A Novel Alkaline Phosphatase/Phosphodiesterase, CamPhoD, from Marine Bacterium Cobetia amphilecti KMM 296.

A novel extracellular alkaline phosphatase/phosphodiesterase from the structural protein family PhoD that encoded by the genome sequence of the marine bacterium Cobetia amphilecti KMM 296 (CamPhoD) has been expressed in Escherichia coli cells. The calculated molecular weight, the number of amino acids, and the isoelectric point (pI) of the mature protein's subunit are equal to 54832.98 Da, 492, and 5.08, respectively. The salt-tolerant, bimetal-dependent enzyme CamPhoD has a molecular weight of approximately 110 kDa in its native state. CamPhoD is activated by Co2+, Mg2+, Ca2+, or Fe3+ at a concentration of 2 mM and exhibits maximum activity in the presence of both Co2+ and Fe3+ ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn2+, Cu2+, and Mn2+, as well as chelating agents EDTA and EGTA, do not have an appreciable effect on the CamPhoD activity. The temperature optimum for the CamPhoD activity is 45 °C. The enzyme catalyzes the cleavage of phosphate mono- and diester bonds in nucleotides, releasing inorganic phosphorus from p-nitrophenyl phosphate (pNPP) and guanosine 5'-triphosphate (GTP), as determined by the Chen method, with rate approximately 150- and 250-fold higher than those of bis-pNPP and 5'-pNP-TMP, respectively. The Michaelis-Menten constant (Km), Vmax, and efficiency (kcat/Km) of CamPhoD were 4.2 mM, 0.203 mM/min, and 7988.6 S-1/mM; and 6.71 mM, 0.023 mM/min, and 1133.0 S-1/mM for pNPP and bis-pNPP as the chromogenic substrates, respectively. Among the 3D structures currently available, in this study we found only the low identical structure of the Bacillus subtilis enzyme as a homologous template for modeling CamPhoD, with a new architecture of the phosphatase active site containing Fe3+ and two Ca2+ ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family.

Synthesis and Biological Potential Assessment of 2-Substituted Quinazolin-4(3H)-ones as Inhibitors of Phosphodiesterase-I and Carbonic Anhydrase-II.

A library of twenty-five derivatives of 2-substituted quinazolin-4(3H)-ones 1-25 was synthesized and evaluated against phosphodiesterase-I (PDE) and carbonic anhydrase-II (CA). Compounds 17 (IC50 = 210.7 ± 2.62 µM), 16 (IC50 = 301.6 ± 1.18 µM), and 13 (IC50 = 458.13 ± 3.60 µM), selectively exhibited PDE inhibition while compounds 22 (IC50 = 61.33 ± 2.38 µM), 1 (IC50 = 108.30 ± 0.93 µM), and 21 (IC50 = 191.93 ± 2.72 µM), discriminatingly exhibited CA inhibition as compared to standards EDTA (IC50 = 277.69 ± 2.52 µM) and acetazolamide (IC50 = 0.12 ± 0.03 µM), for PDE and CA inhibitions, respectively. However, compound 15 was found to be active against both enzymes with the IC50 values 344.33 ± 4.32 µM and 20.94 ± 0.58 µM, for PDE and CA inhibitions, respectively. Remaining compounds were found to be inactive against both the enzymes. Structure-activity relationship studies are discussed herein.

Phosphoproteomic approach to characterize protein mono- and poly(ADP-ribosyl)ation sites from cells.

Poly(ADP-ribose), or PAR, is a cellular polymer implicated in DNA/RNA metabolism, cell death, and cellular stress response via its role as a post-translational modification, signaling molecule, and scaffolding element. PAR is synthesized by a family of proteins known as poly(ADP-ribose) polymerases, or PARPs, which attach PAR polymers to various amino acids of substrate proteins. The nature of these polymers (large, charged, heterogeneous, base-labile) has made these attachment sites difficult to study by mass spectrometry. Here we propose a new pipeline that allows for the identification of mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation sites via the enzymatic product of phosphodiesterase-treated ADP-ribose, or phospho(ribose). The power of this method lies in the enrichment potential of phospho(ribose), which we show to be enriched by phosphoproteomic techniques when a neutral buffer, which allows for retention of the base-labile attachment site, is used for elution. Through the identification of PARP-1 in vitro automodification sites as well as endogenous ADP-ribosylation sites from whole cells, we have shown that ADP-ribose can exist on adjacent amino acid residues as well as both lysine and arginine in addition to known acidic modification sites. The universality of this technique has allowed us to show that enrichment of ADP-ribosylated proteins by macrodomain leads to a bias against ADP-ribose modifications conjugated to glutamic acids, suggesting that the macrodomain is either removing or selecting against these distinct protein attachments. Ultimately, the enrichment pipeline presented here offers a universal approach for characterizing the mono- and poly(ADP-ribosyl)ated proteome.

Purification and characterization of phosphodiesterase i from Walterinnesia aegyptia venom.

A phosphodiesterase I (EC 3.1.4.1; PDE-I) was purified from Walterinnesia aegyptia venom by preparative native polyacrylamide gel electrophoresis (PAGE). A single protein band was observed in analytical native PAGE and sodium dodecyl sulfate (SDS)-PAGE. PDE-I was a single-chain glycoprotein with an estimated molecular mass of 158 kD (SDS-PAGE). The enzyme was free of 5'-nucleotidase and alkaline phosphatase activities. The optimum pH and temperature were 9.0 and 60°C, respectively. The energy of activation (Ea) was 96.4, the V(max) and K(m) were 1.14 µM/min/mg and 1.9 × 10(-3) M, respectively, and the K(cat) and K(sp) were 7 s(-1) and 60 M(-1) min(-1) respectively. Cysteine was a noncompetitive inhibitor, with K(i) = 6.2 × 10(-3) M and an IC(50) of 2.6 mM, whereas adenosine diphosphate was a competitive inhibitor, with K(i) = 0.8 × 10(-3) M and an IC(50) of 8.3 mM. Glutathione, o-phenanthroline, zinc, and ethylenediamine tetraacetic acid (EDTA) inhibited PDE-I activity whereas Mg(2+) slightly potentiated the activity. PDE-I hydrolyzed thymidine-5'-monophosphate p-nitrophenyl ester most readily, whereas cyclic 3'-5'-AMP was least susceptible to hydrolysis. PDE-I was not lethal to mice at a dose of 4.0 mg/kg, ip, but had an anticoagulant effect on human plasma. These findings indicate that W. aegyptia PDE-I shares various characteristics with this enzyme from other snake venoms.

Large scale purification and characterization of recombinant human autotaxin/lysophospholipase D from mammalian cells.

We utilized a mammalian expression system to purify and characterize autotaxin (ATX)/lysophospholipase D, an enzyme present in the blood responsible for biosynthesis of lysophosphatidic acid. The human ATX cDNA encoding amino acids 29-915 was cloned downstream of a secretion signal of CD5. At the carboxyl terminus was a thrombin cleavage site followed by the constant domain (Fc) of IgG to facilitate protein purification. The ATX-Fc fusion protein was expressed in HEK293 cells and isolated from conditioned medium of a stable clone by affinity chromatography with Protein A sepharose followed by cleavage with thrombin. The untagged ATX protein was further purified to essential homogeneity by gel filtration chromatography with a yield of approximately 5 mg/liter medium. The purified ATX protein was enzymatically active and biologically functional, offering a useful tool for further biological and structural studies of this important enzyme.

Identification of small-molecule inhibitors of autotaxin that inhibit melanoma cell migration and invasion.

Autotaxin (ATX) is a prometastatic enzyme initially isolated from the conditioned medium of human melanoma cells that stimulates a myriad of biological activities, including angiogenesis and the promotion of cell growth, survival, and differentiation through the production of lysophosphatidic acid (LPA). ATX increases the aggressiveness and invasiveness of transformed cells, and ATX levels directly correlate with tumor stage and grade in several human malignancies. To study the role of ATX in the pathogenesis of malignant melanoma, we developed antibodies and small-molecule inhibitors against recombinant human protein. Immunohistochemistry of paraffin-embedded human tissue shows that ATX levels are markedly increased in human primary and metastatic melanoma relative to benign nevi. Chemical screens identified several small-molecule inhibitors with binding constants ranging from nanomolar to low micromolar. Cell migration and invasion assays with melanoma cell lines show that ATX markedly stimulates melanoma cell migration and invasion, an effect suppressed by ATX inhibitors. The migratory phenotype can be rescued by the addition of the enzymatic product of ATX, LPA, confirming that the observed inhibition is linked to suppression of LPA production by ATX. Chemical analogues of the inhibitors show structure-activity relationships important for ATX inhibition and indicate pathways for their optimization. These studies suggest that ATX is an approachable molecular target for the rational design of chemotherapeutic agents directed against malignant melanoma.

Scalable purification and characterization of the extracellular domain of human autotaxin from prokaryotic cells.

Autotaxin (ATX) is an approximately 125kDa transmembrane protein known as a tumor progression factor based on its lysophospholipase D (lysoPLD) activity. There are many reports of the biological and biochemical properties of ATX, but crystallographic or structural studies have not been reported because a large-scale production process using prokaryotic cells has not been established. Here we report a bulk purification process and soluble expression of the recombinant human ATX (rhATX S48) from prokaryotic cells. The extracellular domain of human ATX cDNA was cloned into a pET101/D-TOPO vector and transformed to an Escherichia coliBL21 strain which was co-transformed with a pTF16 chaperone plasmid. The rhATX S48 was purified with chaperone and it was removed by Mg(2+)-ATP treatment. The final yield of purified rhATX S48 was approximately 3.5mg/l culture of recombinant strain. The rhATX S48 shows lysoPLD enzymatic activity and effectively stimulates the growth and motile activity of the human tumor cells as well as native ATX. This is a first report for scalable purification of the ATX molecule and the rhATX S48 should be a good tool for immunization of anti-ATX or crystallographic analysis of ATX.

Validation of an autotaxin enzyme immunoassay in human serum samples and its application to hypoalbuminemia differentiation.

BACKGROUND: Autotaxin (ATX), a tumor cell motility-stimulating factor, regulates the blood concentrations of lysophosphatidic acid (LPA), an important and multi-functional bioactive lipid, through its lysophospholipase D activity (lysoPLD). The introduction of ATX measurements into clinical laboratory testing is urgently needed. METHODS: Anti-human ATX monoclonal antibodies were produced by immunization of recombinant human ATX expressed in a baculovirus system. An immunoassay for the quantitative determination of ATX was established, and human serum samples were assayed. RESULTS: The within-run and between-run precision, interference, detection limit, and linearity studies were satisfactory. The central 95 percentile reference interval for the serum ATX antigen concentration in healthy subjects was 0.468-1.134 mg/l (n=120) and was strongly correlated with the serum lysoPLD activity. The ATX concentration was significantly (p<0.001) higher in women (0.625-1.323 mg/l) than in men (0.438-0.914 mg/l). The serum ATX concentrations were increased in patients with chronic liver diseases and decreased in postoperative prostate cancer patients but were not altered in nephrosis patients. Thus, serum ATX antigen concentrations could be used to discriminate these hypoalbuminemia conditions. CONCLUSIONS: The present ATX antigen assay may be useful for clinical laboratory testing.

Measurement of autotaxin/lysophospholipase D activity.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator present in the blood and other biological fluids at physiologically relevant concentrations. In the cardiovascular system, studies using in vitro and in vivo experimental models indicate that LPA stimulates platelet activation, differentiation and migration of vascular smooth muscle cells, and changes in vascular tone. A growing body of evidence suggests that aberrant production and actions of LPA could play an important role in atherothrombotic disease. Hydrolysis of lysophospholipids by the secreted plasma protein autotaxin/lysophospholipase D (lysoPLD) is a major mechanism for generation of LPA in the blood. This chapter describes methods for determining the activity of recombinant autotaxin/lysoPLD using radiolabeled and fluorogenic substrates.

Cyclic phosphatidic acid is produced by autotaxin in blood.

Cyclic phosphatidic acid (cPA), an analog of lysophosphatidic acid (LPA), was previously identified in human serum. Although cPA possesses distinct physiological activities not elicited by LPA, its biochemical origins have scarcely been studied. In the present study, we assayed cPA formation from lysophosphatidylcholine in fetal bovine serum and found significant activity of transphosphatidylation that generated cPA. The cPA-producing enzyme was purified from fetal bovine serum using five chromatographic steps yielding a 100-kDa protein with cPA biosynthetic activity. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of its tryptic peptides revealed that the enzyme shared identical fragments with human autotaxin, a serum lysophospholipase D that produces LPA. Western blot analysis demonstrated that the 100-kDa protein was specifically recognized by an anti-human autotaxin antibody. Moreover, recombinant rat autotaxin was found to generate cPA in addition to LPA. No significant cPA- or LPA-producing activity was detected in autotaxin-depleted serum from bovine or human prepared by immunoprecipitation with an anti-autotaxin monoclonal antibody. These results indicate that the generation of cPA and LPA in serum is mainly attributed to autotaxin.

The yeast Snm1 protein is a DNA 5'-exonuclease.

Interstrand cross-links (ICL) in DNA arise from bifunctional alkylating agents, including nitrogen mustards, mitomycin C and psoralens. Such adducts prevent normal transcription or replication and are mutagenic. Therefore, cellular mechanisms for removing ICL damage are needed to maintain genome stability. Normal ICL repair requires the action of a number of genes, some specific for such damage. The yeast Snm1 protein is one such protein, but its function has been unknown. Incision for ICL repair is normal in mutants lacking Snm1, so it appears to act after the earliest steps. We have used recombinant SNM1 constructs in an Escherichia coli (E. coli) expression system to demonstrate that the yeast gene encodes a 5'-exonuclease. The exonuclease activity is required for Snm1 to be functional in ICL repair.