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PIMT and RBC metabolism
decays approximately 70-85% of the time into an L-isoas- partyl residue, impacting the protein structure (backbone orientation) and function. Clarke, Ingrosso and colleagues identified L-isoaspartyl groups on RBC membrane proteins in response to aging or pathological oxidative stress, such as in glucose 6-phosphate dehydrogenase deficiency or Down syndrome.6–12 Thus, accumulation of L-isoaspartyl groups in essential RBC proteins correlates with RBC dysfunction and pathology.
A specific repair pathway exists for asparagines and aspartates that have been oxidized into L-isoaspartyl groups. The enzyme L-isoaspartyl methyltransferase (PIMT, gene name PCMT1)13 methylates L-isoaspartyl groups to form an isoaspartyl methyl ester, which can then spontaneously decompose into a normal aspartyl group (returning aspartate to its original stucture and converting asparagine into aspartate). However, the isoaspartyl methyl ester can also decompose back into the “damaged” L-isoaspartyl group, which can again be methylated by PIMT. Due to the stochastic nature of isoaspartyl methyl ester decomposition (as well as ongoing oxidation of aspar- tates and asparagines by ROS), multiple and ongoing cycles of PIMT-dependent methylation are required – a process that is constrained both by sufficient PIMT activity and a sufficient pool of methyl donors as co-factors (e.g., of S-Adenosyl-methionine [SAM]).
Recently, we have observed that oxidant stressors from either environmental insults or genetic disease results in the extensive methylation of at least 116 RBC proteins in proximity to their active sites.14 Moreover, tracing experi- ments with 13C15N-methionine (substrate for the synthesis of SAM – the methyl group donor in PIMT-dependent reactions) revealed that the formation of isoaspartyl 13C- methyl-esters was increased in response to oxidative insults. Thus, a model has emerged in which oxidative damage induces formation of L-isoaspartyl groups, which are then methylated by PIMT, resulting in repair of oxi- dized protiens without the need for resynthesis. However, each of the observations that support his model are correl- ative. The goal of this paper is to test the causal role of the PIMT pathway in RBC oxidant-damage repair through genetic ablation of PCMT1.
Methods
Animal studies with mice
All the animal studies described in this manuscript were reviewed and approved by either the BloodworksNW Research Institute IACUC or the University of Virginia Institutional Animal Care and Use Committee. PCMT1+/- founders15 were acquired from the National Institutes of Health mouse embryo repository and were bred with C57BL/6 females. Interbreeding of PCMT1+/- (henceforth PCMT1 heterozygous) mice was designed to gener- ate PCMT1-/- (PCMT1 KO), as confirmed by genotyping. The use of Ubi-GFP and HOD mice have been previously described in prior work from our group.16 Whole blood was drawn by cardiac puncture as a terminal procedure for PCMT1 KO or WT mice, fol- lowed by harvesting of organs (10 mg of tissues from brain, heart, kidney, liver and spleen). Tissues and blood were snap frozen in liquid nitrogen and stored at -80oC until subsequent analysis. For transfusion studies, fresh RBC (never frozen) were used.
Diamide treatments
RBC from WT and PCMT1 KO mice were incubated with
diamide (0.5 mM, Sigma Aldrich) at 37°C for 0, 3 and 6 hours (h), as previously described,17 prior to metabolomics analyses.
Bone marrow transplantation and phenylhydrazine treat- ment
BMT was performed as previously described, but with WT or PCMT1 KO donors and green fluorescence protein (GFP) recipi- ents.18 Engraftment was monitored by the appearance of GFP-neg- ative RBC and the dissappearance of GFP-positive RBC. On aver- age, GFP-positive RBC were undetectable after approximatley 56 days, consistent with the known RBC lifespan of mice.
Prior to treatment with phenylhydrazine (PHZ) [Sigma Aldrich, USA], mice were injected daily for 3 consecutive days with biotin- XSE (ThermoFisher, Waltham, MA, USA Cat# B1582) until 100% biotinylation of RBC was achieved. Each injection consisted of 1 mg biotin-XSE in a 8% solution of dimethyl sulfoxide (DMSO) in phosphate buffered saline. Mice were then given two intraperi- toneal injections (6 h apart) of PHZ to achieve a final dose of 0.01 mg/g body weight. Perpheral blood was then harvested longitudi- nally and RBC stained with avidin-APC to allow enumeration of RBC that had been present at time of PHZ treatment and to distin- guish them from RBC generated by hematopoeisis after PHZ injec- tion.
Storage under blood bank conditions
RBC were collected, processed, stored, transfused and post- transfusion recovery was determined as previously described.19
Tracing experiments with labeled glucose and methionine
RBC from WT and PCMT1 KO mice (100 mL) were incubated at 37oC for 1 h in the presence of 1,2,3-13C3-glucose or 13C-methionine, prior to determination of lactate isotopologues +2/+3 (as markers of pentose phosphate pathway to glycolysis fluxes) and 13C-SAM (as marker of methyltransferase activity14), as previously described.20
Sample processing and metabolite extraction
A volume of 50 mL of frozen RBC aliquots was extracted in 450 mL of methanol:acetonitrile:water (5:3:2, v/v/v). After vortexing at 4°C for 30 minutes (min), extracts were separated from the protein pellet by centrifugation for 10 min at 10,000 RPM at 4°C and stored at −80°C until analysis.
Ultra-high-pressure liquid chromatography-mass spectrometry metabolomics
Analyses were performed using a Vanquish ultra-high-pressure liquid chromatography-mass spectrometry (UHPLC) coupled online to a Q Exactive mass spectrometer (MS) (Thermo Fisher, Bremen, Germany). Samples were analyzed using a 3 min isocratic condition or a 5, 9 and 17 min gradient as described.21 Solvents were supplemented with 0.1% formic acid for positive mode runs and 1 mM ammonium acetate for negative mode runs. MS acqui- sition, data analysis and elaboration was performed as previously described.21–23
Proteomics
Proteomics analyses were performed via filter aided sample preparation (FASP) digestion and nano UHPLC-MS/MS identifica- tion (nanoEasy LC 1000 coupled to a QExactive HF, Thermo Fisher), as previously described.24
Statistical analyses
Graphs and statistical analyses (either t-test or repeated meas- ures ANOVA) were prepared with GraphPad Prism 5.0 (GraphPad
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