Terence L. Kirley

Comparative hydrolysis of P2 receptor agonists by NTPDases 1, 2, 3 and 8

Nucleoside triphosphate diphosphohydrolases 1, 2, 3 and 8 (NTPDases 1, 2, 3 and 8) are the dominant ectonucleotidases and thereby expected to play important roles in nucleotide signaling. Distinct biochemical characteristics of individual NTPDases should allow them to regulate P2 receptor activation differentially. Therefore, the biochemical and kinetic properties of these enzymes were compared. NTPDases 1, 2, 3 and 8 efficiently hydrolyzed ATP and UTP with Km values in the micromolar range, indicating that they should terminate the effects exerted by these nucleotide agonists at P2X1- and P2Y2,4,11 receptors. Since NTPDase1 does not allow accumulation of ADP, it should terminate the activation of P2Y1,12,13 receptors far more efficiently than the other NTPDases. In contrast, NTPDases 2, 3 and 8 are expected to promote the activation of ADP specific receptors, because in the presence of ATP they produce a sustained (NTPDase2) or transient (NTPDases 3 and 8) accumulation of ADP. Interestingly, all plasma membrane NTPDases dephosphorylate UTP with a significant accumulation of UDP, favoring P2Y6 receptor activation. NTPDases differ in divalent cation and pH dependence, although all are active in the pH range of 7.0-.5. Various NTPDases may also distinctly affect formation of extracellular adenosine and therefore adenosine receptor-mediated responses, since they generate different amounts of the substrate (AMP) and inhibitor (ADP) of ecto-5-nucleotidase, the rate limiting enzyme in the production of adenosine. Taken together, these data indicate that plasma membrane NTPDases hydrolyze nucleotides in a distinctive manner and may therefore differentially regulate P2 and adenosine receptor signaling.

Cloning, sequencing, and expression of a human brain ecto-apyrase related to both the ecto-ATPases and CD39 ecto-apyrases

Abstract An extracellular ATPase (E-type ATPase) clone was isolated from a human brain cDNA library and sequenced. The transcript shows similarity to the previously published chicken smooth muscle and rat brain ecto-ATPase cDNAs, human CD39L1 cDNA (putative human ecto-ATPase), and mammalian CD39 (lymphoid cell activation antigen, ecto-apyrase, ATPDase, ATP-diphosphohydrolase) cDNAs. The full-length human brain cDNA encodes a 529 amino acid glycoprotein with a putative membrane spanning region near each terminus, with the majority of the protein found extracellularly. Expression of this clone in mammalian COS-1 cells yielded NaN3-sensitive ATPase and ADPase activity detectable both on intact cells and cell membrane preparations. The nucleotide hydrolysis ratio of the expressed protein is approx. 2.75:1 (ATPase:ADPase activity), classifying it as an ecto-apyrase. However, this hydrolysis ratio is intermediate between that observed for the ecto-ATPases and the CD39 ecto-apyrases (L. Plesner, Int. Rev. Cytol. 158 (1995) 141–214). Quantitative analyses of amino acid identities and similarities between this ecto-apyrase and other vertebrate E-type ATPases suggest that this human brain enzyme is nearly equally related to the ecto-ATPases and the CD39s, and phylogenetic analysis suggests that it could be an ancestral enzyme from which both ecto-ATPases and CD39 ecto-apyrases are derived.

Reduction and fluorescent labeling of cyst(e)ine-containing proteins for subsequent structural analyses

Procedures which allow rapid, quantitative, and selective fluorescent labeling of protein cyst(e)ine residues prior to electrophoresis by reaction with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) under mild conditions are described. After labeling, the protein(s) of interest is easily monitored throughout electrophoresis and subsequent electroblotting or electroelution procedures. The stoichiometry of labeling and therefore the number of cysteine and/or half-cystine residues can be measured spectrophotometrically or fluorometrically and the derived cyst(e)ine adduct can also be quantitated by amino acid analysis and identified in protein sequencing. N-terminal blockage is not observed under the conditions utilized, nor are any other amino acid side chains modified. The procedures described allow complete, rapid, and facile reduction and alkylation of proteins with simultaneous incorporation of a fluorophore, permitting sensitive detection in subsequent manipulation of the proteins. Quantitative fluorescence prelabeling also allows the generation, purification, and sequencing of peptide fragments containing cyst(e)ine residues for determination of internal sequences and residues involved in disulfide bonds.

Complementary DNA Cloning and Sequencing of the Chicken Muscle Ecto-ATPase HOMOLOGY WITH THE LYMPHOID CELL ACTIVATION ANTIGEN CD39

The ecto-ATPase from chicken gizzard (smooth muscle) was solubilized, and the 66-kDa cell membrane ecto-ATPase protein was purified. The protein was then subjected to both enzymatic and chemical cleavage, and the resultant peptides were purified by reverse phase high pressure liquid chromatography and sequenced. Several of these internal peptide sequences were used to design oligonucleotides to screen a chicken muscle library to identify the cDNA encoding the ecto-ATPase. Two overlapping partial clones were sequenced, yielding the complete coding region and a long 3′-untranslated sequence. The deduced amino acid sequence is in agreement with the N-terminal and peptide sequences obtained from the purified protein. The chicken muscle ecto-ATPase is a slightly basic (predicted pI = 7.93) 494-amino acid protein (54.4 kDa), containing a single transmembrane domain at each end of the protein. The majority of the protein is predicted to be extracellular, making it a Type Ia plasma membrane protein. There are four putative N-glycosylation sites, a single potential cAMP/cGMP-dependent protein kinase phosphorylation site, as well as a single putative tyrosine kinase phosphorylation site. Analysis of the sequence using the BLAST programs demonstrated homology with other ecto-ATPases and ecto-apyrases, including those from the parasitic protozoan Toxoplasma gondii, potato tubers, and garden pea, as well as a guanosine diphosphohydrolase from yeast. However, the most striking homology observed was to the human and mouse lymphoid cell activation antigen 39 (CD39), a molecule now known to have apyrase activity. The chicken ecto-ATPase showed considerable amino acid sequence homology with CD39 over the entire length of the sequence, excluding about 30-40 amino acids at the extreme ends of the protein (which include the two membrane-spanning helices). The sequence homology between the gizzard ecto-ATPase and CD39 was confirmed by Western blots demonstrating immunocross-reactivity between mono- and polyclonal antibodies raised against the chicken ecto-ATPase and two commercially available monoclonal antibodies against the human CD39 protein. The results suggest that the muscle ecto-ATPase may be involved in cell adhesion, since the highly homologous CD39 protein is involved in homotypic adhesion of activated B lymphocytes.

Immunolocalization of ecto-nucleoside triphosphate diphosphohydrolase 3 in rat brain: Implications for modulation of multiple homeostatic systems including feeding and sleep–wake behaviors

Three anti-peptide antisera were raised against three distinct amino acid sequences of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), characterized by Western blot analyses, and used to determine the distribution of NTPDase3 protein in adult rat brain. The three antisera all yielded similar immunolocalization data, leading to increased reliability of the results obtained. Unlike NTPDase1 and NTPDase2, NTPDase3 immunoreactivity was detected exclusively in neurons. Immunoreactivity was localized primarily to axon-like structures with prominent staining of presynaptic elements. Specific perikaryal immunostaining was detected primarily in scattered neurons near the lateral hypothalamic area and the perifornical nucleus. High densities of immunoreactive axon-like fibers were present in midline regions of the forebrain and midbrain. Highly scattered NTPDase3 positive fibers were observed in the cerebral cortex, the hippocampal formation, and the basal ganglia. Moreover, very high densities of immunostained fibers were detected in the mediobasal hypothalamus, with the overall mesencephalic pattern of staining associated closely with hormone responsive nuclei. High densities of NTPDase3 positive terminals were also associated with noradrenergic neurons. However, co-immunolocalization studies revealed clearly that NTPDase3 immunoreactivity was not localized within the noradrenaline cells or terminals. In contrast, nearly all of the NTPDase3 immunopositive hypothalamic cells, and most fibers in the mid- and hindbrain, also expressed hypocretin-1/orexin-A. The overall pattern of expression and co-localization with hypocretin-1/orexin-A suggests that NTPDase3, by regulating the extracellular turnover of ATP, may modulate feeding, sleep-wake, and other behaviors through diverse homeostatic systems.

Expression and characterization of soluble and membrane-bound human nucleoside triphosphate diphosphohydrolase 6 (CD39L2).

Ecto-nucleoside-triphosphate diphosphohydrolase-6 (eNTPDase61, also known as CD39L2) cDNA was expressed in mammalian COS-1 cells and characterized using nucleotidase assays as well as size exclusion, anion exchange, and cation exchange chromatography. The deduced amino acid sequence of eNTPDase6 is more homologous with the soluble E-type ATPase, eNTPDase5, than other E-type ATPases, suggesting it may also be soluble. To test this possibility, both the cell membranes and the growth media from eNTPDase6-transfected COS-1 cells were assayed for nucleotidase activities. Activity was found in both the membranes and the media. Soluble eNTPDase6 preferentially exhibits nucleoside diphosphatase activity, which is dependent on the presence of divalent cations. Western blot analysis of eNTPDase6 treated with PNGase-F indicated both soluble and membrane-bound forms are glycosylated. However, unlike some membrane-bound ecto-nucleotidases, the eNTPDase6 activity was not specifically inhibited by deglycosylation with peptideN-glycosidase F. Soluble eNTPDase6 hydrolyzed nucleoside triphosphates poorly and nucleoside monophosphates not at all. Analysis of the relative rates of hydrolysis of nucleoside diphosphates (GDP = IDP > UDP > CDP ≫ ADP) suggests that soluble eNTPDase6 is a diphosphatase most likely not involved in regulation of ADP levels important for circulatory hemostasis.

Cloning, expression, and characterization of a soluble calcium-activated nucleotidase, a human enzyme belonging to a new family of extracellular nucleotidases☆

The salivary apyrases of blood-feeding arthropods are nucleotide-hydrolyzing enzymes implicated in the inhibition of host platelet aggregation through the hydrolysis of extracellular adenosine diphosphate. A human cDNA homologous to the apyrase cDNA of the blood-feeding bed bug was identified, revealing an open reading frame encoding a 371-amino acid protein. A cleavable signal peptide generates a secreted protein of 333 residues with a predicted core molecular mass of 37,193 Da. Expression in COS-1 cells produced a secreted apyrase in the cell media. The ADPase and ATPase activities were dependent upon calcium, with a pH optimum between pH 6.2 and 7.2. Interestingly, the preferred substrate was not ADP, as might be expected for an enzyme modulating platelet aggregation, but rather UDP, followed by GDP, UTP, GTP, ADP, and ATP. The nucleotidase did not hydrolyze nucleoside monophosphates. Size-exclusion chromatography and Western blot analysis revealed a molecular mass of approximately 34-37 kDa. Treatment of the enzyme with peptide N-glycosidase F indicated that the protein is glycosylated. Northern analysis identified the transcript in a range of human tissues, including testis, placenta, prostate, and lung. No traditional apyrase-conserved regions or nucleotide-binding domains were identified in this human enzyme, indicating membership in a new family of extracellular nucleotidases.

Purification and characterization of the ecto-Mg-ATPase of chicken gizzard smooth muscle

The ecto-Mg-ATPase isolated from chicken gizzard smooth muscle was solubilized, purified and characterized. The purification did not require the use of expensive or specialized apparatus. The chromatographic and electrophoretic characteristics of the ecto-Mg-ATPase from chicken are similar to those reported earlier for the ecto-Mg-ATPase isolated from rabbit skeletal muscle transverse tubule membranes [1992, J. Biol. Chem. 267, 11777-11782]. One obvious difference found was that the solubilized chicken ecto-Mg-ATPase can be stimulated approximately 1900% by the lectin Concanavalin A (Con A) under the same conditions that the rabbit enzyme is inhibited by approximately 50%. This stimulatory effect of Con A is useful for following the purification, and also increases the specific activity of the chicken enzyme to a very high level similar to that observed for the rabbit enzyme. After purification of the solubilized chicken ecto-Mg-ATPase by three steps of anion exchange chromatography, as well as Con A and erythroagglutinating Phaseolus vulgaris (PHA-E) lectin affinity chromatographies, a single diffuse glycoprotein band at approximately 66 kDa is observed after SDS-PAGE. This protein could be deglycosylated to a core protein of 53 kDa. Thus, the chicken gizzard protein is very similar in molecular size to the rabbit skeletal muscle ecto-Mg-ATPase both before and after deglycosylation [1992, J. Biol. Chem. 267, 11777-11782]. The N-terminal sequence of the 66 kDa chicken gizzard protein was found to be: Ala-Arg-Arg-Ala-Ala-Ala-Val-Leu-Leu-Leu-Leu-Ala. This is a unique sequence which, while very different from the rabbit ecto-Mg-ATPase N-terminus, exhibits some of the same characteristics, since it contains basic residues as the second and third amino acids, with the remainder of the N-terminus being very hydrophobic in nature. Furthermore, the chicken gizzard ecto-Mg-ATPase can be separated from the adhesion molecule, truncated cadherin (T-cadherin) by anion exchange chromatography, and is therefore not identical to that protein, as had been recently proposed [1993, Arch. Biochem. Biophys. 303, 32-43].

Characterization of a monoclonal antibody as the first specific inhibitor of human NTP diphosphohydrolase‐3

The study and therapeutic modulation of purinergic signaling is hindered by a lack of specific inhibitors for NTP diphosphohydrolases (NTPDases), which are the terminating enzymes for these processes. In addition, little is known of the NTPDase protein structural elements that affect enzymatic activity and which could be used as targets for inhibitor design. In the present study, we report the first inhibitory monoclonal antibodies specific for an NTPDase, namely human NTPDase3 (EC 3.6.1.5), as assessed by ELISA, western blotting, flow cytometry, immunohistochemistry and inhibition assays. Antibody recognition of NTPDase3 is greatly attenuated by denaturation with SDS, and abolished by reducing agents, indicating the significance of the native conformation and the disulfide bonds for epitope recognition. Using site‐directed chemical cleavage, the SDS‐resistant parts of the epitope were located in two fragments of the C‐terminal lobe of NTPDase3 (i.e. Leu220–Cys347 and Cys347–Pro485), which are both required for antibody binding. Additional site‐directed mutagenesis revealed the importance of Ser297 and the fifth disulfide bond (Cys399–Cys422) for antibody binding, indicating that the discontinuous inhibitory epitope is located on the extracellular C‐terminal lobe of NTPDase3. These antibodies inhibit recombinant NTPDase3 by 60–90%, depending on the conditions. More importantly, they also efficiently inhibit the NTPDase3 expressed in insulin secreting human pancreatic islet cells in situ. Because insulin secretion is modulated by extracellular ATP and purinergic receptors, this finding suggests the potential application of these inhibitory antibodies for the study and control of insulin secretion.

The structure of the nucleoside triphosphate diphosphohydrolases (NTPDases) as revealed by mutagenic and computational modeling analyses

Over the last seven years our laboratory has focused on the determination of the structural aspects of nucleoside triphosphate diphosphohydrolases (NTPDases) using site-directed mutagenesis and computational comparative protein modeling to generate hypotheses and models for the hydrolytic site and enzymatic mechanism of the family of NTPDase nucleotidases. This review summarizes these studies utilizing NTPDase3 (also known as CD39L3 and HB6), an NTPDase family member that is intermediate in its characteristics between the more widely distributed and studied NTPDase1 (also known as CD39) and NTPDase2 (also known as CD39L1 and ecto-ATPase) enzymes. Relevant site-directed mutagenesis studies of other NTPDases are also discussed and compared to NTPDase3 results. It is anticipated that many of the results and conclusions reached via studies of NTPDase3 will be relevant to understanding the structure and enzymatic mechanism of all the cell-surface members of this family (NTPDase1–3, 8), and that understanding these NTPDase enzymes will aid in modulating the many varied processes under purinergic signaling control. This review also integrates the site-directed mutagenesis results with a recent 3-D structural model for the extracellular portion of NTPDases that helps explain the importance of the apyrase conserved regions (ACRs) of the NTPDases. Utilizing this model and published work from Dr Guidotti’s laboratory concerning the importance and characteristics of the two transmembrane helices and their movements in response to substrate, we present a speculative cartoon model of the enzymatic mechanism of the membrane-bound NTPDases that integrates movements of the extracellular region required for catalysis with movements of the N- and C-terminal transmembrane helices that are important for control and modulation of enzyme activity.