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