Conserved lysine 79 is important for activity of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3)

The protein family of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDase family) contains multiple members that hydrolyze nucleoside 5 0 -triphosphates and nucleoside 5 0 -diphosphates with varying preference for the individual type of nucleotide. We report the cloning and functional expression of rat NTPDase3. The rat brain-derived cDNA has an open reading frame of 1590 bp encoding 529 amino acid residues, a calculated molecular mass of 59.1 kDa and predicted N-and C-terminal hydrophobic sequences. It shares 94.3% and 81.7% amino acid identity with the mouse and human NTPDase3, respectively, and is more closely related to cell surface-located than to the intracellularly located members of the enzyme family. The NTPDase3 gene is allocated to chromosome 8q32 and organized into 11 exons. Rat NTPDase3 expressed in CHO cells hydrolyzed both nucleoside triphosphates and nucleoside diphosphates with hydrolysis ratios of ATP:ADP of 5:1 and UTP:UDP of 8:1. After addition of ATP, ADP is formed as an intermediate product that is further hydrolyzed to AMP. The enzyme is preferentially activated by Ca 2+ over Mg 2+ and reveals an alkaline pH optimum. Immunocytochemistry confirmed expression of heterologously expressed NTPDase3 to the surface of CHO cells. PC12 cells express endogenous surface-located NTPDase3. An immunoblot analysis detects NTPDase3 in all rat brain regions investigated. An alignment of the secondary structure domains of actin conserved within the actin/HSP70/sugar kinase superfamily to those of all members of the NTPDase family reveals apparent similarity. It infers that NTPDases share the two-domain structure with members of this enzyme superfamily.

Section: Discussionmentioning

confidence: 99%

Cloning and characterization of the ecto-nucleotidase NTPDase3 from rat brain: Predicted secondary structure and relation to other members of the E-NTPDase family and actin

Vorhoff

Zimmermann

Pelletier

et al. 2005

“…The corresponding lysine in human NTPDase3 is K79 and similar results were obtained with the K79A and K79R mutants. Basu et al suggested that a positively charged amino acid in the vicinity of the conserved glycosylation site (N81) of human NTPDase3 is necessary for ConA binding [198]. The effect of mutating this conserved asparagine was studied in three enzymes: rat NTPDase1 [42], human NTPDase2 [43], and human NTPDase3 [40].…”

Section: Mutagenesis Studies Of Cell Surface Ntpdasesmentioning

confidence: 99%

The GDA1_CD39 superfamily: NTPDases with diverse functions

Knowles

2011

145

117

The first comprehensive review of the ubiquitous "ecto-ATPases" by Plesner was published in 1995. A year later, a lymphoid cell activation antigen, CD39, that had been cloned previously, was shown to be an ecto-ATPase. A family of proteins, related to CD39 and a yeast GDPase, all containing the canonical apyrase conserved regions in their polypeptides, soon started to expand. They are now recognized as members of the GDA1_CD39 protein family. Because proteins in this family hydrolyze nucleoside triphosphates and diphosphates, a unifying nomenclature, nucleoside triphosphate diphopshohydrolases (NTPDases), was established in 2000. Membrane-bound NTPDases are either located on the cell surface or membranes of intracellular organelles. Soluble NTPDases exist in the cytosol and may be secreted. In the last 15 years, molecular cloning and functional expression have facilitated biochemical characterization of NTPDases of many organisms, culminating in the recent structural determination of the ecto-domain of a mammalian cell surface NTPDase and a bacterial NTPDase. The first goal of this review is to summarize the biochemical, mutagenesis, and structural studies of the NTPDases. Because of their ability in hydrolyzing extracellular nucleotides, the mammalian cell surface NTPDases (the ecto-NTPDases) which regulate purinergic signaling have received the most attention. Less appreciated are the functions of intracellular NTPDases and NTPDases of other organisms, e.g., bacteria, parasites, Drosophila, plants, etc. The second goal of this review is to summarize recent findings which demonstrate the involvement of the NTPDases in multiple and diverse physiological processes: pathogen-host interaction, plant growth, eukaryote cell protein and lipid glycosylation, eye development, and oncogenesis.

“…The importance of lysine residue 79 located near the conserved glycosylation site near ACR1 was investigated [14]. This residue is conserved in cell surface NTPDases (NTPDase1–3, 8), but not in the soluble (NTPDase5–6) and intracellular (NTPDase4, 7) members of this nucleotidase family (see Table 1 in [14]).…”

Section: Site-directed Mutagenesis Studies (Point Mutations) Of Ntpdasesmentioning

confidence: 99%

“…This residue is conserved in cell surface NTPDases (NTPDase1–3, 8), but not in the soluble (NTPDase5–6) and intracellular (NTPDase4, 7) members of this nucleotidase family (see Table 1 in [14]). All substitutions at this site resulted in a loss of 70%–80% of activity.…”

Section: Site-directed Mutagenesis Studies (Point Mutations) Of Ntpdasesmentioning

confidence: 99%

The structure of the nucleoside triphosphate diphosphohydrolases (NTPDases) as revealed by mutagenic and computational modeling analyses

Kirley

Crawford

Smith³

2006

Self Cite

Over the last seven years our laboratory has focused on the determination of the structural aspects of nucleoside triphosphate diphosphohydrolases (NTPDases) using site-directed mutagenesis and computational comparative protein modeling to generate hypotheses and models for the hydrolytic site and enzymatic mechanism of the family of NTPDase nucleotidases. This review summarizes these studies utilizing NTPDase3 (also known as CD39L3 and HB6), an NTPDase family member that is intermediate in its characteristics between the more widely distributed and studied NTPDase1 (also known as CD39) and NTPDase2 (also known as CD39L1 and ecto-ATPase) enzymes. Relevant site-directed mutagenesis studies of other NTPDases are also discussed and compared to NTPDase3 results. It is anticipated that many of the results and conclusions reached via studies of NTPDase3 will be relevant to understanding the structure and enzymatic mechanism of all the cellsurface members of this family (NTPDase1Y3, 8), and that understanding these NTPDase enzymes will aid in modulating the many varied processes under purinergic signaling control. This review also integrates the site-directed mutagenesis results with a recent 3-D structural model for the extracellular portion of NTPDases that helps explain the importance of the apyrase conserved regions (ACRs) of the NTPDases. Utilizing this model and published work from Dr Guidotti's laboratory concerning the importance and characteristics of the two transmembrane helices and their movements in response to substrate, we present a speculative cartoon model of the enzymatic mechanism of the membrane-bound NTPDases that integrates movements of the extracellular region required for catalysis with movements of the N-and C-terminal transmembrane helices that are important for control and modulation of enzyme activity.Abbreviations: ACR -apyrase conserved region; Con A -Concanavalin A; DTT -dithiothreitol; Endo H -endoglycosidase H; NTPDase3 -nucleotide triphosphate diphosphohydrolase 3; pCMPS -p-chloromercuriphenylsulfonic acid; PDBprotein data base; PNGase-F -peptide N-glycosidase F; PPX/GPPA -exopolyphosphatase/guanosine pentaphosphate phosphohydrolase; TM -transmembrane Site-directed mutagenesis studies (point mutations) of NTPDasesIn this section site-directed mutagenesis studies introducing point mutations in NTPDases are reviewed. Other manipulations of protein structure including chimeras and truncation mutants of NTPDases are not covered in this review.NTPDase3 studies (see Table 1)Over the last seven years, 10 studies have been published using point mutations introduced by site-directed mutagenesis to investigate the structure and function of human NTPDase3 (see Table 1). What follows is a concise summary of those studies, in chronological order.The first report of site-directed mutagenesis of any NTPDase [1] was important for establishing experimentally what had been hypothesized based on limited sequence homology of the NTPDases with the actin/heat shock protein/sugar kinase superfamily o...