The calcium activated nucleotidases: A diverse family of soluble and membrane associated nucleotide hydrolyzing enzymes

It has long been known that the salivary glands of hematophagous (blood-feeding) arthropods secrete soluble apyrases, which are potent nucleotide hydrolyzing enzymes capable of hydrolyzing extracellular ATP and ADP, the latter being a major agonist contributing to platelet aggregation. Only recently, however, has the identification of proteins homologous to these apyrases been reported in non-blood-feeding organisms such as rodents and humans. In this review, we present an overview of the diverse family of apyrases first described in the blood-feeding arthropods, including the identification and characterization of the soluble and membrane-bound vertebrate enzymes homologous to these arthropod apyrases. We also describe the enzymatic properties and nucleotide specificities of the expressed enzymes, and insights gained into the structure and function of this calcium activated nucleotidase (CAN) family from biophysical, mutagenesis and crystallography studies. The potential therapeutic value of these proteins is also discussed.     

Evidence that the caterpillar salivary enzyme glucose oxidase provides herbivore offense in solanaceous plants

The insect salivary enzyme glucose oxidase (GOX) can inhibit wound-inducible nicotine production in tobacco, Nicotiana tabacum. We examined whether salivary gland extracts of Helicoverpa zea lacking active GOX could still suppress nicotine in tobacco, Nicotiana tabacum, and whether GOX could suppress wound-inducible defenses of another Solanaceous plant, tomato Lycopersicon esculentum. Tobacco leaves were wounded with a cork borer and treated with water, salivary gland extracts with active GOX (SxG), or salivary gland extracts with inactive GOX (SxI). After three days, leaves treated with SxG had significantly less nicotine than all other wounded treatments. Neonates that fed on the terminal leaves of tobacco plants treated with SxG had significantly higher survival than neonates that fed on leaves treated with either SxI or water. This evidence supports the assertion that GOX is the salivary factor responsible for the suppression of tobacco plant nicotine production by H. zea saliva. Results for the NahG tobacco plants, which lack salicylic acid (SA) due to a transgene for bacterial SA hydroxylase, indicate that suppression of nicotine by GOX does not require SA. However, tobacco leaves that were wounded and treated with SxG had significantly higher levels of the SA-mediated PR-1a protein than leaves treated with SxI or water. Leaves of tomato plants wounded with scissors and then treated with SxG had trypsin inhibitor levels that were moderately lower than plants wounded and treated with purified GOX, water, or SxI. However, all the wounded tomato leaves irrespective of treatment resulted in lower caterpillar growth rates than the non-wounded tomato leaves. Glucose oxidase is the first insect salivary enzyme shown to suppress wound-inducible herbivore defenses of plants.     

Triatoma infestans apyrases belong to the 5'-nucleotidase family

Apyrases are nucleoside triphosphate-diphosphohydrolases (EC 3.6.1.5) present in a variety of organisms. The apyrase activity found in the saliva of hematophagous insects is correlated with the prevention of ADP-induced platelet aggregation of the host during blood sucking. Purification of apyrase activity from the saliva of the triatomine bug Triatoma infestans was achieved by affinity chromatography on oligo(dT)-cellulose and gel filtration chromatography. The isolated fraction includes five N-glycosylated polypeptides of 88, 82, 79, 68 and 67 kDa apparent molecular masses. The isolated apyrase mixture completely inhibited aggregation of human blood platelets. Labeling with the ATP substrate analogue 5'-p-fluorosulfonylbenzoyladenosine showed that the five species have ATP-binding characteristic of functional apyrases. Furthermore, tandem mass spectroscopy peptide sequencing showed that the five species share sequence similarities with the apyrase from Aedes aegypti and with 5'-nucleotidases from other species. The complete cDNA of the 79-kDa enzyme was cloned, and its sequence confirmed that it encodes for an apyrase belonging to the 5'-nucleotidase family. The gene multiplication leading to the unusual salivary apyrase diversity in T. infestans could represent an important mechanism amplifying the enzyme expression during the insect evolution to hematophagy, in addition to an escape from the host immune response, thus enhancing acquisition of a meal by this triatomine vector of Chagas' disease.     

Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation

Apyrase activity is present in the saliva of haematophagous arthropods. It is related to blood-feeding because of the apyrase ability to hydrolyse ADP, a key component of platelet aggregation. Five apyrases with apparent molecular masses of 88, 82, 79, 68 and 67 kDa were identified in the saliva of the vector of Chagas disease, Triatoma infestans. The large size observed during purification of these enzymes suggested oligomerization. In the present study, we confirmed, using gel-filtration and analytical ultracentrifugation, the presence of apyrase oligomers with molecular masses of 200 kDa in the saliva. Electrophoretic analyses showed that disulphide bonds were involved in homo-oligomerization. In addition, heterogeneity in disulphide bonds and in pI was detected, with the pI ranging from 4.9 to 5.4. The present study gives the first insights into the quaternary structure of soluble apyrases.     

Apyrase Activity and Platelet Aggregation Inhibitors in the Tick Ornithodoros Savignyi (Acari: Argasidae)

Ticks are ectoparasites that cause considerable damage to their hosts while feeding. The feeding process is facilitated by anti-haemostatic factors present in the tick saliva. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is a platelet aggregation inhibitor found in most haematophagous organisms studied. The present study describes the identification and characterization of such an activity in the tick Ornithodoros savignyi. The enzyme conformed to many properties common to apyrases. These included a low substrate specificity, dependence on bivalent metal ions for activity and insensitivity to the classical ATPase inhibitors. Heat denaturation studies, pH optima and similar effects of inhibitors on the enzyme's ATP and ADP hydrolysing activities supported its classification as an apyrase. Salivary gland extracts inhibited the platelet aggregation induced by ADP, collagen and thrombin and disaggregated aggregated platelets. The results suggest the presence of two or more anti-platelet factors present in the salivary glands of this tick species.     

Comparative subcellular distribution of apyrase from animal and plant sources. Characterization of microsomal apyrase

1. Apyrase (ATP: diphosphohydrolase) has been found in the microsomal fraction of rat salivary gland, mammary gland and uterus. 2. This enzyme, already described in plant tissue, is mainly present as a soluble polypeptide in tubers of Solanum tuberosum. 3. A fraction of this enzyme is associated with the microsomal fraction with a higher specific activity than the soluble one, for either ATP or ADP as substrate. 4. Apyrase bound to microsomes from rat and potato tissues was characterized in its substrate specificity and effect of inhibitors. 5. The Km values for ATP and ADP, optimum pH and metal ion requirement were determined. 6. A characteristic common to the microsomal and soluble apyrases is the stimulatory effect of a potato activator protein of soluble plant apyrase. 7. The microsomal-bound apyrase from rat and potato tissues were solubilized and subjected to size-exclusion chromatography. 8. The mammary gland and salivary gland apyrases eluted as molecular aggregates, in contrast to the uterus and potato enzyme.     

Potato tuber isoapyrases: substrate specificity, affinity labeling, and proteolytic susceptibility

Apyrase/ATP-diphosphohydrolase hydrolyzes di- and triphosphorylated nucleosides in the presence of a bivalent ion with sequential release of orthophosphate. We performed studies of substrate specificity on homogeneous isoapyrases from two potato tuber clonal varieties: Desiree (low ATPase/ADPase ratio) and Pimpernel (high ATPase/ADPase ratio) by measuring the kinetic parameters K(m) and k(cat) on deoxyribonucleotides and fluorescent analogues of ATP and ADP. Both isoapyrases showed a broad specificity towards dATP, dGTP, dTTP, dCTP, thio-dATP, fluorescent nucleotides (MANT-; TNP-; ethene-derivatives of ATP and ADP). The hydrolytic activity on the triphosphorylated compounds was always higher for the Pimpernel apyrase. Modifications either on the base or the ribose moieties did not increase K(m) values, suggesting that the introduction of large groups (MANT- and TNP-) in the ribose does not produce steric hindrance on substrate binding. However, the presence of these bulky groups caused, in general, a reduction in k(cat), indicating an important effect on the catalytic step. Substantial differences were observed between potato apyrases and enzymes from various animal tissues, concerning affinity labeling with azido-nucleotides and FSBA (5'-p-fluorosulfonylbenzoyl adenosine). PLP-nucleotide derivatives were unable to produce inactivation of potato apyrase. The lack of sensitivity of both potato enzymes towards these nucleotide analogues rules out the proximity or adequate orientation of sulfhydryl, hydroxyl or amino-groups to the modifying groups. Both apyrases were different in the proteolytic susceptibility towards trypsin, chymotrypsin and Glu-C.     

Kinetics of expression of the salivary apyrases in Triatoma infestans

Apyrases are nucleoside triphosphate-diphosphohydrolases that remove Pi from ATP and ADP. The blood feeding reduviid Triatoma infestans, which transmits the Trypanosoma cruzi agent of Chagas disease to animals and man, presents in its salivary glands five apyrases with molecular masses of 88, 82, 79, 68 and 67 kDa. These triatomine apyrases have been associated with the prevention of ADP induced platelet aggregation in the host. Here we provide biochemical data showing that these apyrases are stored in the lumen of the salivary gland D1 pairs, and that about one half of the pool of the enzyme is consumed during feeding. After the feeding recovery of apyrases to maximal activity level takes days, thus suggesting de novo protein synthesis. This hypothesis is supported by quantitative RT-PCR analysis which shows an upregulation of the 79 kDa apyrase mRNA level after feeding.     

Membrane-bound ATPase of a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius

The membranes of Sulfolobus, a thermoacidophilic archaebacterium showed two types of ATP hydrolyzing activity. One was that of a neutral ATPase at an optimum pH around 6.5. This enzyme was activated by 10 mM sulfate with a shift of optimum pH to 5. In these respects, the enzyme was similar to membrane-bound ATPase of Thermoplasma, another thermoacidophilic archaebacterium, reported by Searcy and Whatley [1982) Zbl. Bakt. Hyg., I. Abt. Orig. C3, 245-257). The enzyme hydrolyzed ATP and other NTPs, but not ADP or AMP. It was highly thermostable, but irreversibly inactivated in 0.1 M HCl. The other activity was that of an acidic apyrase at an optimum pH around 2.5. This enzyme was extremely stable toward high temperature and acid and inhibited by sulfate. Both of these ATP hydrolyzing enzymes were resistant to N,N'-dicyclohexylcarbodiimide (DCCD), azide, oligomycin, N'-ethylmaleimide, p-chloromercuribenzoate, orthovanadate, or ouabain. Sulfolobus ATPases differ from F1 and other transport ATPases so far described.     

Salivary signals of European corn borer induce indirect defenses in tomato

Plants can recognize the insect elicitors and activate its defense mechanisms. European Corn Borer (ECB; Ostrinia nubilalis) saliva, produced from the labial salivary glands and released through the spinneret, is responsible for inducing direct defenses in host plants. Glucose oxidase (GOX) present in the ECB saliva induced direct defenses in tomato. By contrast, GOX activity in ECB saliva was insufficient to trigger defenses in maize, suggesting that host-specific salivary elicitors are responsible for inducing direct defenses in host plants. Our current study further examined whether ECB saliva can trigger indirect defenses in tomato. Relative expression levels of TERPENE SYNTHASE5 (TPS5) and HYDROPEROXIDE LYASE (HPL), marker for indirect defenses in host plants, were monitored. Quantitative real-time PCR analysis revealed that ECB saliva can induce the expression of TPS5 and HPL, suggesting that salivary signals can induce indirect defenses in addition to the direct defenses. Further experiments are required to identify different ECB elicitors that are responsible for inducing direct and indirect defenses in host plants.     

Characterisation of the secreted apyrase family of Heligmosomoides polygyrus

Apyrases are a recurrent feature of secretomes from numerous species of parasitic nematodes. Here we characterise the five apyrases secreted by Heligmosomoides polygyrus, a natural parasite of mice and a widely used laboratory model for intestinal nematode infection. All five enzymes are closely related to soluble calcium-activated nucleotidases described in a variety of organisms, and distinct from the CD39 family of ecto-nucleotidases. Expression is maximal in adult worms and restricted to adults and L4s. Recombinant apyrases were produced and purified from Pichia pastoris. The five enzymes showed very similar biochemical properties, with strict calcium dependence and a broad substrate specificity, catalysing the hydrolysis of all nucleoside tri- and diphosphates, with no activity against nucleoside monophosphates. Natural infection of mice provoked very low antibodies to any enzyme, but immunisation with an apyrase cocktail showed partial protection against reinfection, with reduced egg output and parasite recovery. The most likely role for nematode secreted apyrases is hydrolysis of extracellular ATP, which acts as an alarmin for cellular release of IL-33 and initiation of type 2 immunity.     

Effects of chemical inhibitors and apyrase enzyme further document a role for apyrases and extracellular ATP in the opening and closing of stomates in Arabidopsis

In Arabidopsis leaves there is a bi-phasic dose-response to applied nucleotides; i.e., lower concentrations induce stomatal opening, while higher concentrations induce closure. Two mammalian purinoceptor antagonists, PPADS and RB2, block both nucleotide-induced stomatal opening and closing. These antagonists also partially block ABA-induced stomatal closure and light-induced stomatal opening. There are two closely related Arabidopsis apyrases, AtAPY1 and AtAPY2, which are both expressed in guard cells. Here we report that low levels of apyrase chemical inhibitors can induce stomatal opening in the dark, while apyrase enzyme blocks ABA-induced stomatal closure. We also demonstrate that high concentrations of ATP induce stomatal closure in the light. Application of ATPγS and chemical apyrase inhibitors at concentrations that have no effect on stomatal closure can lower the threshold for ABA-induced closure. The closure induced by ATPγS was not observed in gpa1-3 loss-of-function mutants. These results further confirm the role of extracellular ATP in regulating stomatal apertures.     

Evolution of the salivary apyrases of blood-feeding arthropods

Phylogenetic analyses of three families of arthropod apyrases were used to reconstruct the evolutionary relationships of salivary-expressed apyrases, which have an anti-coagulant function in blood-feeding arthropods. Members of the 5′nucleotidase family were recruited for salivary expression in blood-feeding species at least five separate times in the history of arthropods, while members of the Cimex-type apyrase family have been recruited at least twice. In spite of these independent events of recruitment for salivary function, neither of these families showed evidence of convergent amino acid sequence evolution in salivary-expressed members. On the contrary, in the 5′-nucleotide family, salivary-expressed proteins conserved ancestral amino acid residues to a significantly greater extent than related proteins without salivary function, implying parallel evolution by conservation of ancestral characters. This unusual pattern of sequence evolution suggests the hypothesis that purifying selection favoring conservation of ancestral residues is particularly strong in salivary-expressed members of the 5′-nucleotidase family of arthropods because of constraints arising from expression within the vertebrate host.     

Apyrase,streptavidin-binding proteins,and antimicrobial activity in Pisum sativum

The 49 kD apyrase (EC 3.6.1.5), streptavidin-binding proteins, and antimicrobial activity in the subcellular fractions from different seed parts of Pisum sativum L. var. Alaska were examined. Except cotyledons, all subcellular fractions contained 49 kD apyrase, and a considerable relationship was found between 49 kD apyrase and NTPase activities that increased with increasing time of germination. The bulk of 49 kD apyrase and NTPase activities was found in the nucleus pellets and cytoskeleton-enriched fraction, indicating their physiological importance. At 72 h of germination, all subcellular fractions of primary stems have a greater amount of 49 kD apyrase and NTPase than primary leaves and much more than primary roots and cotyledonary stalks. All seed parts showed antimicrobial activities, and the bulk of inhibition activities was found in the cytoskeleton-enriched and nucleus pellets, which was greater in the primary stems and leaves than in other parts. Current findings reveal that apyrases have important roles in metabolic activities in all parts of the pea plants except cotyledons. Cotyledons contained much streptavidin-binding proteins, which might have different physiological roles than apyrases.