key: cord-1031989-crnmnssm authors: Stevens, Bruce R; Ellory, J Clive; Preston, Robert L title: B(0)AT1 amino acid transporter complexed with SARS-CoV-2 receptor ACE2 forms a heterodimer functional unit: in situ conformation using radiation inactivation analysis date: 2021-05-13 journal: Function (Oxf) DOI: 10.1093/function/zqab027 sha: 806d011636a07d47867caa886c92e4e350209011 doc_id: 1031989 cord_uid: crnmnssm The SARS-CoV-2 receptor, Angiotensin Converting Enzyme-2 (ACE2), is expressed at levels of greatest magnitude in the small intestine as compared to all other human tissues. Enterocyte ACE2 is co-expressed as the apical membrane trafficking partner obligatory for expression and activity of the B(0)AT1 sodium-dependent neutral amino acid transporter. These components are assembled as an [ACE2: B(0)AT1](2) dimer-of-heterodimers quaternary complex that putatively steers SARS-CoV-2 tropism in the gastrointestinal (GI) tract. GI clinical symptomology is reported in about half of COVID-19 patients, and can be accompanied by gut shedding of virion particles. We hypothesized that within this 4-mer structural complex, each [ACE2: B(0)AT1] heterodimer pair constitutes a physiological “functional unit.” This was confirmed experimentally by employing purified lyophilized enterocyte brush border membrane vesicles that were exposed to increasing doses of high-energy electron radiation from a 16 MeV linear accelerator. Based on established target theory, the results indicated the presence of Na(+)-dependent neutral amino acid influx transport activity functional unit with target size mw = 183.7 ± 16.8 kDa in situ in intact apical membranes. Each thermodynamically stabilized [ACE2: B(0)AT1] heterodimer functional unit manifests the transport activity within the whole ∼345 kDa [ACE2: B(0)AT1](2) dimer-of-heterodimers quaternary structural complex. The results are consistent with our prior molecular docking modeling and gut-lung axis approaches to understanding COVID-19. These findings advance the understanding of the physiology of B(0)AT1 interaction with ACE2 in the gut, and thereby potentially contribute to translational developments designed to treat or mitigate COVID-19 variant outbreaks and/or GI symptom persistence in long-haul Post-Acute Sequelae of SARS-CoV-2 (PASC). These seminal studies were obligatory to subsequently assigning the functional properties to an SLC6A19 gene expression product by Broer, Verrey and colleagues, and in implicating ACE2 as indispensable in epithelial cell trafficking/chaperoning 18 and apical membrane expression of B 0 AT1 [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] . Following recommendations made by Halvor Christensen at a 1994 membrane transport symposium in Stowe, Vermont, the Stevens' NBB ("Neutral Brush Border") term 3, 7 was changed to B and then to B 0 , in order to conform to the then-evolving transporter nomenclature convention 30 . This alluded back to Christensen's pioneering Blastocyst classification categories in which the uppercase refers to sodium-dependency and the "0" superscript refers to the zwitterion net zero charge of neutral amino acid substrates 30 . Ultimately, the NBB/B/B 0 amino acid transporter (AT) various interchangeable appellations in the literature [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] 30, 31 were eventually consolidated into the current designation "B 0 AT1" 17, 20, 32 . The small intestine is the human body's site of greatest magnitude expression of both B 0 AT1 and ACE2 [33] [34] [35] [36] [37] [38] [39] [40] [41] The present study addresses this knowledge gap, in order to provide insights that may lead to developing new therapies and treatments for COVID-19 in current or future outbreaks. Our approach was to exploit radiation inactivation analysis and target theory utilizing highenergy ionizing electrons from a 16 MeV linear accelerator. As empirically established by us 57, 58 and others [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] , this technique reveals membrane in situ structure-function relationships, accurately identifying the molecular size of "functional units" entwined within physical structures of complex multi-subunit biological systems such as channels, transporters, enzymes, and receptors. We report that sodium-dependent carrier-mediated B 0 AT1 activity in situ in small intestinal enterocyte purified apical brush border membrane vesicles occurs via an apparent physiological "functional unit" of target size mw ~184 kDa representing the Small intestinal epithelium isolated apical brush border membrane vesicles (BBMV) were prepared using New Zealand white rabbit ileum mucosa, lyophilized and reconstituted for use in radiation inactivation experiments as previously described by us 3, 4, 7, 15, 57, 58, 71 . Briefly, rapidly isolated mucosal scrapings were obtained from 1 meter of ileum proximal to the ileocecal junction, treated with 10 mM MgCl 2 , followed by a series of differential centrifugations and progressively diluted washes using 300 mM to 10 mM D-mannitol in 1 mM HCl/Tris pH 7.6 buffer. The final pellets were suspended in distilled water using a glass homogenizer. BBMVs The irradiated vesicles were stored in their vacuum-sealed ampules at -10 o C until required for assays. Following post-irradiation, BBMVs were reconstituted with 200 mM D-mannitol pH 7.5 buffer as described above, and the vesicles were then allowed to equilibrate for 30 minutes before transport measurements were made. Influx initial rates were measured at 22°C in reconstituted BBMVs, defined as the 5 sec initial uptake of zero-trans (i.e., substrate outside but not inside) unidirectional carriermediated sodium-dependent portion of total uptake of radiolabeled 0.1 mM [ 100 mM NaSCN or 100 mM KSCN in 100 mM D-mannitol pH 7.5. Sodium-dependent carriermediated transport activity was calculated from the total radiotracer uptake in Na + media minus diffusion uptake as measured in K + media replacing Na + in the presence of unlabeled 100 mM L-methionine or 100 mM L-alanine. A rapid-mix/rapid filtration apparatus was employed with ice-cold 200 mM D-mannitol stop buffer to arrest uptake, as described by us 3, 15, 58 . Uptake measurements were replicated N = 6 times. Radiation inactivation target size mw's were obtained by measuring post-irradiation remaining activity of zero-trans unidirectional sodium-dependent initial influx rates in reconstituted lyophilized BBMVs at various radiation doses: where A = activity remaining, A 0 = control initial activity, D = radiation dose in kGy units, k = rate constant dependent on target mw. It has been empirically established by us 57,58 and others [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] , that for activity of biological systems in lyophilized preparations irradiated by high-energy electron beams, then the 'functional unit" radiation target size is calculated by, where D 37 = radiation dose (in kGy units) at which activity = A 0 . e -1 (i.e., 37% of control activity). In practice, target sizes were computed by nonlinear regressions constrained to 100% activity at zero dose radiation, fitting the raw data using the R package 'investr' with objects of class 'nls' using the function, were J represents influx initial rates, J max is the maximal influx rate of a given transport carrier "Other" activity contributed < 5% to maximal sodium-dependent uptake as compared to > 95% of Na + -dependent active attributable to B 0 AT1. It could be speculated that "Other" might potentially represent Systems ASCT2, SNAT2, or the [rBAT:b 0,+ AT1] heterodimer complex 77 . However, unlike B 0 AT1, ASCT2 is an amino acid exchanger/antiporter 77 which mechanistically would be principally unresponsive to the zero-trans initial rate unidirectional sodium-coupled uptake assay conditions employed in the present study Methods. Furthermore, ASCT2 is reportedly expressed in small intestine at levels ~2.4% of B 0 AT1 expression 26, 78 , with ASCT2 prominence dominating ascending colon compared to small intestine. SNAT2 17 is a highly unlikely candidate because it is primarily a basolateral membrane transport system that is Fig. 5 the effect of binding Na + ion in the absence of K + are revealed as shown by the 54.8 kDa monomer structure from atomic coordinates of human alkaline phosphatase PDB ID: 3MK1, with release of p-nitrophenol product 80 . The main finding of this study is that sodium-dependent carrier-mediated B 0 AT1 activity in situ in small intestinal enterocyte purified apical brush border membrane vesicles occurs via an apparent physiological "functional unit" of target size mw = 183. "functional unit" whether as a single polypeptide or as an oligomeric assembly of many individual polypeptide subunits [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] . The technique exploits the loss of measured biological activity surviving a random hit by a high-energy electron from a linear accelerator, with the probability of being knocked out by deposition of the electron's 60 eV (1500 kcal/mol) ionizing energy directly correlated with the mw "target size" of the functioning entity, as described in Methods and extensively discussed elsewhere [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] . In the case of biological activity of a multimer comprised of subunits, a single electron hitting any one of the subunit members within the collective assembly will completely abolish functional activity as the consequence of transferring its ionizing energy to other subunits of the complex via bonds of contact interface amino acid residues [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] . Thus, for a heterodimer with subunits paired by one or more bonds of interface contact residues, and in accordance with radiation inactivation target theory 59-70 an electron direct hit to either one of the subunits will nullify biological activity, even if only one of the subunit entities is responsible for the actual biological activity. The data of Fig. 3 fit the simple exponential relationship of Eqs. 1-3 for the inactivation of membrane in situ B 0 AT1 transport activity. The computed values in Table 1 pairings that each lack residues with bonds able to transfer electron hit energy into the adjoining subunits (Δ i G = +3.8 kcal/mol in the case of [ACE2:ACE2]; and null interfacings between the B 0 AT1 subunits). Thus, a high energy electron direct hit to any ACE2 subunit will transfer its energy to a B 0 AT1 subunit, resulting in annihilating measurable B 0 AT1 transport activity. Based on Eqs. 2 and 3, the above arguments collectively indicate that the high energy electron irradiation "sees" a functional unit target mw ~184 kDa for B 0 AT1 transport activity, which is consistent with radiation target theory describing a multimeric functional unit [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] comprised of the [ACE2:B 0 AT1] heterodimer. The radiation inactivation target size results (Fig. 3) The authors declare no conflict of interest. The data underlying this article will be shared on reasonable request to the corresponding author. Solvation-free energies were calculated for each isolated chain, and also for the interfaces between contact residues of chain combinations within the [ACE2:B 0 AT1] 2 dimer-ofheterodimers complex described in Fig. 4 . The Δ i G values represent solvation free energy gain (kcal/mol) upon formation of a given interface, with P < 0.05 representing statistical significance. Shown are the distances between specific residues responsible for interface contact hydrogen bonds between paired chains shown in Fig. 4 . There were null interactions between the B 0 AT1 chains. 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