Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
Cloning, sequencing, and expression of a human brain ecto-apyrase related to both the ecto-ATPases and CD39 ecto-apyrases1
Introduction
E-type ATPases (consisting of the ecto-ATPases and ecto-ATPDases or apyrases) are a class of ecto-enzymes that have the following characteristics: (1) a highly active nucleotide hydrolysis site situated on the exterior of the cell, (2) a glycosylated catalytic subunit, (3) a strict dependence upon divalent cations (usually calcium or magnesium) for activity, (4) a general insensitivity to the specific inhibitors of the P-, F-, and V-type ATPases, and (5) an ability to hydrolyze a wide range of purine and pyrimidine nucleoside tri- and diphosphates [1, 2]. The ecto-ATPases may be further distinguished from the ecto-apyrases by the fact that they only utilize nucleoside triphosphates as substrates and are insensitive to the inhibitory effects of sodium azide [1]. In contrast, the apyrases can hydrolyze both nucleoside tri- and diphosphates and are substantially inhibited by 10–20 mM sodium azide [3, 4, 5, 6].
The E-type ATPases have been described in a wide range of tissues as both soluble [7, 8, 9] and membrane-associated proteins [6, 10, 11, 12] (for a review, see Plesner [1]). Much research has recently been devoted to the study of both the soluble and membrane-associated E-type ATPases of excitable nervous tissue [13, 14, 15, 16] since extracellular ATP has been well established as a neurotransmitter in the nervous system [17, 18], and several classes of nucleotide purinoceptors (designated P2) have been discovered [19] that serve as the receptors for these nucleotide ligands. It has been postulated that E-type ATPases could serve a functional role in this regard by regulating the concentrations of agonists and antagonists around the sites of the purinoceptors [20]. These effects could be either direct, such as the conversion of an agonist to an inactive compound, or indirect, such as the conversion of an agonist to a secondary compound that can subsequently activate or inhibit other receptors or enzymes associated with the system. For example, in lobster olfactory cells, extracellular ATP may act as an attractant, directing the creature toward a putative food source [21]. Dephosphorylation of the nucleotide by an E-type ATPase alone, or in concert with other ecto-nucleotidases, could serve to clear the stimulus from the receptor environment or generate an inhibitory signal (e.g. adenosine) [22]. E-type ATPase activity has also been found intimately associated with a neural cell adhesion molecule (NCAM) of nervous tissue [23, 24], suggesting the possibility of involvement of similar ecto-enzymes in cell adhesion functions.
Recently, a CD39-like gene (CD39L1) was isolated from human tissue [25] that showed great sequence similarity on the amino acid level to the previously described chicken smooth muscle ecto-ATPase antigen [26] and human CD39 lymphoid cell activation antigen [11]. However, no protein expression of the cDNA clone was performed to analyze the biochemical characteristics of that putative enzyme to confirm that the gene isolated actually encodes a human ecto-ATPase. We have cloned and sequenced a different E-type ATPase gene present in human brain and have studied the expressed protein product in mammalian tissue culture cells. Enzymatic characterization shows that the expressed protein functions as an ecto-apyrase. Analysis of the deduced amino acid sequence, however, suggests that this enzyme is equally related to both the ecto-ATPases and ecto-apyrases and could perhaps be a progenitor from which these members were derived. This report describes the discovery and expression of this novel human brain E-type ATPase, and the possibility that the enzyme may be regulated by cytoplasmic phosphorylation from either cAMP- and cGMP-dependent protein kinase or protein kinase C.
Section snippets
Materials
The GeneTrapper cDNA positive selection system kit and human brain pCMV-SPORT cDNA library, generated from a 36-year-old Caucasian female, were obtained from Life Technologies. Synthetic oligonucleotides were produced at the DNA Core Facility at the University of Cincinnati. The 3′-oligo labeling system and the ECL detection reagents were from Amersham. Sequenase Version 2.0 kits were obtained from US Biochemical Corp. Epicurian coli ultracompetent bacteria were purchased from Stratagene.
Cloning and sequencing of the human brain E-type ATPase
A 2809 base cDNA clone (designated HB6) was isolated from a human brain cDNA library through the use of the GeneTrapper cDNA positive selection system and a biotinylated oligonucleotide primer designed from a putative human E-type ATPase cDNA. The HB6 insert was fully sequenced from both the 3′ and 5′ directions revealing an open reading frame extending from nucleotide 80 to 1666 (Fig. 1), with 1143 bases of 3′ untranslated sequence. The amino acid sequence deduced from this cDNA is a 529 amino
Discussion
This paper reports the cloning, sequencing, and expression of a human brain cDNA belonging to the class of enzymes known as the E-type ATPases. This was accomplished through the use of the GeneTrapper ‘affinity enrichment’ technique of screening a human brain cDNA library with an oligonucleotide probe designed against a partial putative E-type ATPase cDNA clone obtained from human endometrial tissue. The isolated cDNA consisted of 2809 bases (Fig. 1) with an open reading frame coding for a
Acknowledgements
The authors would like to thank Drs. Lois Lane for supplies and technical support in mammalian cell tissue culture and expression. This work was supported by Grant 96013960 from the American Heart Association (to T.L.K.).
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The nucleotide sequence reported in this paper has been submitted to the GenBank database and assigned Accession No. AF034840.