oxidant stress-associated hepatotoxicity. Initially, 100 genes were chosen that responded significantly different to oxidant stressors/reactive metabolites (OS/RM) compared to other samples in the database, then a 25-gene subset was selected by multivariate analysis. Many of the selected genes (e.g., aflatoxin aldehyde reductase, diaphorase, epoxide hydrolase, heme oxgenase and several glutathione transferases) are well-characterized oxidant stress/Nrf-2-responsive genes. Less than 10 other compounds co-cluster with our training and testing set compounds and these are known to generate OS/RMs as part of their mechanisms of toxicity. Using OS/RM signature gene sets, compounds previously associated with macrophage activation formed a distinct cluster separate from OS/RM and other compounds. A 69-gene set was chosen to maximally separate compounds in control, macrophage activator, peroxisome proliferator and OS/RM classes. The ease with which these 'oxidative stressor' classes can be separated indicates a role for microarray technology in early prediction and classification of hepatotoxicants. The ability to rapidly screen the oxidant stress potential of compounds may aid in avoidance of some idiosyncratic drug reactions as well as overtly toxic compounds.

Identification of macrophage/microglia activation factor (MAF) associated with late endosomes/lysosomes in microglial cells.

Damage to the central nervous system triggers rapid activation and specific migration of glial cells towards the lesion site. There, glial cells contribute heavily to secondary neuronal changes that take place after lesion. In an attempt to identify the molecular cues of glial activation following brain trauma we performed differential display reverse transcription-polymerase chain reaction screenings from lesioned and control hippocampus. Here we report on the identification of the macrophage/microglia activation factor (MAF), a new membrane protein with seven putative transmembrane domains. Expression analysis revealed that MAF is predominantly expressed in microglial cells in the brain, and is upregulated following brain lesion. Overexpression of MAF in non-glial cells shows an intracellular codistribution with the lysosomal marker endosome/lysosome-associated membrane protein-1 (lamp-1). Furthermore, MAF-transfected cells show that MAF is primarily associated with late endosomes/lysosomes, and that this association can be disrupted by activation of protein kinase C-dependent pathways. In conclusion, these results imply that MAF is involved in the dynamics of lysosomal membranes associated with microglial activation following brain lesion.

Homologue of macrophage-activating lipoprotein in Mycoplasma gallisepticum is not essential for growth and pathogenicity in tracheal organ cultures.

While the genomes of a number of Mycoplasma species have been fully determined, there has been limited characterization of which genes are essential. The surface protein (p47) identified by monoclonal antibody B3 is the basis for an enzyme-linked immunosorbent assay for serological detection of Mycoplasma gallisepticum infection and appears to be constitutively expressed. Its gene was cloned, and the DNA sequence was determined. Subsequent analysis of the p47 amino acid sequence and searches of DNA databases found homologous gene sequences in the genomes of M. pneumoniae and M. genitalium and identity with a gene family in Ureaplasma urealyticum and genes in M. agalactiae and M. fermentans. The proteins encoded by these genes were found to belong to a family of basic membrane proteins (BMP) that are found in a wide range of bacteria, including a number of pathogens. Several of the BMP family members, including p47, contain selective lipoprotein-associated motifs that are found in macrophage-activating lipoprotein 404 of M. fermentans and lipoprotein P48 of M. agalactiae. The p47 gene was predicted to encode a 59-kDa peptide, but affinity-purified p47 had a molecular mass of approximately 47 kDa, as determined by polyacrylamide gel analysis. Analysis of native and recombinant p47 by mass peptide fingerprinting revealed the absence of the carboxyl end of the protein encoded by the p47 gene in native p47, which would account for the difference seen in the predicted and measured molecular weights and indicated posttranslational cleavage of the lipoprotein at its carboxyl end. A DNA construct containing the p47 gene interrupted by the gene encoding tetracycline resistance was used to transform M. gallisepticum cells. A tetracycline-resistant mycoplasma clone, P2, contained the construct inserted within the genomic p47 gene, with crossovers occurring between 73 bp upstream and 304 bp downstream of the inserted tetracycline resistance gene. The absence of p47 protein in clone P2 was determined by the lack of reactivity with rabbit anti-p47 sera or monoclonal antibody B3 in Western blots of whole-cell proteins. There was no difference between the p47(-) mutant and wild-type M. gallisepticum in pathogenicity in chicken tracheal organ cultures. Thus, p47, although homologous to genes that occur in many prokaryotes, is not essential for growth in vitro or for attachment and the initial stages of pathogenesis in chickens.

Baculovirus-expressed vitamin D-binding protein-macrophage activating factor (DBP-maf) activates osteoclasts and binding of 25-hydroxyvitamin D(3) does not influence this activity.

Vitamin D-binding protein (DBP) is a multi-functional serum protein that is converted to vitamin D-binding protein-macrophage activating factor (DBP-maf) by post-translational modification. DBP-maf is a new cytokine that mediates bone resorption by activating osteoclasts, which are responsible for resorption of bone. Defective osteoclast activation leads to disorders like osteopetrosis, characterized by excessive accumulation of bone mass. Previous studies demonstrated that two nonallelic mutations in the rat with osteopetrosis have independent defects in the cascade involved in the conversion of DBP to DBP-maf. The skeletal defects associated with osteopetrosis are corrected in these mutants with in vivo DBP-maf treatment. This study evaluates the effects of various forms of DBP-maf (native, recombinant, and 25-hydroxyvitamin D(3) bound) on osteoclast function in vitro in order to determine some of the structural requirements of this protein that relate to bone resorbing activities. Osteoclast activity was determined by evaluating pit formation using osteoclasts, isolated from the long bones of newborn rats, incubated on calcium phosphate coated, thin film, Ostologic MultiTest Slides. Incubation of osteoclasts with ex vivo generated native DBP-maf resulted in a dose dependent, statistically significant, activation of the osteoclasts. The activation was similar whether or not the vitamin D binding site of the DBP-maf was occupied. The level of activity in response to DBP-maf was greater than that elicited by optimal doses of other known stimulators (PTH and 1,25(OH(2)D(3)) of osteoclast function. Furthermore, another potent macrophage activating factor, interferon--gamma, had no effect on osteoclast activity. The activated form of a full length recombinant DBP, expressed in E. coli showed no activity in the in vitro assay. Contrary to this finding, baculovirus-expressed recombinant DBP-maf demonstrated significant osteoclast activating activity. The normal conversion of DBP to DBP-maf requires the selective removal of galactose and sialic acid from the third domain of the protein. Hence, the differential effects of the two recombinant forms of DBP-maf is most likely related to glycosylation; E. coli expressed recombinant DBP is non-glycosylated, whereas the baculovirus expressed form is glycosylated. These data support the essential role of glycosylation for the osteoclast activating property of DBP-maf.

ImmTher, a lipophilic disaccharide derivative of muramyl dipeptide, up-regulates specific monocyte cytokine genes and activates monocyte-mediated tumoricidal activity.

ImmTher, a liposome-encapsulated lipophilic disaccharide tripeptide derivative of muramyl dipeptide, previously showed activity against liver and lung colorectal metastases in a phase I trial. The purpose of the current studies was to investigate whether ImmTher could up-regulate specific cytokine gene expression and protein production, as well as activate the tumoricidal or cytostatic activity of human monocytes. ImmTher induced the expression and production of interleukin(IL)-1alpha IL-1beta, IL-6, IL-8, IL-12, macrophage chemotactic and activating factor, and tumor necrosis factor alpha but not IL-2 or IL-10. Cytostatic or cytotoxic monocyte activity was stimulated against human Ewing's sarcoma, osteosarcoma, and melanoma cells but not breast cancer cells. Production and secretion of these cytokine proteins may play a role in the antitumor activity of ImmTher.

Immunotherapy of BALB/c mice bearing Ehrlich ascites tumor with vitamin D-binding protein-derived macrophage activating factor.

Vitamin D3-binding protein (DBP; human DBP is known as Gc protein) is the precursor of macrophage activating factor (MAF). Treatment of mouse DBP with immobilized beta-galactosidase or treatment of human Gc protein with immobilized beta-galactosidase and sialidase generated a remarkably potent MAF, termed DBPMAF or GcMAF, respectively. The domain of Gc protein responsible for macrophage activation was cloned and enzymatically converted to the cloned MAF, designated CdMAF. In Ehrlich ascites tumor-bearing mice, tumor-specific serum alpha-N-acetylgalactosaminidase (NaGalase) activity increased linearly with time as the transplanted tumor cells grew in the peritoneal cavity. Therapeutic effects of DBPMAF, GcMAF, and CdMAF on mice bearing Ehrlich ascites tumor were assessed by survival time, the total tumor cell count in the peritoneal cavity, and serum NaGalase activity. Mice that received a single administration of DBPMAF or GcMAF (100 pg/mouse) on the same day after transplantation of tumor (1 x 10(5) cells) showed a mean survival time of 35 +/- 4 days, whereas tumor-bearing controls had a mean survival time of 16 +/- 2 days. When mice received the second DBPMAF or GcMAF administration at day 4, they survived more than 50 days. Mice that received two DBPMAF administrations, at days 4 and 8 after transplantation of 1 x 10(5) tumor cells, survived up to 32 +/- 4 days. At day 4 posttransplantation, the total tumor cell count in the peritoneal cavity was approximately 5 x 10(5) cells. Mice that received two DBPMAF administrations, at days 0 and 4 after transplantation of 5 x 10(5) tumor cells, also survived up to 32 +/- 4 days, while control mice that received the 5 x 10(5) ascites tumor cells only survived for 14 +/- 2 days. Four DBPMAF, GcMAF, or CdMAF administrations to mice transplanted with 5 x 10(5) Ehrlich ascites tumor cells with 4-day intervals showed an extended survival of at least 90 days and an insignificantly low serum NaGalase level between days 30 and 90.