These effects are mainly reported through case reports. We conducted an individual patient data meta-analysis to assess the prevalence of clinical, laboratory, and magnetic resonance findings in association with N2O exposure in medical and recreational settings. We calculated the pooled estimates for the studied outcomes and assessed the potential bias related to population stratification using principal component analysis. Eighty-five publications met the inclusion criteria and reported on 100 patients with a median age of 27 years and 57% of recreational users. The most frequent outcomes were subacute combined degeneration (28%), myelopathy (26%), and generalized demyelinating polyneuropathy (23%). A T2 signal hyperintensity in the spinal cord was reported in 68% (57.2–78.8%) of patients. The most frequent clinical manifestations included paresthesia , unsteady gait , and weakness . At least one hematological abnormality was retrieved in 71.7% (59.9–83.4%) of patients. Most patients had vitamin B12 deficiency: vitamin B12 <150 pmol/L , homocysteine >15 µmol/L , and methylmalonic acid >0.4 µmol/L . Consistently, 85% of patients exhibited a possibly or probably deficient vitamin B12 status according to the cB12 scoring system. N2O can produce severe outcomes, with neurological or hematological disorders in almost all published cases. More than half of them are reported in the setting of recreational use. The N2O-related burden is dominated by vitamin B12 deficiency. This highlights the need to evaluate whether correcting B12 deficiency would prevent N2O-related toxicity, particularly in countries with a high prevalence of B12 deficiency.The risk of adverse effects of nitrous oxide (N The first use of N2O as an anesthetic agent was reported on December 1844 by Dr. Horace Wells, an American dentist who demonstrated insensitivity to pain from a dental extraction after N2O inhalation  AND (“nitrogen”[MeSH Terms] OR “nitrogen”[All Fields])) OR “Nitrous oxide”[All Fields] OR “nitrogen protoxide”[All Fields] OR “laughing gas”[All Fields] OR whippet[All Fields] OR whippets[All Fields]) AND  OR (“homocysteine”[MeSH Terms] OR “homocysteine”[All Fields]) OR “methylmalonic acid”[All Fields] OR (“methionine”[MeSH Terms] OR “methionine”[All Fields]) OR “folic Acid”[All Fields]). Additional articles were retrieved from primary search references. EndNote X7.8 was used for reference management ; We calculated the combined indicator of vitamin B12 status score (cB12) by combining vitamin B12, homocysteine and methylmalonic acid according the Fedosov et al. . The cB12O exposure was defined as repeated exposure to N2O, especially in a recreational setting, pain management or occupational exposure, with minimum consumption of one cartridge per month. For each patient, we estimated the average number of cartridges consumed per day and the duration of exposure in years. In the recreational setting, N2O was commonly available in the form of small pressurized cartridges which can deliver the equivalent of 8 liters of N2O gas at standard temperature and pressure (8 g) [2O using the following formula: Amount of N2O exposure = (average number of cartridges consumed per day × duration of exposure expressed in years).Regular Nre (8 g) . We quan2O in medical and recreational settings. Main clinical findings: Paresthesia in extremities, numbness, tingling; Unsteady gait, walking difficulty; Weakness; Fallings or equilibrium disorders; Lhermitte’s sign; and Ataxia. Laboratory findings: Hemoglobin; Hematocrit; MCV; Vitamin B12; Folate; Homocysteine; and Methylmalonic acid. Magnetic resonance finding: Presence of T2 signal hyperintensity in the spinal cord. We assessed two secondary outcomes: 1) to look for predictors of regular N2O exposure; 2) to assess the potential association between N2O exposure and health outcomes.The primary outcome of the systematic review was to report on the clinical, laboratory, and magnetic resonance findings of subjects who were exposed to Nth and 75th percentiles). We compared proportions using the chi-square test or Fisher’s exact test as appropriate. Medians were compared using the Mann–Whitney U test. We assessed correlations using Spearman’s rank correlation coefficient. To derive predictors of short N2O exposure, we performed univariate logistic regression analysis on binary and continuous variables, using the “Short N2O exposure” item as a dependent variable. When a continuous variable was significantly associated with a short N2O exposure, we carried out receiver operating characteristic (ROC) analysis for defining its optimal cut-off using the “Short exposure to N2O” item as a classification variable [p-value using the exact binomial method. In multivariate analysis, all significant items resulting from the univariate logistic regression were integrated into a multivariate logistic regression model using the stepwise method. All variables with p < 0.1 were included in the model and variables with P <0.05 were retained in the model. Results were shown as odds ratios (ORs) and 95% CI. We assessed model discrimination using ROC analysis and model calibration using the Hosmer and Lemeshow goodness-of-fit test. All statistical analyses were conducted using the SAS® 9.4 platform  and MedCalc for Windows v16.8.4  based on a two-sided type I error with an alpha level of 0.05.Categorical variables were summarized as frequency counts and percentages with the 95% confidence interval (95% CI). Quantitative variables were expressed as medians and interquartile range .We assessed the potential bias related to population stratification using principal component analysis. We used all the variables related to clinical findings, laboratory findings, reported diagnoses, and NThe systematic search generated 513 citations of which 132 appeared to be relevant to the systematic review. Of these 132 studies, 47 were not retained by the selection criteria , leaving2O in the setting of recreational use (57%) or surgery (25%) . Most patients included in the meta-analysis were exposed to Nry (25%) .2O. Among them, recreational use , occupational exposure , and pain management  represented the three main N2O exposure modes. Among regular users, the median N2O exposure was 18.5 cartridge-years, corresponding to a total amount of 54,020 g of N2O. The three most frequently reported diagnoses were subacute combined degeneration (28%), myelopathy (26%), and generalized demyelinating polyneuropathy (23%). A T2 signal hyperintensity in the spinal cord was reported in 68% of patients who underwent magnetic resonance imaging of the spinal cord (Seventy-six percent (76/100) of patients had regular exposure to Nnal cord .At least one neurological symptom was reported in 96% (92.1–99.9%) of patients and included the following clinical manifestations by decreasing order of frequency: paresthesia in the extremities (80%), unsteady gait or walking difficulties (58%), weakness (43%), fallings or equilibrium disorders (24%), Lhermitte’s sign (15%), and ataxia (12%) .Patients had a high risk of macrocytic anemia with a median MCV of 100 fL (IQR: 94–103) and median values of hemoglobin of 12.8 g/dL (IQR: 10.8–14.2) and 10.7 g/dL (IQR: 8.3–12.4) in males and females, respectively. At least one hematological abnormality was retrieved in 71.7% of cases (59.9–83.4%). The proportions of patients with low hemoglobin level  with 70.7% of patients exhibiting a vitamin B12 level <150 pmol/L, considered as the threshold of vitamin B12 deficiency. The median plasma concentrations of homocysteine and methylmalonic acid were 55 µmol/L  and 5.0 µmol/L (IQR: 1.1–6.6), respectively, with a vast majority of patients exhibiting high homocysteine (>15 µmol/L) and methylmalonic acid (>0.4 µmol/L) levels . According to the cB12 scoring system, 90.9% of patients exhibited at least a decreased vitamin B12 status and 84.8% of patients exhibited a possibly or probably deficient vitamin B12 status . The ser2O exposure, namely: age, vitamin B12 concentration, and MCV ; vitamin B12 ≤74 pmol/L , and MCV >100 fL   only three were significantly associated with a short N and MCV . In ROC exposure . The dic3–49.15) .2O exposure, namely: age ≥40 years  and vitamin B12 ≤74 pmol/L  .Among the variables retained in univariate logistic regression, two remained significant in multivariate logistic regression and were independently associated with a short N = 0.01) . The mul2O exposure was available in 28 patients. In exploratory analyses, the amount of N2O exposure was not significantly correlated with any biological variable. Furthermore, the amount of N2O exposure was not significantly associated with the most frequently reported diagnoses , the most commonly reported clinical findings , or the presence of T2 signal hyperintensity in the spinal cord . The N2O-related burden is dominated by vitamin B12 deficiency and the associated alterations in one-carbon metabolism markers.The present meta-analysis highlights the potential side-effects related to N2O-related toxicity exhibited a low vitamin B12 status (<150 pmol/L). Consistently, 85% of patients exhibited a possibly or probably deficient vitamin B12 status according to the cB12 scoring system. In the human body, vitamin B12 is physiologically active in two forms: 1) methylcobalamin which represents a cofactor for the methyltransferase enzyme 5-methyltetrahydrofolate-homocysteine methyltransferase, also known as methionine synthase (MTR), and 2) adenosylcobalamin which serves as a cofactor for the enzyme methylmalonyl coenzyme A mutase (MMCoAM) [2O-related toxicity. N2O exposure is well described as the most effective way to produce vitamin B12 deficiency in animal models [2O induces oxidation of the active cobalt center contained in the vitamin B12 chemical structure. It also induces a displacement of the vitamin B12 molecule from MTR with a parallel loss of MTR activity [Three-quarter of patients with N status <0 pmol/L.(MMCoAM) ,21,22,23(MMCoAM) . In the l models ,25,26,27activity .2O exposure and the occurrence of clinical and biochemical abnormalities associated with vitamin B12 deficiency, notably subacute combined degeneration of the spinal cord in association with hyperhomocysteinemia and elevated levels of methylmalonic acid. Hathout & El-Saden nicely described these hypotheses in their review paper regarding N2O-induced myelopathy [2O occurred primarily through the MMCoAM pathway. However, this was challenged by clinical observations in patients with monogenic inherited disorders associated with methylmalonic acidemia who do not develop subacute combined degeneration [2O exposure and neurological complications is the effect of N2O on the methylcobalamin-MTR pathway. Indeed, inherited disorders in MTR activity induce a decrease of methionine and a low methyl-donor status with defective methylation and resulting instability of the myelin sheath. However, this hypothesis was hampered by experiments using the B12-deficient fruit bat animal model. These experiments did not show significant alterations in S-adenosyl-methionine and S-adenosyl-homocysteine in the brain and spinal cord after induction of severe vitamin B12 deficient myelopathy following a combination of dietary deprivation and N2O exposure [2O-induced myelopathy could be explained by an imbalance between cytokines and growth factors exhibiting myelinotoxic  or myelinotrophic  effects [Several mechanistic hypotheses have been advanced for explaining the association between Nelopathy . Three melopathy , the altelopathy ,28,29, aelopathy ,32,33,34neration . The secexposure ,28,29. D effects ,32,33,342O exposure exhibited high methylmalonic acid concentrations. In an experimental rat model, the exposure to 50% N2O and 50% O2 mixture during 48 h was associated with a 70% reduction of MTR activity in the liver, kidney, and brain with no significant changes in the activity of MMCoAM [2O induced a further decrease in both MTR and MMCoAM activities . This experiment demonstrates the time-dependent effect that N2O exerts on the reduction of enzymatic activity of MTR and MMCoAM. The effect of N2O on MTR is evident from the first hours of exposure, while the effect on the MMCoAM requires longer exposure times [2O causes a decrease in the activity of both MTR and MMCoAM with harmful effects on one-carbon metabolism markers [The present meta-analysis reports that patients with regular Nf MMCoAM . Prolongre times . Prolong markers .2O exposure, mostly associated with surgery and a more severe clinical picture. These data are in keeping with those of the ENIGMA trial that assessed the effects of N2O on patients’ outcome after major surgery [2O-containing general anesthesia were compared with 179 patients undergoing N2O-free general anesthesia [2O group exhibited a significantly increased risk of postoperative hyperhomocysteinemia defined by rising to the 90th percentile of the preoperative homocysteine concentration (>13.5 µmol/L)  [In the present meta-analysis, age ≥40 years, a vitamin B12 concentration ≤74 pmol/L, and an MCV >100 fL were associated with a short N surgery . In thisesthesia . The N2O82–8.40) . Importa82–8.40) .2O were recreational users. The use of N2O as a “laughing gas” became popular in the early 1800s during aristocratic laughing gas parties. Over time, the social image of N2O as a recreational agent has shifted from a “high-society drug” to a “psychedelic drug”, ending up during the last decade as a "hippie crack drug.” The 2016/17 Crime Survey for England and Wales reported that 2.6% of adults aged 16–59  had used N2O in the preceding year [2O use was dramatically higher with 9.3% of 16–24 year olds reported using N2O  in the preceding year [2O as a recreational drug with a lifetime prevalence of 38.6% and 29.4% in the UK and US, respectively [2O users, the reported rates for persistent numbness and accidental injury were 4.3% and 1.2%, respectively [2O users had symptoms of nerve damage [2O represents a significant public health issue. Awareness campaigns among high-risk populations are eagerly needed, particularly for the 16–24 year olds. Most cases of N2O-related toxicity were reported in world areas and countries with a low prevalence of B12 deficiency, including North America and Western Europe. Few reports originated from Turkey and Iran, two Middle Eastern countries with an intermediate-to-high prevalence of vitamin B12 deficiency. This highlights the lack of published data that would allow analyzing the risk of N2O-related outcomes in exposed subjects from countries with a high prevalence of vitamin B12 deficiency, including India, sub-Saharan Africa, and Mexico  [2O-related disorders.The medical community, particularly physicians who are involved in the emergency departments, anesthesiologists, and surgeons, should be aware of the magnitude of recreational Npulation . The eva03–0.28) . Thus, a2O-related toxicity in recreational use and general anesthesia settings. Garakani et al. reported the main findings related to chronic N2O abuse with a focus on neurological sequelae and psychiatric disorders [2O abuse represents a potentially difficult-to-diagnose condition which could lead to death and that physicians should be aware of N2O-related toxicity. In the setting of anesthesia, a meta-analysis summarized the evidence from randomized clinical trials associating N2O with serious cardiovascular complications [2O used as part of general anesthesia affects mortality and cardiovascular complications [Two systematic reviews assessed Nisorders . In thisications . This meications .2O toxicity. This approach has also allowed performing univariate and multivariate analyses. Second, the present meta-analysis reported quantitative evidence about the status of one-carbon metabolism markers in the setting of N2O-related toxicity and highlighted the risk of hyperhomocysteinemia and high methylmalonic acidemia. Third, the meta-analysis of case reports allowed the compilation of unselected patients, thereby reducing the risk of population heterogeneity. The analysis of bias did not reveal a significant stratification of the analyzed population. We acknowledge several limitations. First, we used data extracted from available case reports through a systematic retrospective search. This strategy carries the risk of missing data. Second, laboratory findings were not available for all patients, which resulted in a decrease in the statistical power for univariate and multivariate analyses. Third, in the surgical setting, thromboembolic events potentially associated with N2O-induced hyperhomocysteinemia could have been mistakenly attributed to the postoperative context, leading to a potential underestimation of the thromboembolic risk associated with N2O exposure [2O-induced toxicity. These events may be relatively rare as N2O is widely used. Our data point out the need to perform large population studies to better report on N2O-induced toxicity.The present meta-analysis has several strengths. First, we report an individual patient data meta-analysis that collected original data from 100 patients to describe the global burden related to Nexposure ,45,46,47exposure . Fifth, 2O exposure with severe neurological and hematological manifestations and altered one-carbon metabolism markers. These findings lead to raising awareness among the medical community urgently about the risk of N2O exposure and particularly in the recreational setting among young people. Population studies are warranted to evaluate whether the correction of vitamin B12 deficiency prevents N2O-related toxicity in the context of anesthesia and recreational use, particularly in countries with a high prevalence of vitamin B12 deficiency.This meta-analysis points out the association of N


Escherichia coli D-galactonate/H+ symporter D-galactonate transporter (DgoT) in 2 states: one open to the cytoplasmic side and the other open to the periplasmic side with substrate bound. The structures suggest a mechanism that couples H+ flux to substrate recognition. A transition in the role of H+ from flux coupling to allostery may confer regulation by trafficking to and from the plasma membrane.Members of the solute carrier 17 (SLC17) family use divergent mechanisms to concentrate organic anions. Membrane potential drives uptake of the principal excitatory neurotransmitter glutamate into synaptic vesicles, whereas closely related proteins use proton cotransport to drive efflux from the lysosome. To delineate the divergent features of ionic coupling by the SLC17 family, we determined the structure of  The first structures of a family of organic anion transporters reveal an interaction with protons that is conserved from bacterial transporters to the mammalian proteins that transport glutamate into synaptic vesicles. Secondary active transporters couple the movement of ions down their electrochemical gradient to the concentration of substrate. Differences in the mechanism of energy coupling enable them to meet diverse physiological requirements. In general, transporters related in sequence exhibit similar ionic coupling. However, a highly conserved transporter family contains members that use distinct mechanisms of energy coupling to transport substrate in opposite directions, suggesting divergence from a common underlying mechanism.H+ (= Δψ − 2.3× (RT/F) × ΔpH, where R is the gas constant, T is temperature, and F is Faraday’s constant), with contributions from both electrical potential Δψ and proton gradient ΔpH (pHo/pHi). To take advantage of environmental conditions, members of the SLC17 family have tuned their response to the available driving force, illustrating an unusual form of adaptive divergence.The concentration of organic anions has a crucial role in diverse biological processes, from nutrient uptake by cells to the packaging of neurotransmitter into secretory vesicles for subsequent release by exocytosis. Members of the solute carrier 17 (SLC17) family catalyze the coupled transport of anions driven by a proton electrochemical gradient Δμ+ symport . Se. Se+ to + binding site of bacterial relatives (Glu133 in DgoT) and sialin. The VGLUTs function in an acidic environment, and protonation of Glu133 could be the titratable site for allosteric activation by H+ that we have previously observed  (as a percentage), with galactonate added at t = 0. To assess coupling between galactonate and H+, we used the same conditions but with lumenal pH 7.2 and external pH 7.5 (both buffered with Hepes rather than MES), 5 mM K+ in both lumenal and external solutions, with or without 0.2 μM valinomycin. The fluorescence measurements at the low pH were performed in triplicate on multiple occasions. The fluorescence measurements at the higher pH were performed using 2 reconstitutions, and the initial rates were determined by subtracting the drift in fluorescence before galactonate addition from the time points after. Subtracted data were fitted to the equation y = A + B(x) + Ce−D(x), which contains a single exponential decay component [Ce−D(x)] plus a linear term [A + B(x)] that better fits the rate of decay in liposome controls.To measure transport by DgoT, an inwardly directed HE. coli strain dgoT727(del)::kan  was lysogenized with IPTG-inducible T7 RNA polymerase using the λDE3 kit . DgoT variants were subcloned into pQE60 vector containing a C-terminal deca-histidine tag and transformed into the lysogenic strain. One colony was picked to inoculate a 150 ml LB culture (+100 μg/mL carbenicillin and 35 μg/ml kanamycin) and grown at 37°C with shaking until the OD600 reached 1.0. Basal protein expression from the T7 promoter was sufficient for uptake assays, and no IPTG was added during growth. After harvesting the cells at 3,800g using a table-top centrifuge, the pellet was washed twice with 5 mM MES (pH 6.5), 150 mM KCl (MK buffer), and resuspended to an OD600 of 2.0. To measure substrate transport, 150 μl cell suspension was pre-incubated at 25°C for 5 minutes; 50 μl MK solution containing 10 μM 14C-galactonate  was added to initiate the reaction, the mixture was incubated 30 seconds or 2 minutes at 25°C, the reaction was terminated by filtration through 0.45 μm nitrocellulose (Millipore), and the filters were washed 3 times with 2 ml cold MK buffer before measurement of the bound radioactivity by scintillation counting. Incubation for 30 seconds was used for competition and saturation experiments, and 2 minutes was used for the comparison of different DgoT constructs. All transport assays were performed in triplicate on at least 3 independent cell preparations.DgoT-deficient The expression of DgoT protein was monitored by immunoblotting with a polyhistidine antibody conjugated to horseradish peroxidase  and the chemiluminescent detection of HRP monitored with a ChemiDoc MP imaging system . The absolute amounts of WT and mutant DgoT expression were estimated by comparing the band intensities of each to a serial dilution of purified DgoT (100 to 3 ng) on the same western blot.E. coli DgoT knock-out strain (see previous section). One colony was picked to inoculate a 150 ml LB culture (+100 μg/mL carbenicillin and 35 μg/mL kanamycin) and grown at 37°C with shaking overnight. The overnight culture (3 ml) was used to inoculate a 150 ml LB culture (+carbenicillin/kanamycin) and grown at 37°C with shaking until the OD600 reached 1.0. At that point, 25 ml culture was sedimented at 3,800 rpm for 10 minutes using a table-top centrifuge, the pellet was washed twice in 30 mM Tris  and was resuspended in 30 mM Tris (pH 8) with 20% sucrose (approximately 80 ml buffer per g cell pellet), lysozyme was added (to 20 μg/m), and the mixture was incubated for 5 to 10 minutes. After 1:1 dilution with 30 mM Tris (pH 8) buffer, EDTA was added (to 1 mM), and the resulting spheroplasts were incubated for 10 to 15 minutes with gentle nutation. The spheroplasts were then sedimented at 16,000g for 20 minutes and resuspended in 5 mM MES (pH 5.5), 150 mM KCl, and 20% (w/v) sucrose (MKS-5.5 buffer) supplemented with 20 mM glycerol and 5 mM MgCl2 to an OD600 of 2.0.Spheroplast generation was adapted from previously established methods . DgoT co14C-galactonate was added, and the mixture was incubated for 2 minutes. After incubation, the spheroplasts were subjected to filtration through 0.45 μm nitrocellulose and were washed 3 times with 2 ml cold MK buffer before measurement of the bound radioactivity by scintillation counting. For the analysis of ionophores, 150 mM KCl was replaced by K acetate in both internal and external solutions.To measure net flux, 150 μl spheroplast solution was pre-incubated at 25°C for 5 minutes, 50 μl MKS-5.5 with 10 μM g, washed 3 times with 10 ml MKS-5.5, and resuspended in MKS-5.5 containing 20 mM glycerol to an OD600 of approximately 1.1. Transport was measured as previously described for net uptake by spheroplasts, with or without nigericin or valinomycin (both at 2 μM). As described above, both net flux and exchange were measured in triplicate using at least 3 independent preparations. We subtracted the value for empty vector from WT and mutant DgoT and normalized the exchange rate by mutants for their reduced expression relative to WT  Silver stain of individual fractions after sucrose gradient fractionation (5%–60%) of DgoT proteoliposomes. Proteoliposomes containing reconstituted DgoT appear in fractions F7 to F9. Aggregated and/or nonincorporated DgoT appears at the bottom of the gradient in F12. Purified DgoT (lane 2) was added for molecular weight comparison. (C) Traces (left) show representative fluorescence ratios of proteoliposomes containing pyranine with (left) and without DgoT (right), both formed at pH 7.2, in external solution at pH 7.5, with or without 0.2 µM valinomycin. Yo is the ratio recorded at the addition of 10 mM D-galactonate, and Yf is the ratio recorded at the end of the recording. Both internal and external solutions contained 5 mM K+. (D) Statistical analysis of the results from 9 DgoT proteoliposome and 4 liposome experiments fitted to the equation Y = A + B(x) + C−D(x) that contains both linear and exponential terms to account for the proton leak (linear) and galactonate-driven H+ flux . A is the y-intercept, B the linear slope between Yo and Yf, C the difference between Yo and Yf, and D the rate of exponential decay. The curve fitting is shown in C. Best-fit values of each component are listed along with regression (R2). DgoT, D-galactonate transporter.(A) Size exclusion chromatography (TSK 3000) and Coomassie stain of purified (TIF)Click here for additional data file.S2 Fig(Top) Cylinder representations of the 2 polypeptide chains in the inward-facing conformation of DgoT. β-NG (magenta) appears in the aqueous cavity of both chain A (A) and chain B (B) but at different locations in each. Chain A also contains a molecule of gluconate (yellow). (Bottom) Cross section of the corresponding electrostatic surface potential representations for chains A (A) and B (B). (C) Stick representation of residues interacting with gluconate and the nearby β-NG molecule. (D) Closest interatomic distances between β-NG and gluconate shown in angstroms. (E) Close-up of β-NG located within the aqueous cavity of chain B. Electron density map for C through E shown as 2Fo-Fc map contoured at 1σ. β-NG, β-Nonylglucoside; DgoT, D-galactonate transporter; Fo-Fc, observed structure factor–calculated structure factor.(TIF)Click here for additional data file.S3 FigE. coli DgoT (accession number: AKK15832.2), human VGLUT1 , human VGLUT2 , human VGLUT3 , and human sialin . Amino acid numbering above corresponds to E. coli DgoT. Strongly conserved residues are highlighted in a red box with white text; Asp31 in E. coli DgoT and other less well conserved but important residues are highlighted in a purple box with black text. The secondary structure (above) includes 12 TM domains and 2 ICHs, with the outward-occluded helices shown above and the inward-open below. N- and C-terminal helices are highlighted in blue and green, respectively. Substrate-binding residues are labeled with an asterisk (*). Asp31, Aspartate-31; DgoT, D-galactonate transporter; ICH, intracellular helix; SLC17, solute carrier 17; TM, transmembrane domain; VGLUT, vesicular glutamate transporter.Sequence alignment of (TIF)Click here for additional data file.S4 Fig+ tunnel. Acidic residues including E59 and E180 contribute to the strong negatively charged electrostatic surface. (B) Cross section through electrostatic surface potential representation, viewed from the plane of the membrane. The entrance (near E180) has a diameter of 3.6 Å, a constriction (near Pro279 and Thr297) of 2.4 Å, and an exit (near Asp46) of 5.0 Å. Asp46, Aspartate-46; DgoT, D-galactonate; Pro279, Proline-279; Thr297, Threonine-297.(A) DgoT viewed from the periplasm is shown in electrostatic surface potential representation, with arrow indicating the entrance to the putative H(TIF)Click here for additional data file.S5 Fig(A) TM10 separates from TM4 in the inward-facing conformation (colored), breaking the helix in TM10 but re-establishing the continuity of TM4 that was disrupted in the outward-facing conformation (gray) (B) For each panel, the N- and C-terminal domains were aligned from the other structure for comparison. 2Fo-Fc density map (1σ) of residues from TM4 and TM10 from the inward-facing conformation (C) and outward-facing conformation (D). Fo-Fc, observed structure factor–calculated structure factor; TM, transmembrane domain.(TIF)Click here for additional data file.S6 Fig(A) View of the substrate binding site showing residues and substrate. Alignment of N- and C-terminal domains from the inward-facing conformation (gray) onto the outward-facing conformation (colored). (B) Close-up of the alignment between Arg47 (TM1) and glutamate (inward-facing) or glutamine (outward-facing) at position 133. (C) A composite, simulated annealing “omit map” (1σ) is shown in a close-up stereo view of the substrate recognition site. Residues Gln164, Gln264, Ser370, and Asn393 form hydrogen bonds (orange dashes) with the 5 hydroxyl groups in D-galactonate. Tyr44 and Tyr79 form hydrogen bonds, and Arg47 forms a salt bridge with the carboxyl group of D-galactonate. Arg, Arginine; Asn, Asparagine; Gln, Glutamine; Ser, Serine; TM, transmembrane domain; Tyr, Tyrosine.(TIF)Click here for additional data file.S7 Fig+ tunnel, ending at Asp46. (Right panel) Cross section of the N-terminal domain in the outward-facing conformation shows the remnant of the H+ tunnel with an occlusion. Asp46, Aspartate-46; DgoT, D-galactonate transporter.(Left panel) Cross section of inward-facing DgoT viewed from the periplasm shown in surface representation . Arrow indicates the entrance to the putative H(TIF)Click here for additional data file.S8 Fig−E. coli transformed with WT E. coli DgoT, empty pQE60 vector, D46N, E133Q, or the double D46N/E133Q DgoT detected using a polyhistidine antibody and chemiluminescence. Standards of WT purified E. coli DgoT are shown to the right. (B) Immunoblot of extracts from DgoT−E.coli transformed with empty pQE60 vector, WT, and R47K DgoT. DgoT, D-galactonate transporter; WT, wild type.(A) Immunoblot of extracts from DgoT(TIF)Click here for additional data file.S1 TableMR, molecular replacement.(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


N-linked protein glycosylation and fucosylation. The screens also identified lysosomal-associated protein transmembrane 4 alpha (LAPTM4A), a poorly characterized four-pass membrane protein, as a factor specifically required for Stxs. Mass spectrometry analysis of glycolipids and their precursors demonstrates that LAPTM4A knockout (KO) cells lack Gb3 biosynthesis. This requirement of LAPTM4A for Gb3 synthesis is not shared by its homolog lysosomal-associated protein transmembrane 4 beta (LAPTM4B), and switching the domains between them determined that the second luminal domain of LAPTM4A is required, potentially acting as a specific “activator” for the GT that synthesizes Gb3. These screens also revealed two Golgi proteins, Transmembrane protein 165 (TMEM165) and Transmembrane 9 superfamily member 2 (TM9SF2), as shared factors required for both Stxs and ricin. TMEM165 KO and TM9SF2 KO cells both showed a reduction in not only Gb3 but also other glycosphingolipids, suggesting that they are required for maintaining proper levels of glycosylation in general in the Golgi. In addition, TM9SF2 KO cells also showed defective endosomal trafficking. These studies reveal key Golgi proteins critical for regulating glycosylation and glycolipid synthesis and provide novel therapeutic targets for blocking Stxs and ricin toxicity.Glycosylation is a fundamental modification of proteins and membrane lipids. Toxins that utilize glycans as their receptors have served as powerful tools to identify key players in glycosylation processes. Here, we carried out Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9–mediated genome-wide loss-of-function screens using two related bacterial toxins, Shiga-like toxins (Stxs) 1 and 2, which use a specific glycolipid, globotriaosylceramide (Gb3), as receptors, and the plant toxin ricin, which recognizes a broad range of glycans. The Stxs screens identified major glycosyltransferases (GTs) and transporters involved in Gb3 biosynthesis, while the ricin screen identified GTs and transporters involved in  Shigella dysenteriae and enterohemorrhagic Escherichia coli (EHEC), which cause food poisoning throughout the world. Ricin is a plant toxin and a potential bioterrorism agent. Stxs recognize the host receptor glycolipid Gb3, while ricin utilizes a broad range of glycans as receptors. Here, we carried out genome-wide loss-of-function CRISPR-Cas9 screens using human cells to identify factors required for Stxs and ricin. Besides host factors previously known to be involved in the action of these toxins, our screens revealed three previously poorly characterized Golgi proteins: LAPTM4A, which is specifically required for Stxs, and TMEM165 and TM9SF2, which are required for both Stxs and ricin. Further characterization demonstrates that LAPTM4A is specifically required for biosynthesis of the glycolipid Gb3, potentially acting as an “activator” protein for the glycosyltransferase that synthesizes Gb3, whereas TMEM165 and TM9SF2 are likely required to maintain a proper environment within the Golgi for optimal activity of glycosyltransferases. These findings provide mechanistic insights to glycolipid biosynthesis and regulation of glycosylation levels in the Golgi and also reveal novel therapeutic targets for preventing Stxs and ricin intoxication.Shiga and Shiga-like toxins (Stxs) are a family of bacterial toxins and key virulence factors for the bacteria  Shigella dysenteriae, and related Shiga-like toxins Stx1 and Stx2, produced by Shigatoxigenic strains such as enterohemorrhagic E. coli (EHEC) [The plant toxin ricin is derived from castor oil plant seeds. It has been utilized as a poison in criminal cases and is classified as a potential bioterrorism agent . Shiga ai (EHEC) . EHEC isi (EHEC) .N-glycosidase and inhibit protein synthesis by cleaving the same adenine of 28S rRNA. Ricin is composed of an A chain (32 kDa) and a B chain (34 kDa), connected via a disulfide bond. The A chain is an N-glycosidase and the B chain is the receptor-binding domain. Stxs are A-B5 bacterial toxins [N-glycosidase, and a receptor-binding domain consisting of five identical B chains (about 7.7 kDa each). These B chains form a ring, and the A chain connects to the B chain by inserting its C-terminus into the center pore of the B chain ring. Stx1 has only one single amino acid difference from Stx, while Stx2 represents a distinct serotype, with about 56% sequence identity to Stx.Ricin and Stxs are structurally and evolutionarily distinct but share the same mode of action: both act as ribosomal RNA  A chain 2 kDa andRicin and Stxs also share similar entry pathways into cells ,3,5,6. ON-acetylgalactosamine [The major difference between ricin and Stxs is their receptor recognition. Ricin binds broadly to glycan moieties containing galactose and tosamine . In conttosamine ,3,9. Crytosamine . Expresstosamine .N-linked protein glycosylation.Glycosylation is one of the most common modifications of proteins and membrane lipids ,13. It iHere, we identified a human bladder cancer cell line that is highly sensitive to Stxs. Utilizing this cell line, we carried out CRISPR-Cas9–mediated KO screens for Stx1 and Stx2. In addition, we also carried out a genome-wide screen for ricin. These screens identified lysosomal-associated protein transmembrane 4 alpha (LAPTM4A) as a novel Golgi protein specifically required for Gb3 biosynthesis. The screens also revealed two Golgi proteins, Transmembrane protein 165 (TMEM165) and Transmembrane 9 superfamily member 2 (TM9SF2), as key factors for maintaining proper glycosylation levels in cells.50. Most cell lines are insensitive to Stx1 and Stx2, with IC50 > 10,000 ng/mL (50 at about 0.028 (Stx1) and 0.007 (Stx2) ng/mL. The human kidney adenocarcinoma cell line ACHN is also quite sensitive to Stx. This level of sensitivity is not shared by two other bladder cancer cell lines (T24 and RT4), suggesting that it is not a general feature of bladder cancer cells. Consistently, immunostaining analysis using a polyclonal anti-Stx1 antibody showed robust binding of Stx1 to 5637 and ACHN cells, whereas binding to HeLa cells was undetectable  and the total number of NGS reads (x axis) , targeting 19,052 human genes with six single guide ribonucleic acids (sgRNAs) per gene ,21. Thes(x axis) . The fulA4GALT, B4GALT5, SLC35A2, UGCG, and SPTSSA [β-1,4-galactosyltransferase 5 (B4GALT5) then catalyzes the transfer of a galactose from UDP-galactose onto GlcCer to generate lactosylceramide (LacCer), which is the precursor for both globo-series and ganglio-series of glycosphingolipids. α-1,4-galactosyltransferase (A4GALT) finally produces Gb3 by catalyzing the transfer of galactose to LacCer [Most top-ranked hits overlapped between Stx1 and Stx2 screens. Five of the top eight genes are known factors in the Gb3 synthesis pathway : A4GALT,d SPTSSA . Serine o LacCer ,23. A4GALAPTM4A, TMEM165, and TM9SF2. LAPTM4A is a poorly characterized small four-pass membrane protein with unknown function, previously proposed to be localized mainly on lysosomes [2+ hemostasis in the Golgi, as Mn2+ is an essential metal cofactor for many GTs [TMEM165 and TM9SF2 have been previously identified as genes involved in heparan sulfate (HS) biosynthesis in two independent genetic screens using haploid cells, suggesting that TMEM165 and TM9SF2 deficiency affects multiple glycosylation pathways [The other three of the top eight genes are ysosomes –27. It imany GTs –30. TM9Smany GTs . Both TMpathways ,33. Intepathways . Furtherpathways .A4GALT, SLC35A2, UGCG, B4GALT5, LAPTM4A, TMEM165, and TM9SF2. Cell viability assays were carried out to determine their sensitivity to Stx1 and Stx2 (5-fold increase in resistance to Stx1 and Stx2 compared to wild-type (WT) cells. TM9SF2 KO and TMEM165 KO cells showed about 10-fold and 780-fold smaller increases in resistance compared with LAPTM4A KO and A4GALT KO cells.To validate our screen results, we generated mixed (uncloned population) KO 5637 cells using the CRISPR-Cas9 approach for and Stx2 . A4GALT,N-linked protein glycosylation, fucosylation, membrane trafficking, and ER-associated protein degradation (ERAD)/quality-control pathways  and transferring oligosaccharides to an asparagine residue as an N-linked glycan (OST4), as well as enzymes located in the Golgi that convert oligo-mannose to galactose-containing complex N-linked glycans . These results are consistent with the established view that galactose on N-linked glycans is the primary receptor for ricin.In contrast to Stxs, most cells are sensitive to ricin . We thuspathways . Top hitN-acetyllactosamine to generate fucosylated carbohydrates, such as the non-sialylated carbohydrate antigen, Lewis X. The critical role of fucosylation in ricin toxicity has been previously reported and validated, possibly because fucosylation promotes the exposure of terminal galactoses by preventing their sialylation [Our screens also identified key players in the fucosylation pathway, including TSTA3 and GMDS, which catalyze the synthesis of GDP-fucose; SLC35C1, which transports GDP-fucose from the cytosol into the Golgi lumen; and FUT4, which catalyzes the transfer of fucose to lylation ,35,36.The screens revealed a series of proteins involved in intracellular vesicular transport, including members of the Golgi-associated retrograde protein (GARP) complex , which is a tethering complex involved in retrograde transport from the early endosome to the Golgi, as well as a few proteins involved in Golgi-ER trafficking . There are also two ER proteins among the top-ranked hits: ER resident protein 44 (ERP44), which is a member of the protein disulfide isomerase family, and ubiquitin conjugating enzyme E2 G2 (UBE2G2), which is an E2 ubiquitin-conjugating enzyme involved in ERAD.Ricin has been previously subjected to shRNA-mediated KD screens and a CRISPR-Cas9–mediated loss-of-function screen utilizing a K562 cell line (a human bone marrow lymphoblast) and a retroviral mutagenesis screen using haploid cells –19. AmonMGAT2, SLC35C1, ERP44, and TAPT1, which are among the top hits reported from previous screens but have not been experimentally validated. We also generated mixed KO cells for the three new factors GOSR1, JTB, and NBAS. These KO cells all showed modest increases (about 10–200-fold) in resistance to ricin compared with WT cells  and cholera toxin (Ctx). A-Dtx is composed of the N-terminal part of anthrax toxin lethal factor (LFn) fused to the enzymatic domain of diphtheria toxin (DTA) and the receptor-binding/translocation domains of Anthrax toxin (PA) . PA mediTo determine which step of toxin action is affected in LAPTM4A KO cells, we first examined binding of Stx1 to cells via immunofluorescent staining. WT and Mut-9 cells showed robust binding of Stx1, while there was no detectable binding to KO-10 or KO-12 cells . As expeLoss of Stx1 binding implies that LAPTM4A is critical for Gb3 expression. To examine this possibility directly, we sought to quantify the levels of Gb3 and its precursors LacCer, GlcCer, and Cer in cells using mass spectrometry analysis. Total lipids were extracted from cell lysates. Samples were then analyzed using an ultra-pressure liquid chromatograph (UPLC) coupled to a mass spectrometer. Quantification of glycolipids was normalized based on endogenous phosphatidylcholine (PC) as an internal standard . We founTo understand the function of LAPTM4A, we next examined its subcellular localization. As there is no suitable antibody to detect endogenous LAPTM4A, we utilized a LAPTM4A tagged with a triple HA tag on its C-terminus. This tagged version restored binding of Stx1 when expressed in KO-10 and KO-12 cells . ExpressTo further examine whether LAPTM4A interacts with A4GALT, we co-expressed HA-tagged LAPTM4A and FLAG-tagged A4GALT in HEK293T cells and then carried out co-immunoprecipitation (co-IP) assays using a FLAG tag antibody. The FLAG-tagged A4GALT is correctly localized to the Golgi and can rescue binding of Stx1 to A4GALT KO cells . In addiWe next analyzed whether the absence of LAPTM4A affects the Golgi localization and/or stability of A4GALT. We found that A4GALT is still localized to the Golgi in KO-10 and KO-12 cells and the expression levels of A4GALT in KO-10 and KO-12 cells are similar to those of WT and LAPTM4B KO cells . ImmunobLAPTM4A and LAPTM4B are small four-pass transmembrane proteins  whose ovTaking advantage of the observation that LAPTM4B cannot restore Stx1 binding to LAPTM4A KO cells, we generated a series of chimeric proteins between these two homologs in order to map the region responsible for their functional difference. These homologs were expressed in KO-10 and KO-12 cells and binding of Stx1 was analyzed by immunostaining, flow cytometry, and immunoblot . The resWe next investigated the role of TMEM165 and generated single clones of TMEM165 KO 5637 cells via the CRISPR-Cas9 approach. We obtained one KO line (TM-KO-3) that contains frameshift mutations on all chromosomes, and one mutation line (TM-Mut-1) that contains a frameshift and an insertion of six base pairs . The lat50 values were not significantly changed under our assay conditions  contain frameshift mutations in all three chromosomes . A singlty assay , but thety assay . TM9SF2 ty assay .We found that binding of Stx1 and CtxB was largely abolished in two TM9SF2 KO cells, but not in the SF2-WT-5 control cells . BindingPrevious studies have showed that Myc-tagged TM9SF2 is localized mainly on endosomes . More reInterestingly, both SF2-KO-8 and SF2-KO-9 cells appear to form many large vacuole-like structures within the cytosol, which are labeled with Rab7 . To furt5-fold), further demonstrating that binding to Gb3 is the key rate-limiting step for Stx intoxication. In contrast, top hits from the ricin screen are distributed across multiple pathways, including protein glycosylation, fucosylation, retrograde trafficking, and ERAD pathway. Mutations in these ricin host factors elicited only low levels of resistance (<200-fold). These results are consistent with the view that various galactose-containing moieties and multiple trafficking pathways can mediate entry of ricin redundantly.Here, we found that the human bladder cancer cell line 5637 is highly sensitive to Stxs. Cell line 5637 is easy to culture and well suited for cell biology studies; thus, it can serve as a useful human cell model for studying Stxs. The top eight hits from Stx1 and Stx2 screens are all involved in Gb3 synthesis and cells lacking Gb3 are highly resistant to Stxs  is a component of the coat protein complex I (COPI) complex, a protein complex that mediates budding of vesicles from the Golgi membranes. COPI plays a critical role in vesicular transport between the Golgi and the ER [Other notable top-ranked hits shared between Stx1 and Stx2 screens include d the ER , but itsd the ER ,40. UDP-d the ER ,42. SPPLOur screening protocol relies on the cell viability assay after 72 h incubation. Such a long incubation could mask minor reductions in toxin trafficking within cells. It is possible that a few trafficking-related factors, such as GARP and STX5, were identified because they potentially may also affect glycosylation processes. Our Stx1 screen did not identify GPP130, a protein previously reported to be important for retrograde transport of Stx1 (but not Stx2) . HoweverOur major finding is identification of LAPTM4A, a poorly characterized small four-pass transmembrane protein, as a novel factor critical for Gb3 biosynthesis. Endogenous LAPTM4A protein has been detected in both the Golgi and lysosome fractions of rat liver membrane lysates ,25. PrevThe exact function of LAPTM4A remains to be established. It has been shown that LAPTM4A expression in yeast confers a multidrug-resistance phenotype and altered subcellular distribution of steroids, suggesting that LAPTM4A is involved in the transport of nucleosides or some small molecules ,25,46. LMany GTs require other proteins for their stability/trafficking/activity. For instance, the core 1 β3galactosyltransferase (C1GALT1) requires the ER-localized membrane protein Cosmc (C1GALT1C1) as a molecular chaperone, which is essential for its proper folding and trafficking –52. Howeα-lactalbumin can serve as an “add-on” domain for β4-galactosyltransferase, and formation of the complex changes the specificity of the acceptor [Drosophila β-1,4-N-acetylgalactosaminyltransferase B, which is responsible for synthesis of glycolipids and requires a six-pass membrane protein GABPI for its activity [The soluble enzymatic domain of GTs involved in glycolipid biosynthesis often showed no activity in vitro, suggesting that their activity requires a proper membrane environment and/or “activator” proteins to present the lipid acceptor to GTs. This requirement of an “activator” might be because the headgroups of lipid acceptors are difficult for the soluble enzymatic domain of GTs to reach. This is analogous to the well-established finding that degradation of sphingolipid requires cofactor proteins in addition to glycosidases. These cofactors are known as sphingolipid activator proteins, which are small enzymatically inactive proteins that present glycolipid substrates to glycosidases . This acacceptor . We propactivity ,57. Furtactivity . It is p2+ homeostasis within the Golgi, although its exact function remains to be fully established. Mn2+ is an essential cofactor for many Golgi GTs involved in both glycoprotein and glycolipid synthesis. It has been shown that TMEM165 deficiency leads to severe reduction in galactose and GalNAc in both glycoproteins and glycolipids [2+ and partially by galactose supplementation [2+ supplementation restored binding of Stx to TMEM165 KO cells. Furthermore, TMEM165 KO cells become more sensitive to toxicity from high concentrations of Mn2+. Together, these findings further support that TMEM165 plays a critical role in Mn2+ homeostasis in the Golgi.Our screens also identified TMEM165 and TM9SF2 as two host factors required for both Stx and ricin intoxication. TMEM165 has been proposed to maintain Mncolipids . This reentation ,58. ConsDrosophila (TM9SF2 to TM9SF4), and Dictyostelium discoideum (Phg 1A to 1C) and four members in mammals (TM9SF1 to TM9SF4). The sequence features of this family suggest that they could function as channels or small-molecule transporters. Defects in TM9SF2 family members have been associated with two types of defects: (1) disruption in phagocytosis and endosomal trafficking: it has been shown that the absence of Phg 1A strongly impaired both cell adhesion and phagocytosis of bacteria in Dictyostelium, and both TM9SF4 and TM9SF2 are required for phagocytosis in Drosophila as well [2+ [The function of TM9SF2 remains a mystery. TM9SF2 is expressed in all tissues and is evolutionarily conserved. It belongs to a family of highly conserved membrane proteins, with three members in yeast (Tmn1 to Tmn3),  as well ,60. Furt as well . Consistwell [2+ . This diGlobo-series glycosphingolipids are unique in that their expression is highly restricted. Our studies revealed that biosynthesis of Gb3 not only requires its GT but also a membrane protein, LAPTM4A, which may provide an additional layer of regulation to ensure the restricted expression of Gb3. Our studies also revealed two Golgi-localized factors required for maintaining a proper environment for GT activities within the Golgi. These findings further indicate that the glycosylation process in the Golgi is tightly controlled and regulated. Further research into the function of these factors will contribute to our understanding of the glycosylation process and CDG. Finally, our findings have also provided new potential therapeutic targets for treating Stxs and ricin intoxication.The following reagents were purchased from commercial vendors: ricin , Ctx , CtxB Alexa Flour 555 conjugate , MTT , NBD-Sph , NBD-C6-Cer , and NBD-PS . Antibodies for the following antigens were obtained from the indicated vendors: the A domain of Stx1 , Actin , HA-tag , FLAG-tag , Rab5 , Rab7 , Giantin , TGN46 , Sec61A , Lamp1 , and A4GALT . A chicken polyclonal anti-TM9SF2 antibody was generously provided by Dr. Yusuke Maeda  . The folE. coli (BL21 strain) and purified as His-tagged proteins. The cDNAs of LAPTM4A (2958870), A4GALT (3913851), LAPTM4B (5264567), TMEM165 (8143981), TM9SF2 (40146324), EMC1 (4831005), and B4GALT5 (30915234) were purchased from GE Dharmacon. SgRNA-resistant full-length LAPTM4A, LAPTM4B, TM9SF2, and EMC1 with triple-HA tag at their C-termini (with EFGSGSGS as linker); full-length LAPTM4A, TMEM165, and EMC1 with triple-HA tag at their N-termini (with GSGSGSEF as linker); full-length A4GALT and B4GALT5 with triple-FLAG tag at their N-termini (with GSGSGSEF as linker) were cloned into ether pcDNA3.1 vector  or pLenti-Hygro vector . The LAPTM4A (LA)-LAPTM4B (LB) chimeric proteins were constructed as the following: AB1 (LB1–173–LA181–233), AB2 (LB1–153–LA161–233), AB3 (LB1–120–LA129–233), AB4 (LB1–120–LA129–160–LB154–226), AB5 (LA1–29–LB26–226), AB6 (LB1–25–LA30–49–LB47–71–LA83–102–LB93–99–LA109–128–LB121–152–LA161–180–LB174–226), AB7 (LB1–25–LA30–180–LB174–226), AB8 (LB1–46–LA50–160–LB153–226), and AB9 (LB1–71–LA83–128–LB121–226), with triple-HA tag at their C-termini. These constructs were cloned into pLenti-Hygro vector via Gibson Assembly . The A4GALT (A4)-B4GALT5 (B4) chimera construct A43B352 (A41–43–B436–388) with triple-FLAG tag at the N-termini (with GSGSGSEF as linker) was cloned into pcDNA3.1 vector via Gibson Assembly.Shiga toxin clones (pLPSH3 for Stx1 and pJES120 for Stx2) were generously provided by Dr. Alison O’Brien . Protective antigen (PA) of Anthrax toxin was cloned into pET21a vector (Novagen) with a His6-tag at C-terminal. pET15b-LFn-DTA was obtained from Addgene (#11075). Recombinant PA and LFn-DTA were expressed in 7 (for sub-library A) or 2.9 × 107 (for sub-library B) cells were either saved as Round 0 (R0) samples, or seeded onto four 15-cm culture dishes and exposed to toxins. The survival cells were reseeded and cultured with normal medium without toxins until about 70% confluence. Cells were then subjected to the next round of selection. The remaining cells were harvested. The genomic DNA was extracted using a commercial kit . DNA fragments containing the sgRNA sequences were amplified by PCR using primers lentiGP-1_F (AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG) and lentiGP-3_R (ATGAATACTGCCATTTGTCTCAAGATCTAGTTACGC). NGS was performed by a commercial vendor .Lentiviral sgRNA plasmid libraries were generated using the human GeCKO-V2 sgRNA library . The GeCKO-V2 library is composed of two sub-libraries (sub-library A and sub-library B). Each sub-library contains three unique sgRNA per gene and was independently packed into lentiviral libraries. The titer of lentiviral libraries was calculated as colony-forming units (CFU) per mL. The cell representation was 500, so each sgRNA on average is distributed into 500 cells. The multiplicity of infection (MOI) was 0.2. Polybrene  was also added to the medium to increase viral transduction efficiency. Cells were cultured in virus-containing medium for 2 d. Infected cells were selected with Puromycin ; 3.3 × 10E. coli (DH5α strain) and plated onto agar plates. Twenty single clone colonies were selected, and their plasmids were extracted and sequenced.The selected sgRNA sequences  were cloCells were seeded in 96-well microplates and incubated overnight until about 70% confluence. The medium was replaced with 100 μL toxin-containing medium, with a total of 10 serial dilutions (10-fold). The cells were further incubated for 72 h in toxin-containing medium and then 10 μL MTT (5 mg/mL in PBS) was added to each well and incubated at 37 °C for 4 h. A total of 100 μL solubilization solution (10% SDS in 0.01 M HCl) was then added to each well and incubated overnight. The absorbance of formazan formed was then measured at 580 nm by a microplate reader . A vehicle control without toxins and a blank of PBS were analyzed in parallel. The cytotoxicity curves were analyzed and fitted using Origin software (version 8.5).Cells were seeded onto glass coverslips in 24-well plates and incubated for 24 h until about 70% confluence. Cells were washed three times with ice-cold PBS, fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature (RT), permeabilized with 0.3% Triton X-100 for 30 min, and blocked with 10% goat serum for 40 min, followed by incubation with primary antibodies (1 h) and fluorescence-labeled secondary antibodies (1 h). Slides were sealed within DAPI-containing mounting medium . We note that for labeling endogenous TM9SF2, cells were fixed in methanol at −20 °C for 10 min, as this antibody did not work on samples fixed by PFA. For the toxin surface binding assay, cells were incubated with toxin-containing medium  on ice for 60 min. Cells were washed, fixed, and subjected to immunostaining without permeabilization. Stx1 polyclonal antibody was used to recognize surface-bound Stx1. Fluorescent images were captured with the Olympus DSU-IX81 Spinning Disk Confocal System. Images were pseudocolored and analyzed using ImageJ.Cells were harvested and washed three times with PBS. The cell pellets were lysed with RIPA buffer . Cell lysates were centrifuged and the protein amounts in supernatants were measured by BCA assay . The supernatants were heat denatured for 5 min, subjected to SDS-PAGE, and transferred onto a nitrocellulose membrane. The membrane was blocked with a TBST buffer  containing 5% skim milk at RT for 1 h. Then, the membrane was incubated with the primary antibodies for 1 h, washed, and incubated with secondary antibodies for 1 h. Signals were detected using the enhanced chemiluminescence method by a Fuji LAS3000 imaging system.For Stx and Ctx binding, cells were incubated with toxin-containing medium  on ice for 60 min, washed three times with ice-cold PBS, and collected. Cell pellets were fixed with 4% PFA for 20 min at RT and blocked with 10% goat serum for 40 min. Stx1 polyclonal antibody was used to recognize surface-bound Stx1 (1 h). Cells were washed and incubated with Alexa488-labeled secondary antibody for 1 h, washed twice, and subjected to single-cell sorting using a Canto II FACS system (BD Biosciences). Cells not treated with toxins were used as controls. For cell surface HS, cells were collected with 1 mM EDTA in PBS and subsequently resuspended in PBS with 1% BSA. Cells were incubated with either the 10E4 monoclonal antibody against HS  or mouse IgM  for 1 h on ice. Cells were washed twice with PBS and incubated with Alexa488-labeled secondary antibody for 1 h on ice and washed twice, followed by single-cell sorting. Single cells were gated and analyzed using FlowJo software .Cellular concentrations of cAMP were examined using the Direct Immunoassay Detection Kit . Briefly, cells were treated with Ctx , lysed, and the protein amounts were measured. The cell lysates were loaded into anti-cAMP antibody-coated plates. The HRP-linked cAMP was added to compete with the cellular cAMP. The activity of HRP-cAMP conjugate was measured using a microplate reader. A calibration curve of free cAMP was analyzed in parallel. The cAMP concentrations in different cells were normalized by the total protein amount in cell lysates.HEK293T cells were co-transfected with plasmids encoding FLAG-tagged A4GALT, B4GALT5, or a chimeric protein A43B352, together with HA-tagged LAPTM4A, LAPTM4B, or AB4 at a 1:1 ratio. Cells were harvested 48 h later and cell lysates subjected to co-IP assays using anti-FLAG magnetic beads . Briefly, cells were lysed with RIPA buffer and incubated with anti-FLAG beads overnight at 4 °C. Beads were then washed (0.1% Triton X-100 in PBS), pelleted, and boiled in SDS sample buffer . After centrifugation, the supernatant (Pull-down) as well as the whole cell lysates (Input) were subjected to immunoblot analysis.6 cells from each cell line were washed with 0.1% ammonium acetate and suspended in 0.35 mL of deionized water. A mixture of methanol (1.25 mL) and chloroform (0.675 mL) was then added, together with 3,000 ng lipid standards d3-GM2 . The sample was vortexed for 1 min, followed by incubation for 30 min and centrifugation at 2,000g for 30 min at RT. Supernatant (the methanol/chloroform fraction) was collected. The remaining cell pellet was resuspended in 250 μL of water, and lipids were extracted again using a methanol/chloroform mixture . The supernatant fractions were combined, dried under nitrogen gas, and stored at −20 °C until analysis.Briefly, 3.5×10Lipid extracts were resuspended in 100 μL of 60% methanol and 40% water. For each sample, a total of 5 μL was injected into a Kinetex column  using a UPLC  coupled to a Waters Synapt G2-si quadrupole time-of-flight mass spectrometer fitted with an electrospray ionization source operating in positive ion mode. LC separation was carried out at a flow rate of 0.27 mL/min using mobile phase A: 0.1% formic acid, 5 mM ammonium formate in water, and mobile phase B: 0.1% formic acid, 5 mM ammonium formate in methanol, using the gradient conditions as follows: 0–1 min (60% B), 1–2 min (60%–70% B), 2–40 min (70%–100% B), 40–43 min (100% B), 43–43.1 min (100%–60% B), and 43.1–50 min (60% B). Gangliosides were analyzed in negative ion mode. For each sample, a total of 10 μL was injected onto a Kinetex column and LC-MS system described above. Mobile phase A, 0.1% (v/v) formic acid in water, and mobile phase B, 0.1% (v/v) formic acid, methanol, isopropanol (5/47.5/47.5) at a flow rate of 0.23 mL/min were used for elution using the following gradient conditions: 0–1 min (60% B), 1–12 min (100% B), 12–14 min (100% B), 14–14.1 min (60% B), and 14.1–20 min (60% B). Mass calibration and external lock mass correction were carried out using Glu-1-Fibrinopeptide B. For each lipid detected in positive ion mode , protonated, sodium adduct, and dehydrated ions were detected. Extracted ion chromatograms obtained using a 15-ppm window centered on the theoretical ionic mass of glycosphingolipids were integrated using TargetLynx XS (Waters Corporation); summing of peak areas of the corresponding adducts and further data processing was carried out in Excel . Endogenous PC was used as an internal standard to obtain peak area ratios for Gb3, LacCer, GlcCer, and Cer. The corrected peak areas and that of the internal standard d3-GM2 were used to obtain peak area ratios of GM2. Relative standard deviations (RSDs) for the replicate analyses were within 15%–20%.Cells were precooled on ice and exposed to fluorescently labeled lipids  in serum-free medium containing DF-BSA (5 μM) for 40 min at 4 °C. Cells were washed twice with PBS and incubated for the indicated time in serum-free medium containing DF-BSA (5 μM). Cells were then washed three times with ice-cold PBS and fixed with 4% PFA for 20 min at RT. Slides were sealed within DAPI-containing mounting medium.2, 1 mM EDTA, 10 mM HEPES, pH 6.9) for 15 min at RT. Cells were then subjected to immunofluorescent staining analysis.HEK293T cells were transfected with HA-tagged LAPTM4A or EMC1. After fixation in 4% PFA, cells were permeabilized with either Saponin buffer  for 30 min at RT or Digitonin buffer , quantified, and subjected to a reverse transcription reaction . The cDNA was quantified and subjected to a qPCR reaction. The reactions were run in triplicate on 96-well plates with an ABI Prism 7700 Sequence Detection System (Applied Biosystems). SYBR Green  was used to monitor dsDNA synthesis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping control to normalize the relative mRNA level. The primers used were as follows: A4GALT_RT_F (GAGACTTCAGACCGGACCAA), A4GALT_RT_R (AAGCCCTTTCATCAGGACCA), LAPTM4A_RT_F (CAAGTGGGTTGGCTGATTCC), LAPTM4A_RT_R (AGGCCAGGAGGTCATCTTTG), GAPDH_RT_F (AGGGCTGCTTTTTAACTCTGGT), and GAPDH_RT_R (CCCCACTTGATTTTGGAGGGA).S1 FigN = 3. (B) The sensitivities of the four indicated cell lines to ricin were determined using cell viability assays. HeLa was the most sensitive one among the four cell lines. Error bars indicate mean ± SD, N = 3. (C) Schematic diagram of the ricin screen. HeLa cells stably expressing Cas9 were transduced with the human GeCKO-V2 sgRNA library and then selected by increasing concentrations of ricin . The survival cells were recovered and their sgRNAs were analyzed by NGS. (D) Binding of Stx1 to the cell surface of 5637, ACHN, and HeLa cells was examined by immunostaining using a polyclonal Stx1 antibody. Cells were exposed to Stx1 (4.8 μg/mL) on ice for 60 min, washed, and fixed. Nuclei were labeled with DAPI. ACHN and 5637 cells showed robust binding of Stx1, while binding of Stx1 to HeLa cells was not detectable. Scale bar, 5 μm. Representative images are from one of the three independent experiments. (E) Top genes were enriched in Stx1, Stx2, and ricin screens. For each gene, the number of NGS reads and the number of unique sgRNAs identified from sub-library A and sub-library B were combined. The top 1,000 genes with the highest NGS reads identified in Stx1_R2 (orange circles), Stx2_R2 (purple circles), and Ricin_R4 (green circles) were plotted versus their numbers in R0 (gray circles). The full lists of identified genes were shown in (A) Cell viability assays were carried out as described in (TIF)Click here for additional data file.S2 Fig50 values are listed in N = 3.  Mixed LAPTM4A and A4GALT KO ACHN cells were generated via the CRISPR-Cas9 approach and subjected to cell viability assays for Stx1 and Stx2. Both LAPTM4A and A4GALT KO cells showed increased resistance to Stx1 (C) and Stx2 (D). Error bars indicate mean ± SD, N = 3. (E) Mixed KO HeLa cells for the selected hits in ricin screen  were generated via the CRISPR-Cas9 approach. These cells were subjected to cell viability assays. The IC50 values are listed in N = 3. Mixed stable 5637 KO cells for the indicated genes were generated via the CRISPR-Cas9 approach. For LAPTM4A, two independent KO cell lines using two different sgRNAs were generated (LAPTM4A-KO-Mix and LAPTM4A-KO-II-Mix). We also generated and tested a KO cell line lacking LAPTM4B, a homolog of LAPTM4A. These cells were subjected to cell viability assays for Stx1 (A) or Stx2 (B). The IC(TIF)Click here for additional data file.S3 Fig(A) WT and mutant 5637 cells lacking LAPTM4A (LA-KO-10 and LA-KO-12), A4GALT (A4-KO-Mix), or LAPTM4B (LB-KO-Mix) as well as a cell line that expresses a mutated form of LAPTM4A (LA-Mut-9) were exposed to Stx1 (4.8 μg/mL) on ice for 60 min. Cells were washed and cell lysates were subjected to immunoblot analysis detecting bound Stx1 using a polyclonal anti-Stx1 antibody. The A domain of Stx1 (Stx1A) is shown. Actin served as a loading control. Representative images are from one of the three independent experiments. (B) Experiments were carried out as described in panel A, except that cells were analyzed by flow cytometry using Stx1 and Ctx labeled with antibody or fluorescent dyes (Alexa 555), respectively. Cells not exposed to toxins were used as a control (Ctrl). The percentages of cells showing positive toxin binding signals are marked. Representative histograms are from one of the three independent experiments.(TIF)Click here for additional data file.S4 Fig(A) The levels of LacCer, GlcCer, and Cer in cells were quantified using mass spectrometry analysis. Ion chromatograms for LacCer, GlcCer, and Cer in indicated cell lines are shown using their respective protonated ion mass centered within 15 ppm for the most abundant fatty acyl chains . Quantification was normalized based on using PC as an internal standard. The quantification data are listed in (TIF)Click here for additional data file.S5 Fig LAPTM4A with a triple C-terminal HA tag (LAPTM4A-HA-C) was expressed in HEK293T (A) and HeLa (B) cells via transient transfection. Cells were subjected to immunostaining detecting the HA tag and six common organelle markers. LAPTM4A-HA-C is colocalized with the Golgi markers Giantin and TGN46. Scale bar, 5 μm. Arrow, colocalization. Representative images are from one of the three independent experiments.(TIF)Click here for additional data file.S6 FigN = 3. *Student’s t test, p < 0.01. (F) Co-IP experiments were carried out for HEK293T cells co-transfected with HA-tagged LAPTM4A, LAPTM4B, AB4, and FLAG-tagged A4GALT. Samples were analyzed by immunoblot using anti-FLAG and anti-HA antibodies. LAPTM4A, LAPTM4B, and AB4 were co-immunoprecipitated with A4GALT. Scale bar, 5 μm. Arrow, colocalization. Representative images are from one of the three independent experiments.(A) Endogenous A4GALT in 5637 cells showed a high degree of colocalization with the Golgi marker Giantin. Endogenous A4GALT was detected by immunofluorescent staining using a polyclonal A4GALT antibody. A4GALT KO cells were utilized as a control to confirm the specificity of the A4GALT antibody.  A4GALT with an N-terminal triple FLAG tag (A4GALT-FLAG-N) was correctly localized in the Golgi when it was expressed in 5637 (B) and HEK293T (C) cells via transient transfection. (D) Ectopic expression of A4GALT-FLAG-N in A4GALT KO cells (A4-KO-Mix) restored binding of Stx1. (E) The mRNA level of A4GALT and LAPTM4A in WT, LA-KO-10, LA-KO-12, A4-KO-Mix, and LB-KO-Mix cells, as well as in LA-KO-10 and LA-KO-12 cells that express LAPTM4A via lentiviral transduction, were determined by qRT-PCR. A4GALT mRNA level is reduced in A4GALT KO cells but remains similar in LAPTM4A KO cells compared to WT cells. Error bars indicate mean ± SD, (TIF)Click here for additional data file.S7 Fig(A) The protein sequences of LAPTM4A (UniPortKB: Q15012) and LAPTM4B isoform 2 (UniPortKB: Q86VI4-2) were aligned by CLC Sequence Viewer (Version 7.7). The same amino acid residues are labeled in red. LAPTM4A and LAPTM4B have the same domain arrangement with four transmembrane domains , two lumen domains (L1 and L2), N-terminal and C-terminal cytosolic domains (C1 and C3), and a short cytosolic linker between the second and third transmembrane domains (C2). The two sgRNA targeting regions are also marked. (B) LAPTM4A with an N-terminal triple HA tag or a C-terminal triple HA tag was expressed in HEK293T cells. Cells were permeabilized with either Saponin, which permeabilizes all the membranes, or Digitonin, which only permeabilizes the plasma membrane. The accessibility of the HA tag was assessed by immunostaining using an anti-HA antibody. Both HA-N and HA-C were detected with either Saponin or Digitonin, suggesting that both the C- and N-termini of LAPTM4A are localized in the cytosol. EMC1 with an N-terminal HA tag or a C-terminal HA tag was analyzed in parallel as a control, which showed no signaling for the N-terminal tagged version under Digitonin treatment. Scale bar, 10 μm. Representative images are from one of the three independent experiments. (C) Schematic drawings depicting the topology of LAPTM4A and EMC1.(TIF)Click here for additional data file.S8 Fig(A) Indicated chimeric proteins between LAPTM4A and LAPTM4B were expressed in LAPTM4A KO cells. Binding of Stx1 to these cells was examined by flow cytometry. The percentages of cells showing positive toxin-binding signals are marked. Representative histograms are from one of the three independent experiments. (B) Experiments were carried out as described in panel A, except that binding of Stx1 was assessed by immunoblot analysis of cell lysates. All chimeric proteins contain a triple HA tag on their C-terminal, and their expression was confirmed using a HA antibody. Stx1 was detected using a polyclonal Stx1 antibody, and the A domain was shown (Stx1A). Actin served as a loading control. Representative images were from one of the three independent experiments.(TIF)Click here for additional data file.S9 Fig(A) Experiments were carried out as described in (TIF)Click here for additional data file.S10 Fig(A) WT, TM9SF2 KO cells (SF2-KO-8 and SF2-KO-9), and a cell line that still expresses WT TM9SF2 (SF2-WT-5) were exposed to Stx1. Surface-bound Stx1 was detected by immunoblot analysis of cell lysates. Representative images are from one of the three independent experiments. (B) Experiments were carried out as described in A, except that cells were also exposed to fluorescently labeled CtxB, and binding of Stx1 and CtxB were analyzed by flow cytometry. Representative histograms are from one of the two independent experiments.(TIF)Click here for additional data file.S11 Fig(A) The specificity of the TM9SF2 polyclonal antibody is validated, as it showed no signal on TM9SF2 KO 5637 cells and HeLa cells (mixed KO cells were generated via CRISPR-Cas9 approach).  The endogenous TM9SF2 in HEK293T (B) and HeLa (C) cells was colocalized with the Golgi marker Giantin and TGN46. Scale bar, 5 μm. Arrow, colocalization. Representative images were from one of the three independent experiments.  TM9SF2 KO cells showed dysfunction in trafficking of internalized Cer and PS. WT, SF2-WT-5, SF2-KO-8, and SF2-KO-9 cells were loaded with NBD-labeled six-carbon Cer  or PS  on ice and then incubated at 37 °C for the indicated time. For WT and SF2-WT-5 cells, the NBD-C6-Cer and NBD-PS fluorescent signals showed as punctate dots at 10 min or 0.5 h, respectively. The fluorescent signals were then disbursed throughout the cells. For SF2-KO-8 and SF2-KO-9 cells, the NBD-PS fluorescent signals remain as punctate dots after 5 h, while the NBD-C6-Cer fluorescent signals remain as punctate dots even 24 h later. Scale bar, 5 μm. Representative images are from one of the two independent experiments. (F) Mixed TM9SF2 KO HeLa cells also showed dysfunction in trafficking of NBD-labeled Sph, Cer, and PS similar to TM9SF2 KO 5637 cells. Scale bar, 5 μm. Representative images are from one of the two independent experiments.(TIF)Click here for additional data file.S12 FigThe colocalization of C-terminal HA-tagged LAPTM4A , N-termi(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.S5 Data(XLSX)Click here for additional data file.


Children with loss-of-function (LOF) mutations exhibit Epileptiform Encephalopathy 17 (EIEE17). Gain-of-function (GOF) mutations or those with normal function are found in patients with Neurodevelopmental Disorder with Involuntary Movements (NEDIM). There is no animal model with a human mutant GNAO1 allele.Infants and children with dominant GNAO1 mutation (G203R) and determine whether the clinical features of patients with this GNAO1 mutation, which includes both epilepsy and movement disorder, would be evident in the mouse model.Here we develop a mouse model carrying a human Gnao1 knock-in GOF mutation (G203R) was created by CRISPR/Cas9 methods. The resulting offspring and littermate controls were subjected to a battery of behavioral tests. A previously reported GOF mutant mouse knock-in (Gnao1+/G184S), which has not been found in patients, was also studied for comparison.A mouse Gnao1+/G203R mutant mice are viable and gain weight comparably to controls. Homozygotes are non-viable. Grip strength was decreased in both males and females. Male Gnao1+/G203R mice were strongly affected in movement assays (RotaRod and DigiGait) while females were not. Male Gnao1+/G203R mice also showed enhanced seizure propensity in the pentylenetetrazole kindling test. Mice with a G184S GOF knock-in also showed movement-related behavioral phenotypes but females were more strongly affected than males.Gnao1+/G203R mice phenocopy children with heterozygous GNAO1 G203R mutations, showing both movement disorder and a relatively mild epilepsy pattern. This mouse model should be useful in mechanistic and preclinical studies of GNAO1-related movement disorders. GNAO1. It is characterized by “hypotonia, delayed psychomotor development, and infantile or childhood onset of hyperkinetic involuntary movements” (OMIM 617493). NEDIM is monogenetic and associated with GOF mutations in GNAO1 [GNAO1 gene has also been associated with early infantile epileptic encephalopathy 17 . However, 36% of patients showed both epilepsy and movement disorder phenotypes  [Neurodevelopmental Disorder with Involuntary Movements (NEDIM) is a newly defined neurological disorder associated with mutations in GNAO1 encodes Gαo, the most abundant membrane protein in the mammalian central nervous system [o is the α-subunit of the Go protein, a member of the Gi/o family of heterotrimeric G proteins. Gi/o proteins couple to many important G protein-coupled-receptors (GPCRs) involved in movement control like GABAB, dopamine D2, adenosine A1 and adrenergic α2A receptors [o and Gβγ separate from each other and modulate separate downstream signaling pathways. Gαo mediates inhibition of cyclic AMP (cAMP), and Gβγ mediates inhibition of cAMP and N-type calcium channels and activation of G-protein activated inward rectifying potassium channels (GIRK channels) [o is expressed mainly in the central nervous system and it regulates neurotransmitter release by modulating intracellular calcium concentrations in pre-synaptic cells [o plays a role in neurodevelopmental processes like neurite outgrowth and axon guidance [o is an important modulator of neurological functions.s system . Gαo is eceptors –7. Upon hannels) . Go is eic cells . It has guidance , 11. ConGNAO1 [GNAO1 mutations expressed in HET293T cells, which lack a complex physiological content. Therefore, it would be important to see whether mouse models with GNAO1 mutations would share clinical characteristics of the human patients. Such a result would verify the previously reported genotype-phenotype correlation and would provide a preclinical testing model for possible new therapeutics. Previously, we studied heterozygous Gnao1+/G184S mice carrying a human-engineered GOF mutation (G184S). This mutation blocks the binding of the regulation of G protein signaling (RGS) proteins and results in GOF [Gnao1+/G184S mice also exhibit movement disorders. The G184S is a GOF mutation but has not been found in human.Previously, we defined a functional genotype-phenotype correlation for GNAO1 . The exps in GOF , 13. Thos in GOF . In thisGNAO1 mutations found clinically [Gnao1+/G203R) to see if it replicated the clinical phenotype of GNAO1 G203R-associated neurological disorders. If so, it would be a valuable tool to understand neural mechanisms underlying the complex phenotypic spectrum of patients with GNAO1 mutations.G203R is a GOF mutation that is one of the more common inically –19. Mostinically –19. We wo GOF mutations Gnao1+/G203R and Gnao1+/G184S have sex-specific motor impairment and seizures. These two mouse models present the possibility of studying GNAO1-associated neurological defects in animal models.In this report, we show that mice carrying two GαAnimal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals established by the National Institutes of Health. All experimental protocols and personnel were approved and trained by the Michigan State University Institutional Animal Care and Use Committee. Mice were housed on a 12-h light/dark cycle and had free access to food and water. They were studied between 8–12 weeks old.Gnao1+/G184S mutant mice were generated as previously described [escribed , 14, 20 Gnao1G203R mutant mice were generated using CRISPR/Cas9 genome editing on the C57BL/6NCrl strain. gRNA targets within exon 6 of the Gnao1 locus (ENSMUSG00000031748) were used to generate the G203R mutation  to rank gRNAs by specificity with respect to off-target modifications occurring –24. The TGCAGGCTGTTTGACGTCGG GGG) that occur within coding regions. One potential off-target site with 4 mismatches and a score of 0.52 was validated for locus ENSMUSG00000041390. We also analyzed two other off-target candidates with 4 mismatches ENSMUSG00000086805 and ENSMUSG00000097637 and scores of 0.15 and 0.069 respectively. They were predicted to occur on the same chromosome (chr 8) as Gnao1. To test these 3 off-target candidates, DNA from WT and founder animals was analyzed by PCR and sequencing and we found that no off-target effects had occurred for all 3 off-target loci analyzed .Nevertheless, we directly validated several predicted off-target loci for the G203 gRNA target (Gnao1+/G203R mutant founder mice were crossed against C57BL/6J mice to generate Gnao1+/G203R heterozygotes (N1 backcross). Further breeding was done to produce N2 backcross heterozygotes while male and female N1 heterozygotes were crossed to produce homozygous Gnao1G203R/G203R mutants. Studies were done on N1 or N2 G203R heterozygotes with comparisons to littermate controls.Heterozygous o was identified by Sac II digests (wt 462 Bp and G203R 320 & 140Bp) of genomic PCR products generated with primers . Reaction conditions were: 0.8μl template, 4μl 5x Promega PCR buffer, 0.4μl 10mM dNTPs, 1μl 10μM Forward Primer, 1μl 10μM Reverse Primer, 0.2μl Promega GoTaq and 12.6 μl DNase free water . Samples were denatured for 4 minutes at 95 oC then underwent 32 cycles of PCR  followed by a final extension (7 minutes at 72°C). After PCR, samples were incubated with Sac II restriction enzyme for 2 hrs.All mice had ears clipped before weaning. DNA was extracted from earclips by an alkaline lysis method . The G20Researchers conducting behavioral experiments were blinded until the data analysis was completed. Before each experiment, mice were acclimated in the testing room for at least 10 min. The timeline of behavioral protocols is described in The Open Field test was conducted in a Fusion VersaMax 42 cm x 42 cm x 30 cm arenas . Mice and their littermate controls were placed in the arena for 30 minutes to observe spontaneous activities. Using the Fusion Software, distance traveled (cm) was evaluated for novel (first 10 minutes), sustained (10–30 minutes), and total (0–30 minutes) activity. Center Time was also measured. Center Time was defined as the time spent in the center portion (20.32cm x 20.32cm) of the Open Field cage.Motor skills were assessed using an Economex accelerating RotaRod . The entire training and testing protocol took two days. On day 1, mice were trained for three 2-minute sessions, with a 10-minute rest between each training period. During the first two sessions, the RotaRod was maintained at a constant speed of 5 rpm. In the third training session, the rod was started at 5 rpm and accelerated at 0.1 rpm/sec for 2 minutes. On day 2, mice were trained with two more accelerating sessions for 2 minutes each with a 10-minute break in between. The final test session was 5 minutes long, starting at 5 rpm then accelerating to 35 rpm (0.1 rpm/sec). For all training and test trials, the time to fall off the rod was recorded. RotaRod learning curves were done on a separate group of mice with 10 tests in one day with a 5-min rest between each test. The learning rate of each group of animals was calculated as described .Mouse grip strength data was collected following a protocol adapted from Deacon et al  using seMouse gait data were collected using a DigiGait Imaging System  . The tesA PTZ kindling protocol was performed as described before  to asses2 immediately after kindling or after 12 PTZ injections and observation. In total, 40 animals were used for this study, among which 27 died of tonic-clonic seizures and 13 were euthanized after 12 doses of PTZ injections.Animals were humanely euthanized with COAll data was analyzed using GraphPad Prism 7.0 . Data are presented as mean ± SEM and a p value less than 0.05 was considered significant. All statistical tests are detailed in Figure Legends. Multiple comparison correction of the dataset from DigiGait was performed via a false discovery rate (FDR) correction at a threshold value of 0.01 in an R environment using the psych package.Gnao1+/G203R x WT crosses (N1—C57BL/6NCrl x C57BL/6J) were observed at the expected frequency (29 WT and 27 heterozygous). All three homozygous mice from Gnao1+/G203Rx Gnao1+/G203R crosses died by P1. The small numbers of offspring observed from these crosses so far, however, were not significantly different from expected frequencies . Heterozygous Gnao1+/G203R mice did not show any growth abnormalities compared to Gnao1+/+ mice  and males showed 8  analysis with a Q of 1% as described in Methods to reduce the probability of Type I errors  Tables. showed 8 . For Gna from WT  Tables. Gnao1+/G184S mice showed significantly reduced stride length , not in stride length  that we reported previously to have a mild seizure phenotype [In this report, we describe the first mouse model carrying a human ovements , 51, 53.henotype . This muhenotype , the GnaGnao1+/G203R mutant mouse, neither the seizure propensity nor the movement abnormality was obvious without a stress being applied. Male Gnao1+/G203R mice showed decreased motor ability on RotaRod, decreased fore paw strength, and gait abnormalities at higher speeds of walking/running. No spontaneous seizures were observed but there was a substantial increase in sensitivity to PTZ-induced seizures in the kindling model in males. This very closely replicates the mild seizure phenotype of female Gnao1+/G184S mice [Gnao1+/G184S mice also exhibit gait and motor abnormalities.In mouse models of movement disorders, the mouse phenotype is usually not as striking or as easily observed as the clinical abnormalities in the patients , 55, how84S mice . We now GNAO1 G203R and the G184S mutations show a definite but modest GOF phenotype in biochemical measurements of cAMP regulation [2A adrenergic agonist, used in those studies to reduce cAMP levels, was increased about 2-fold. This effectively doubles signaling through these two mutant G proteins at low neurotransmitter concentrations . This, however, does not prove that cAMP is the primary signal mechanism involved in pathogenesis of the disease. The heterotrimeric G protein, Go, of which the GNAO1 gene product, Gαo, is the defining subunit, can signal to many different effector mechanisms [GNAO1 mechanisms that seem highly likely to account for the pathophysiology of GNAO1 mutants [o signaling effectors (including cAMP and neurotransmitter release) can be mediated by the Gβγ subunit released from the Go heterotrimer, other effectors could also be involved in the disease mechanisms. A recent hypothesis has also been raised that intracellular signaling by Gαo may be involved [209) does not markedly alter cAMP signaling in in vitro models, does suggest that the mechanism is more complex than a simple GOF vs LOF distinction at cAMP regulation.Both the chanisms , 56. We involved . The obsGnao1+/G184S mice and male Gnao1+/G203R mice showed much more prominent movement abnormalities than male G184S and female G203R mutants. However, the patterns of changes in the behavioral tests did not exactly overlap. Only G184S mutants showed significant changes in open field tests while only the G203R mutants showed the striking reduction in ability to walk/run at higher treadmill speeds. For both mutant alleles, the seizure phenotype was also worse in the sex with the more prominent movement disorder. GNAO1 encephalopathy is slightly more prevalent (60:40) in female than male patients [i/o coupled estrogen receptor, GPR30, also contributes to estrogen physiology and pathophysiology [GNAO1-associated movement disorders [We observed a striking sex difference in the phenotypes of our two mouse models. Female patients . The Gi/ysiology . PD is mrevalent :40 in feisorders , 61, 62,GNAO1 encephalopathy is often associated with developmental delay and cognitive impairment [Gnao1+/G184S and male Gnao1+/G203R mice is due to a neurodevelopmental malfunction or to ongoing active signaling alterations. Go coupled GPCRs play an important role in hippocampal memory formation [Gnao1 mutant mice.Since pairment , it woulormation , 64. AddGNAO1-associated neurological disorders, it is important to learn about the role of Go in the regulation of central nervous system. The novel Gnao1 G203R mutant mouse model reported here, and further models under development, should facilitate our understanding of GNAO1 mechanisms in the in vivo physiological background rather simply in in vitro cell studies. The animal models can also be used for preclinical drug testing and may permit a true allele-specific personalized medicine approach in drug repurposing for the associated movement disorders.With the increasing recognition of S1 FigGnao1+/G184S mice showed significantly motor abnormalities in test trial at day 2 . (B) Male Gnao1+/G184S mice did not show any significance in any training or test session. (C) Female Gnao1+/G203R mice did not exhibit any motor abnormalities in any RotaRod trial or test session. (D) Male Gnao1+/G203R mice showed significantly decreased capability in motor balance .(A) Female (TIFF)Click here for additional data file.S2 FigGnao1+/G184S mice and their littermate controls. B) No significant differences were observed between Gnao1+/G203R mice and their littermate controls.A) No significant differences were observed between (TIFF)Click here for additional data file.S3 FigGnao1+/+ and Gnao1+/G203R in both sexes. (A & C) Both male and female Gnao1+/G203R mice showed reduced capability of keeping balance on RotaRod. (E) No significant difference in either sexes between Gnao1+/+ and Gnao1+/G203R mice was observed comparing the rate of learning.  Short-term learning curve comparison between Gnao1+/+ and Gnao1+/G184S in both sexes. (B & D) Both male and female Gnao1+/G184S mice showed reduced capability of keeping balance on RotaRod. (F) No significant difference in either sexes between Gnao1+/+ and Gnao1+/G184S mice was observed comparing the rate of learning. Short-term learning curve comparison between (TIFF)Click here for additional data file.S4 FigGnao1+/G184S and their littermate controls showed parameters with significance detected by the FDR analysis. C&D) Male Gnao1+/G184S and their littermates controls showed parameters with significance detected by the FDR analysis. FDR is calculated by a two-stage step-up method of Benjamini, Krieger and Yekutiel. Significant values are defined as q < 0.01.All parameters showed significance at belt speed 25 cm/s are plotted. A&B) Female (TIFF)Click here for additional data file.S5 FigGnao1+/G203R and their littermate controls showed 9 parameters with significance detected by the FDR analysis. C&D) Male Gnao1+/G203R and their littermates controls exhibited 27 parameters with significance detected by the FDR analysis in fore and hind limb data combined. FDR is calculated by a two-stage step-up method of Benjamini, Krieger and Yekutiel. Significant values are defined as q < 0.01.All parameters that showed significance are plotted here. A&B) Female (TIFF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 TableRow 1 includes the on-target gRNA for the Gnao1 G203 site. Off-target hits are scored and ranked by an inverse likelihood of off-target binding. If an off-target is predicted to occur within a coding region of a gene, the Ensembl number of the affected locus is listed in the Gene column. Analysis was performed on the Benchling platform using reference genome GRCM38 , guide length of 20bp, and an NGG PAM.(PDF)Click here for additional data file.