Structural Biochemistry/Membrane Proteins/Ecto-nucleoside tri-Phosphate diphosphohydrolase (E-NTPDases)

 Introduction 

E-NTPDases are the main extracellular ATPases involved in the degradation of extracellular ATP. It currently includes six subfamilies, E-NTPDase1 through E-NTPDase6. As Table 1 indicates, all E-NTPDases contain five Apyrase Conserved Regions (ACRs), four Conserved Regions (CRs), several conserved cysteine residues and three to thirteen N-glycosylation sites (1 - 4). Some of the ACR sequences are similar to the β and γ-phosphate binding motifs of actin-hsp70- hexokinase superfamily, suggesting a possible role of ACRs in nucleotide binding (2, 5).



 E-NTPDase Subfamilies 

E-NTPDase currently includes six subfamilies. NTPDase1, 2, 3 and 4 have two short transmembrane domains at the C- and N-termini whereas E-NTPDase5 and 6 lack the C-terminal transmembrane domain. E-NTPDase1, 2, and 3 are expressed on cell surfaces having molecular size of 66 - 80 kDa (4). E-NTPDase4 is expressed in the Golgi and lysosomes depending on cell type (6, 7). E-NTPDase5 is expressed in ER (8), and E-NTPDase6 is expressed in the Golgi apparatus (8, 9). The different E-NTPDases also have different tissue distribution (Table 1). All E-NTPDases require Ca or Mg divalent cations for the nucleotide hydrolysis reaction (4), however, they have different substrate specificities. E-NTPDase1 hydrolyzes ATP and ADP equally well whereas E-NTPDase2 has a 30-fold preference for the hydrolysis of ATP over ADP (10 - 13). E-NTPDase3 prefers ATP approximately three times better than ADP (14). The physiological functions of the E-NTPDases are not completely understood. However, convincing evidence demonstrating that E-NTPDase1 of endothelial cells reduces ADP-induced platelet aggregation has been presented (15, 16). Sesti et al. have also shown that E-NTPDase1 regulates ATP and norepinephrine secretion in cardiac synaptosomes (17). Therefore, E-NTPDase1 is a candidate for potential therapeutics for occlusive diseases and cardiac dysfunctions.

Table 1. Nomenclature, Cellular and Tissue Distribution, and Substrate Specificity of E-NTPDase Subfamilies. Human ecto-ATPase belongs to the E-NTPDase2 subfamily. The classification and nomenclature of E-NTPDases were decided during the Second International Workshop on “Ecto-nucleotidases” held in Diepenbeek, Belgium in 1999 (3, 4)

 E-NTPDase2 

E-NTPDase2 is also called ecto-ATPase (Figure 1). The research of ecto-ATPase was initiated with intense study of the cell surface of tumor cells. Ecto-ATPase was found as one of the cell surface proteins which were expressed at high levels on some tumor cells. Using cytochemical staining, Karasaki et al. showed that cell surface ATPase activity increased during tumorigenesis of rat hepatoma (18 - 21). However, the ATPase activity in the normal rat hepatocytes and the rat hepatoma cells were not characterized. Knowles, et.al. characterized the cell surface ATPase activity of small cell lung carcinoma (SCLC) and a human hepatoma cell line, Li-7A (22 - 25). Similar to the rat hepatoma cell surface ATPase, the ecto-ATPases of Li-7A and SCLC cells are inhibited by mercurials, e.g., pCMB and pCMPS, but it is unaffected by high concentrations of azide, an inhibitor of a membrane bound ATP diphosphohydrolase (25). The enzyme is inactivated by most non-ionic detergents and bile salts and can only be partially solubilized in an active form by digitonin (22). In addition to inhibition by pCMPS, the partially purified enzyme was also inhibited after prolonged incubation with p-fluorosulfonylbenzoyl-5’- adenosine, an ATP analog, and dithiothreitol, a disulfide reducing agent (22). The human ecto-ATPase has been cloned from the SCLC NCIH69 (13), and its sequence is identical to that cloned from a human bladder tumor cell line (26). There are three splice variants of the human ecto-ATPase, which consist of 495, 472, and 450 amino acids (26, 27). Mateo et al. reported that the splice variants containing 472 and 450 amino acids do not have biological activity (26).



 Roles of Glysosylation in ecto-APTase 

Reserach Plan

This research was undertaken in Aileen F. Knowles lab at San Diego State University to further characterize the human ecto-ATPase, and is concerned specifically with the role of glycosylation in ecto-ATPase function. Oligosaccharides in glycoproteins have been shown to be important for proper protein folding and intracellular trafficking (28 - 30), quality control in the secretory pathway (31), topogenesis (32) and protein-protein and protein-ligand interactions (28). Additionally, glycosylation may affect protein structure, stability, or activity. The oligosaccharides of a glycoprotein tend to project away from the protein surface, and some oligosaccharaides may play structural roles by limiting the conformational freedom of their attached polypeptide chains. Cell surface carbohydrates also provide biological information such as in ABO blood group antigens on cell surfaces whose carbohydrates are the best known immunochemical markers. While the general effects of glycans attached to asparagines (N-glycans) are known, the role of a particular N-glycan in a given protein has been unpredictable. It is possible that an N-glycosylation site is not used or that glycosylation of a particular asparagine is not essential for function. Therefore, this study was conducted to determine the contribution of individual N-glycan to ecto-ATPase activity. N-glycosylation occurs on asparagine residues in the consensus sequence Asn-X-Ser/Thr, where X is any amino acid except proline and aspartic acid (33). According to this criterion, there are five asparagines in human ecto-ATPase that can be potentially glycosylated, and there is one asparagine that may not be glycosylated because it has proline in the X position (Figure 2). The goal of this study was to determine which asparagine residues in human ecto-ATPase are important for function and protein expression. To this end, the asparagine residues in the six putative glycosylation sites were mutated individually to glutamine, and each mutant ecto-ATPase was characterized.

Methods

Preparation of Mutagenic Primers

Twelve oligonucleotides for mutagenesis, substituting the codon of Gln (CAG) for the six Asn (AAC) residues were designed. The preparative acrylamide gel was run to obtain the purified oligonucleotides. Samples containing 300 µg primers in 60 µL were mixed with 60 µL of 2X denaturing loading dye and loaded on 15 % acrylamide gel containing 7 M urea. After electrophoresis for 2 h at 350 V and 40 mA, the target oligonucleotide bands were detected with a UV lamp and were excised by a razor blade. The gel slices were cut into small pieces and transferred to a 15 mL round-bottomed tube containing 3.5 mL of 0.1X SSC (15.0 mM NaCl and 1.5 mM Na-citrate, pH 7.7), and the tube was rocked overnight. The supernatant was transferred into a new tube to which was added 0.1 volume of 3 M sodium acetate (pH 4.8) and 2 volumes of ice cold 100 % ethanol. The tube was then left in a dry ice-methanol bath for 2 h and then centrifuged in HB-6 swinging bucket rotor at 10,000 rpm for 1 h. The precipitated oligonucleotides were dried and resuspended with 100 µL H2O. The solution was transferred into a microcentrifuge tube and spun to remove any residual acrylamide. The concentrations of purified primers were measured by the absorbance at 260 nm.

Site-Directed Mutagenesis of Human Ecto-ATPase

Human ecto-ATPase cDNA cloned from the SCLC cell line (NCIH69), and inserted in the mammalian expression vector pcDNA3 (Invitrogen), was used as a template in site-directed mutagenesis. Using the QuikChange Site-Directed Mutagenesis kit purchased from Stratagene, mutant cDNAs were prepared by PCR. The 50 µL of PCR mixture contained 5 µL of 10x reaction buffer, 1 ng/µL template cDNA, 0.25 µg/µL forward primer, 0.25 µg/µL reverse primer, 0.2 mM dNTP mixture, and 1 µL Pfu TURBO DNA polymerase. The cycle of denaturing (95℃ for 30 sec), annealing (55℃ for 1 min) and extension (68℃ for 14 min) was repeated sixteen times. One µL of DpnI (20 units/µL) was added to the PCR product and incubated for 1 h at 37℃ to digest the template cDNA. One µL of this mixture was used for transformation of 50 µL of XL-1 blue cells. The six mutagenized cDNAs were prepared from the transformed cells using the Qiagen Maxiprep kit and were sequenced. For the construction of the N88Q_N129Q double mutant, N88Q ecto-ATPase cDNA was used as a template with the N129Q forward and reverse primers.

Stable Transfection of HeLa Cells

HeLa cells were maintained in a humidified incubator with 5 % CO2 at 37℃ in Dulbecco’s modified Eagle’s medium supplemented with 0.5 % penicillin and streptomycin, 5 % fetal bovine serum, and 5 % newborn bovine serum. HeLa cells were plated in 6-well plates and grown to 50-70 % confluency. Cells in each well were transfected with 1 µg of wild type and mutant ecto-ATPase cDNAs in pcDNA3 using LipofectAMINE (5µL). The culture media in the plates were changed after 24 h. The transfected cells were harvested two days after transfection using 200 µL of 0.05 % trypsin per well and were transferred into a T-25 flask in 5 mL of culture media. After allowing the cells to attach to the flask, geneticin (160 µg/mL) was added. Cell growth in the flask was observed every day under a microscope. The amount of geneticin was gradually increased to 320 µg/mL. When the cells in the flask became confluent, the cells were propagated in 10-cm plates. The harvested cells were washed twice with 2 mL of isotonic buffer (0.1 M NaCl, 0.01 M KCl, and 25 mM TrisCl, pH 7.5) and resuspended in 300 µL of the suspension buffer (0.25 M sucrose, 20 mM Mops and 0.01 M EDTA, pH 7.0). Two days after transfection, cells harvested from one well

Membrane Preparation

Membranes were prepared from 5 10-cm plates of HeLa cells stably transfected with either wild type ecto-ATPase cDNA or mutant human ecto-ATPase cDNAs. Cells were harvested by trypsinization and washed twice with isotonic buffer to remove residual serum and trypsin. The cell pellet was homogenized in 10 mL of ice-cold wash buffer (20 mM Mops, 140 mM NaCl, and 5 mM KCl, pH 7.4) with a Potter-Elvejem homogenizer and centrifuged in a Beckman 65 rotor for 60 min at 48,000 rpm at 4℃. The membrane pellet was homogenized with 1.5 mL tissue homogenization buffer (0.25 M sucrose, 20 mM MOPs, 2 mM EDTA, pH 7.4) and layered on top of 10 mL of 40 % sucrose containing 20 mM MOPs, pH 7.4 in a Beckman SW41 rotor tube. The tube was centrifuged in a Beckman SW41 rotor for 60 min at 14,000 rpm at 4℃. The membrane fraction that partitioned at the interface of the two sucrose layers was collected and stored at -20℃.

Ecto-ATPase Assay by Colorimetric Method

ATPase assays were carried out in 0.5 mL reaction mixture (25 mM Tris-Cl, pH 7.5, 5 mM MgCl2, 0.1 mM azide, and 5 mM ATP) with 100-150 µg cell proteins or 20-30 µg membrane proteins. Reactions were initiated by the addition of 25 µL of 0.1 M ATP, and allowed to proceed at 37℃ for 10 min (standard condition), and then terminated by the addition of 0.1 mL of 10% trichloroacetic acid. After centrifugation to remove denatured proteins, an aliquot of the supernatant solution was used for determination of Pi using a colorimetric reagent consisting of 1 volume of 10 mM ammonium molybdate, 1 volume of 5N H2SO4 and 2 volumes of acetone. Absorbance at 355 nm was determined. A standard curve of Pi was constructed with 0, 50, 100, 150, 200 and 250 nmoles of potassium phosphate. The protein concentrations were determined using DC protein assay reagents (Bio-Rad Laboratories) using BSA as the protein standard.

SDS-PAGE and Western Blot Analysis

SDS-PAGE was performed in 10 % polyacrylamide gel in TrisCl, pH 6.8. The protein in the acrylamide gel was transferred to a PVDF membrane (Millipore) by electrophoresis for 55 min at 100 V in the transfer buffer (0.025 M Tris, 0.192 M glycine, 20 % methanol, pH 8.5) at 4℃. The membrane was blotted dry, then wetted with methanol, and blocked in Tris-buffered saline (TBS: 0.02 M Tris and 0.5 M NaCl, pH 7.4) containing 5 % non-fat milk for 1 h. The blocked membrane was incubated with primary antibody (1000-fold dilution) for 2 h at room temperature, washed 4 times with TBS containing 0.05 % Tween 20, and then incubated with goat anti-rabbit IgG conjugated to alkaline phosphatase (5000-fold dilution) for 1.5 h. After 3 washes with TBS containing 0.05 % Tween 20 and a final wash with TBS, immunoreactive proteins were detected by incubating the blot with 10 mL of alkaline phosphatase substrate (NBT/BCIP) solution.



Northern Blot Analysis

Total cellular RNA was isolated from a 10-cm plate of stably transfected HeLa cells by 2 mL of Trizol reagent. Integrity of RNA was confirmed by the presence of the RNAs of the large and small ribosomal subunits in 1.25 % analytical agarose gel. For Northern blot analysis, electrophoresis of RNA was carried out with 20 µg of RNAs for 3-4 h at 80 V. The gel was transferred to nitrocellulose (NC) membrane by capillary transfer in 20X SSC. RNA was immobilized by heating the membrane at 80℃ for 2 h. To obtain the probe for hybridization, the human ecto-ATPase cDNA in pcDNA3 was subjected to double digestion by StuI and Sac II (Figure 3). Stu I cut the human ecto-ATPase cDNA at 403 bp and the vector at 2069 bp. Sac II cut the human ecto-ATPase cDNA at 50 bp and 1289 bp. The four resulting fragments (353 bp, 886 bp, 1271 bp and 4270 bp) were separated by agarose gel electrophoresis and the 886 bp fragment was used as a probe. The 886 bp partial cDNA was purified and labeled with [α-32P]dCTP by random-primed labeling. Prehybridization (2 h) and hybridization (20 h with probes of approximately 1 x 107 cpm/ml) were carried out at 60℃. After washing with SSC solution containing SDS at 60℃, the blot was used for autoradiography.

Deglycosylation by PNGase F

Peptide N-glycosidase F (PNGase F) purchased from New England BioLabs was used to deglycosylate the expressed human ecto-ATPases. PNGase F is an amidase that cleaves oligosaccharides between the innermost N-acetylglucosamine (GlcNAc) and asparagine residues in N-linked glycoproteins. Deglycosylation of denatured membrane proteins (20 µg) was carried out overnight at 37℃ with 500 units of PNGase F according to the manufacture’s instruction. Deglycosylated membranes were analyzed by Western blot analysis.

Results

The results were summarized and published in the paper; Javed, R., Yarimizu, K., Pelletier, N., Li, Cheryl., and Knowles, A. F. (2007) Mutagenesis of Lysine 62, Asparagine 64, and Conserved Region 1 Reduces the Activity of Human Ecto-ATPase (NTPDase 2), Biochemistry 46, 6617.

In summary, this study showed that human ecto-ATPase, a membrane-bound cell surface glycoprotein, has five N-glycosylation sites. Of the six putative glycosylation sites, N378 is not glycosylated. Glycosylation of N88 and N129, both in the non-conserved region is not essential for protein expression and enzyme activity. However, glycosylation of N64 and N443, both in conserved regions, and N294 in a non-conserved region appear to be important for protein folding and protein stability

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