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The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality

Band 3 (anion exchanger 1; AE1) is the most abundant membrane protein in red blood cells, which in turn are the most abundant cells in the human body. A compelling model posits that, at high oxygen saturation, the N-terminal cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during red blood cell aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of the quality of stored blood and efficacy of transfusion, a life-saving intervention for millions of recipients worldwide. Here we leveraged two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of the role of this protein in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and crosslinking proteomics, we provide a map of the red blood cell interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase. As a proof-of-principle and to provide further mechanistic evidence of the role of AE1 in the regulation of redox homeo stasis of stored red blood cells, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recovery of stored red blood cells from healthy human donors and genetically ablated mice.


Methods
Animal studies with mice All the animal studies described in this manuscript were reviewed and approved by the University of Virginia Institutional Animal Care and Use Committee (protocol n: 4269). Band 3 mouse founders, including huB3, HA Del and BS KO mice -originally generated by Low's group 1 -were acquired from the National Institutes of Health mouse embryo repository and were bred with C57BL/6 females. The use of Ubi-GFP and HOD mice have been previously described in prior work from our group. 2 Whole blood was drawn by cardiac puncture as a terminal procedure for the mice. Blood was snap frozen in liquid nitrogen and storaged at -80ºC until subsequent analysis.
For transfusion studies, fresh RBCs (never frozen) were used.

Band 3 Neapolis
RBCs (100 ul) were obtained from a subject carrying a rare mutation resulting in the lack of amino acids 1-11 in the N-terminus of band 3, as extensively described by Perrotta et al. 3 Methylene blue treatments RBCs from WT and band 3 KO mice were incubated with methylene blue (100 uM, Sigma Aldrich) at 37°C for 1h, as described, 4 prior to metabolomics analyses.

Storage under blood bank conditions
RBCs from the four main strains investigated in this study, along with 13 different mouse strains described before 5 were collected, processed, stored, transfused and post-transfusion recovery was determined as previously described. 5 Tracing experiments with labeled glucose, citrate, glutamine, arachidonic acid and methionine RBCs from all the mouse strains investigated in this study (100 ul) or RBC lysates from healthy donor volunteers (n=3) or the individual carrying band 3 Neapolis RBCs were incubated at 37ºC for 1h in presence of 1,2,3-13 C3-glucose (5 mM -Cambridge Isotopes -product no. CLM-4673), prior to determination of lactate isotopologues +2/+3 (as markers of pentose phosphate pathway to glycolysis fluxes), as described. 6 Additional tracing experimetns were performed by incubating RBCs with 13 C 15 N -methionine (1 mM -Sigma Aldrich -product no. 608106), 13 C 15 N -glutamine (1 mM -Cambridge isotope -product no. CNLM-1275-H) or D8-arachidonic acid (Cayman -product no. 390010), as previously described. 7,8 Sample processing and metabolite extraction: A volume of 50μl of frozen RBC aliquots was extracted in 450μl of methanol:acetonitrile:water (5:3:2, v/v/v). After vortexing at 4°C for 30 min, extracts were separated from the protein pellet by centrifugation for 10 min at 10,000g at 4°C and stored at −80°C until analysis.

Ultra-High-Pressure Liquid Chromatography-Mass Spectrometry metabolomics:
Analyses were performed using a Vanquish UHPLC coupled online to a Q Exactive mass spectrometer (Thermo Fisher, Bremen, Germany). Samples were analyzed using a 3 minute isocratic condition or a 5, 9 and 17 min gradient as described. [9][10][11] Solvents were supplemented with 0.1% formic acid for positive mode runs and 1 mM ammonium acetate for negative mode runs. MS acquisition, data analysis and elaboration was performed as described. [9][10][11] Proteomics Proteomics analyses were performed via FASP digestion and nanoUHPLC-MS/MS identification (nanoEasy LC 1000 coupled to a QExactive HF, Thermo Fisher), as previously described. 12
All Band 3 and GAPDH protein constructs contained an N-or C-terminal 6xHis-SUMO. Constructs were cloned into the bacterial expression vector, pET21b, using the NdeI restriction site. Growth media with proper antibiotics was inoculated with a colony from a freshly transformed LB agar plate and shaken at 37 °C for 16 hours. Fresh media was inoculated with 2.5% of the overnight growth and shaken at 37 °C until an OD of 0.6 was reached. Protein expression was induced by the addition of 1 mM isopropyl-beta-D-thiogalactoside (IPTG). Induced cultures were shaken for 4 hours at 37 °C before being harvested by centrifugation at 4,500 rpm for 10 min.
The purification strategy of all protein constructs was similar. Bacterial pellets were resuspended in Lysis Buffer (50 mM Tris pH 7.5, 300 mM NaCl, 3% glycerol, 10 mM imidazole, 10 mM DTT) and lysed by sonication with 7 cycles of 30 sec on, 30 sec off. Cellular debris was removed by centrifugation at 13,000 rpm for 30 min and 4 °C. Lysate was applied to a column packed with Ni Sepharose Excel resin (Cytiva) and washed with 7 column volumes of Lysis Buffer. Bound protein was eluted with 4 column volumes of Elution Buffer (50 mM Tris pH 7.5, 300 mM NaCl, 400 mM imidazole, 5% glycerol). The elution fractions were pooled and SUMO protease (ulp1, in-house) was added. The sample was dialyzed overnight into Lysis Buffer at 4 °C using dialysis tubing with a 3.5K molecular weight cut-off (MWCO). The sample was applied to a column packed with Ni-NTA His•Bind Resin (Sigma) and the flow-through material was collected. The flow-through fraction was concentrated and applied to a Superose 6 Increase column (Cytiva) equilibrated with Storage Buffer (20 mM Bis-Tris pH 6.5, 150 mM NaCl). Fractions containing target protein were pooled, concentrated, and stored at -80 °C.
For the membrane penetrating version of the peptide, three sequences were used based on previous studies: 13,14 a) Internalization sequence -RQIKIWFQNRRMKWKKGG b) Poly Arg -CRRRRRRRRC c) TAT -YGKKRRQRRG Thermal proteome profiling Thermal proteome profiling (TPP) were performed as previously described. 15 Lysates from red blood cells were adjusted to 1 mg/mL and divided into 60 aliquotes of 150 µL. Treated lysate samples were µm, spherical solid core). Samples were run at 400 nL/min over a 70-min linear gradient from 4 to 32% acetonitrile with 0.1% formic acid. The mass spectrometer was operated in positive ion datadependent mode. MS1 scans were run in the orbitrap from 375 to 1,500 m/z at 120,000 resolution.
Full scan automatic gain control (AGC) and maximum injection time was set to 4 x 10 5 ions and 50 ms. Ions above an intensity threshold of 1 x 10 4 were selected for MS2 analysis with fragmentation by collision induced dissociation (CID). Filtering was performed by the quadrupole with an isolation window of 1.2 m/z. AGC and maximum injection time was set to 1 x 10 4 and 50 ms. Fragmentation was performed by CID with a normalized collision energy of 35%. Detection was set to the ion trap operating in rapid mode. Synchronous precursor selection (SPS) was utilized to co-select 10 MS2 fragments for MS3 analysis. Excluding the precursor, 10 SPS ions were selected within the 400-1200 m/z window. AGC and maximum injection time was set to 1 x 10 5 and 105 ms. The co-selected SPS ions were fragmented by high-energy collisional dissociation (HCD) with a normalized collision energy of 65%, and analyzed in the orbitrap with a resolution of 50,000 and scan range of 100-500 m/z. SPS-MS3 scan frequency was determined by a 3 s total duty cycle.
Proteome Discoverer 2.2 (Thermo Fisher Scientific) was used for the database search and TMT quantification. Raw data was searched against the human Swiss-prot database. Search parameters included carbamidomethylation-C, TMT 10plex-K, and TMT 10plex-peptide N-terminus as fixed modifications, oxidation-M and pyro-Q N-terminus were set as variable modifications. Up to two missed cleavages were allowed with MS1 and MS2 tolerances set to 20 ppm and 0.5 Da. Reporter ion quantification was set at the MS3 level with an integration tolerance of 0.003 Da. A 65% threshold was set for SPS-MS3 mass match.
Melting curve analysis utilized the lowest temperature condition as the reference. Curve fitting and data normalization was performed as previously described. 16 Briefly, the equation

= +
was used in Prism (GraphPad) for curve fitting and protein Tm determination. The melting point (Tm) is described as the temperature at which the melting curve intercepts 50% protein abundance. Complexes were precipitated by the addition Ni-NTA (88831, Thermo Fisher) or anti-FLAG (A36797, Thermo Fisher) magnetic agarose. The samples were mixed by rotation for 1 hr at 4 °C.

Band 3 Co-IP and chemical cross-linking
The beads were settled in a magnetic tube rack and unbound material was removed by aspiration.
Beads were resuspended with IP Buffer and washed by mixing for 10 min. A total of 6 washes were performed. Bound protein was eluted in 2 rounds with the addition of FLAG peptide (A6002, APExBIO Technology) or 400 mM imidiazole.
The eluted protein was proteolytically digested according to the FASP protocol as previously described 17 . In brief, 300 µg of each sample was reduced, alkylated, and digested at 1:50 with sequencing grade trypsin and mass spectrometry grade rAsp-N (Promega) by incubating at 37 °C for µm, spherical solid core) and run at 400 nL/min over a 70-min gradient from 4 to 32% acetonitrile with 0.1% formic acid. Non-crosslinked and DMTMM treated samples used a MS2 acquisition method. In brief, MS1 scans were run in the orbitrap from 300 to 1,600 m/z at 120,000 resolution.
MS2 was performed in a stoichiometric fashion on top ions from each precursor scan and fragmented at a HCD collision energy of 30%. MS2 scan frequency was determined by a 5 s total duty cycle.
DSSO treated samples utilized an MS3 acquisition scheme. DSSO treated samples were analyzed as previously described. 18 In brief, MS1 scans were run in the orbitrap from 375 to 1,500 m/z at 60,000

The Red Blood Cell Interactome as gleaned by cross-linking proteomics
The human body contains 25 trillion red blood cells (RBCs -83% of all human cells) 23,24 Each erythrocyte contains ~250-270 million molecules of hemoglobin and allows the transport of up to 1 billion molecules of oxygen/cell 23,24 . These numbers also explain why hemoglobin makes up for ~92% of the total protein content in a mature RBC. 25 To maximize intracellular hemoglobin content, erythroid precursors progressively lose nuclei and organelles during maturation to RBC. 26 Another unique characteristic of non-nucleated RBCs is that the plasma membrane, its only structural component, and membrane proteins account for all of its diverse antigenic, transport, and mechanical characteristics. 27 Over the decades, careful biochemical studies have elucidated the relevance of membrane protein interactions in the regulation of the RBC cytoskeletal properties, which allow the stabilization of the erythrocyte membrane during RBC turbulent flow from the lungs into the capillary beds as small as 3 μm. 1 Based on biochemical data and imaging approaches, 28 biophysical models of RBC membrane structure and function have been generated that allow to simulate RBC responses to energy-induced cytoskeletal defects, 29 as well as mechanical and osmotic stress. 30 Despite the apparent simplicity, advancements in the field of mass spectrometry-based proteomics have catalogued over 2000 proteins in highly-purified erythrocytes, 31 which are closer to ~3000 protein entries when reports from multiple studies are merged. 12,32-34 Based on these lists, in silico approaches have tried to predict protein-protein interactions 35,36 -the interactome -in an attempt to update our understanding of RBC physiology. To follow up on these predictions, biochemical approaches were devised to investigate protein-protein interactions in the form of multimeric complexes by means of native gel electrophoresis studies. 37 Unfortunately, these techniques were affected by slow throughputness and lack of precision in the identification of such complexes, other than validation of actual direct interaction based on close structural proximity of protein components of the putatively assigned complexes.
To overcome these limitations, in the present study we adopted a novel cross-linking proteomics approach, based on multiple cross-linking agents. This strategy, coupled to high-resolution MS 3 mass spectrometry, allows to chemically stabilize protein-protein interactions by bridging epsilon amine residues of K residues and carboxylic groups in the side chains of acidic amino acids D and E, provided they face each other within a given range (e.g., 11.4 to 24 Å for the zero-length cross-linking agents we used in this study). 18,20,38 Through this approach we mapped over 5597 peptide-peptide unique inter-or intra-molecular cross-links, involving a total of 134 unique RBC proteins, generating a preliminary map of the RBC experimental interactome.

Red Blood Cell experimental interactome as gleaned by cross-linking proteomics experiments
RBC cytosols and membranes were incubated with two different cross-linking agents, DSSO and DMTMM. This approach affords determination of protein-protein interactions by cross-linking free epsilon-amines of adjacent residues of interacting proteins between each other (DSSO- Supplementary Figure 6.A) or carboxylic group of D/E residues (DMTMM) within a range spanning from 11.4 to 24 Å. 18,38 Cross-linked proteins were thus enzymatically digested with trypsin, which cleave at the C-term of basic K or R residues, prior to high-pH reversed phase fractionation to enrich

Cross-linking proteomics studies recapitulate structural elucidation of hemoglobin tetramers
One of the advantages of the cross-linking proteomics approach is that it not only highlights the intermolecular interactions between two distinct proteins, but also affords resolution of intramolecular interactions whenever basic and acidic residues that are substrates for the cross-linker face each other within the same protein/protein complex in the linker range. This approach is thus useful to refine knowledge on the structure of molecular complexes, which may be relevant in case the structure or oligomeric conformation of a molecule are unknown. Vice versa, since this is the first report adopting this method in RBCs, we decided to test the reliability of our findings against protein structures that are extremely well-established. The paradigmatic example of the statement above is hemoglobin.
First of all, we graphed intra-(purple lines) and inter-molecular interactions (teal) between hemoglobin chains alpha and beta -HBA and HBB as bar plots (Supplementary Figure 7.A). We and HBB (teal) or within HBB (purple for intra and teal for inter-chain cross-links).

Experimental interactome of red blood cell membrane proteins
The RBC membrane interactome is a critical lynchpin for the erythrocyte cytoskeleton. The dynamic nature of membrane protein interaction is also critical to RBC capacity to respond to shear and mechanical stress when traversing narrow capillaries in the peripheral vasculature. 28 Integral membrane protein components not only participate in anchoring the membrane to the cytoskeleton, but also contribute to blood groups and other RBC membrane immunomodulators/antigens that impact untimely removal from the bloodstream, such as B3AT oligomeric state. 39 As such, RBC membrane protein interactions have represented one of the early focuses of structural biologist when tackling erythrocyte complexity. [40][41][42][43] By focusing on the membrane fraction of RBC lysates, we managed to overcome some of the limitations noted above with respect to the overwhelming abundance of hemoglobin. This approach allowed us to map the intra-and intermolecular interactome of RBC structural protein components, to expand upon existing literature. Caveats in the interpretation of the present results is that the experimental findings reported here are not meant to overwrite decades of careful biochemistry focused on isolated protein components, rather to complement such information based on evidence generated with a brand new tool that affords mapping of many more such intra and inter-molecular interactions in a single experiment.

Cross-linking proteomics evidence of the interactions between the N-terminus of band 3 and hemoglobin or glycolytic enzymes
The interactome of RBC membrane proteins has been suggested to play functional role in erythrocyte physiology beyond cytsokeletal homeostasis. One paradigmatic example of such interaction is found in the N-terminus cytosolic domain of B3AT. The two most abundant cytosolic and membrane proteins in RBCs -hemoglobin and B3AT, have long been shown to interact 47,48 Hemoglobin, especially under deoxygenated conditions, has been shown to outcompete glycolytic enzymes, among which glyceraldehyde 3-phosphate dehydrogenase (GAPDH - Figure 6), which are otherwise bound to the N-term of B3AT and inhibited. Through this mechanism, RBCs regulate metabolic fluxes through glycolysis and the pentose phosphate pathway to generate energy or reducing equivalents to counteract oxidant stress in response to hypoxia or high oxygen saturationinduced oxidant stress, respectively 1,49 .
Hemoglobin and glycolytic enzyme interactions to B3AT are now supported by a plethora of data, based on immunological assays (co-immunoprecipitation, western blot on everted membranes, immunofluorescence), enzymatic activity assays, determination of metabolic fluxes via nuclear magnetic resonance and mass spectrometry-based metabolic flux analysis as determined via stableisotope labeled tracers. [50][51][52][53][54][55][56] Such interactions were further determined mechanistically by studies in mice carrying genetic ablation of specific sequences (residues 1-11 and 12-23) of B3AT. 1,49 However, the actual structural underpinning of B3AT binding to hemoglobin or glycolytic enzymes is difficult to investigate experimentally through classic structural approaches such as nuclear magnetic resonance, owing to the dynamic conformational changes of the B3AT N-terminus. Indeed, while the structure of the N-terminus of AE1 beyond residue K56 has been solved through crystallography 44 , the very first residues 1-56 of the N-terminus B3AT arrange into an intrinsically disorder domain that has hitherto eluded structural studies. Besides, structural studies aimed at determining B3AT N-term structural interactions has so far eluded also early proteomics studies reliant on cross-linking agents and previous generation mass spectrometers. 50 In addition, the B3AT N-terminus is extremely acidic and the first trypsin cleavable residue (K56) generates a very long peptide that often times eludes detection at the mass spectrometry level. In the present study we overcame those limitations by and 56-356.

Discussion
Mapping RBC protein-protein interactions has critical implications for expanding our understanding of RBC physiology in health and disease. Being devoid of de novo protein synthesis capacity, to quickly respond to environmental stimuli, RBCs rely on post-translational modifications (e.g., phosphorylation) and the impact these have on protein-protein interactions and protein function. 42,58 Competitive interactions between hemoglobin and glycolytic enzymes with the N-terminus of band 3 have been investigated over the years as a critical pathway in response to environmental oxygen tension and oxidant stress. 42,50,52,[59][60][61][62] Interactions between the band 3-spectrin-ankyrin cytoskeleton, along with actin, adducin and protein 4.1 have been extensively described as key regulators of RBC membrane stability and deformability in the classic model of the RBC cytoskeleton. 27 On top of intermolecular interactions, changes in the multimeric state of RBC structural proteins may have immunogenic potential, such as in the case of band 3 oligomerization triggering RBC recognition by naturally occurring antibodies 63 and subsequent removal from the bloodstream.
Advances in the field of proteomics have revealed the unanticipated complexity of the RBC proteome, 12,[31][32][33][34] and informed in silico studies to predict how these proteins may interact in a way that could impact RBC physiology. 35,36 Here we leveraged a novel cross-linking proteomics approach to provide the first map of the experimental cytosolic and membrane RBC interactome. To validate some of our key findings, we mapped the experimentally-identified intramolecular interactions against available protein structures for the hemoglobin tetramer. We thus focus on the RBC membrane interactome, with an emphasis on band 3 and its interactions with structural components, hemoglobin and glycolytic enzymes.
This study holds several limitations. First of all, in this proof of principle study we used hemoglobin intra-and inter-molecular interactions as an internal validation for the reliability of the approach. However, the overwhelming abundance of hemoglobin, despite the high pH-RP fractionation approach aimed at enriching cross-linked peptides, has hampered the detection of other cross-links involving low abundance protein components -limiting the depth of the analysis. In addition, the abundance of hemoglobin may have favored the identification of spurious cross-links, simply by stochastically increasing the likelihood of solvent accessible K or D/E residues on hemoglobin to be exposed to other cross-linkable residues on other proteins. These sub-stoichiometric events either eluded detection at the mass spectrometry level, their signal covered by more abundant hits, or resulted in low score events that were filtered out upon curation of the data in Supplementary fluxes through glycolysis and decreased fluxes through the pentose phospahte pathway (A). At high oxygen saturation, hemoglobin is displaced from the membrane and glycolytic enzymes bind to the N-term of band 3, resulting in their partial inhibition, decreases in fluxes through glycolysis and increased fluxes through the pentose phosphate pathway to generate the reducing equivalent NADPH necessary to counteract oxidant stress, which in turn increases as a function of Fenton chemistry in presence of oxygen (B). In C, in light of this model and the existing literature from our group and others, we predict that RBCs from mice lacking the N-term of band 3 would suffer from increased oxidant stress during storage under refrigerated conditions. We anticipate that RBCs from these mice would de facto phenocopy G6PD deficiency owing to the incapacity to redirect glucose oxidation fluxes towards the pentose phosphate pathway (C). In D, Re-elaboration of TAILS data from Lange et al. showing N-term band 3 residues that are fragmented at the end of the shelf-life of human packed red blood cells (storage day 42).

Supplementary Figure 5 -Immuno-precipitation proteomics studies with recombinantly expressed band 3 1-56.
A peptide coding for the amino acids 1-56 of the N-term of band 3 was recombinantly expressed with a SUMO-tag at either the N-or C-term terminus of the peptide, prior to incubation with plasma, red blood cell cytosols and membrane in independent experiments, pull-down against the SUMO tag, fractionation and nanoUHPLC-MS/MS-based identification of band 3 interacting partners (A). Pathway analyses of the hits from this analysis revealed a widespread interaction of band 3 with up to 63 proteins involved in metabolic regulation, as mapped against the KEGG pathway map of human metabolism (B). In C, enzymes identified from the pull down of SUMO-tagged band 3 1-56 mapped against the Metabolic pathways map (KEGG map: hsa01100).

Supplementary Figure 6 -Red Blood Cell experimental interactome as gleaned by crosslinking proteomics experiments.
In A, an overview of the cross-linking strategy with DSSO. This approach affords determination of protein-protein interactions by cross-linking free amines of adjacent residues of interacting proteins within a range spanning from 11.4 to 24 Å. In the present study, DSSO and DMTMM (E/D residues to K) were combined to investigate the RBC interactome (B). In C, circos plot view of the proteinprotein cross-links identified in this study (full list in Supplementary Table 1). In D, the same inputs were used to provide a network view of the results.

Supplementary Figure 8 -Experimental interactome of red blood cell membrane proteins.
In A, highlighted network of RBC membrane proteins according to the canonical models (proteins are abbreviated as Uniprot gene names). In B, an overview of the interactions across these proteins in bar plot format. In C, expansion of the bar plots focusing on band 3 (B3AT), spectrin alpha and beta (SPTA1 and SPTB1), ankyrins 1 and 2 (ANK1 and ANK2), carbonic anhydrase 1 and 2 (CAH1 and CAH2), erythrocyte protein band 4.1 and 4.2 (41 and EPB42). In D and E, highlights of the intramolecular interaction of band 3 (residues 56-356 -1HYN.pdb and 381-886 -4YZF.pdb, respectively). In F, zoom in on a sub-network of structural proteins interacting with STPB1. Zoom in on a subset of intramolecular cross-links in SPTB1 (1S35.pdb -G) are shown in H. Figure 9 -Cross-linking proteomics provides direct evidence of the interactions between hemoglobin and the N-terminus of band 3. These interactions are shown in bar plot format for band 3 (B3AT) with several hemoglobin chains (including alpha, beta, delta and epsilon -HBA, HBB, HBD and HBE -A), with a zoom on the cross-links between B3AT and HBB in B. In C and D, the interactions in B are highlighted against hemoglobin structure and B3AT cytosolic regions 1-15 and 56-356.