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

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Abstract

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.

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Materials

The human SCAN-1 cDNA I.M.A.G.E. clone was obtained from Incyte Genomics (GenBank EST Accession No. AA632390; Clone ID 1131402). The Epicurian coli ultracompetent bacteria were purchased from Stratagene. Plasmid purification kits were purchased from Qiagen. Lipofectamine Plus Reagent, Dulbecco’s modified Eagle’s medium (DMEM), calf serum, and antibiotics/antimycotics were all obtained from Life Technologies. Falcon tissue culture treated plates were from Becton–Dickinson. The mammalian

Identification and analysis of the human SCAN-1 cDNA clone

A human cDNA clone, homologous to the C. lectularius apyrase cDNA [31], was discovered in the human EST GenBank database. Sequencing of this clone revealed an insert of 1691 bases with an open reading frame which extends from nucleotide 492 to 1604, and the amino acid sequence deduced from this cDNA encodes a 371-amino acid protein (Fig. 1). Analyzing the protein sequence using the PSORT II program predicts a cleavable signal peptide at residues 37 and 38 (amino acids GR). Cleavage of this

Discussion

This paper reports the cloning, expression, and analysis of the first human soluble calcium-activated nucleotidase (referred to as SCAN-1) belonging to the blood-feeding arthropod family of apyrases. This was accomplished through the identification of a human EST clone homologous to the apyrases cloned from the salivary glands of the blood-feeding C. lectularius, P. papatasi, and L. longipalpis organisms. The human EST cDNA clone consists of 1691 bases (Fig. 1) with an ATG start codon at

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