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

Abstract

The protein family of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDase family) contains multiple members that hydrolyze nucleoside 5'-triphosphates and nucleoside 5'-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.

Figure 1

DNA sequence and predicted protein sequence of rat NTPDase3. The five ‘apyrase conserved regions’ (ACR) are indicated by boxes and numbered. Cysteine residues and potential N-glycosylation sites are indicated by arrow heads and filled circles, respectively. Predicted N- and Cterminal hydrophobic sequences are shaded. Amino acid residues conserved from NTPDase1 and NTPDase8 are in bold. In ACR 1 and ACR 4 these include the DXG-containing phosphate-binding motif and a conserved glycine in ACR 5. (GenBank accession number AJ437217.)

Figure 2

Chromosomal localization of rat NTPDase3 and intron-exon structure. The upper part of the figure depicts the position of the NTPDase3 gene in chromosome 8q32. The lower part enlarges the genomic sequence of NTPDase3. Upper line: Exons are numbered and indicated by black boxes in relation to the length of the encoded sequences (gDNA fragment = 31 kb). The lower line indicates the length of individual exons. The ORF is shaded. The position of ACRs 1 to 5 is indicated by white boxes, that of the two putative transmembrane domains by black boxes.

Figure 4

Alignment of potential N-glycosylation sites and of cysteine residues in surface-located NTPDases. All sequences from Rattus norvegicus. Potential N-glycosylation sites are indicated by arrow heads, cysteine residues by arrows, and the five ‘apyrase conserved regions’ (ACR) by shading. The asterisk marks the only potential glycosylation site conserved between NTPDase1, NTPDase2 and NTPDase3. Predicted N- and C-terminal hydrophobic sequences are indicated by black boxes. For GenBank accession numbers see legend of Figure 3.

Figure 3

Hypothetical phylogenetic tree derived for 22 selected members of the E-NTPDase family (NTPDase1 to NTPDase8) from rat (r), human (h) and mouse (m). Amino acid sequences were aligned using ClustalX 1.81 and the dendrogram was prepared using TreeView 1.6.6. The length of the lines indicates the differences between amino acid sequences. The graph depicts a clear separation between intracellular (top) and surface-located NTPDases (bottom). Protein sequences are derived from mRNA sequences. The GenBank accession numbers of the sequences are as follows: (NTPDase1, Homo sapiens (S73183), Mus musculus (AF037366), Rattus norvegicus (U81295); NTPDase2, Homo sapiens (U91510), Mus musculus (AF042811), Rattus norvegicus (Y11835); NTPDase3, Homo sapiens (AF034840), Mus musculus (AY376710), Rattus norvegicus (AJ437217); NTPDase4, Homo sapiens (AF016032), Mus musculus (AK004761); NTPDase5, Homo sapiens (AF039918), Mus musculus (AF006482), Rattus norvegicus (BC62044); NTPDase6, Homo sapiens (AF039916), Mus musculus (NM_172117), Rattus norvegicus (AJ277748); NTPDase7, Homo sapiens (AK055540), Mus musculus (AF288221); NTPDase8, Homo sapiens (AY430414), Mus musculus (AY364442), Rattus norvegicus (AY536920).

Figure 5

Comparison of the secondary structure conserved between members of the actin/HSP70/sugar kinase superfamily (as depicted for rabbit actin, Swiss-Prot accession P29751) with the predicted secondary structure of the E-NTPDase family members. Secondary structure prediction was performed with the SSpro tool. α-Helices are indicated by black boxes, β-strands by arrows and putative transmembrane domains by white boxes. ACRs 1 to 5 are indicated by shading. The alignment of the first domain is oriented around the DXG motif in ACR 1 of NTPDases and the corresponding motif in actin. Correspondingly, the second domain is aligned to the DXG motif in ACR 4 and the corresponding motif in actin. In order to emphasize the repetition in topology, the sequence has been interrupted by dots. The conserved a-helices and b-strands in actin are consecutively numbered for each of the two domains. All NTPDase sequences are from rat with the exception of NTPDase4 and NTPDase7 which are from mouse. For GenBank accession numbers see legend of Figure 3.

Figure 6

Ion dependence of NTPDase3 catalytic rate. ATPase (top) or ADPase (bottom) activity of membrane fractions derived from transfected CHO cells was analyzed in the presence of increasing concentrations of Ca²⁺ (closed symbol) or Mg²⁺ (open symbol) (500 µM ATP or ADP). The 100% values (± S.D., n = 3) correspond to 452 ± 23 and 89 ± 2 nmol/min/mg protein for ATPase and ADPase activity, respectively.

Figure 7

pH dependence of NTPDase3 catalytic rate. ATPase activity (500 µM ATP) of membrane fractions derived from transfected CHO cells was analyzed at varying pH in the presence of either Ca²⁺ (closed symbol) or Mg²⁺ (open symbol) (500 µM each). The 100% values ( ± T S.D., n = 3) correspond to 641 ± 602 and 370 ± 339 nmol/min/mg protein for Ca²⁺- and Mg²⁺-ATPase activity, respectively.

Figure 8

ATP hydrolysis and product formation. ATPase activity (250 µM ATP) of membrane fractions derived from transfected CHO cells was analyzed at varying time points in the presence of Ca²⁺ (250 µM). ATP hydrolysis and product formation was monitored by HPLC. The contribution of each nucleotide is expressed as percentage of total nucleotides present in the sample. Values of 100% correspond to the initial amount of ATP substrate. Values represent means ± SD of three experiments. Equal amounts of starting ATPase activity (7.9 nmol Pi/min) were employed for each assay.

Figure 9

Immunolocalization of NTPDase3. a–d: Viable CHO cells were incubated with the anti-NTPDase3 antibody two days after transfection with the cDNA encoding the NTPDase indicated at the left or with the empty vector (mock). Antibody binding was visualized by immunofluorescence (left column). Total cells are depicted by DAPI staining of the nuclei (right column). The anti-NTPDase3 antibody bound only to NTPDase3-transfected cells (d). e: PC12 cells were cultured for 14 days and surface-located immunoreactivity for NTPDase3 was analyzed as for CHO cells. Arrow heads depict an accumulation of immunoreactivity at growth cones. Bars = 10 µm.

Figure 10

Western blot analysis of NTPDase3 in transfected CHO cells and brain tissue. CHO cells were transfected with NTPDase3 two days before analysis. The protein amount of ConA-purified membrane fraction loaded per lane corresponded to 25 mg of starting tissue for hippocampus, mesencephalon, diencephalon, striatum and spinal cord, to 10 mg for total brain and 3.3 mg for pituitary.