Structure and function of the natural-resistance-associated macrophage protein (Nramp1), a candidate protein for infectious and autoimmune disease susceptibility

The ability of macrophages to become activated is central to antimicrobial immunity. Microbial stimuli can elicit a cascade of gene-inductive events mediating inflammation, elimination of the invading organism and induction of T-cell memory aganst reinvasion. Nramp1, a gene originally identified as Ity/Lsh/Bcg for its role in controlling Salmonella typhimurium, Leishmania donovani and Mycobacterium bovis infections in mice, regulates this cascade. Here we examine how the structure of the Nramp1 protein might relate to its function, and how variable expression of the human homologue (NRAMP1) might mediate enhanced resistance to infection but cause susceptibility to autoimmune disease.     

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.     

Functional Consequences of the Macrophage Stimulating Protein 689C Inflammatory Bowel Disease Risk Allele

Background

Macrophage stimulating protein (MSP) is a serum growth factor that binds to and activates the receptor tyrosine kinase, Recepteur d''Origine Nantais (RON). A non-synonymous coding variant in MSP (689C) has been associated with genetic susceptibility to both Crohn''s disease and ulcerative colitis, two major types of inflammatory bowel disease (IBD) characterized by chronic inflammation of the digestive tract. We investigated the consequences of this polymorphism for MSP-RON pathway activity and IBD pathogenesis.

Methods

RON expression patterns were examined on mouse and human cells and tissues under normal and disease conditions to identify cell types regulated by MSP-RON. Recombinant MSP variants were tested for their ability to bind and stimulate RON and undergo proteolytic activation. MSP concentrations were quantified in the serum of individuals carrying the MSP 689R and 689C alleles.

Results

In intestinal tissue, RON was primarily expressed by epithelial cells under normal and disease conditions. The 689C polymorphism had no impact on the ability of MSP to bind to or signal through RON. In a cohort of normal individuals and IBD patients, carriers of the 689C polymorphism had lower concentrations of MSP in their serum.

Conclusions

By reducing the quantities of circulating MSP, the 689C polymorphism, or a variant in linkage disequilibrium with this polymorphism, may impact RON ligand availability and thus receptor activity. Given the known functions of RON in regulating wound healing and our analysis of RON expression patterns in human intestinal tissue, these data suggest that decreased RON activity may impact the efficiency of epithelial repair and thus underlie the increased IBD susceptibility associated with the MSP 689C allele.     

Engineering superactive granulocyte macrophage colony‐stimulating factor transferrin fusion proteins as orally‐delivered candidate agents for treating neurodegenerative disease

Intravenously injected granulocyte macrophage colony‐stimulating factor (GM‐CSF) has shown efficacy in Alzheimer's Disease (AD) and Parkinson's Disease (PD) animal studies and is undergoing clinical evaluation. The likely need for dosing of GM‐CSF to patients over months or years motivates pursuit of avenues for delivering GM‐CSF to circulation via oral administration. Flow cytometric screening of 37 yeast‐displayed GM‐CSF saturation mutant libraries revealed residues P12, H15, R23, R24, and K72 as key determinants of GM‐CSF's CD116 and CD131 GM‐CSF receptor (GM‐CSFR) subunit binding affinity. Screening combinatorial GM‐CSF libraries mutated at positions P12, H15, and R23 yielded variants with increased affinities toward both CD116 and CD131. Genetic fusion of GM‐CSF to human transferrin (Trf), a strategy that enables oral delivery of other biopharmaceuticals in animals, yielded bioactive wild type and variant cytokines upon secretion from cultured Human Embryonic Kidney cells. Surface plasmon resonance (SPR) measurements showed that all evaluated variants possess decreases in CD116 and CD131 binding K_D values of up to 2.5‐fold relative to wild type. Improved affinity led to increased in vitro bioactivity; the most bioactive variant, P12D/H15L/R23L, had a leukocyte proliferation assay EC₅₀ value 3.5‐fold lower than the wild type GM‐CSF/Trf fusion. These outcomes are important first steps toward our goal of developing GM‐CSF/Trf fusions as orally available AD and PD therapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:668–677, 2015     

Protein kinase C levels and protein phosphorylation associated with inhibition of proliferation in a murine macrophage tumor

Treatment of M5076 tumor cells with the phorbol estes 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13 dibutyrate (PdBu) inhibited cellular proliferation, whereas 1,2-dioctanoyl-glycerol (DiC8) and 1-oleoyl2-acetyl-glycerol (OAG) did not affect cell growth. Inhibition of cellular proliferation in this cell line appears to be a consequence of protein kinase C (PKC) down-regulation since phorbol esters, but not a single application of diacylglycerols (DGs) down-regulated cellular PKC levels. By repeated application of DGs, PKC down-regulation was achieved and correlated with inhibition of proliferation. Phorbol ester-induced PKC down-regulation was reversible, upon removal of the phorbol ester, and the reappearance of PKC was associated with resumption of proliferation. The mitogenic responsiveness of these cells to added serum depended upon cellular PKC levels. Phorbol esters also caused the phosphorylation of two proteins which were not phosphorylated in response to DG treatment. Inhibition of growth of M5076 cells appears to be associated with phosphorylation of two novel proteins and/or PKC down-regulation.     

The regulation of macrophage protein turnover by a colony stimulating factor (CSF-1)

CSF-1 is a hemopoietic growth factor that specifically regulates the survival, proliferation, and differentiation of mononuclear phagocytic cells. A homogeneous population of mononuclear phagocytes, bone marrow derived macrophages (BMM), were used to study the regulation of protein turnover by CSF-1. Removal of CSF-1 (approximately 0.4 nM) from exponentially growing BMM cultured in 15% fetal calf serum containing medium decreases the rate of DNA synthesis by more than 100-fold. Addition of CSF-1 to these cells causes them to resume DNA synthesis within 12 h. More immediate effects of CSF-1 were observed on BMM protein metabolism. BMM cultured for 24 h in the absence of CSF-1 reduce their protein synthetic rate by 50-60%. The protein synthetic rate commences to decrease at 2-3 h after CSF-1 removal. Readdition of CSF-1 to BMM previously incubated in its absence causes a return to the protein synthetic rate of exponentially growing cells within 2 h. In the presence of CSF-1, BMM synthesize protein at a rate of approximately 8.7%/h and degrade it at a rate of approximately 0.9%/h. Removal of CSF-1 results in a decrease in the protein synthetic rate to approximately 3.4%/h and an increase in the rate of protein degradation to approximately 3.4%/h. The rate of protein synthesis by BMM increases linearly with CSF-1 concentration over the range of concentrations stimulating both survival and proliferation, while the rate of protein degradation decreases exponentially over the range of concentrations stimulating survival without proliferation. Therefore, it appears that the stimulation of the rate of protein synthesis and inhibition of the rate of protein degradation are two distinct effects of CSF-1, both part of the pleiotropic response to this growth factor. The inhibition of the rate of protein degradation by CSF-1 may be most significant for its survival inducing effect.     

Crystal structure of the beta-chain of human hepatocyte growth factor-like/macrophage stimulating protein

Hepatocyte growth factor like/macrophage stimulating protein (HGFl/MSP) and hepatocyte growth factor/scatter factor (HGF/SF) define a distinct family of vertebrate-specific growth factors structurally related to the blood proteinase precursor plasminogen and with important roles in development and cancer. Although the two proteins share a similar domain structure and mechanism of activation, there are differences between HGFl/MSP and HGF/SF in terms of the contribution of individual domains to receptor binding. Here we present a crystal structure of the 30 kDa beta-chain of human HGFl/MSP, a serine proteinase homology domain containing the high-affinity binding site for the RON receptor. The structure describes at 1.85 Angstrom resolution the region of the domain corresponding to the receptor binding site recently defined in the HGF/SF beta-chain, namely the central cleft harboring the three residues corresponding to the catalytic ones of active proteinases (numbers in brackets define the sequence position according to the standard chymotrypsinogen numbering system) [Gln522 (c57), Gln568 (c102) and Tyr661 (c195)] and an adjacent loop flanking the S1 specificity pocket and containing residues Asn682 (c217) and Arg683 (c218) previously shown to be essential for binding of HGFl/MSP to the RON receptor. The study confirms the concept that the serine proteinase homology domains of HGFl/MSP and HGF/SF bind their receptors in an 'enzyme-substrate' mode, reflecting the common evolutionary origin of the plasminogen-related growth factors and the proteinases of the clotting and fibrinolytic pathways. However, analysis of the intermolecular interactions in the crystal lattice of beta-chain HGFl/MSP fails to show the same contacts seen in the HGF/SF structures and does not support a conserved mode of dimerization of the serine proteinase homology domains of HGFl/MSP and HGF/SF responsible for receptor activation.     

SNP cherry picker: maximizing the chance of finding an association with a disease SNP

SUMMARY: The high cost of genotyping single nucleotide polymorphisms (SNPs) generally prohibits the systematic mapping of entire genetic linkage regions in order to find the polymorphisms associated with increased risk of disease. In practice, SNPs are selected at approximately equal spacing across the linkage region to try to locate a SNP lying in the haplotype block of the disease SNP. The size of the haplotype block may not be known, however, and SNPs taken from public domain sources may not in fact be polymorphic. Our program will choose a subset of the SNPs in a linkage region so as to maximize the expected proportion of the sequence that lies within a given distance of a real SNP. AVAILABILITY: The software is available, free of charge, for academic use on request from the authors. SUPPLEMENTARY INFORMATION: www.oxagen.co.uk     

Cystatin C: a candidate biomarker for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurologic disease characterized by progressive motor neuron degeneration. Clinical disease management is hindered by both a lengthy diagnostic process and the absence of effective treatments. Reliable panels of diagnostic, surrogate, and prognostic biomarkers are needed to accelerate disease diagnosis and expedite drug development. The cysteine protease inhibitor cystatin C has recently gained interest as a candidate diagnostic biomarker for ALS, but further studies are required to fully characterize its biomarker utility. We used quantitative enzyme-linked immunosorbent assay (ELISA) to assess initial and longitudinal cerebrospinal fluid (CSF) and plasma cystatin C levels in 104 ALS patients and controls. Cystatin C levels in ALS patients were significantly elevated in plasma and reduced in CSF compared to healthy controls, but did not differ significantly from neurologic disease controls. In addition, the direction of longitudinal change in CSF cystatin C levels correlated to the rate of ALS disease progression, and initial CSF cystatin C levels were predictive of patient survival, suggesting that cystatin C may function as a surrogate marker of disease progression and survival. These data verify prior results for reduced cystatin C levels in the CSF of ALS patients, identify increased cystatin C levels in the plasma of ALS patients, and reveal correlations between CSF cystatin C levels to both ALS disease progression and patient survival.     

Purification and characterization of a ubiquitin-like peptide with macrophage stimulating,antiproliferative and ribonuclease activities from the mushroom Agrocybe cylindracea

A peptide, with a molecular mass of 9.5kDa and demonstrating an N-terminal sequence similar to ubiquitin, was isolated from fruiting bodies of the mushroom Agrocybe cylindracea. The peptide was isolated with a purification protocol involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, FPLC-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex 75. The peptide was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and Mono S. It showed antiproliferative activity on leukemia cell line (M1) and hepatoma cell line (HepG2), and enhanced nitric oxide production in murine peritoneal macrophages with a potency comparable to that of lipopolysaccharide. A pH of 6.0 was required for optimal RNase activity. Its RNase activity was stable over the temperature range of 0-60 degrees C. It exerted ribonucleolytic activity preferentially on polyC, much lower activity on polyU, and negligible activity on polyA and polyG.     

Molecular identification and characterization of a protein lymphokine exhibiting macrophage migration inhibition activity

Lymphocytes activated specifically with antigen or nonspecifically with lectins produce lymphokines, which modulate the immune response. Few lymphokines have been identified at the molecular level or purified to homogeneity. These studies describe our procedures to identify a protein responsible for macrophage migration inhibition activity (MIF) produced by concanavalin A-stimulated murine spleen cell cultures. MIF-active material was adsorbed onto insolubilized hog gastric mucin and specifically eluted with a solution of d-glucose and l-fucose. This eluate, derived from a stimulated leukocyte culture supernatant which exhibited molecular weight heterogeneity, displayed two zones of MIF activity when subjected to polyacrylamide gel electrophoresis. The MIF activity in the zone of slower mobility with an R_f equal to 1.2 times that of horse heart myoglobin, was obtained by preparative electrophoresis as a single radioactive labeled component. Electrophoresis in the presence of sodium dodecyl sulfate demonstrated that this component is composed of a single polypeptide of 21,000 daltons.     

A partial characterization of a Sertoli cell-secreted protein stimulating Leydig cell testosterone production.

To examine whether immature rat Sertoli cells in culture secrete a factor(s) which stimulates testosterone production by mature mouse Leydig cells, Sertoli cell-enriched cultures were prepared from 3-week-old male rats with trypsin and collagenase. Sertoli cells were plated at an initial density of 3-5 x 10(6) cells/35 mm well and cultured in 3 ml serum free media supplemented with insulin (10 micrograms/ml). Sertoli cell culture medium (SCCM) collected every 3rd day was added to Leydig cells (10(6) cells in 1 ml of MEM with 2% steroid-free FCS) prepared from 10-week-old mice by mechanical separation and incubated for 3 h at 34 degrees C. Secreted testosterone was determined by RIA. SCCM 15 times concentrated by Amicon YM10 membrane demonstrated a dose-dependent stimulation of testosterone production, whereas there was no effect on testosterone secretion when Leydig cells were maximally stimulated by LH. Leydig cell stimulating activity was retained by both a dialysis membrane with a pore size of 24 A and an ultrafiltration membrane with a molecular weight cut-off of 10 kDa. However, activity was reduced by heating at 60 degrees C for 30 min and almost lost after incubation with 0.1% trypsin for 1 h at 37 degrees C. This activity was not retained by means of a Con A-Agarose column and was demonstrated only in break-through fractions. HPLC gel filtration of a 15 times concentrated SCCM preparation on a TSK gel G3000SW revealed Leydig cell-stimulating activity at approximately 13 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity-dependent neuroprotector homeobox protein: A candidate protein identified in serum as diagnostic biomarker for Alzheimer's disease

Alzheimer's disease (AD) is the most common cause of dementia of late life. To enhance our understanding of AD proteome, the serum proteins were analyzed using two-dimensional gel electrophoresis (2DE) combined with nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (nano-HPLC-ESI-MS/MS) followed by peptide fragmentation patterning. In this study, six protein spots with differential expression were identified. Five up-regulated proteins were identified as actin, apolipoprotein A-IV (Apo A-IV), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-1-antitrypsin (AAT), and antithrombin-III (AT-III); one protein, activity-dependent neuroprotector homeobox protein (ADNP) was down-regulated in AD patients. These proteins with differential expression in the serum may serve as potential indicators of AD. Our results suggested that ADNP may play an important role in slowing the progression of clinical symptoms of AD.     

alpha 1-Antichymotrypsin is the human plasma inhibitor of macrophage ectoenzymes that cleave pro-macrophage stimulating protein

Macrophage stimulating protein (MSP) is secreted as 78-kDa single chain pro-MSP, which is converted to biologically active, disulfide-linked alphabeta chain MSP by cleavage at Arg(483)-Val(484). Murine resident peritoneal macrophages have two cell surface proteolytic activities that cleave pro-MSP. One is a pro-MSP convertase, which cleaves pro-MSP to active MSP; the other degrades pro-MSP. The degrading protease is inhibited by soybean trypsin inhibitor or by low concentrations of blood plasma, which allows the convertase to cleave pro-MSP to MSP. Using pro-MSP cleavage as the assay, we purified the inhibitor from human plasma. The bulk of the plasma protein was removed by salting out and by isoelectric precipitation of albumin. Highly purified inhibitor was then obtained in three steps: dye-ligand binding and elution, ion exchange chromatography, and high performance liquid chromatography gel filtration. After SDS-polyacrylamide gel electrophoresis and transfer to a polyvinylidene membrane, N-terminal sequencing of the product identified it as alpha(1)-antichymotrypsin. The mean concentration of alpha(1)-antichymotrypsin in human plasma is 7 micrometer. At this concentration, alpha(1)-antichymotrypsin inhibits both macrophage enzymes. A concentration of 0.4 micrometer, which is in the expected concentration range in extracellular fluid, preferentially inhibits the degrading enzyme, which allows for cleavage to active MSP by the pro-MSP convertase.     

Linkage relationships of the protein kinase C gamma gene which exclude it as a candidate for myotonic dystrophy

Using a cDNA probe for the gamma gene of protein kinase C (PKCG), an informative RFLP with a PIC value of 0.62 has been identified with the enzyme MspI. The polymorphic bands have been assigned to chromosome 19. Analysis of the segregation of alleles for this probe in myotonic dystrophy families show several recombinants between PKCG and myotonic dystrophy (DM) and exclude this gene as a candidate for DM. Linkage relationships between PKCG and other loci on chromosome 19 are presented which exclude PKCG from the proximal region of chromosome 19 and which are consistent with the localization being at 19q13.2----qter.     

Identification and characterization of C6orf37, a novel candidate human retinal disease gene on chromosome 6q14

We have identified a novel human gene, chromosome 6 open reading frame 37 (C6orf37), that is expressed in the retina and maps to human chromosome 6q14, a genomic region that harbors multiple retinal disease loci. The cDNA sequence contains an open reading frame of 1314 bp that encodes a 437-amino acid protein with a predicted molecular mass of 49.2 kDa. Northern blot analysis indicates that this gene is widely expressed, with preferential expression observed in the retina compared to other ocular tissues. The C6orf37 protein shares homology with putative proteins in R. norvegicus, M. musculus, D. melanogaster, and C. elegans, suggesting evolutionary conservation of function. Additional sequence analysis predicts that the C6orf37 gene product is a soluble, globular cytoplasmic protein containing several conserved phosphorylation sites. Furthermore, we have defined the genomic structure of this gene, which will enable its analysis as a candidate gene for chromosome 6q-associated inherited retinal disorders.     

The molecular mechanism of acylation stimulating protein regulation of adipophilin and perilipin expression: involvement of phosphoinositide 3-kinase and phospholipase C

The novel adipokine acylation stimulating protein (ASP) is involved in lipid metabolism and obesity‐related disorders. Adipophilin and perilipin, two members of the lipid droplet protein family, participate not only in fat storage within adipocytes, but also in ectopic lipid deposition in the form of cytoplasmic triglyceride (TG) droplets within many types of mammalian cells. During differentiation to mature adipocytes, mechanisms controlling the synthesis and turnover of these lipid droplet proteins are only partially understood, the mechanisms regulating gene/protein expression as yet unidentified. In our previous study, ASP has been shown to regulate adipophilin and perilipin expression to facilitate TG synthesis during 3T3‐L1 cell differentiation. Our aim in this study was to provide insight into the physiological importance of phosphoinositide 3‐kinase (PI3K) and phospholipase C (PLC) in ASP‐triggered alteration of adipophilin and perilipin expression. We found that acute (2.5 h) inhibition of PLC or PI3K results in a decrease in mRNA and protein of perilipin and adipophilin at any time during differentiation. The fact that there is such a rapid change even with mRNA levels suggests a rapid turnover of both mRNA and protein independent of a direct ASP effect. Also, the presence of these inhibitors blocked the ASP stimulatory effects with a maximal decrease in gene and protein expression of adipophilin (?45% and ?60%, respectively, P < 0.01) and perilipin (?96% and ?63%, respectively, P < 0.01 and P < 0.05). These findings provide further understanding of the adipogenic properties of ASP in adipocytes. J. Cell. Biochem. 112: 1622–1629, 2011. © 2011 Wiley‐Liss, Inc.