The calcium activated nucleotidases: A diverse family of soluble and membrane associated nucleotide hydrolyzing enzymes
- First Online:
- Cite this article as:
- Smith, T.M. & Kirley, T.L. Purinergic Signalling (2006) 2: 327. doi:10.1007/s11302-005-5300-7
- 274 Downloads
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.
Key wordsapyraseblood-feeding insectscalciumnucleotidasesite-directed mutagenesisstructural determination
calcium activated nucleotidase
expressed sequence tag
nucleotidase conserved region
nucleoside triphosphate diphosphohydrolase
soluble calcium activated nucleotidase
Overview of the ‘soluble’ apyrase family
Hemostasis (the prevention of blood loss) is a vertebrate process with three main components: The plasma protein clotting factor cascade; vasoconstriction; and platelet aggregation. To facilitate feeding, hematophagus (blood-feeding) arthropods such as fleas , ticks , biting midges , and sand flies [4, 5] have evolved mechanisms to disarm all three components and thereby inhibit hemostasis in their hosts. Contained within the saliva of such blood-feeding organisms are a variety of potent bioactive compounds, including anticoagulants, vasodilators, and antiplatelet factors. For recent reviews on the antihemostatic strategies of blood-feeding arthropods, see [6–8].
We are particularly interested in one mechanism utilized by these blood-feeding organisms to inhibit hemostasis: The injection of soluble salivary apyrases (ATP and ADP hydrolyzing enzymes, EC 188.8.131.52) at the site of host skin puncture. These apyrases are members of a family of calcium-dependent proteins that rapidly hydrolyze extracellular nucleotides such as adenosine tri- and diphosphate . Lacerations produced by the arthropod’s mouth parts whilst probing for a blood vessel in the host likely expose platelets to copious amounts of extracellular ADP, which is released from injured cells and from the dense granules of activated platelets. ADP represents one of the most important physiological agonists for platelet recruitment, aggregation and plug formation via activation of platelet purinergic receptors (recently reviewed in [10–13]), and the apyrases facilitate the blood-meal acquisition for these organisms by inhibiting the ADP-mediated activation of host platelets through an increase in ADP catabolism .
Cloning, expression and enzymatic characterization of the vertebrate homologues of the arthropod salivary apyrases
The same year that the characterization of the rat homologue was reported, Smith et al. described the cloning and expression of the human homologue of the bed bug apyrase (first referred to as SCAN-1, for soluble calcium activated nucleotidase-1 ). When expressed in COS-1 cells, many similarities were found to the rat nucleotidase, including the strict requirement for calcium for activity, a pH optimum around 7, and a nucleotide preference of UDP > GDP ≫ ADP, with no detectable hydrolysis of nucleoside monophosphates. Unlike the expressed rat enzyme however, which was shown to be membranebound , the human enzyme that was characterized was a soluble protein found in the conditioned media of transfected cells, likely produced by cleavage at the predicted signal peptide cleavage site (see Figure 2) .
Further enzymatic characterization of the human nucleotidase was performed by Murphy et al. . Those investigators expressed the soluble portion of the human enzyme in E. coli, and refolded the enzyme from the resultant bacterial inclusion bodies. The bacterially expressed human apyrase was similar in enzymatic properties to the mammalian cell produced soluble form, indicating that a transmembrane domain and post-translational modifications that do not occur in bacteria (such as glycosylation) are not necessary for proper folding of the protein or nucleotidase activity. Many divalent cation chloride salts were tested (CaCl2, MgCl2, MnCl2, SrCl2, CuCl2, CoCl2, and NiCl2), but only calcium was able to support nucleotide hydrolysis. Interestingly, those authors also found that the human protein was a calcium-binding protein (as detected by changes in protein absorbance, fluorescence, and activity as a function of calcium concentration) with a calcium-triggered transition from an enzymatically inactive to an active form with halfsaturation at about 90 µM CaCl2 at low ionic strength .
Another important characteristic of the human calcium-activated nucleotidase that distinguishes it from other unrelated nucleotidases such as the naturally occurring soluble forms of human NTPDases (i.e., NTPDase5 and NTPDase6) is its remarkable stability. For example, Valenzuela et al.  reported that the P. papatasi apyrase was successfully renatured after SDS-PAGE, while the bed bug apyrase was resistant to denaturation by the 0.1% trifluoroacetic acid and acetonitrile used for purification by reverse-phase chromatography . The wildtype human enzyme has been reported to be stable for over 24 h at 37°C in the presence of calcium, and for more than one week at room temperature . Indeed, the enzymatic activity of this human CAN protein is retained for at least six months when stored in buffer at 4°C under sterile conditions (T.M. Smith, personal observations). Thus, the human calcium activated nucleotidase is considerably more stable than the naturally occurring soluble NTPDase apyrases, NTPDase5 and NTPDase6 (T.L. Kirley, personal observations). In fact, in stark contrast to the human CAN, purified NTPDase5 (also known as CD39L4) was noted to be unstable and prone to aggregation .
Mutagenesis and crystallographic analysis of the human calcium activated nucleotidase
Extensive mutagenesis studies have recently been performed on the human enzyme in an effort to evaluate key amino acids in this family [18, 20]. Yang and Kirley  used computational analysis of multiple sequence alignments of invertebrate and vertebrate members of the apyrase family to identify eight highly conserved regions of amino acids. The conserved regions were designated as nucleotidase conserved regions 1–8 (NCR 1–8) and they were used to direct a mutagenesis approach to identify amino acids important for enzymatic function. Four amino acids (Asp or Arg residues) contained in the NCRs were mutated to alanine and, consistent with their degree of conservation, all four mutations had substantial detrimental effects on the enzyme activity. Several of these mutants failed to bind the nucleotide analog Cibacron blue and were sensitive to limited tryptic digestion, suggesting alterations in their nucleotide binding pockets and/or their ability to bind calcium and undergo the calcium-induced conformational change. One mutation in the human enzyme was reported (E130Y), which was targeted for mutagenesis due to differences found between the sequences of the vertebrate and invertebrate sequences at that residue (Figure 2). This E130Y mutation increased GDPase activity two-fold and ADPase activity five-fold, which led the authors to suggest that this amino acid was important for determining the nucleotidase specificity for this family.
Interestingly, it was found that either CaCl2 or SrCl2 could induce the conformational change detected by the increases in tryptophan fluorescence, confer resistance to proteolysis, and provide thermal stability of the enzyme at 37°C, although only calcium could support nucleotide hydrolysis . This suggested that calcium plays an important role not only as a nucleotide co-substrate, but also for maintaining the conformation and structural stability of the enzyme.
The platelet is a major contributor to occlusive thrombus formation in acute coronary syndromes and stroke in humans, which are the primary causes of death and disability in the industrialized world. The major platelet inhibitors currently approved for use in thrombotic disorders by definition target the platelet itself, with a predominant side effect often being bleeding complications (reviewed in [22–24]). Achieving a potent anti-thrombotic effect without undermining hemostasis would be a ‘magic bullet’ against arterial thrombosis , thus new approaches for anti-platelet therapy utilizing soluble apyrases are being examined. One potential advantage of soluble nucleotidases that preferentially hydrolyze adenosine-based nucleotides is that they remove one of the major factors of the platelet release reaction (ADP), effectively limiting further platelet aggregation in a manner that does not involve binding to the platelet surface or disruption of intracellular protein machinery. Thus, serious bleeding complications may be minimized using this approach. It is important to point out however, that the vertebrate CAN proteins described in this review are primarily UDP/GDP hydrolyzing enzymes, with little endogenous ADPase activity (hence, their physiological function likely does not involve control of hemostasis). However, the bloodfeeding arthropod enzymes and also the HB mutant constructed by Dai et al.  are potent ADPases, capable of preventing ADP-mediated platelet aggregation.
There have been several recent reviews on the utilization of ADP hydrolyzing enzymes (usually a soluble derivative of the endothelial cell CD39/NTPDase1 apyrase, unrelated to the calcium activated nucleotidase (CAN) family members described in this report) in occlusive vascular disorders [25–30]. The therapeutic utility of these apyrases has been demonstrated in a variety of cardio- and cerebrovascular disorders, including middle cerebral artery occlusion [31, 32], endothelial denudation-induced arterial thrombosis , ATP and ischemia-induced norepinephrine release from heart synaptosomes , intestinal ischemia-reperfusion injury , arterial balloon injury , and cardiac xenograft transplantation [36, 37]. Hence, the unique anti-platelet actions of the ADP hydrolyzing enzymes make them particularly attractive as soluble protein therapeutics for the inhibition of platelet-mediated thrombotic pathologies.
Summary and conclusions
In this review, we have summarized the characterization of a distinct new family of nucleotidases with representative members found in both vertebrates and invertebrate bloodfeeding arthropods. Due to the strict dependency on calcium reported for all characterized family members to date, the designation CAN (calcium activated nucleotidase) has been proposed for this family. Enzymatic studies of the proteins isolated from different species have made it very clear however that the invertebrate and vertebrate members of this family do not share common nucleotide substrate preferences, presumably related to their distinct physiological functions. It is also interesting to note that the amino acid sequences of this novel family of enzymes have no homology to the NTPDase family of nucleotidases (e.g., CD39/NTPDase1) and their ‘apyrase conserved regions’ (ACRs ). Thus, the CANs are enzymatically analogous, rather than homologous in sequence, to the NTPDases, performing similar enzymatic functions despite their evolutionary distance. The differences in amino acid sequences between these two families of nucleotidases are also reflected in dramatic differences in their determined/predicted tertiary structures (see the article on NTPDase3 mutagenesis and structure by Kirley et al. appearing in this volume). Thus, we extend the observation made by Valenzuela et al. , that the CAN and the NTPDase families of nucleotide hydrolyzing enzymes are the product of convergent evolution, having evolved proteins of similar biochemical function from unrelated genes, which give rise to unrelated 3-D protein structures.
This work was partially supported by Wyeth Discovery Research (T.M.S.) and by NIH grants HL59915 and HL72882 to T.L.K.