Adenosine monophosphate deaminase 3 activation shortens erythrocyte half-life and provides malaria resistance in mice

Abstract

Key Points• AMPD3 activation reduces red blood cell half-life, which is associated with increased oxidative stress and phosphatidylserine exposure.• AMPD3 activation causes malaria resistance through increased RBC turnover and increased RBC production.The factors that determine red blood cell (RBC) lifespan and the rate of RBC aging have not been fully elucidated. In several genetic conditions, including sickle cell disease, thalassemia, and G6PD deficiency, erythrocyte lifespan is significantly shortened. Many of these diseases are also associated with protection from severe malaria, suggesting a role for accelerated RBC senescence and clearance in malaria resistance. Here, we report a novel, N-ethyl-N-nitrosourea-induced mutation that causes a gain of function in adenosine 59-monophosphate deaminase (AMPD3). Mice carrying the mutation exhibit rapid RBC turnover, with increased erythropoiesis, dramatically shortened RBC lifespan, and signs of increased RBC senescence/eryptosis, suggesting a key role for AMPD3 in determining RBC half-life. Mice were also found to be resistant to infection with the rodent malaria Plasmodium chabaudi. We propose that resistance to P. chabaudi is mediated by increased RBC turnover and higher rates of erythropoiesis during infection. (Blood. 2016; 128(9):1290-1301) IntroductionThe lifespan of red blood cells (RBC) is tightly regulated, lasting some 120 days in humans and 51 days in mice. 1 Senescent RBCs are cleared from the bloodstream by macrophages of the reticuloendothelial system and are replaced with new erythrocytes.2-4 This destruction is not random, and it is highly dependent on RBC age. 4 The factors determining the rate of RBC aging, and thereby RBC lifespan, have not been fully elucidated.In several genetic conditions, including pyruvate kinase deficiency, sickle cell disease, thalassemia, hereditary spherocytosis, and G6PD deficiency, 5,6 erythrocyte lifespan is significantly shortened. In most cases, cells show signs of increased senescence, including increased oxidative stress and phosphatidylserine (PS) exposure.2,7-9 Intriguingly, many of these diseases are also associated with protection from severe malaria, 10,11 which has led some authors to propose a role for RBC senescence and clearance in malaria resistance. 15,16 For example, the shortened lifespan of RBCs in pyruvate kinase-deficient individuals and associated pathology has been linked to reduced RBC ATP levels. Similarly, ATP loss contributes to pathology in sickle cell disease.17 Erythrocytic ATP levels are controlled by adenosine monophosphate deaminase (AMPD3), which converts AMP to inosine 59-monophosphate (IMP) and plays an important role in maintaining the adenylate energy charge or the ratio of ATP to AMP. Thus, ATP loss may be mediated through AMPD3, which converts AMP to IMP. In most cells, the conversion of ITP back to AMP by adenylsuccinate synthetase and adenylsuccinate lyase balances AMPD3 activity. However, in RBCs, the machinery for conversion of IMP back to AMP is absent, and to maintain AM...

Analysis of Ca 2 + mediated signaling regulating Toxoplasma infectivity reveals complex relationships between key molecules

“…Tachyzoite cell pellets were extracted in 10‐volumes of ice cold acetonitrile containing 2 μM 13 C, 15 N–AMP and 13 C, 15 N–UMP and extracts vortex mixed (1 min), then sonicated in an ice‐water bath (5 min), before centrifugation (20,000 rpm, 10 min, 4°C). The supernatants were transferred into HPLC vials, and cyclic nucleotides detected using LC‐QTOF as previously described Hortle et al, with the following modifications (Hortle et al, ). LC analysis was performed on an Agilent 1260 HPLC system using a ZIC®‐pHILIC column 5 μm particle size, 150 × 4.6 mm, Merck SeQuant®), a 23 minute gradient (containing a 6 min washout and equilibration) with mobile phases A (20 mM ammonium carbonate pH 9) and B (100% acetonitrile) eluted at a constant flow rate of 300 μL/min (25°C) and a sample injection of 5 μL.…”

“…Tachyzoite cell pellets were extracted in 10-volumes of ice cold acetonitrile containing 2 μM 13 C, 15 N-AMP and 13 C, 15 N-UMP and extracts vortex mixed (1 min), then sonicated in an ice-water bath (5 min), before centrifugation (20,000 rpm, 10 min, 4°C). The supernatants were transferred into HPLC vials, and cyclic nucleotides detected using LC-QTOF as previously described Hortle et al, 2016 with the following modifications (Hortle et al, 2016). LC analysis was performed on an Agilent 1260 HPLC system using a ZIC®-pHILIC column 5 μm particle size,…”

A novel ENU-induced ankyrin-1 mutation impairs parasite invasion and increases erythrocyte clearance during malaria infection in mice

“…DNA from two G2 mice carrying the abnormal red blood cell parameters (MCV < 48 fL) were extracted with Qiagen DNeasy blood and tissue kit (Qiagen, Venlo, Netherlands) for exome sequencing as previous described46. Briefly, at least 10 μg of DNA was prepared for exome enrichment with Agilent Sure select kit paired-end genomic library from Illumina (San Diego, CA), followed by high throughput sequencing using a HiSeq 2000 platform.…”

Ankyrin-1 gene exhibits allelic heterogeneity in conferring protection against malaria

“…DNA from two G2 mice per strain carrying the abnormal red blood cell parameters (MCV <48fL) were extracted with Qiagen DNeasy blood and tissue kit (Qiagen, Venlo, Netherlands) for exome sequencing as previously described [37]. Briefly, 10μg of DNA was prepared for exome enrichment with Agilent Sure select kit paired-end genomic library from lllumina (San Diego, CA), followed by high throughput sequencing using a HiSeq 2000 platform.…”