High population frequencies of MICA copy number variations originate from independent recombination events

MICA is a stress-induced ligand of the NKG2D receptor that stimulates NK and T cell responses and was identified as a key determinant of anti-tumor immunity. The MICA gene is located inside the MHC complex and is in strong linkage disequilibrium with HLA-B. While an HLA-B*48-linked MICA deletion-haplotype was previously described in Asian populations, little is known about other MICA copy number variations. Here, we report the genotyping of more than two million individuals revealing high frequencies of MICA duplications (1%) and MICA deletions (0.4%). Their prevalence differs between ethnic groups and can rise to 2.8% (Croatia) and 9.2% (Mexico), respectively. Targeted sequencing of more than 70 samples indicates that these copy number variations originate from independent nonallelic homologous recombination events between segmental duplications upstream of MICA and MICB. Overall, our data warrant further investigation of disease associations and consideration of MICA copy number data in oncological study protocols

The biochemical properties of the Arabidopsis ecto-nucleoside triphosphate diphosphohydrolase AtAPY1 contradict a direct role in purinergic signaling.

The Arabidopsis E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) AtAPY1 was previously shown to be involved in growth and development, pollen germination and stress responses. It was proposed to perform these functions through regulation of extracellular ATP signals. However, a GFP-tagged version was localized exclusively in the Golgi and did not hydrolyze ATP. In this study, AtAPY1 without the bulky GFP-tag was biochemically characterized with regard to its suggested role in purinergic signaling. Both the full-length protein and a soluble form without the transmembrane domain near the N-terminus were produced in HEK293 cells. Of the twelve nucleotide substrates tested, only three--GDP, IDP and UDP--were hydrolyzed, confirming that ATP was not a substrate of AtAPY1. In addition, the effects of pH, divalent metal ions, known E-NTPDase inhibitors and calmodulin on AtAPY1 activity were analyzed. AtAPY1-GFP extracted from transgenic Arabidopsis seedlings was included in the analyses. All three AtAPY1 versions exhibited very similar biochemical properties. Activity was detectable in a broad pH range, and Ca(2+), Mg(2+) and Mn(2+) were the three most efficient cofactors. Of the inhibitors tested, vanadate was the most potent one. Surprisingly, sulfonamide-based inhibitors shown to inhibit other E-NTPDases and presumed to inhibit AtAPY1 as well were not effective. Calmodulin stimulated the activity of the GFP-tagless membranous and soluble AtAPY1 forms about five-fold, but did not alter their substrate specificities. The apparent Km values obtained with AtAPY1-GFP indicate that AtAPY1 is primarily a GDPase. A putative three-dimensional structural model of the ecto-domain is presented, explaining the potent inhibitory potential of vanadate and predicting the binding mode of GDP. The found substrate specificity classifies AtAPY1 as a nucleoside diphosphatase typical of N-terminally anchored Golgi E-NTPDases and negates a direct function in purinergic signaling

The Arabidopsis apyrase AtAPY1 is localized in the Golgi instead of the extracellular space

<p>Abstract</p> <p>Background</p> <p>The two highly similar Arabidopsis apyrases AtAPY1 and AtAPY2 were previously shown to be involved in plant growth and development, evidently by regulating extracellular ATP signals. The subcellular localization of AtAPY1 was investigated to corroborate an extracellular function.</p> <p>Results</p> <p>Transgenic Arabidopsis lines expressing <it>AtAPY1</it> fused to the SNAP-(O⁶-alkylguanine-DNA alkyltransferase)-tag were used for indirect immunofluorescence and AtAPY1 was detected in punctate structures within the cell. The same signal pattern was found in seedlings stably overexpressing <it>AtAPY1-GFP</it> by indirect immunofluorescence and live imaging. In order to identify the nature of the AtAPY1-positive structures, <it>AtAPY1-GFP</it> expressing seedlings were treated with the endocytic marker stain FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)-pyridinium dibromide) and crossed with a transgenic line expressing the <it>trans</it>-Golgi marker <it>Rab E1d</it>. Neither FM4-64 nor Rab E1d co-localized with AtAPY1. However, live imaging of transgenic Arabidopsis lines expressing <it>AtAPY1-GFP</it> and either the fluorescent protein-tagged Golgi marker <it>Membrin 12</it>, <it>Syntaxin of plants 32</it> or <it>Golgi transport 1 protein homolog</it> showed co-localization. The Golgi localization was confirmed by immunogold labeling of AtAPY1-GFP. There was no indication of extracellular AtAPY1 by indirect immunofluorescence using antibodies against SNAP and GFP, live imaging of AtAPY1-GFP and immunogold labeling of AtAPY1-GFP. Activity assays with AtAPY1-GFP revealed GDP, UDP and IDP as substrates, but neither ATP nor ADP. To determine if AtAPY1 is a soluble or membrane protein, microsomal membranes were isolated and treated with various solubilizing agents. Only SDS and urea (not alkaline or high salt conditions) were able to release the AtAPY1 protein from microsomal membranes.</p> <p>Conclusions</p> <p>AtAPY1 is an integral Golgi protein with the substrate specificity typical for Golgi apyrases. It is therefore not likely to regulate extracellular nucleotide signals as previously thought. We propose instead that AtAPY1 exerts its growth and developmental effects by possibly regulating glycosylation reactions in the Golgi.</p

Influence of divalent metal ions on AtAPY1 activity.

<p>Enzyme activities were determined in the presence of 3 mM UDP using the discontinuous apyrase activity assay. The activity of AtAPY1 (1 U = 1 μmol P_i /min) was measured in the absence or presence of either 1 mM CaCl₂, CuCl₂, MgCl₂, MnCl₂, NiCl₂ or ZnCl₂. The control (-) shows the activity without the addition of any divalent ions. The means <u>+</u> SD of duplicates from one assay are shown. Different letters above the columns indicate mean values that are significantly different from one other (one-way ANOVA and Tukey test; p < 0.01). Data are representative of two activity assays.</p

K_m values of AtAPY1-GFP for GDP, UDP and IDP.

<p>Michaelis-Menten plots of the initial reaction velocities (v) for different concentrations of substrate are shown. Different amounts of AtAPY1-GFP enzyme were used in (A), (B) and (C) as a result of different starting material. The enzyme velocities were determined by the continuous assay. For each substrate concentration, the mean velocity calculated from two parallel reactions was plotted. The initial velocities were linear over time for ≥ 30 min. Each initial velocity was determined from a minimum of 24 data points from this linear phase. The error bars represent the standard deviations of the velocity means. The data set is representative of six (A, B) and three experiments (C), respectively.</p

Detection of extracted AtAPY1-GFP.

<p>A Western blot analysis of two different crude protein extracts before (= in) and after (= out) the immobilization by GFP-multiTrap is shown. Equal volumes of extract (15 μL each) were loaded per lane, its proteins subjected to 10% SDS-PAGE, transferred to a nitrocellulose membrane and incubated with antibodies to GFP. The arrows mark the signals of the AtAPY1-GFP fusion protein and free GFP, respectively. The explanation of the colors in the schematic representation of AtAPY1-GFP can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115832#pone.0115832.g002" target="_blank">Fig. 2</a>. Recombinant GFP (19 ng) served as a positive control and quantitative reference for densitometric evaluation of signal intensities. With this, the total amounts of bound AtAPY1-GFP from 100 μL extract 1 and 2 were calculated as 130 ng and 22 ng, respectively. The image shows bands from the same exposure of the same membrane, but non-pertinent lanes were cropped as indicated by vertical lines. The shown signals are representative of at least five separate Western blot analyses of different GFP-multiTrap immobilization experiments.</p

Substrate specificities of AtAPY1 and AtAPY1-δTM.

<p>Activities (1 U = 1 μmol P_i /min) of AtAPY1 (A) and AtAPY1-δTM (B) were determined in the presence of various substrates (3 mM each) using the discontinuous apyrase activity assay. The means <u>+</u> SD of duplicates from one assay are shown. Different letters above the columns indicate mean values that are significantly different from one other (one-way ANOVA and Tukey test; p < 0.05). Each data set is representative of three independent activity assays.</p

K_m values.

<p>The mean K_m values are listed ± SD. The K_m values for GDP, UDP and IDP were all significantly different from each other (p < 0.0001; one-way ANOVA test and Tukey test).</p><p>¹The mean of the K_m value was calculated from six separate experiments. The means were not statistically different from each other (p < 0.001; one-way ANOVA). AtAPY1-GFP purified from three different protein extracts (biological repeats) was analyzed. One, two and three separate experiments were run with each protein extract, respectively.</p><p>²The mean of the K_m value was calculated from six separate experiments. The means were not statistically different from each other (p < 0.001; one-way ANOVA). AtAPY1-GFP purified from two different protein extracts (biological repeat) was analyzed. Two and four separate experiments were run with each protein extract, respectively.</p><p>³The mean of the K_m value was calculated from three separate experiments. The means were not statistically different from each other (p < 0.01; one-way ANOVA). AtAPY1-GFP purified from two different protein extracts (biological repeat) was analyzed resulting in one technical and one biological repeat.</p><p>K_m values.</p

Purification of AtAPY1 and AtAPY1-δTM.

<p>The explanation of the colors in the schematic representations of AtAPY1 and AtAPY1-δTM can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115832#pone.0115832.g002" target="_blank">Fig. 2</a>. (A) Total proteins from 1.6 x 10⁵ HEK293 cells were harvested at each of the indicated time points post transfection with <i>AtAPY1</i> DNA, separated in a 4–12% gradient gel under denaturing conditions, transferred onto a PVDF membrane and successively incubated with anti-APY1 and anti-His antibodies (left panel). The black arrows mark the signal specific for AtAPY1, while the gray arrowheads indicate unspecific bands. The right panel shows the total protein extract from 1.4 x 10⁸ HEK293 cells harvested at 89 h after transfection with <i>AtAPY1</i> DNA subjected to Ni²⁺-affinity chromatography. Various fractions were separated in a 4–12% gel under denaturing conditions and either stained with Coomassie or transferred onto a PVDF membrane for Western blot analysis. The black arrow indicates the signal detected with antibodies against AtAPY1. The volumes loaded were 1/480 of the flow through (FT) fraction, 1/50 of each of the final two wash fractions W3 and W4 and 1/100 of the elution fraction E. (B) The left panel shows samples representing equal volumes (1/3,000) of the culture medium of 1 x 10⁸ HEK293 cells taken at the indicated time points post transfection with <i>AtAPY1-δTM</i> DNA and separated in a 4–12% gradient gel under denaturing conditions. Subsequently, the proteins were either stained with Coomassie or blotted onto a PVDF membrane for Western blot analysis. The right panel depicts the culture medium of 4 x 10⁷ HEK293 cells at time point 88 h after transfection with <i>AtAPY1-δTM</i> DNA subjected to Ni²⁺-affinity chromatography. A gradient gel (4–12%) was loaded with 20 μL of supernatant (S) and 20 μL of flow through (FT), 10 μL of each wash 1–5 and 10 μL of each eluate 1–2. For total volumes of the individual fractions see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115832#sec002" target="_blank">Materials and Methods</a>. The protein amount loaded for eluate 1 equals about 70 ng. Following SDS-PAGE, the gel was silver-stained.</p