Salivary Apyrase Research Articles

Xenopus apyrase ( xapy), a secreted nucleotidase that is expressed during early development

We have characterized a cDNA encoding a Xenopus laevis apyrase (XAPY) that is expressed during embryogenesis. XAPY is highly homologous to two recently described mammalian apyrases, human SCAN-1 and rat Ca 2+-NDPase, and to a lesser extent the salivary apyrase of the blood-feeding arthropod Cimex lectularis. RT-PCR analysis shows that Xapy is expressed at all the developmental stages tested, from oocytes through to tadpoles. Xapy transcripts are widely distributed in the embryo, but from late neurulae through to late tailbud stages they are highly enriched in the cement gland, an adhesive organ in the epidermis of the head. When expressed in HEK 293 cells, XAPY is largely retained in the endoplasmic reticulum, although some is also secreted. XAPY conditioned media hydrolyses UDP and UTP, confirming that it is a functional apyrase.

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.

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.

Structure and Protein Design of a Human Platelet Function Inhibitor

Hematophagous arthropods secrete a salivary apyrase that inhibits platelet activation by catabolizing ADP released from damaged tissues and blood cells. We report the X-ray crystal structures of a human enzyme of the soluble apyrase family in its apo state and bound to a substrate analog. The structures reveal a nucleotide binding domain comprising a five-blade β propeller, binding determinants of the substrate and the active site, and an unusual calcium binding site with a potential regulatory function. Using a comparative structural biology approach, we were able to redesign the human apyrase so as to enhance its ADPase activity by more than 100-fold. The engineered enzyme is a potent inhibitor of platelet aggregation and may serve as the basis for the development of a new class of antithrombotic agents.

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.

Disaggregation of aggregated platelets by apyrase from the tick, Ornithodoros savignyi (Acari: Argasidae).

Apyrase, secreted by ticks during feeding, is a platelet aggregation inhibitor that functions as a regulator of the host's hemostatic system. This present study concerns the disaggregation effect of salivary gland apyrase from the tick Ornithodoros savignyi. Secondarily aggregated platelets, disaggregated by apyrase, exhibited a reversal of shape from a spherical (aggregated) form to a discoid form, reminiscent of reversible aggregation at low ADP concentrations in citrated platelet-rich plasma. However, they showed a dilatory open canaliculary system and an absence of granules indicating disaggregation after degranulation had taken place. In contrast, disaggregation by the fibrin(ogen)olytic enzyme, plasmin, showed that platelets degranulated, but retained a spherical form with numerous extended pseudopods. While thrombin had no effect on aggregation or clotting of platelets disaggregated with plasmin, it did activate those platelets disaggregated with apyrase and clotted the plasma. This is the first study to describe the disaggregating effects of tick derived apyrase on aggregated platelets. It also shows that apyrase can disaggregate platelets even after secondary aggregation and degranulation of platelets has taken place. Platelet aggregation is one of the main barriers encountered by ticks during feeding and counteraction of this process by ticks is an important factor for successful feeding.

Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly Lutzomyia longipalpis.

The saliva of blood-sucking arthropods contains powerful pharmacologically active substances and may be a vaccine target against some vector-borne diseases. Subtractive cloning combined with biochemical approaches was used to discover activities in the salivary glands of the hematophagous fly Lutzomyia longipalpis. Sequences of nine full-length cDNA clones were obtained, five of which are possibly associated with blood-meal acquisition, each having cDNA similarity to: (i) the bed bug Cimex lectularius apyrase, (ii) a 5'-nucleotidase/phosphodiesterase, (iii) a hyaluronidase, (iv) a protein containing a carbohydrate-recognition domain (CRD), and (v) a RGD-containing peptide with no significant matches to known proteins in the BLAST databases. Following these findings, we observed that the salivary apyrase activity of L. longipalpis is indeed similar to that of Cimex apyrase in its metal requirements. The predicted isoelectric point of the putative apyrase matches the value found for Lutzomyia salivary apyrase. A 5'-nucleotidase, as well as hyaluronidase activity, was found in the salivary glands, and the CRD-containing cDNA matches the N-terminal sequence of the HPLC-purified salivary anticlotting protein. A cDNA similar to alpha-amylase was discovered and salivary enzymatic activity demonstrated for the first time in a blood-sucking arthropod. Full-length clones were also found coding for three proteins of unknown function matching, respectively, the N-terminal sequence of an abundant salivary protein, having similarity to the CAP superfamily of proteins and the Drosophila yellow protein. Finally, two partial sequences are reported that match possible housekeeping genes. Subtractive cloning will considerably enhance efforts to unravel the salivary pharmacopeia of blood-sucking arthropods.

Inhibition of mosquito salivary gland apyrase activity by antibodies produced in mice immunized by bites of Anopheles stephensi mosquitoes.

Mice (BALB/c) were immunized to mosquito saliva by repeated bites of Anopheles stephensi mosquitoes. Studies were conducted on the ability of these mice to develop antibodies against the apyrase component of the saliva. By means of immunoprecipitation procedures and Western blot analysis, we demonstrated the presence of antiapyrase antibodies to the mosquito saliva. Furthermore, these antibodies were able to inhibit apyrase activity. Serum titers of 1:20 were able to inhibit approximately 90% of salivary gland apyrase activity, while titers of 1:160 retained the ability to inhibit more than 50% of apyrase activity. Parallel inhibition assays with immunoglobulin G (IgG) from immunized versus nonimmunized mice showed that the inhibitory activity of serum from immunized mice could be accounted for by its IgG component. Mosquito salivary gland apyrase has previously been shown to facilitate mosquito feeding by inhibiting aggregation of platelets at the mosquito bite site. However, our studies have shown that mosquitoes feeding on immunized mice had no deficiency in probing these mice for a blood meal, even in the face of high titers of anti-apyrase antibodies.

Apyrase and anti-platelet activities from the salivary glands of the bed bug Cimex lectularius

Apyrase activity was detected in the salivary gland of Cimex lectularius. Kinetic, HPLC chromatography, isoelectric focusing gel electrophoresis and thermal sensitivity assays indicated that the enzymatic activity is the result of a true apyrase and not the result of independent or combined different enzyme activities. The apyrase activity was dependent on calcium and not on magnesium, manganese or zinc, and was inhibited in the presence of EDTA. The pH optimum for both, ATPase and ADPase activities was 8.5. A high specific activity of Cimex salivary apyrase for hydrolysis of both ADP and ATP was found. Cimex salivary gland apyrase activity decreased by 43 percent right after insects fed on blood as compared with insects before feeding. The apparent molecular mass of apyrase activity as determined by HPLC molecular sieving chromatography was 79 600 daltons. Additionally, a peak showing apyrase activity was detected with a molecular mass of 21 000. However, the small molecular mass activity shifted to a high molecular mass activity when the salivary gland homogenate was prepared in more alkaline buffer and frozen, thus suggesting that the high molecular weight form of Cimex apyrase may result from aggregation of active small units. The saliva of Cimex lectularius inhibited platelet aggregation induced by ADP; one salivary gland pair was sufficient to completely inhibit the aggregation of 0.1 ml of citrated platelet rich plasma induced by 5 μM ADP. The inhibitor was shown to be a component different than nitric oxide. Altogether the data suggest that Cimex salivary apyrase can act to prevent an important step on hemostasis, platelet aggregation, and thus play an important role during the insect feeding to overcome the vertebrate host hemostatic defense mechanism.

Salivary apyrase in New World blackflies (Diptera: Simuliidae) and its relationship to onchocerciasis vector status.

Salivary gland apyrase is believed to be critical to blood-feeding in arthropod vectors. This enzyme was measured in six New World blackflies representing three taxonomic pairs of non-vectors and vectors of Onchocerca volvulus. In Simulium (Psilopelmia) ochraceum, a highly anthropophilic vector in Mexico and Guatemala, apyrase exhibited maximum activity between pH 8.0 and 9.0, mean 39.8 +/- 4.7 milliUnits/pair of gland equivalents (mU), and was enhanced when ATP was used as a substrate. In the zoophilic non-vector Simulium (Psilopelmia) bivittatum maximum activity was significantly less (5.1 +/- 0.7 mU) under all conditions examined. Preference for ADP or ATP as substrate was a function of the pH of the reaction for this species. Apyrase activity in Simulium (Simulium) metallicum Bellardi (29.5 +/- 11.5 mU), a zoophilic secondary vector in Mexico and Guatemala, resembled that of S. (Ps.) ochraceum (24.8 +/- 13.7 mU at pH 8.5) with ADP as substrate, but showed reduced activity with ATP. Both these Central American vectors had higher apyrase activity than found in Simulium (Notolepria) exiguum, a vector of O. volvulus in Ecuador and Colombia. However, maximum apyrase activity, measured at pH 8.0 with ADP as substrate, was greater in S. (N.) exiguum (10.9 +/- 0.6 mU) than in Simulium (Notolepria) gonzalezi (5.9 +/- 1.9 mU), a non-vector species widespread in Central America. Therefore, for the consubgeneric species pairs examined, a positive association was detected between higher concentrations of apyrase activity and their vector status for O.volvulus.

The salivary gland-specific apyrase of the mosquito Aedes aegypti is a member of the 5'-nucleotidase family.

The saliva of hematophagous insects contains a variety of pharmacologically active substances that counteract the normal hemostatic response to injury in vertebrate hosts. The yellow-fever mosquito, Aedes aegypti, secretes an apyrase that inhibits ADP-dependent platelet aggregation. Apyrase was purified as an active enzyme from adult female salivary glands and subjected to tryptic digestion, and the resulting peptides were sequenced. The amino acid sequences obtained match the conceptual translation product of a cDNA clone isolated from an adult female salivary gland library. Sequence comparisons indicate similarities with a ubiquitous family of 5'-nucleotidases. The mosquito protein differs from other members of the family by lacking a carboxyl-terminal hydrophobic domain. The apparent conversion of a gene encoding an enzyme involved in a common metabolic event at the cellular level to a gene involved in the antihemostatic response of mosquitoes illustrates one way this particular insect has adapted to the challenges of bloodfeeding.

Salivary Apyrase in African and New World Vectors of Plasmodium Species and its Relationship to Malaria Transmission

The salivary gland activities of apyrase, an enzyme that prevents platelet aggregation by eliminating ADP, were compared among five members of the Anopheles gambiae species complex and An. albimanus. Within the An. gambiae group, An. quadriannulatus exhibited the lowest amount of enzyme activity at all pH levels measured. Apyrase activity could be separated into three groups at pH 7.5 and 8.0. The two most anthropophilic species (An. gambiae and An. arabiensis) exhibited higher activity at pH 9.0. Anopheles merus and An. melas, both saltwater taxa, and An. albimanus, a New World species, exhibited peak apyrase activity at pH 8.0. When the effects of divalent cations (Ca++, Mg++) on enzyme activity were compared at pH 8.5, apyrase activity in the presence of Mg++ could be separated into three levels. Anopheles gambiae and An. quadriannulatus exhibited reduced activity in the presence of Mg++. Anopheles arabiensis, An. merus, and An. melas displayed the highest relative levels of activity. Anopheles albimanus, with a Mg:Ca ratio of 0.80, was most similar to An. arabiensis. These biochemical differences suggest that different isoenzymes of apyrase have developed within the genus Anopheles.

Salivary gland apyrase in black flies ( Simulium vittatum)

Apyrase enzyme activity was demonstrated in the salivary glands of a colonized strain of Simulium vittatum. Activity was maximum (8.5 ± 0.7 mU/pair of gland equivalents) at pH 8.0, with ADP as substrate and Ca 2+ as the divalent cation. Activity was minimal in newly emerged females (1.6 ± 0.5 mU/pair of gland equivalents) but increased by 48 h. Activity in male salivary glands was marginally detectable (0.7 ± 0.8 mU/pair of gland equivalents), even 72 h after emergence. When newly emerged females were maintained at 4°C, salivary apyrase activity accumulated at a slow rate. Transferring females to warmer temperatures increased the rate of apyrase accumulation, but 27°C did not yield greater activity than 20°C. Apyrase activity was decreased when females engorged on whole bovine blood or on a simulated blood meal. Activity remained low 6 h after feeding, but increased to prefeeding levels by 48 h. During the second, anautogenous gonotrophic cycle, apyrase activity was not greater than during the first, autogenous gonotrophic cycle. Apyrase activity was not related to long term colonization as total salivary gland apyrase activity and pH profile in wild S. vittatum was not different from colonized S. vittatum.

Blood vessel location time by Anopheles stephensi (Diptera: Culicidae).

Probing behavior of Anopheles stephensi Liston is characterized by the following: the first probe is longer than subsequent ones, the probability of blood location rises initially and then falls, and blood vessel location is deterministic. The overall probing behavior of An. stephensi, therefore, is similar to that of Aedes aegypti (L.); i.e., differences between them were quantitative and may be accounted for by different levels of salivary apyrase and different experimental vertebrate hosts.

Saliva of the soft tick, ornithodoros moubata, contains anti-platelet and apyrase activities

1. 1. Pilocarpine-induced saliva of the soft tick Ornithodoros moubata inhibits platelet aggregation induced by ADP or collagen, even when diluted 2000 times into platelet rich plasma. 2. 2. Saliva contains apyrase (ATP-diphosphohydrolase) activity, which has an optimal pH of 7.0 for ADP and of 8.0 for ATP hydrolysis, respectively. Both Ca 2+ and Mg 2+ activate the reactions. 3. 3. The mean specific activities for ATP and ADP hydrolysis at pH7.5 were 0.97 and 0.74/moles orthophosphate/min/mg protein. 4. 4. These results, which demonstrate for the first time such activities in the saliva of soft ticks, support the hypothesis that the saliva of blood sucking arthropods serves an anti-hemostatic role during feeding and that large amounts of salivary apyrase activity have evolved independently in hematophagous arthropods.

Characterization of the salivary apyrase activity of three rodent flea species

1. 1. Salivary gland lysates of the adult female fleas Oropsylla bacchi, Orchopea howardi and Xenopsylla cheopis hydrolyse ATP and ADP, but not AMP, thus characterizing the existence of a salivary apyrase activity. 2. 2. In all species Mg ++ or Ca ++ function as activators, and a pH optimum between 7 and 8 is observed. 3. 3. Salivary gland lysates of male fleas contain significantly smaller amounts of the enzyme activity than do those of female fleas. 4. 4. Immediately following a blood meal, apyrase activity and protein content of female X, cheopis salivary glands are 2–3-fold less than that of unfed fleas, indicating that salivary apyrase activity is secreted during feeding. 5. 5. It is suggested that, as in other arthopods, salivary apyrase may facilitate blood location and blood feeding by preventing ADP-induced platelet aggregation at the site of the bite.

Diet and salivation in female Aedes aegypti mosquitoes

Salivary glands of the female mosquito, Aedes aegypti, contain an α-glucosidase in their proximal lobes and an apyrase in the distal lobes. Following a liquid sugar meal, there is a significant reduction in the activity of the salivary glucosidase, but no significant reduction in the activity of the apyrase. Following a blood meal, both enzyme activities are significantly reduced. The rate of accumulation of both enzymes after a blood meal differs. The glucosidase activity returns to nonfed control values within 1 h of the blood meal and increases to 3 times the control values after 2 days. Salivary apyrase activity remains significantly lower than control values for up 3 h after the blood meal, reaches control values by 24 h and becomes significantly (30%) higher than control values 2 days after the blood meal. Total salivary gland protein levels remain significantly lower than control values for up to 3 h after a blood meal and return to control values at 24 h. A neuro-humoral model is postulated to explain the results.

Immunoreactivity of salivary gland apyrase of Aedes aegypti with antibodies against a similar hydrolase present in the pancreas of mammals

Eucaryotes have the ubiquitous enzyme apyrase or ATP diphosphohydrolase, known to catalyze the hydrolysis of the α and β phosphate groups of di- and triphosphonucleosides. In hematophagous arthropods, the salivary glands are the main source of this enzyme that helps the insects to locate blood in hosts by preventing platelet aggregation. A structural comparison between mosquito salivary gland and pig pancreas apyrase was performed using immunoblot analysis with specific polyclonal antibodies raised to the pancreatic enzyme. Strong reactivity was observed with a polypeptide of mol. wt close to 60 kDa in the Aedes aegypti salivary glands, providing evidence for structural homology between the apyrase present in vertebrates and invertebrates.

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

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

Salivary apyrase activity of some old world phlebotomine sand flies

Salivary gland homogenates of three Old World phlebotomine sand flies ( Phlebotomus papatasi, P. argentipes and P. perniciosus) contained abundant ATPase and ADPase activities, indicating the presence of an apyrase activity. These activities had an optimum pH around 8.0 and were activated by Ca 2+ but not Mg 2+. Both hydrolytic activities and salivary protein content were significantly reduced after the female sand fly took a blood meal indicating a secretory fate for the enzymic activities and salivary gland contents during the feeding process. In contrast to the above mentioned species, the salivary apyrase activity of P. colabaensis is much less abundant. Salivary gland homogenates of P. papatasi, P. argentipes and P. perniciosus inhibited ADP-induced platelet aggregation of citrated rabbit platelet rich plasma. It is suggested that salivary apyrase activity, as in some other blood-sucking arthropods, helps the blood-feeding process by preventing host platelet aggregation.