GNAO1 mutations cause an emerging group of childhood-onset neurological disorders characterized by developmental delay, intellectual disability, movement disorders, drug-resistant seizures and neurological deterioration. GNAO1 encodes the α-subunit of an inhibitory GTP/GDP-binding protein regulating ion channel activity and neurotransmitter release. The pathogenic mechanisms underlying GNAO1-related disorders remain largely elusive and there are no effective therapies. Here, we assessed the functional impact of two disease-causing variants associated with distinct clinical features, c.139A > G (p.S47G) and c.662C > A (p.A221D), using Caenorhabditis elegans as a model organism. The c.139A > G change was introduced into the orthologous position of the C. elegans gene via CRISPR/Cas9, whereas a knock-in strain carrying the p.A221D variant was already available. Like null mutants, homozygous knock-in animals showed increased egg laying and were hypersensitive to aldicarb, an inhibitor of acetylcholinesterase, suggesting excessive neurotransmitter release by different classes of motor neurons. Automated analysis of C. elegans locomotion indicated that goa-1 mutants move faster than control animals, with more frequent body bends and a higher reversal rate and display uncoordinated locomotion. Phenotypic profiling of heterozygous animals revealed a strong hypomorphic effect of both variants, with a partial dominant-negative activity for the p.A221D allele. Finally, caffeine was shown to rescue aberrant motor function in C. elegans harboring the goa-1 variants; this effect is mainly exerted through adenosine receptor antagonism. Overall, our findings establish a suitable platform for drug discovery, which may assist in accelerating the development of new therapies for this devastating condition, and highlight the potential role of caffeine in controlling GNAO1-related dyskinesia.Dominant  GNAO1 gene  underlie a complex constellation of infantile/childhood-onset severe neurological disorders characterized by global developmental delay (GDD), intellectual disability (ID), hypotonia, movement disorders (MD), drug-resistant seizures and neurological deterioration  of a heterotrimeric guanine nucleotide-binding protein (Go), which is highly expressed in the mammalian brain, particularly in cerebral cortex, hippocampus and striatum , including GABAB, dopamine D2, α2A adrenergic and adenosine A1, regulating neuronal excitability and neurotransmission (o negatively controls the production of cyclic adenosine monophosphate (cAMP) by inhibiting adenylyl cyclase (AC), directly prevents neurotransmitter release, inhibits N-type and P/Q-type calcium channels and activates G protein-coupled inward rectifying potassium (GIRK) channels. Gαo is part of a complex network of proteins regulating intracellular cAMP levels involved in the pathophysiology of hyperkinetic MD ). The resulting nematodes, homozygous for the desired variant, showed ‘slow growth’ and a ‘protruding vulva’ (Pvl) phenotype , resembling features reported following RNAi-mediated knockdown of goa-1 (goa-1(sa734) null allele ). This strain was available at the CGC (https://cgc.umn.edu/strain/COP1863); mutant animals were described as hyperactive and show a Pvl phenotype.of goa-1  as well  p.Q52*; ,34]. Sim p.Q52*; . These go function and to distinguish between GOF and LOF effect, we counted the number of eggs retained in the uterus of gravid hermaphrodites and assessed acetylcholine release at the neuromuscular junction (NMJ). Unlaid eggs are easily visible inside the body of adult animals . In C. elegans, body-wall muscles receive signals from both excitatory (cholinergic) and inhibitory (GABAergic) motor neurons to generate an alternating wave of contraction and relaxation, allowing proper locomotion  null mutants showed a more penetrant phenotype compared with both knock-in animals, pointing to a strong hypomorphic rather than a complete LOF effect of the p.S47G and p.A221D disease-causing variants.To explore the impact of the p.S47G and p.A221D amino acid changes on Gα animals . Wild-tyr uterus , suggestned eggs . Given t muscles , LOF/hypic cleft . In cont the NMJ . Knock-i the NMJ . It is wcomotion . PTZ is y to PTZ , indicatgoa-1 mutants, increased acetylcholine release from the ventral cord motor neurons leads to hyperactive locomotion  (goa-1(sa734) null mutants, further supporting the hypomorphic effect of both variants.In null comotion ,24. Herecomotion . Computacrawling  and tendcrawling . In linecrawling . Furthercrawling  than conenotype) . Again, goa-1 allele. Our data showed that goa-1(+/sa734) null heterozygotes display a clear phenotype in terms of both number of unlaid eggs and increased reversal rate, although less severe than that observed in homozygous null mutants  nematodes displayed a weaker phenotype compared with heterozygous null mutants, which was similar to wild-type in the number of reversals per minute (goa-1(+/sa734) in the number of unlaid eggs (goa-1(+/A221D) mutants exhibited a slightly more severe phenotype compared with heterozygous null mutants in terms of number of unlaid eggs. More importantly, the prevalence of this phenotype was similar to that observed in goa-1[A221D] homozygous animals. Specifically, the percentage of rescue in heterozygous versus homozygous animals was 35, 36 and 11% in goa-1(+/sa734), goa-1(+/S47G) and goa-1(+/A221D), respectively (P < 0.001 between goa-1(+/A221D) and the other mutants; Student’s t-test), suggesting a DN effect of p.A221D on egg laying.To explore a possible DN effect of p.S47G and p.A221D, we analyzed the F1 progeny after crossing homozygous mutant hermaphrodites to control males carrying the wild-type  mutants . This ser minute  and simiaid eggs , indicato function. Based on our findings, C. elegans has emerged as a suitable platform for drug screening or validation in GNAO1-related disorders. As a proof-of-concept, we assessed the effect of caffeine on abnormal locomotion of goa-1 mutant animals. Caffeine is a psychotropic agent acting as an antagonist of adenosine GPCRs in mammals  was shown to rescue aberrant locomotion in terms of number of reversals per minute in a dose-dependent manner  on the hyperactive crawling of goa-1[S47G] and goa-1[A221D] mutants as well as of animals lacking goa-1 . Phenotypic profiling of homozygous and heterozygous animals establish that p.S47G and p.A221D behave as strong hypomorphic rather than complete LOF defects, with the latter exhibiting a partial DN effect. Our findings also reveal the potential role of caffeine and other AR antagonists in controlling GNAO1-related dyskinesia.We report on the functional consequences of two de novo GNAO1 mutations has rapidly grown up. According to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), 60 pathogenic/likely pathogenic variants have been reported so far  to the plasma membrane and inhibition of calcium currents by norepinephrine was variably affected in heterologous cells, suggesting a LOF behavior of these variants on Gαo signaling. Interestingly, mutations associated with both seizures and MD (p.T191_F197del and p.G203R) were characterized by a more severe LOF effect compared with variants underlying epilepsy without MD (p.D174G and p.I279N). More recently, a different scenario was proposed by Feng and coworkers  and GOF  variants appeared to be primarily associated with EIEE17 and NEDIM, respectively. Several recurrent disease-causing variants, however, including those affecting the mutational hot spot Arg209, did not affect cAMP levels at all. Therefore, as correctly recognized by the authors, assessment of cAMP inhibition does not represent a fully informative readout for understanding the functional consequences of GNAO1 mutations, at least in in vitro models or under the used experimental conditions. Finally, a recent paper by Muntean et al.  were shown to behave as DN mutations interfering with the function of normal Gαo via receptor trapping (p.G203R) or an alternative mechanism still to be defined (p.R209C). Conversely, variants involving residues located within or proximal to the P-loop  were LOF changes with no DN activity; rather these changes affect association with the βγ dimer. Of note, LOF and DN effects are not mutually exclusive: their relative degree of manifestation depends on the specific residue and substitution involved.In the original paper, Nakamura and colleagues  providedoworkers  who poinn et al.  providedgoa-1[S47G] is a strong hypomorphic allele that does not act as a DN in vivo. Based on the localization of Ser47 in the P-loop motif, the LOF effect of the serine-to-glycine substitution is likely because of defective interaction with Mg2+ ions that, in turn, may lead to inefficient GTP binding. Interestingly, reduced affinity for Mg2+ has been described for other GNAO1 variants affecting residues located far from the P-loop region, including Gly203. The LOF role of these changes was mainly attributed to a reduced ability of the switch II region to undergo the allosteric transition upon GTP binding needed to release the βγ dimer  heterozygous animals revealed that p.A221D, which had not previously been functionally characterized in in vitro models, has a partial DN effect in vivo, at least on neurotransmitter release by HSN motor neurons mediating egg laying. Considering the possible effect of p.A221D on the conformation of the α5 helix, a key region mediating the interaction between the α-chain and the cognate receptor, the DN effect could be mediated by a more stable GPCR association, as previously reported for the p.G203R, p.G204R and p.Q233P mutants  null heterozygotes display a residual phenotype in terms of both unlaid eggs and reversal rate. This is in line with the observation that in the human population GNAO1 is intolerant to LOF variation in the heterozygous state, according to gnomAD (pLI = 0.99). The reason why no frank LOF mutations including frameshift, stop-gain and out-of-frame splice site changes, have been reported so far in GNAO1-realated disorders remain elusive. These variants could be associated with a more severe phenotype causing embryonic lethality or, alternatively, to weaker clinical features. Mouse models do not help in this case. Although Gnao1−/− mice show multiple neurological abnormalities including seizures and MD  by modulating the effects of dopamine .Caffeine is the most widely used psychoactive stimulant, known to influence sleep, cognition, learning and memory . In mammly A2ARs . These Gly A2ARs . Of notetric use . Worth mtric use . Specifidopamine . Caffeindopamine  further dopamine . Finallyutations ,41, whicutations . A relatC. elegans, caffeine extends worm’s lifespan (m caffeine (the same concentration used in our experiments) to C elegans protects dopaminergic neurons from neurodegeneration elicited by excessive biosynthesis of endogenous L-DOPA  concentrations used in our assays. Indeed, caffeine may act as a non-specific phosphodiesterase inhibitor  variant according to the local institutional policy. Structural modelling was developed by using the UCSF Chimera software .goa-1(sa734) and COP1863 goa-1(knu751) strains were provided by the CGC .Culture, maintenance, injections and genetic crosses were performed using standard techniques ,75. The goa-1 locus was carried out by CRISPR/Cas9, as previously described by the authors (dpy-10 crRNA (5′-CTCGTGGTGCCTATGGTAGC-3′), 37.5 ng/μl ssODN dpy-10 (5′-ATAGGCTGTTGGTCTGAAGCCATGTGAAGCTCCGCTACCATAGGCACCG CATGCGGTACGGTTTCCAGTCATTCTCATCTTGCCGTATTGAAGTTCAAGTG-3′), 350 ng/μl goa-1 crRNA (5′-AAACTGCTGCTACTTGGTGC-3′) and 175 ng/μl ssODN goa-1[S47G] (5′-TTAAAGAAGACGGCATGCAAGCGGCAAAAGATATCAAGCTTCTCCTTCTCGGAGCTGGTGAGTCTGGTAAGGGAACAATCGTAAAACAGATGAAGTGAGATTTTTTTAAATTTCT-3′). Injected worms were recovered on nematode growth medium (NGM) at 20°C, each in a separate plate. Animals with a roller  or a dumpy  phenotype were isolated, as well as pools of five wild-type hermaphrodites from jackpot plates . To isolate worms harboring the goa-1 c.139_141TCG > GGA (p.S47G) nucleotide change, PCR amplification was performed using a single forward primer (5′- GAGGATATCAAGTGGAGACC -3′) and two reverse primers annealing with the wild-type (5′- CGTGTTACTGTAGACAACC -3′) or the modified (5′- CCTTACCAGACTCACCAGCT -3′) sequence. Homozygosity was confirmed by Sanger sequencing using standard techniques. Two independent lines holding the desired change were generated; both were out-crossed twice to the N2 strain to remove any potential off-target mutation and were then used for further analyses and crosses. These two lines showed an equivalent phenotype and were designated as goa-1(pan5[S47G]).The engineered change to the endogenous  authors ,77. Twenm Potassium phosphate buffer pH 6.0, 1 mm CaCl2, 1 mm MgSO4) to a final concentration of 1, 10 or 20 mm. Plates were prepared the day before use, seeded with 12.5 μl of E. coli OP50 bacteria to obtain a thin and homogeneous lawn of food. The reversal rate was assessed in L3 animals moved on the assay plate following exposure to caffeine (1–4 h or overnight). To assess the effect of chronic exposure, young adults were moved to the assay plate to let them lay eggs, and the number of reversals per minute was assessed in the F1 progeny at the L3 stage. Sensitivity to subtype-selective ARs agonists/antagonists (Tocris) was assessed as described previously after 2 h exposition. DPCPX  was freshly dissolved in ethanol. Istradefylline (8-[(1E)-2-ethenyl]-1,3-diethyl-3,7-dihydro-7-methyl-1H-purine-2,6-dione), ZM241385 (4-(2-triazolotriazin-5-ylamino]ethyl)phenol), CPA (N-cyclopentyladenosine) and CGS21680 (4-[2-[[6-Amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride) were freshly dissolved in DMSO. Since ethanol and DMSO were shown to affect the reversal rate in goa-1[S47G]  and N2 animals , worms were treated in parallel with the drug or the solvent at the corresponding concentration. The CPA/caffeine competition assay was performed by adding both drugs into just-agar plates . The percentage of rescue is defined as [1 − (Δ1/Δ2)∙100], where Δ1 and Δ2 represent the difference between the number of reversals per minute in the presence and absence of the drug, respectively, and the number of reversals per minute of wild-type nematodes treated with the solvent at the corresponding concentration. In other words, the percentage of rescue is 0% when the reversal rate remains the same before and after drug exposure and 100% when the reversal rate following drug exposure corresponds to that of wild-type animals.Sensitivity to aldicarb, levamisole and PTZ  was determined as previously described . Stock sC. elegans have been recorded using an in-house automated tracking system under uniform and steady illumination, on 35 mm petri dishes spread with a thin lawn of E. coli OP50 bacteria, with a greyscale camera , at 3 frames per second for 10 min. The petri dishes were placed on a transparent plexiglass panel. The light illuminates the bottom of the petri dish and is emitted by a matrix of white light LEDs (12 W Panel SMD LED Ceiling Down Light Bulb Lamp) under the plexiglass panel. The camera is placed at a calibrated height over the petri dish and collects the light passing through the sample with a zoom lens with 18/108 range and F2.5 that conveys the light on the camera sensor (1280 × 1024 pixels). A filter film  is placed between the plexiglass and the petri dish to increase contrast and image quality. Videos were post-processed with a custom-made MATLAB software  through a gaussian filter and then thresholded. Caenorhabditis elegans were detected based on the size of the objects in the thresholded image. Too large or too small objects were discarded. Trajectories were created based on the proximity of the centroid (corresponding to the mean position of the points belonging to the object) in all consecutive frames, and then validated visually one by one. Velocity was calculated based on the distance between the centroids of objects of the same trajectory in consecutive frames, whereas reversals were defined as portions of the trajectory that present a bend with an angle greater than 50°, as described in  equipped with DIC optics on live animals mounted on 2% agarose pads containing 10 mGenotype blinding was used for all experiments except for the acquisition of microphotographs shown in t-test at the 5% significance level with MATLAB .Significant differences were calculated using the GraphPad Prism 8.4.2 software. To compare the speed distributions associated with different strains we performed the two-sample Supplementary_Material_ddab296Click here for additional data file.


High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (auto-HSCT) represents a standard treatment regime for multiple myeloma (MM) patients. Common and potentially fatal side effects after auto-HSCT are infections due to a severely compromised immune system with hampered humoral and cellular immunity. This study delineates in depth the quantitative and functional B cell defects and investigates underlying extrinsic or intrinsic drivers.ex vivo using flow cytometry. Additionally, B cell function was assessed with T cell dependent (TD) and T cell independent (TI) stimulation assays, analyzing proliferation and differentiation of B cells by flow cytometry and numbers of immunoglobulin secreting cells in ELISpots.Peripheral blood of MM patients undergoing high-dose chemotherapy and auto-HSCT (before high-dose chemotherapy and in early reconstitution after HSCT) was studied. Absolute numbers and distribution of B cell subsets were analyzed Quantitative B cell defects including a shift in the B cell subset distribution occurred after auto-HSCT. Functionally, these patients showed an impaired TD as well as TI B cell immune response. Individual functional responses correlated with quantitative alterations of CD19+, CD4+, memory B cells and marginal zone-like B cells. The TD B cell function could be partially restored upon stimulation with CD40L/IL-21, successfully inducing B cell proliferation and differentiation into plasmablasts and immunoglobulin secreting cells.Quantitative and functional B cell defects contribute to the compromised immune defense in MM patients undergoing auto-HSCT. Functional recovery upon TD stimulation and correlation with CD4+ T cell numbers, indicate these as extrinsic drivers of the functional B cell defect. Observed correlations of CD4+, CD19+, memory B and MZ-like B cell numbers with the B cell function suggest that these markers should be tested as potential biomarkers in prospective studies. The TD activation is initiated by protein antigens, which are presented to follicular naïve B cells by antigen presenting cells (APC). Interaction with CD4+ T cells, that previously encountered the same antigen, is necessary in order for naïve B cells to differentiate and synthesize antibodies  and B1 cells  has been established as a standard treatment regime for patients suffering of multiple myeloma (MM) who are not weakened by major comorbidities , 7. Afteuto-HSCT , 17–19. uto-HSCT .in vitro stimulations especially within the first three months are described , poke weed mitogen (PWM) and oligonucleotides with cytosine and guanin motives (CpG) or CD40L, (IL-21) and CpG that were used in this study have been shown to recover B cell functions in other B cell deficiencies including common variable immunodeficiency (CVID) and systemic autoimmune diseases  and 14 MM patients undergoing high-dose chemotherapy and auto-HSCT. Patients’ characteristics are shown in ex vivo and after a seven-day stimulation. PBMCs were stained extracellular with fluorochrome-conjugated antibodies  were cultured in a 6-well plate. They were diluted in 3ml RPMI medium + 10% fetal calf serum + 1% penicillin/streptomycin (culture medium) and stimulated at 37°C and 5% CO2 for seven days. Stimulation protocols were adapted from Crotty et al. and Muir et al. , IgA 1:500 (Thermo Fisher Scientific)) overnight. The development of spots was induced by one-hour incubation with 2,5 µg/ml streptavidin-HRP (Biolegend) at room temperature, washing and finally by a combination of 3-amino-9-ethyl-carbazole, Dimethylformamid (1:30) and 3% H2O2 in acetate buffer  added for three minutes. The reaction was stopped by rinsing the plates with water. Plates were read and analyzed using the AID ELISpot Reader 7.0.0.0 and the ELISpot software.B cell ELISpot assays were performed after seven-day stimulations to quantify immunoglobulin secreting cells (ISCs). 96-well multiscreen filter plates were coated overnight with 1,2 µg/ml goat anti-human IgG , 15 µg/ml goat anti-human IgA (Dianova) or 10 µg/ml goat anti-human IgM (Dianova) primary antibodies in Dulbecco’s phosphate-buffered saline (PBS) . Control wells were coated with PBS only. After blocking of the plates with culture medium and washing, cells were added at concentrations ranging from 1.56x10Statistical analysis was performed using GraphPad Prism 9.0.1. As data was not normally distributed median and interquartile range (IQR) were calculated for summary statistics. Comparative analysis was performed using nonparametric tests. Patient data to HDs was compared using two-tailed Mann-Whitney-U tests. For dependent samples pre- to post-HSCT and changes upon S/P/C to C/I/C the Wilcoxon test was applied. Groups of three were compared using Kruskal-Wallis-Tests. Correlations were analyzed using the Spearman’s rank correlation test. The correlation coefficient was applied to define weak (0.3<r<0.5), moderate (0.5<r<0.7) and strong (r>0.7) correlations. Exact two-sided statistical significance was defined as a p value <0.05.In our work the B cell compartment of MM patients undergoing high-dose chemotherapy and auto-HSCT was analyzed and compared to HDs. Patients’ characteristics are shown in ex vivo by flow cytometry using a B cell panel adapted from Wehr et al. . An exception was a reduction to the S/P/C stimulus post-HSCT (x0.86) . Post-HSCT the initial deficit was too extensive for either stimulation to compensate, causing the median CD19+ counts to remain significantly reduced in comparison to HDs (C/I/C and S/P/C p<0.001). However, there was a great difference between the two stimulation assays. C/I/C provoked a strong proliferation to a median of 41.5 [9.2-212.5] CD19+/10,000 PBMCs and led to normalized CD19+ counts in 4 patient samples. The S/P/C stimulation in contrast did not provoke any proliferation and remained on a median of 4.4 [1.2-10.9] CD19+/10,000 PBMCs. Overall C/I/C showed a 10 times higher CD19+ count post-HSCT than S/P/C (p<0.001)  Table 1.A second parameter to assess B cell function was differentiation into PBs and ISCs upon stimulation.To measure differentiation into PBs the change of distribution of B cell subpopulations within the CD19+ compartment upon stimulation was determined. In HDs a successful B cell activation was marked by a reduction of memB, naïve, transitional and MZ-like cells through apoptosis and differentiation into PBs, which consequently increased Table 1 The overall count of PB/10,000 CD19+ cells was compared between HDs and patients to determine a successful differentiation into PBs . The S/PDifferentiation into ISCs, measured in B cell ELISpots, confirmed an IgM weighted response upon S/P/C in HD and pre-HSCT and IgG and IgA weighted upon C/I/C in all groups with no significant differences in the overall ISCs (data not shown). Post-HSCT ISCs were reduced compared to HD, which could be attributed to the lesser amount of B cells. However, a deficient differentiation upon S/P/C was confirmed with significantly less overall ISCs compared to C/I/C , less IgG  and less IgA  ISCs upon S/P/C than C/I/C. In fact even IgM ISCs were reduced upon S/P/C  indicating a strongly deficient immunoglobulin production upon S/P/C stimulation  defect contributing to the quantitative deficit. Because ex vivo stimulation was able to trigger efficient proliferation of patients’ B cells, an extrinsic rather than intrinsic cause seems likely.Quantitative B cell defects contributing to the increased susceptibility to infections in treated MM patients have previously been described and are most prominent after auto-HSCT . The preAll measured B cell subpopulations were significantly reduced post-HSCT. A shift in the distribution of subpopulations within the B cell compartment contributed further to the B cell deficiency. The major proportion of the B cell compartment post-HSCT consisted of PBs and DN B cells while populations that can respond to new pathogens such as naïve, MZ-like and transitional B cells comprised a smaller share. Gernert et al. observed a similar composition of the B cell compartment after the first month following auto-HSCT in patients with systemic sclerosis . Gernertin vitro an intrinsic B cell defect was previously assumed , Octapharma (grant number: SAP-Nr. 125051) and the Berlin Institute of Health Johanna Quandt Professorship. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. The library of Charité University Medicine Berlin supports the open access publication.I-KN declares a conflict of interest through financial support from Shire/Takeda and Octapharma.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Currently, there are no approved drugs for the treatment of flavivirus infection. Accordingly, we tested the inhibitory effects of the novel θ-defensin retrocyclin-101 (RC-101) against flavivirus infection and investigated the mechanism underlying the potential inhibitory effects. First, RC-101 robustly inhibited both Japanese encephalitis virus (JEV) and Zika virus (ZIKV) infections. RC-101 exerted inhibitory effects on the entry and replication stages. Results also indicated that the nonstructural protein NS2B-NS3 serine protease might serve as a potential viral target. Furthermore, RC-101 inhibited protease activity at the micromolar level. We also demonstrated that with respect to the glycoprotein E protein of flavivirus, the DE loop of domain III (DIII), which is the receptor-binding domain of the E protein, might serve as another viral target of RC-101. Moreover, a JEV DE mutant exhibited resistance to RC-101, which was associated with deceased binding affinity of RC-101 to DIII. These findings provide a basis for the development of RC-101 as a potential candidate for the treatment of flavivirus infection.IMPORTANCE Retrocyclin is an artificially humanized circular θ-defensin peptide, containing 18 residues, previously reported to possess broad antimicrobial activity. In this study, we found that retrocyclin-101 inhibited flavivirus (ZIKV and JEV) infections. Retrocyclin-101 inhibited NS2B-NS3 serine protease activity, suggesting that the catalytic triad of the protease is the target. Moreover, retrocyclin-101 bound to the DE loop of the E protein of flavivirus, which prevented its entry. Flavivirus and family Flaviviridae. These viruses include more than 70 different pathogens and are transmitted mostly by arthropods. Emerging and reemerging flaviviruses, such as Zika virus (ZIKV), Japanese encephalitis virus (JEV), dengue virus (DENV), West Nile virus (WNV), and yellow fever virus, cause public health problems worldwide (–363SSAN367), are responsible for receptor binding . It contains 18 residues, including three disulfide bonds and four positively charged residues (Retrocyclin (RC) is an artificially humanized θ-defensin that has been reported to possess broad antimicrobial activity –21. RC-1–residues  and B, wresidues , influenresidues , and herresidues , thus prresidues .In this study, we tested the inhibitory effect of RC-101 against flavivirus infection. As flaviviruses possess only one conserved N-linked glycan on the E protein , whether50) of RC-101. Notably, the ZIKV PRVABC 59 strain, belonging to the Asian lineage ZIKV strains, contains one N-linked glycosylation site (N-X-S/T) at residue N154 of E, which is conserved among the flaviviruses, whereas the stocks of the African lineage MR766 may or may not lack the E glycosylation motif due to their extensive passaging (–MK105975.1) was used in this study. The cytotoxicity of RC-101 was initially tested on Vero cells, which showed a marginal response even at 100 μM  value was 11.77 μM, indicating that the enzyme kinetic assay was robust and suitable to investigate the inhibitory effect. As shown in 50 of 7.20 μM, indicating that this protease serves as a viral target of RC-101.To investigate the potential viral target of RC-101, we tested the inhibitory effect of RC-101 on ZIKV NS2B-NS3 protease activity. It has been reported that RC-1, which possesses the same residue sequence as RC-101, except for one lysine (K) instead of arginine (R) in RC-101, might dock at the NS2B-NS3 interface and thus inhibit DENV-2 replication by interfering with the activity of the NS2B-NS3 serine protease . Conside peptide , 34, 35.in vitro enzyme kinetic assays and the experiments in infected cells, it was concluded that RC-101 inhibits flavivirus NS2B-NS3 serine protease activity.Inhibition of the protease activity of NS3 by RC-101 was further supported by the detection of the unprocessed polyprotein precursor (PP) and NS3 in the infected cells . As showAs RC-101 was found to inhibit ZIKV infection at both the entry and replication stages , we furt22–26–37–363SSAN367 to A363AAAA367) impaired receptor binding by the virus, H144A and H319A abrogated the interaction between DI and DIII, and Q258A and T410A resulted in failure of the E protein to refold to form its postfusion conformation  of 1.46 × 104 M−1 s−1, kinetic dissociation (dK) of 1.18 × 10−4 s−1, and an equilibrium dissociation constant (DK) of 8.10 × 10−9 M, indicating that RC-101 has high affinity for DIII. The binding affinity of RC-101 to the DE mutant was decreased by 1 order of magnitude, to a DK of 2.37 × 10−8 M, which suggested that the DE loop might be the binding site of RC-101 and that the DE mutant would disrupt this interaction.To test the possibility that the DE loop is the target of RC-101, and to test whether the DE mutant would disrupt the binding of RC-101 to DIII, the binding affinities of the wild-type (WT) and the DE mutant DIII to RC-101 were examined by biolayer interferometry. The interactions between DIII and RC-101 were calculated using a 1:1 binding model at three different concentrations . The res2LZI [GICRCICGRRICRCICGR]) (5ZMS)   with ZIK) (5ZMS) , we foun) (5ZMS) . RC-101 ) (5ZMS) , 42.154YS glycosylation motif has been reported to interact with DC-SIGN, which is a candidate flavivirus receptor  and Tong Cheng  (GenBank accession no. KU501215), while the MR-776 strain (GenBank accession no. MK105975.1) was obtained from The Microorganisms and Viruses Culture Collection Center, Wuhan Institute of Virology, Chinese Academy of Sciences. The genome sequence of ZIKV strain SZ-WIV001 (GenBank accession no. KU963796) was used as the template for the construction of the ZIKV replicon , respectively. The ZIKV PRVABC 59 strains were kindly provided by Jean K Lim  (GenBank accession no. replicon . JEV AT3RC-101 was synthesized by solid-phase synthesis and purified by reversed-phase high-performance liquid chromatography (HPLC) to homogeneity (98% purity) . The effCells in 96-well plates were infected with ZIKV PRVABC 59, ZIKV MR-766, and JEV AT31 at the indicated MOI in the presence of RC-101 at different concentrations for 48, 72, and 24 h, respectively. The antiviral effects were evaluated by IFA assay and plaque assay.Escherichia coli BL21 using a pET30a expression vector; purified protein was injected into rabbits to obtain the antiserum  mouse monoclonal antibody was purchased from ABclonal . The anti-JEV prM polyclonal antibody was prepared by expressing full-length prM in ntiserum .Cells were fixed with 4% paraformaldehyde, permeabilized using phosphate-buffered saline (PBS) containing 0.2% Triton X-100 for 15 min, and blocked with 5% fetal bovine serum , followed by treatment with the primary antibody: anti-ZIKV NS3 or anti-JEV prM. After six rinses with PBS, the cells were stained with the secondary antibody, DyLight 488-labeled anti-rabbit IgG . Nuclei were then stained with DAPI  according to the manufacturer’s instructions . Nine fields per well were imaged using an Operetta high-content imaging system (PerkinElmer), and the percentages of infected and DAPI-positive cells were calculated using the associated Harmony 3.5 software.JEV-infected BHK-21 cell lysates were analyzed at 23 h postinfection using rabbit prM antiserum, anti-JEV NS3 antibody , and the anti-GAPDH mouse monoclonal antibody as primary antibodies.ZIKV and JEV were propagated in Vero cells and titrated in BHK-21 cells. The plaque assay was carried out by adding the serially diluted virus stock into semiconfluent monolayers of cells for 1 h. Then, the supernatant was discarded, and the cells were overlaid with medium containing 1% methylcellulose and incubated for the indicated time. The cells were then fixed with 4% formaldehyde and stained with 0.1% crystal violet for plaque visualization.KU963796) and then incubated with RC-101. Renilla luciferase activity in the cell lysates was measured using the Rluc system   .GGGGSGGGG-NS3 protein, the ZIKV replicon was used as the template, and the NS2B fragments were amplified by PCR using a forward (5′-TCCATGGGCGTGGACATGTACATTGAAAGAGTAAGAAGGAGATATA-3′) and reverse (5′-CTCCACCTCCACCCGATCCACCTCCACCGATCTCTCTCATGGGGGGACCACCACT-3′) primer pair, and NS3 was also amplified using a forward (5′-GGGTGGAGGTGGATCGGGTGGAGGTGGAAGTGGTGCTCTATGGGATGTGCAGATC-3′) and reverse (5′-CTCGAGCTTCTTCAGCATCGAAGGCTCGAAGCTCAGTGGTGGTGGTGGTGGTG-3′) primer pair. The underlined letters represent restriction endonuclease sites, and the italic letters represent the GGGGSGGGG linker sequence (E. coli BL21(DE3), and the cell lysates were loaded onto a nickel column. The protein was eluted with a gradient concentration of imidazole buffer  (To produce NS2B-sequence . The PCRpH 7.0]) .mK was calculated from a combination of enzyme kinetics and velocity as a function of the substrate model using GraphPad Prism 8.0. The inhibitory effects of RC-101 against protease activities were assessed at 37°C for 30 min, with mixtures of 100 μl consisting of 12 μM fluorogenic peptide substrate, 1.25 μM NS2B-NS3pro, and RC-101 at concentrations ranging from 0 to 100 μM, buffered at pH 8.5 with 200 mM Tris-HCl. The IC50 value of RC-101 was evaluated using the nonlinear regression model in GraphPad Prism 8.0.The proteolytic activity of NS2B-NS3pro was measured using a fluorescence resonance energy transfer-based assay with a fluorogenic peptide substrate  as the substrate. The relative fluorescence units were measured using an EnSpire multimode plate reader with the emission at 440 nm upon excitation at 350 nm. The kinetic parameter of NS2B-NS3pro was obtained using substrate from 2.5 to 20 μM in the fluorescent assay after a 30-min incubation at 37°C . The Km AGTGAACCCCTTCGTCGCGGCGGCGGCGGCGGCGTCAAAGGTGC-3′; reverse, 5′-CGCCGCCGCCGCCGCCGCGACGAAGGGGTTCACTGTCACCAGCCG-3′ (E. coli BL21(DE3); the supernatant of the bacterial pellets was loaded onto a nickel column, and the bound protein was eluted with a gradient concentration of imidazole buffer. DE mutant DIII, expressed as inclusion bodies, was solubilized in 8 M urea . Refolding was carried out by titration dialysis at 4°C against refolding buffer -arginine, 1 mM glutathione, 5% glycerol [pH 7.4]) until the concentration of urea was <2 M. Then, the supernatant was passed through a nickel column as described previously herein.The WT DIII expression vector was constructed using pET-22b(+) and preserved in our laboratory . The DE AGCCG-3′ . WT DIIIdK rate constant divided by the aK rate constant.Real-time binding assays between RC-101 and WT or the DE mutant DIII were performed using biolayer interferometry on an Octet QK system  according to previously reported methods . Binding2ZLI) and ZIKV NS3 (PDB no. 5ZMS) were used to build the complex using the ZDOCK 3.0.2 program (http://zdock.umassmed.edu)  . The resmed.edu) .


Streptoalloteichus hindustanus involves an unprecedented cofactor independent stereoinversion of Dab catalyzed by PddB, which shows weak homology to diaminopimelate epimerase (DapF). Enzymological properties and mechanistic details of this enzyme, however, had remained to be elucidated. Here, through a series of biochemical characterizations, structural modeling, and site-directed mutageneses, we fully illustrate the first Dab-specific PLP-independent racemase PddB and further provide an insight into its evolution. The activity of the recombinant PddB was shown to be optimal around pH 8.5, and its other fundamental properties resembled those of typical PLP-independent racemases/epimerases. The enzyme catalyzed Dab specific stereoinversion with a calculated equilibrium constant of nearly unity, demonstrating that the reaction catalyzed by PddB is indeed racemization. Its activity was inhibited upon incubation with sulfhydryl reagents, and the site-directed substitution of two putative catalytic Cys residues led to the abolishment of the activity. These observations provided critical evidence that PddB employs the thiolate-thiol pair to catalyze interconversion of Dab isomers. Despite the low levels of sequence similarity, a phylogenetic analysis of PddB indicated its particular relevance to DapF among PLP-independent racemases/epimerases. Secondary structure prediction and 3D structural modeling of PddB revealed its remarkable conformational analogy to DapF, which in turn allowed us to predict amino acid residues potentially responsible for the discrimination of structural difference between diaminopimelate and its specific substrate, Dab. Further, PddB homologs which seemed to be narrowly distributed only in actinobacterial kingdom were constantly encoded adjacent to the putative poly-D-Dab synthetase gene. These observations strongly suggested that PddB could have evolved from the primary metabolic DapF in order to organize the biosynthesis pathway for the particular secondary metabolite, poly-D-Dab. The present study is on the first molecular characterization of PLP-independent Dab racemase and provides insights that could contribute to further discovery of unprecedented PLP-independent racemases.We recently disclosed that the biosynthesis of antiviral γ-poly-D-2,4-diaminobutyric acid (poly-D-Dab) in  N-methyl-D-aspartate (NMDA) receptor co-agonists in mammals -like enzyme, poly-D-Dab synthetase (PddA)  and behaves similarly to the other racemases harboring a thiolate-thiol pair in the active site. Further, we provide insights into the evolution and structure-function relationships of DabRs.A novel PLP-independent racemase that catalyzes the racemization of a non-canonical amino acid  strains . This dee (PddA) . In thisddA gene . PddB exddA gene . In the ddA gene , whereasectivity . Since, Escherichia coli Rosetta 2 (DE3) host cells. E. coli NEB10β  was used for routine cloning. S. hindustanus DSM44523 (=NBRC15115) and Actinokineospora enzanensis DSM44649 (=NBRC16517) obtained from the NITE Biological Resource Center (NBRC) of Japan were maintained on ISP4 agar as described previously (E. coli strains were routinely grown in Luria-Bertani (LB) medium. Carbenicillin  and Chloramphenicol  were used to select E. coli recombinant strains.pET-21b (+)  was used for heterologous expression of the recombinant PddB and its homolog in combination with eviously . E. coliA. enzanensis (PID: WP_018680613.1) was similarly prepared. Briefly, the gene C503_RS0102450 coding for the PddB homolog was PCR-amplified from the genome of A. enzanensis NBRC16517 with the primers 018680613-ndeF (5′-AGATATACATATGGACATCGACCCGCGCGCGCTCGAACT C-3′) and -xhoR (5′-GGTGGTGCTCGAGGGGTGTCGCGCTC ACCGGGGCGTCCATGGTG-3′). The resulting PCR fragment was double digested with NdeI and XhoI and was subsequently ligated with similarly digested pET21b(+) vector, yielding the PddB homolog expression vector. After sequence verifications, this vector was introduced into E. coli Rosetta 2 (DE3) by electroporation, and the resulting cells were streaked out onto LB agar plates supplemented with Carb and Chl. A transformant was inoculated into 100 ml LB medium supplemented with Carb, Chl, and 0.4% (w/v) glucose and grown at 37°C with shaking until the OD600 reached around 0.6. After dropping the cultivation temperature down to 18°C, expression of the PddB homolog was induced by 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 16 h. Cells harvested were resuspended in buffer A consisting of 50 mM potassium phosphate buffer (pH 7.5), 300 mM NaCl, 10% (w/v) glycerol, 0.05% (w/v) triton X-100, and 10 mM imidazole, and were sonicated and centrifuged to yield the cell free extract. The supernatant was applied onto a Ni-NTA agarose column  pre-equilibrated with buffer A. Fractions eluted with buffer A containing 50 to 200 mM imidazole were pooled. The purity-verified recombinant enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was dialyzed against 50 mM potassium phosphate buffer (pH 7.5) containing 10% (w/v) glycerol. The protein concentration was determined using a Bio-Rad Protein assay kit (Bio-Rad) with BSA as the standard. The purified enzyme was stored at −80°C until use.The recombinant PddB (PID: WP_073484545.1) was expressed and purified as reported previously . The recpddB, which we previously constructed (pddB gene was generated by PCR using a set of overlapping oligo nucleotide primers pddB-C101S-F (5′-ACGCCCAGATGAGCGGCAACGCGCTGCGCTGCCTG-3′) and -C101S-R (5′-GCGCGTTGCCGCTCATCTGGGCGTGCGT TCCGTC-3′). Similarly, the mutation corresponding to C233S was introduced with the primers pddB-C233S-F (5′-AGACCAAGGCCAGTGGGACCGGCGTCGCCGCGAC-3′) and -C233S-R (5′-CGCCGGTCCCACTGGCCTTGGTCTCGTC CTCCAC-3′). Overlapping sequences for self-annealing are underlined and the substituted nucleotides are in bold. After sequence verifications, the mutated plasmids were individually introduced into E. coli Rosetta (DE3), resulting in the expression of two single point mutants of PddB under the established cultivation/expression conditions. The mutants were purified using Ni-NTA agarose resin as described above.The single point mutations were introduced into the expression plasmid pET21b-structed , by the structed . The plaThe molecular weight of PddB was determined by size-exclusion chromatography using an ÄKTA purifier system equipped with a Superdex 200 10/300 GL column . The mobile phase was an isocratic flow of 50 mM potassium phosphate buffer (pH 7.5) containing 0.15 M KCl at a rate of 0.75 ml/min, and eluents were spectrophotometrically monitored at 210 nm. The calibration curve used for the molecular weight determination was established with aprotinin (6.5 kDa), ribonuclease (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (44 kDa), conalbumin (75 kDa), aldolase (158 kDa), and ferritin (440 kDa).The UV-vis absorption spectrum of PddB dissolved in 50 mM potassium phosphate buffer (pH 7.5) containing 0.15 M KCl was analyzed using V-730BIO spectrophotometer .3 were added. To yield the di-FDLA-labeled product, the mixture was incubated at 37°C for 24 h under dark condition. After quenching the derivatization by addition of 20 μl of 1 M HCl, the resulting solution was diluted with 810 μl of methanol and placed into an amber glass vial prior to the quantitative enantioresolution in the Shimadzu LC-MS-IT-TOF instrument equipped with the Prominence HPLC system. The LC separation was performed using a C18 reverse-phase column  at 40°C with a binary solvent system consisting of 0.03% trifluoroacetic acid plus 0.07% formic acid in H2O (A) and in acetonitrile (B). FDLA-derivatized enantiomers separated with a linear gradient of 45–65% B over 15 min at a flow rate of 0.3 ml/min were monitored at 340 nm and were eluted into the electrospray ionization (ESI) interface in positive mode. LC-MS Solution software  was used for system control and data analysis. Authentic standards of D- and L-Dab were used to establish standard curves for quantification. The racemase activity was determined from the initial velocity of the stereoinversion reaction, and one enzyme unit (U) was defined as the amount of the enzyme that catalyzes the conversion of one micromole of substrate per minute. All the assays were performed in triplicate.All the DabR assays were performed with 0.2 ml reaction mixtures. In the standard assay, the reaction mixture was simply comprised of 100 mM TAPS-NaOH (pH 8.5) and 20 mM enantiopure Dab. Reactions were initiated by addition of 5 μg purified enzyme, and after incubation at 30°C, 20 μl of 50% (w/v) trichloroacetic acid was added to terminate the reaction. The supernatant was neutralized with 100 μl of 0.5 M NaOH and subjected to the quantitative enantio-resolution employing Marfey’s method  with sliThe substrate specificity of PddB was examined using 20 mM concentration of L-arginine, L-lysine, L-ornithine, L-2,3-diaminopropionic acid, LL-2,6-diaminopimelate, and their corresponding enantiomers under the standard assay conditions. The effect of pH on PddB activity was evaluated by measuring the racemization from L-Dab under various pH conditions at 30°C. The reaction mixtures for this evaluation contained 100 mM HEPES-NaOH (pH 7.0–8.0), TAPS-NaOH (pH 8.0–9.0), or CHES-NaOH (pH 8.5–9.5). To evaluate the inhibitory effect, PddB was individually incubated with chelating agents, metal ions, sulfhydryl reagents, or other compounds in 0.2 ml of 100 mM TAPS buffer (pH 8.5) at 30°C. After incubation for 20 min, residual activity was evaluated by measuring the racemization from L-Dab under the standard condition. For determination of kinetic parameters, measurements obtained under the standard condition with various concentration of each Dab isomer were processed using a Lineweaver-Burk plot .1  (The SWISS-MODEL server1  was usedD: 2GKE)  as templ6-tag fused recombinant PddB (PID: WP_073484545.1) was expressed in E. coli mostly as a soluble protein and was confirmed to catalyze the stereoinversion of Dab  for each reaction direction were shown to be nearly equal. In addition, based on the conclusive observation that the equilibrium constant Keq of the PddB reaction calculated from the Vmax/Km values between both reaction directions was near unity (1.07), the stereoinversion reaction catalyzed by PddB was demonstrated to indeed be the racemization .Mycobacterium tuberculosis (Mt-DapF) (As summarized in Mt-DapF) , which eMt-DapF) .via the thiolate-thiol pair within the active site similar to the canonical PLP-independent racemases, we assessed the impact of sulfhydryl reagents on PddB activity. As shown in 2 and 4-chloromercuribenzoic acid (PCMB) at concentrations of 10 μM, and was dramatically decreased with 20 μM of iodoacetic acid (IAA) or iodoacetamide (IAM). These observations thus strongly suggested that PddB most likely relies on Cys residues for the catalysis like the other PLP-independent racemases/epimerases , the other protein was revealed to exhibit higher homology , and thus this protein was presumed to function as the primary metabolic DapF in S. hindustanus. Despite such disparity in their primary structures, a PROSITE motif search  further identified the other putative catalytic Cys conserved within the C-terminal side of PddB  and a hydroxyl (pKa ≈ 16), the dramatically decreased activities observed in the mutants well supported the proposed reaction mechanism of PddB shown in In order to assess whether the Cys residues are involved in PddB catalysis, two single-point mutants (C101S and C233S) were individually generated. Like the wild-type PddB, the mutant enzymes were mostly expressed as soluble proteins, and the purification in an identical fashion resulted in a comparable yield . These oOur recent investigation into the biosynthesis of the antiviral cationic homo poly-amino acid poly-D-Dab revealed that the DapF homolog PddB encoded in its biosynthetic gene cluster catalyzes stereoinversion of Dab in a cofactor-independent manner. The fact that cofactor-independent racemization of Dab had yet to be reported strongly motivated us to fully characterize this enzyme. In this study, through a series of biochemical characterizations, structural modeling, and site-directed mutageneses, we successfully demonstrated that PddB is indeed an unprecedented DabR, which employs a two-base mechanism like the other PLP-independent racemases/epimerases .P. graveolens has been reported to modestly catalyze the racemization of Dab because of its broad substrate specificity  homolog  at the N-terminal of DabRs . The 3D via hydrogen bonding. The structural model of PddB generated using the X-ray crystal structure of Hi-DapF as a template revealed the conservation of all the five residues (via a salt bridge appeared to be substituted by hydrophobic Val in PddB. Since, all these substitutions are universally conserved in the other DabRs (de novo creation of novel racemases by evolutionary engineering approaches. Our current study should thus provide valuable insights into PLP-independent racemases.As noted, despite PddB’s weak sequence similarity to DapF enzymes, a nearly conserved DapF active site signature and a small number of clearly conserved residues around its active site can be seen . In Hi-Dresidues . As shower DabRs , these dThe original contributions presented in the study are included in the article/KY designed the experiments, analyzed the experimental data, and organized the manuscript. TO and YH assisted in the experimental design and performed the data analysis. RO conducted the experiments. Each author substantially contributed to the work reported herein.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


E/Z)-citral to (R)-citronellal. As one of the most performing old yellow enzymes, the enzyme OYE3 from Saccharomyces cerevisiae S288C exhibited complementary enantioselectivity for the reduction of (E)-citral and (Z)-citral, resulting in lower e.e. value of (R)-citronellal in the reduction of (E/Z)-citral. To develop a novel approach for the direct synthesis of enantio-pure (R)-citronellal from the reduction of (E/Z)-citral, the enzyme OYE3 was firstly modified by semi-rational design to improve its (R)-enantioselectivity. The OYE3 variants W116A and S296F showed strict (R)-enantioselectivity in the reduction of (E)-citral, and significantly reversed the (S)-enantioselectivity in the reduction of (Z)-citral. Next, the double substitution of OYE3 led to the unique variant S296F/W116G, which exhibited strict (R)-enantioselectivity in the reduction of (E)-citral and (E/Z)-citral, but was not active on (Z)-citral. Relying on its capability discriminating (E)-citral and (Z)-citral, a new cascade reaction catalyzed by the OYE3 variant S296F/W116G and glucose dehydrogenase was developed, providing the enantio-pure (R)-citronellal and the retained (Z)-citral after complete reduction of (E)-citral.The importance of yeast old yellow enzymes is increasingly recognized for direct asymmetric reduction of ( R)-citronellal serves as the key intermediate in the synthesis of L-menthol, which is one of the most popular flavors in the world [R)-citronellal is currently synthesized through chemical approaches starting from myrcene (Takasago company in Japan) or citral (BASF company in Germany). In the Takasago method, myrcene is converted to geranylamine, then geranylamine is isomerized and hydrolyzed to form (R)-citronellal [E/Z)-citral is distilled to obtain high-purity (E)-citral and then (R)-citronellal is synthesized through asymmetric hydrogenation of (E)-citral [R)-citronellal through enzymatic asymmetric hydrogenation of (E/Z)-citral is the most desired in the industry [E)-citral and (Z)-citral [E/Z)-citral was the crude mixture of ~60% (E)-citral and ~40% (Z)-citral [E)-citral and (Z)-citral by the same native OYE usually yielded the enantio-complementary products, so the e.e. value of the product from the reduction of (E/Z)-citral was often relatively low [-citral ,8. Chemi)-citral ,10. Specindustry . Old yel)-citral ,13. Natu)-citral . Unfortuvely low ,15.Gluconobacter oxydans (Gox0502); and W66 in NCR ene reductase [R)-enantioselectivity to >99% e.e. in the reduction of (E)-citral or (E/Z)-citral [Z)-citral to bind with a flipped orientation in the active site, and thus reversed (S)-enantioselectivity (32.66% e.e.) to (R)-enantioselectivity (~75% e.e.). To the best of our knowledge, no OYE variant was reported to reverse (S)-enantioselectivity to strict (R)-enantioselectivity (>99% e.e.), yet, demonstrating that direct synthesis of enatio-pure (R)-citronellal from the asymmetric reduction of (E/Z)-citral remained challenging. Besides the change of the substrate-binding mode, a novel strategy relying on the utilization difference of (E)-citral and (Z)-citral could be attempted to improve (R)-enantioselectivity. Yeast OYEs exhibited the opposite enantioselectivity in the asymmetric reduction of (E)-citral and (Z)-citral, demonstrating (R)-enantioselectivity and (S)-enantioselectivity, respectively [R)-enantioselectivity of yeast OYEs would be increased if the utilization of (Z)-citral was diminished or even abolished. On the other hand, disabling the reduction of (Z)-citral could simultaneously lead to two isomer: enantio-pure (R)-citronellal from the reduction of (E)-citral and the retained (Z)-citral, both of which are valuable for perfume production and organic synthesis [Protein engineering has been proved to be powerful for overcoming the deficiencies of native OYEs, and successful examples of the improvement of enantioselectivity are accumulating ,17,18,19eductase ,26. The )-citral . Remarkaectively . It is rynthesis .S)-enantioselectivity, yeast OYEs are usually afforded to (R)-enantiomer in the reduction of (E/Z)-citral [R)-citronellal from the reduction of (E/Z)-citral, the enzyme OYE3 was engineered through the modification of both the substrate-binding mode and the substrate-utilization mode. The resulting OYE3 variant discriminating (E)-citral and (Z)-citral was used to cascade asymmetric reduction of (E)-citral and glucose dehydrogenase (GDH)-catalyzed NADPH regeneration (R)-citronellal and the retention of (Z)-citral.In contrast to bacterial OYEs with preference of -citral . Yeast O)-citral , whereasneration , affordiE)-citral and (Z)-citral, whose corresponding e.e. values were 63.45% (R) and 47.22% (S), respectively -citral was lower than that for the reduction of (E)-citral. To improve the (R)-enantioselectivity in the reduction of (E/Z)-citral, site-directed mutagenesis was conducted to test whether key residues critical for other OYEs determined the enantioselectivity of OYE3 [Escherichia coli and purified for the evaluation of catalytic performance (R)-enantioselectivity, whereas the variants I75C, P77C, and V113A resulted in improved (S)-enantioselectivity, suggesting that the enantioselectivity of OYE3 was not determined by a single amino acid residue. Besides, all the other tested variants showed similar enantioselectivity to that of OYE3. The enzyme OYE3 had the opposite enantioselectivity in the asymmetric reduction of -enantioselectivity in the reduction of (E)-citral, and reversed the (S)-enantioselectivity in the reduction of (Z)-citral. The e.e. value in W116A-mediated reduction of (E/Z)-citral reached up to >99% despite lower conversion. To get detailed insights into the molecular mechanism, the substrate and FMNH2 were docked in silico into the crystal structure of OYE3 and its variants W116A and S296F. In the docking of OYE3, a conserved H191/N194 pair formed hydrogen bonds with the carbonyl oxygen of citral; a hydride was enantioselectively transferred to the substrate Cβ atom from FMNH2; and the Y196 residue provided a proton to the substrate Cα atom as an electron acceptor [Z)-citral was due to the flipped binding orientation that placed the opposite face of the alkene above the si face of the FMNH2 cofactor (E)-citral with preserved (R)-enantioselectivity derived from the same binding orientation as wild-type OYE3 -enantioselectivity in comparison with S296F, suggesting that subtle change at the site S296 would result in significantly different enantioselectivity [R)-enantioselectivity in the reduction of (E)-citral, (Z)-citral, or (E/Z)-citral. The (R)-enantioselectivity of W116V and W116I fell between OYE3 and the variant W116A. In the case of W116A, the increase of available free space around W116 by the insertion of Ala, which is less hindered than Trp, seemed to favor a flipped binding mode for (Z)-citral and promote the (R)-enantioselectivity up to >99% [R)-enantioselectivity as W116A, but had no activity on the substrate (Z)-citral, suggesting that the site W116 was not only critical for the determination of enantioselectivity but also the discrimination of (E)- and (Z)-citrals.To explore whether another substitution is superior to S296F, the residue S296 was replaced to be the amino acids with bulky side groups such as W and Y, or those with small side groups such as A and G . As showectivity . Similar to >99% . The varR)-enantioselectivity in the reduction of (E)-citral, and had no catalytic activity on the reduction of (Z)-citral (E/Z)-citral reduction, demonstrating the subtle control on the discrimination of (E)-citral and (Z)-citral. In contrast to the S296 variants, the catalytic activities of the W116 variants were severely decreased. To investigate the effect of the double substitutions of W116 and S296 on catalytic activity, we conducted site-directed mutagenesis of S296 to F and W116 to V, S, A, and G, resulting in the four variants . All fou)-citral . Among tE/Z)-citral, which was supported by the determination of apparent kinetic parameters. OYE3 and its variant S296F/W116G obeyed the substrate-inhibition kinetics (E)-citral was tested as substrate, the values of Km, Ki, and Kcat/Km for the variant S296F/W116G were 0.09 mM, 5.85 mM, and 1.32 mM−1s−1, respectively -enantioselectivity and activity. The docking analyses indicated that the accommodation of (E)-citral in the active site of OYE3 and the variant S296F/W116G was similar, whereas the activity loss in the variant S296F/W116G-mediated reduction of (Z)-citral might be attributed to the elongated distance of the hydroxyl group of W196 and the substrate Cα atom as well as that of the substrate Cβ atom and FMNH2 (E)-citral and (Z)-citral was so subtle, crystal structures of OYE3 and the variant S296F/W116G would be required for further interpretation on its molecular mechanism.The variant S296F/W116G exhibited lower catalytic efficiency than OYE3 in the reduction of -citral, and the pair of GDH from Exiguobacterium sibiricum and glucose were used to drive the regeneration of NADPH [R)-citronellal and simultaneous resolution of (E)-citral and (Z)-citral, it is mandatory to achieve complete reduction of (E)-citral and then cause the retention of (Z)-citral. In the cascade reaction, the optimization of various factors affecting the reduction of (E/Z)-citral would be significantly beneficial for the conversion improvement [2. During the cascade reaction, the enzyme-bound FMN is first reduced at the expense of NADPH, then a hydride is transferred from the reduced FMNH2 to the electronically activated Cβ position of the substrate [+ significantly increased the catalytic efficiency, and the NADP+ concentration (>0.4 mM) was required to maintain high catalytic activity (E/Z)-citral, the optimized parameters for NADPH regeneration were equal activity ratio of GDH and the variant S296F/W116G -citral catalyzed by either OYE3 or OYE3 S296F/W116G was depicted (E/Z)-citral, the conversions to both (R)-citronellal and (S)-citronellal increased as (E)-citral and (Z)-citral were gradually utilized till complete consumption at 12 h. Since OYE3 had the complementary enantioselectivity for (E)-citral and (Z)-citral, the e.e. value of (R)-citronellal was no greater than 40%. The catalytic performance of OYE3 S296F/W116G was notably different from that of OYE3. It took 18 h for the variant OYE3 S296F/W116G to completely reduce (E)-citral, demonstrating its lower catalytic activity than that of OYE3. Most of (Z)-citral was retained and the slight decrease might be attributed to the isomerization of (Z)-citral to (E)-citral. As anticipated, (S)-citronellal was not detectable and the e.e. values of (R)-citronellal remained >99%, verifying that the proposed approach for asymmetric synthesis of (R)-citronellal and simultaneous resolution of (E)-citral and (Z)-citral was feasible.Under optimized conditions, the time course of the reduction of  (E. coli strain BL21(DE3) was used as the host. E. coli cultures were grown routinely in Luria Bertani (LB) medium at 37 °C for 12 h. The genes encoding OYE3 and glucose dehydrogenase from , China) . The pETZ)-citral and (E)-citral were prepared by a previously-described procedure [ competent cells, resulting in the strain E. coli BL21(DE3)/pET28b-oye3. The cells containing pET28b-oye3 were cultured in the LB medium with 100 µg/mL kanamycin at 37 °C and 200 rpm until the OD600 of 0.6, and 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was supplemented for the induction at 25 °C and 160 rpm. After 12 h of induction, E. coli cells were harvested by centrifugation and further washed using 50 mM Tris-HCl buffer (pH 8.0). The productivity of OYE3 was 2.42 U per gram of wet cells. The cells were disrupted through ultrasonication for 10 min, and the cell debris was removed by centrifugation to result in a clear cell extract. The crude cell extracts containing OYE3 was applied to a Ni-NTA chelating affinity column equilibrated with the binding buffer . Unbound proteins were washed off by the application of the binding buffer. The OYE3 was eluted with 100 mM imidazole in 50 mM Tris-HCl (pH 8.0), desalted with 50 mM Tris-HCl buffer (pH 8.0) by ultrafiltration, and then stored at −20 °C for further study.The gene encoding OYE3  was inseNde I/Xho I of the plasmid pET28b, resulting in the plasmid pET28b-gdh. Following the similar procedure to OYE3, the cells harboring pET28b-gdh was induced and the enzyme GDH was purified for further use. The purity of the purified enzymes was visualized through SDS-PAGE analysis using a 12% resolving gel [The gene encoding GDH  was inseving gel .oye3 was used as template DNA in the single substitution, while the creation of double substitutions was based on the gene encoding the variant OYE3 S296F as the template. The primer sets for single or double substitutions were shown in Dpn I at 37 °C for 1 h, and then the digested DNA was directly introduced into E. coli strain BL21(DE3). Each constructed plasmid was confirmed by sequencing. Expression and purification of the resulting OYE3 variants were conducted using the same procedure as OYE3. Specifically, the productivity of OYE3 S296F/W116G was 1.74 U per gram of wet cells. The site-directed mutagenesis was performed as previously described . PCR ampE)-citral and 0.2 mM NADPH in 100 mM PIPES buffer (pH 7.0). The enzyme assay for GDH was carried out in triplicate in a reaction mixture (1 mL) composed of 5 mM glucose and 0.4 mM NADP+ in 100 mM PIPES buffer (pH 7.0). Both reactions were started by the addition of the enzyme . One unit of the activity is defined as formation or oxidation of 1 μmol NADPH per min. The protein concentrations of all samples were determined using the BCA reagent with bovine serum albumin as the standard protein [Activities of the purified OYEs and GDH were measured at 30 °C by monitoring the change of the absorbance at 340 nm. The enzyme assay for OYEs was carried out in triplicate in a reaction mixture (1 mL) composed of 2 mM ( protein . E)-citral and (Z)-citral. Concentrations of (E)-citral and (Z)-citral were 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, and 50 mM. According to substrate-inhibition kinetics, apparent values of Km, Ki, and Vmax were calculated using the curve fittings of the software Prism 7.2 .Apparent kinetic parameters were determined using different concentrations of , molecular docking simulation was performed using AutoDock Vina under the default docking parameters [E)-citral or (Z)-citral acted as a ligand to molecular docking with OYE3, and the calculation of geometric parameters and ligand structure was performed by ChemBioDraw 12.0 . All homology models of OYE3 variants were built using the “BuildModel” tool implemented in FoldX software with the model of OYE3 as a template [Using the crystal structure of OYE3 from rameters . Point crameters . (E)-cittemplate ,43,44. Ttemplate .+, 0.15 U/mL GDH, 50 mM glucose, 0.15 U/mL OYE3 or its variant, and 50 mM PIPES buffer solution (pH 7.0). The substrate included (E)-citral, (Z)-citral, or (E/Z)-citral, whose stock solution was 200 mM substrate in isopropanol. The reaction mixture was incubated in an orbital shaker  for 11 h, and then extracted using an equal volume of ethyl acetate at 30 °C and 200 rpm for 2 h. After extraction and centrifugation , the solvent phase was collected, dried over anhydrous sodium sulfate, and subjected to GC analyses as previously described [To determine catalytic performance of OY3 and its variants, the standard reaction mixture (1 mL) contained 20 mM citral, 0.6 mM NADP2 as the carrier gas were set as 2 mL/min and 1:49, respectively. The temperatures for both injector and detector were set as 250 °C. The column temperature program was listed as follows: initial temperature of 90 °C for 25 min, 20 °C/min ramp to 160 °C for 2 min, and 20 °C/min ramp to 180 °C for 3 min. The injection volume was 1 μL. The retention times of (S)-citronellal, (R)-citronellal, (Z)-citral and (E)-citral were 22.5, 23.0, 29.2, and 30.2 min  equipped with an FID detector and chiral capillary BGB-174 column . The flow rate and split ratio of N30.2 min . E/Z)-Citral, the standard reaction mixture (10 mL) contained 20 mM (E/Z)-citral, 0.6 mM NADP+, 0.15 U/mL GDH, 50 mM glucose, 0.15 U/mL the variant S296F/W116G, and 50 mM PIPES buffer solution (pH 7.0). The reaction was conducted at 400 rpm and 30 °C for 10 h (OYE3) or 18 h (the variant S296F/W116G) in a reactor with pH-constant auto-titration system. The solution for pH auto-titration was 1 M NaOH. The optimal temperature of (E)-citral reduction was determined at a series of temperatures ranging from 20 to 45 °C. The optimal pH was determined over a range of pH 6.0 to 8.0 at 30 °C. The concentrations of NADP+ were explored within the range of 0 to 1 mM. The optimal glucose concentration was determined over a range of 1 to 100 mM. The activity ratio of GDH and the variant S296F/W116G was tested to be 1:4, 2:4, 4:4, 6:4, or 8:4. After optimization, the time courses OYE3 and the variant OYE3 S296F/W116G-mediated reduction of (E/Z)-citral were investigated in the reaction mixture as below: 20 mM (E/Z)-citral, 0.4 mM NADP+, 0.15 U/mL GDH, 60 mM glucose, 0.15 U/mL OYE3, or the variant S296F/W116G, and 50 mM PIPES buffer solution (pH 7.0). The reactions for OYE3 and the variant OYE3 S296F/W116G mediated reduction of (E/Z)-citral were conducted at 400 rpm and 30 °C for 12 h and 18 h, respectively. The reaction mixture was sampled every 2 h. The samples were extracted using an equal volume of ethyl acetate at 30 °C and 200 rpm for 2 h. Then, the solvent phase was collected, dried over anhydrous sodium sulfate, and subjected to GC analyses as described in the To identify key factors for S296F/W116G mediated-reduction of (R)-enantioselectivity in the reduction of (E/Z)-citral to (R)-citronellal. The substitution of S296 to F resulted in the remarkable improvement of (R)-enantioselectivity. The variant W116A showed strict (R)-enantioselectivity in the reduction of (E)-citral, (Z)-citral or (E/Z)-citral. To the best of our knowledge, this is the first report to completely reverse (S)-enantioselectivity to (R)-enantioselectivity in the OYE family. Interestingly, the further double substitution variant W116A/S296F was capable of discriminating (E)-citral and (Z)-citral, indicating that the sites W116 and S296 were pivotal for not only the determination of enantioselectivity, but also the discrimination of citral isomers. Using the variant OYE3 S296F/W116G and GDH as biocatalyst, a novel cascade reaction was achieved to offer two isomers in the (E/Z)-citral reduction: enantio-pure (R)-citronellal and the retained (Z)-citral after complete consumption of (E)-citral. The enzyme OYE3 was engineered through semi-rational design to improve (


In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients.The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed, particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction.  in vitro in VeroE6/TMPRSS2 cells and human lung organoids. When delivered through the nose to infected Syrian hamsters, this antibody prevented the typical symptoms caused by SARS-CoV-2. Our results are significant because delivering simple drugs directly to infected lungs with a nebulizer could increase the potency of the drugs while reducing the risk of immune reaction that could occur if the drugs escape or are delivered through the blood stream.Vaccines for COVID-19 are now available, but therapeutic drugs are still needed to treat life-threatening cases and those who cannot be vaccinated. We discovered a new heavy-chain single-domain antibody that can effectively neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19. Unlike other drug candidates, which prevent the virus from attaching to the receptor on the host cell, this new antibody acts by blocking the virus membrane from fusing with the host cell membrane. We studied the behavior of the new antibody  Coronaviruses (CoV) are enveloped, single-stranded positive-sense RNA viruses. They are divided into four genera: alpha, beta, gamma and delta. Betacoronaviruses are significant interest because they are responsible for the severe acute respiratory syndrome (SARS) and middle eastern respiratory syndrome (MERS) epidemics in the past and now, the coronavirus disease 2019 (COVID-19) pandemic. These viruses can cause mild to severe respiratory tract infections and death in some cases. All betacoronaviruses feature a spike protein on their surface that assists entry into cells. The spike (S) protein has two distinct subunits, S1 and S2, and the receptor binding domain (RBD) in the S1 subunit interacts with the host cell receptor. The S proteins of SARS-CoV-1 (virus causing SARS) and SARS-CoV-2 (virus causing COVID-19) bind to the same host cell receptor, angiotensin-converting enzyme 2 (ACE2). After binding to ACE2 via the RBD, a protease on the host cell surface cleaves and activates the S protein, allowing the virus membrane to fuse with the host cell membrane. Blocking this viral fusion is thought to be a promising therapeutic strategy , direct In vitro studies show ALX-0171 binds to the F protein in RSV with higher affinity than the approved human monoclonal antibody prophylactic drug, Palivizumab  (Abcam) mouse monoclonal antibody and anti-mouse IgG goat polyclonal antibody-HRP (Abcam). The o-phenylenediamine dihydrochloride substrate tablet (Thermo Fisher Scientific) dissolved with Stable Peroxide Substrate Buffer (Thermo Fisher Scientific) was used as a substrate. The absorbance at 450 nm was measured with GloMax Explorer System (Promega).4 TCID50 (MOI = 0.5) of SARS-CoV-2 was incubated with 150 μg/ml of VHHs for 2 hr at 37°C and then 24 hr at 4°C and inoculated to 5.0 x104 VeroE6/TMPRSS2 cells for an hr. Similarly, to compare early RNA replication in the infected cells, infected cells were collected at 3 or 6 hr after infection. Total RNA was extracted from the collected cells with Nucleospin RNA (TAKARA). Numbers of RNA copies of the virus were determined by qRT-PCR , according to manufacturer’s protocol.2.5 x10To quantify the cell fusion induced by S protein, HiBiT technology (Promega) was applied. HiBiT was linked to the C-terminal of ZsGreen (TAKARA) and transduced by lentivirus vector LVSIN-IRES-puro, and LgBiT was transduced by lentivirus vector LVSIN-IRES-Hyg . Equal n2SO4. Signaling was quantified by Ensight (Perkin Elmer).Diluted recombinant S protein (2 μg/mL)  with PBS was incubated to immobilize on the maxisorp plate . After blocking with 1% BSA/PBS, 1 μg of K-874A diluted in 1%BSA/PBS was incubated to bind to S protein. Then, serial diluted recombinant ACE2 (from 1 to 0.06 μg/ml) , subsequent rabbit anti-ACE2 antibody  and HRP-conjugated anti-rabbit IgG  were incubated. To detect K-874A binding to immobilized S protein, serial diluted K-874A (1 to 0.002 μg/ml) in 1% BSA/PBS was incubated, and K-874A which bound to S protein was detected by HRP-conjugated anti-VHH antibody . Between each incubation, wells were washed with PBST. To color, o-phenylenediamine dihydrochloride  was used as a substrate of HRP and reaction was stopped with 2 M HVeroE6/TMPRSS2 cells were infected SARS CoV-2 and fixed at 24 hr after infection. After treatment with 0.05% Triton-X and 3% BSA/PBS for permeabilization and blocking, cells were incubated with anti-NSP8  or anti-dsRNA  for 1 hr and subsequently with Alexa 568-conjugated goat anti-mouse IgG  and Hoechest for an hour at room temperature. Images were taken by BZX800 (Keyence).Human alveolar-derived organoids were established . Briefly3 TCID50 of SARS-CoV-2 was nasally inoculated to 7 weeks age of Syrian hamsters. Then 3 mg of K-874A was nasally administrated just before, 1 day or 1 and 2 days after virus inoculation. Each hamster was weighed every day up to 4 days after inoculation. Lung tissues were homogenized at 4 days after inoculation, and RNA was extracted with Nucleospin RNA (TAKARA) for qRT-PCR.2x10Primers and probes for hamsters were designed as reported . The pri2. A GIF-quantum energy filter (Gatan) was used with a slit width of 20 eV to remove inelastically scattered electrons. Individual movies were subjected to per-frame drift correction by MotionCor2 [S protein trimers of SARS-CoV-2  and K-874A (11 mg/mL) were mixed at 10:1 and kept at room temperature for 1 day. Aliquot (2.5 μL) of the sample was placed onto a holey-carbon copper grid , previously glow-discharged in a plasma ion bombarder . The grid was then blotted  and plunge-frozen at a condition of 95% humidity and 4°C by using a Vitrobot Mark IV (Thermo Fisher Scientific). The cryo-EM data were acquired with a Titan Krios at 300 kV (Thermo Fisher Scientific) and a Gatan K3 camera (Gatan) at a nominal magnification of 64,000, corresponding to 1.11Å per pixel on specimen. Each micrograph was recorded as a movie of 58 frames at the total dose of approximately 40 electrons per ÅtionCor2 . The contionCor2  and the tionCor2 . A totaltionCor2 , the regtionCor2 . The ext2 for a 3-sec exposure. The single particle analysis was performed with RELION3.1. The 3D map reconstructed from 14,235 particles was estimated to be 6.9Å, based on a gold standard FSC. The entire procedure and the processing data are summarized in The cryo-EM images of S protein trimer without K-874A were collected with a JEM2200FS electron microscope (JEOL) using a side-entry Gatan 626 cryo-specimen holder (Gatan). For image formation, a field-emission gun operated at 200 kV and an in-column (Omega-type) energy filter operating in zero-energy-loss mode with a slit width of 20 eV were used. Images of the frozen hydrated particles were recorded on a direct-detector CMOS camera  at a microscope magnification of 40,000×, corresponding to 1.422Å per pixel on the specimen. Using a low-dose method, the total electron dose on the specimen was about 20 electrons per ÅThe SWISS-MODEL  server wS1 Table(TIF)Click here for additional data file.S1 FigGold-standard Fourier shell correlation (FSC) curves show the global resolutions of the entire complex of the S protein trimer with the solid lines and the resolution of the focused refinement maps only, including RBD and NTD.(TIF)Click here for additional data file.S2 FigType 1 cryo-EM structure showed that K-874A bound to both RBD and NTD, and approximately 90% of the particles were classified into this structure. The remaining approximately 10% of particles was classified into the Type 2 cryo-EM structure, where took upward state and K-874A bound to RBD only, not NTD. The focused refinement was performed in the regions RBD, NTD, and K-874A for Types 1 and 2 structures. Gold-standard Fourier shell correlation (FSC) curves show the global resolutions of the entire complex of the S protein trimer and K-874A with the solid lines and the resolution of the focused refinement maps only, including RBD, NTD, and K-874A with dotted lines.(TIF)Click here for additional data file.S3 FigA-C) Side views of the cryo-EM maps of the S protein trimer (A), type 1 structure (B) and type 2 structure (C) of the complex withK-874A. (B) and (C) are composite maps. (D-F) The fitted atomic models of the S protein monomer (D) or the complex with K-874A ((E) type 1 structure; (F) type2 structure). The NTD, RBD and K-874 are colored blue, green and magenta, respectively. (G-I) The atomic models of S protein trimer (G) or the complexes with K-874A  fitted into the maps. Scale bar equals 50Å.((TIF)Click here for additional data file.S4 FigA) A ribbon model of S protein trimer and K-874A. The coloring follows the standard designation of K-874A (magenta), NTD (blue) and RBD (green). (B) An enlarged view of RBD, NTD and K-874A in (A). (C) A view of the structure in (B) rotated 90 degrees. Residues that interact with ACE2 are shown in yellow. N501 and E484 (cyan) are amino acid residues frequently mutated.((TIF)Click here for additional data file.S5 FigLight green and light blue are the positions of RBD and NTD of S protein when K-874A does not bind. (A) Type 1 structure of the complex with K-874A (B) Type 2 structure of the complex with K-874A. In the type 1 structure (approximately 90% of the S protein and K-874A complex), the K-874A bound moved the RBD sideways and the NTD down. In the case of type 2 structure (approximately 10% of the S protein and K-874A complex), the K-874A bound to the upward state of the RBD and the NTD did not interact directly with K-874 but moved slightly aside.(TIF)Click here for additional data file.S6 FigThe secondary-structural elements are indicated over the sequences as a spiral (α-helix) or an arrow (β-sheet). The NTD and the RBD are colored by blue and green, respectively. Letters on a red background indicate identical amino acids. Figure is drawn by ESPript .(TIF)Click here for additional data file.S7 FigThe secondary-structural elements are indicated over the sequences as a spiral (α-helix) or an arrow (β-sheet). Letters on a red background indicate identical amino acids. Figure is drawn by ESPript .(TIF)Click here for additional data file.S8 FigA) RNA copies in the culture supernatant at 0 or 2 days post-infection with or without K-874A. Calu3 cells were infected with each SARS-CoV-2 variant for 1 hr. After infection, cells were washed twice with fresh medium and cultured with or without 150 μg/ml of K-874A for 2 days. RNA copies at day 2 were measured by qRT-PCR. Wuhan, KUH003 (D614G) and B.1.1.7 (Alpha) were slightly reduced in K-874A-treated, but B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta) were not. (B) RNA copies in each culture supernatant were adjusted to 10,000 copies based on qRT-PCR results in (A), and each variant with or without K-874A was infected to VeroE6/TMPRSS2 cells to determine TCID50. A significant difference between with or without K-874A was observed in culture supernatant with Wuhan, KUH003 (D614G) and B.1.1.7 (Alpha).  (C) ELISA data showing direct binding of recombinant S protein from each variant and K-874A. Immobilized K-874A was incubated with Rho1D4-tagged recombinant S protein from each variant and detected by anti-rhodopsin IgG. K-874A bound to Wuhan, and B.1.1.7 (Alpha) but not others.((TIF)Click here for additional data file.S9 FigA) Time schedule for inoculation and K-874A administration to Syrian hamsters. (B) Weight changes in K-874A-treated and -untreated hamsters after SARS-CoV-2 infection as indicated. Weight at Day 0 is 100%. (C) Amounts of viral RNA in the lung homogenates at Day 4 were determined by qRT-PCR. (D) qRT-PCR results showing inflammatory cytokine expression. Expression levels of IL-6, IL-10, TNF-α and IL-1β were assessed in lung homogenates collected at Day 4. ((TIF)Click here for additional data file.S10 FigSchematic diagram indicating K-874A binding domain (A) and ACE2-blocking VHHs binding domain (B). Trimer of S proteins, ACE2 and VHHs were depicted and ACE2 binding domain of RBD was depicted in red. K-874A binds NTD and RBD, while previous reported VHHs binds the surface of ACE2 binding interface.(TIF)Click here for additional data file.


Scylla paramamosain) allergen. This study aimed to assess Maillard-reacted TM (TM-G) induction of allergenic responses with cell and mouse models. We analyzed the difference of sensitization and the ability to induce immune tolerance between TM and TM-G by in vitro and in vivo models, then we compared the relationship between glycation sites of TM-G and epitopes of TM. In the in vitro assay, we discovered that the sensitization of TM-G was lower than TM, and the ability to stimulate mast cell degranulation decreased from 55.07 ± 4.23% to 27.86 ± 3.21%. In the serum of sensitized Balb/c mice, the level of specific IgE produced by TM-G sensitized mice was significantly lower than TM, and the levels of interleukins 4 and interleukins 13 produced by Th2 cells in spleen lymphocytes decreased by 82.35 ± 5.88% and 83.64 ± 9.09%, respectively. In the oral tolerance model, the ratio of Th2/Th1 decreased from 4.05 ± 0.38 to 1.69 ± 0.19. Maillard reaction masked the B cell epitopes of TM and retained some T cell epitopes. Potentially, Maillard reaction products (MRPs) can be used as tolerance inducers for allergen-specific immunotherapy.Tropomyosin (TM) is an important crustacean ( Food allergy, a conceptual term which covers many clinical implications, usually refers to an allergic reaction such as a type I hypersensitivity mediated by immunoglobulin E (IgE) . About 8+ helper T cells (th) associated with allergy after binding to MHCII molecules [Shellfish is also considered to be one of the most common aquatic products, which can induce severe food allergy diseases, tropomyosin (TM) is the pan-allergen . TM witholecules ,13. In aolecules . Basophiolecules .Oral tolerance is a state of systemic unresponsiveness, which is the default response to food antigens . FailureAntigenic epitopes are divided into two groups based on the cells which can be recognized: T cell epitopes and B cell epitopes . ModifyiThere is little data about the MRPs absorption and presentation of DCs, although it is important to explain the allergic responses induced by MRPs. Less information reported that MRPs could be as tolerance inducers. Whether the crab TM treated by MR has the potential to induce oral tolerance requires further exploration. Glycation sites of MRPs should be identified and the relation of epitopes and oral tolerance require further exploration.In the present study, galactose could effectively decrease allergenicity in TM through MR in vitro assays. Besides, the mechanism of lower allergenicity and induced oral tolerance of Maillard-reacted TM (TM-G) was evaluated with cell and mouse models. Finally, the glycation sites of TM-G were identified to analyze the modification side on the T cell epitopes or B cell epitopes. Overall, these results would form the foundation to contribute to the future development of the Maillard reaction products as a functional food for tolerance induction.Galactose was purchased from Macklin . Rabbit anti-crab TM IgG pAb was prepared in our laboratory previously and goat anti-rabbit IgG-HRP antibody was from Abmart . Granulocyte macrophage colony stimulating factor (GM-CSF), IL-4 were obtained from Sigma-Aldrich . RPMI 1640 and fetal bovine sera (FBS) were purchased from Invitrogen . CD11c for flow cytometry analysis were purchased from Biolegend . Imject Alum was purchased from Thermo Fisher Scientific Inc. . Goat anti-mouse IgE, IgG1, and IgG2a antibodies were purchased from Abcam . The ELISA kits of IL-4, IL-10, IL-13, interferon (IFN)-γ, mouse mast-cell protease-1 (mMCP-1) and transforming growth factor-β (TGF-β) were purchased from R&D Systems . An ELISA kit of histamine was purchased from IBL .S. paramamosain) was purchased from a local aquatic products market in Xiamen. Specific pathogen-free female Balb/c mice weighing 14–16 g (4–6-weeks-old) were obtained from Shanghai Laboratory Animal Center of Chinese Academy of Sciences . The housing environment for mice under specific pathogen-free conditions was maintained at 20–25 °C and 60 ± 10% relative humidity with a 12 h light/dark cycle. Protocols were approved by the Institutional Animal Care and Use Committee of Animal Laboratory Center of Jimei University , and all animals were used for academic research.Live crab , and signed informed consent was obtained from all individuals. Sera IgE antibodies  to crab S. paramamosain and preparing TM-G were accorded as described previously [4 dilution and HRP-labeled goat anti-rabbit IgG with 1:2 × 104 dilutions were used as the primary and secondary antibody, respectively, for immunoblot analysis. TM and TM-G were adjusted to 1 mg/mL to dot blot, the primary antibody was human sera (dilution 1:3), the secondary antibody was HRP-labeled goat anti-human IgE antibody (dilution 1:1 × 104).The purification of TM from eviously . All samMouse bone marrow-derived dendritic cells (BMDCs) isolated from BALB/c mice were referred to the classical method by Perusko et al. . After b5 cells per well in 24-well plates and incubated gradient time with 50 μg FITC-protein for 0, 10, 20, 30, 40, 50, 60, 90, 120, and 180 min at 37 °C. Then, the collected cells were stained with APC anti-mouse CD11c for 30 min at 4 °C for BMDCs uptake analysis by flow cytometry.TM and TM-G were labeled with FITC according to the method of Liu et al. . Briefly5 cells per well in 24-well plates and incubated 48 h with 50 μg proteins . The total RNA kit and TIANScript RT Kit  was used to extract the cDNA from BMDCs for RT-PCR, according to the instructions with 20 μL final volume. The changes of CD86 and H2-Ob transcription levels in BMDCs stimulated by PBS, TM, and TM-G were detected by RT-PCR. The following primers were used for RT-PCR: CD86-F, 5′-ATG TCA CAA GAA GCC GAA TC-3′. CD86-R, 5′-TTC AGT GCT CTT GGC CTA TG-3′. H2-Ob-F, 5′-GTG ACC TGG GGA TGT TTG TTG-3′. H2-Ob-R, 5′-CAG GAG ATC CAG GCG TTT GTT-3′. β-actin-F, 5′-ACA GCA CCG TGT TGG CGT AGA G-3′. β-actin-R, 5′-TGC GGG ACA TCA AGG AGA AGC-3′. The result was chosen 2−ΔΔCt method calculation the relative quantitative of CD86 and H2-Ob.Similarly, immature BMDCs were cultured at 1 × 10570 nm to assess cytotoxicity. Assessment of the β-hexosaminidase release rate, the serum of TM sensitized mice with RBL-2H3 cells was incubated for 16 h. Then allergic reactions were induced by co-incubation with PBS, TM-G, and TM [RBL-2H3 was purchased from the American Type Culture Collection  and cultured as described previously . MTT ass, and TM .n = 8). For the sensitization model, one week of adaptive culture female Balb/c mice were, respectively, exposed to TM (150 μg) or TM-G (150 μg) with alum adjuvant (6 mg) by injection on days 0 and 14. Mouse blood and spleen lymph cells were collected on day 15. The levels of TM-specific IgE, IgG1, and IgG2a in sera were measured using ELISA. After 3 days of culture, the cytokine assay of spleen lymph cells was measured by ELISA. For oral tolerance, exposed to TM (150 μg) with alum adjuvant (6 mg) by injection on days 0 and 14; next, on days 21 and 28, mice received 2 mg TM or TM-G via intragastric gavage. On day 33, 10 mg TM was given to the mouse. The diarrhea rate within one hour after three times  of gavage was recorded, and the anaphylactic score and rectal temperature of mice were measured one hour after gavage on day 33. The method used for the anaphylactic score was according to Liu et al. [Injection sensitization and oral tolerance tests were based on Han et al.  with modu et al. . Furtheru et al. ,28,29 Thhttp://tools.iedb.org/mhcii/ (accessed on 12 January 2022)) were analyzed based on Gouw et al. [The amino acid sequence of crab of TM was obtained from NCBI (GenBank number: ABS12233.1). The predicted T cell epitopes’ results from the Prediction System . Statistical significance was assessed using a two-way ANOVA. The bands of TM and TM-G are represented in + and FITC-positive cells were time-dependent, while the mean fluorescence intensity continuously increased up to 180 min , without significant difference to the PBS group (p < 0.01), compared with the PBS group, the expression level of H2-Ob decreased by 6.24 ± 1.02% after stimulation in the TM group , and the degranulation rate of the TM-G group was significantly decreased compared with the TM group (p < 0.01); meanwhile, the degranulation rate of the TM-G group was significantly increased compared with the PBS group (p < 0.05).MTT assay was used to assess the cytotoxic effects of TM-G by RBL-2H3 cells. It was found that there was no effect on cell viability after TM and PBS stimulation. The cell viability rate of TM-G was more than 90% at different concentrations without significant difference to TM group A. The sep < 0.01), and there was no difference in the PBS group and the TM-G group, whereas the specific IgE level in the TM-G group was only one-tenth of that in the TM group (p < 0.01). Additionally, the level of IgG1 and IgG2a in the TM and TM-G group was higher than the PBS group, but the level of IgG1 and IgG2a in the TM-G group was significantly lower than the TM group (p < 0.01). Meanwhile, the released level of the Th1-type cytokines IFN-γ in spleen cells was similar to both the PBS group and the TM-G group; however, the IFN-γ value of the TM group was significantly lower than the TM-G group (p < 0.01) (p < 0.01), whereas the TM-G group was significantly decreased compared with the TM group (p < 0.01). Overall, it is further demonstrated that MR could reduce the immunogenicity of allergens.In vivo experiments further proved our hypothesis. The effect of TM and TM-G on mice can be most intuitively analyzed by using the detection of sera antibodies. A 15-day mouse model was used to test the allergenicity of TM-G in vivo A. On the < 0.01) C. The leFurthermore, the potential of TM-G supporting the development of oral tolerance in mice was explored with a mouse model A. AnaphyFurther, assessing the tolerance potency of TM-G at sera level, normally the activation of mast cells was evaluated by specific antibody, histamine, and mMCP-1 E–G. CompThe results of the prediction of T cell epitopes can support information . The maiTo compare the epitopes and modification sites, the glycated residues of TM-G were marked by a solid triangle, the predicted T cell epitopes of TM are marked by single-underline, and reported B cell epitopes of TM are marked by double-underline . It was MR usually occurrs in processed food to improve food quality and taste, and now MR is gradually used to reduce the sensitivity of food allergens. In the previous study, we found that both galactose and arabinose could reduce the allergenicity of TM, while galactose worked better than arabinose . In this+ T cells. To analyze the immunogenic properties of TM-G, the influences on the uptake activity of DCs by TM-G were investigated. Perusko et al.  found th+ B cells do not compete with IgE+ B cells as IgE+ B cells cannot switch towards IgG1; moreover, allergen-specific IgG1 might block IgE binding to the allergen [Furthermore, the potential of TM-G to support the development of oral tolerance in mice was explored. It has been previously shown that MRPs could promote the balance of Th2 cells and Th1 cells in mouse spleen lymphocytes, making them reach the cell level of normal mice . Treg ceallergen .Meanwhile, we explained the molecular mechanism of inducing tolerance of TM-G. Though the molecular weight of amino acids on the peptide can be analyzed by mass spectrometry , the amiIn this study, the glycosylation for TM could reduce its sensitivity and preserve immunogenicity, aimed at improving the efficacy of TM tolerance induction and reduce TM occurrence of serious side effects. In theory, immunogenicity and a low sensitivity allergen vaccine can be used in high doses regardless of the rapid response mediated by IgE, for the significant decrease in the effect of TM-G on the degranulation of mast cells, but still retains the ability to induce Th1 cells to produce IFN-γ . T cell In summary, due to specific amino acid residues on the T cell epitopes and the B cell epitopes of TM being glycated, the TM-G could reduce the degranulation release rate, and inhibit the uptake and presentation function of BMDCs. Additionally, the IgE levels in mice sera of the TM-G group were significantly decreased in the mouse sensitized model. The TM-G could activate Treg to produce TGF-β and IL-10 without causing allergic symptoms. TM-G positively modulates the Th1/Th2 immune balance, which can decrease the specific IgE antibody and histamine in sera. Potentially, the MRPs as tolerance inducers are used for allergen-specific immunotherapy.