As no experimental structure of TRPM3 is available, we built a homology model of the channel in complex with PIP2via molecular modeling. We identified putative contact residues for PIP2 in the pre-S1 segment, the S4–S5 linker, and the proximal C-terminal TRP domain. Mutating these residues increased sensitivity to inhibition of TRPM3 by decreasing PIP2 levels. Changes in ligand-binding affinities via molecular mechanics/generalized Born surface area (MM/GBSA) showed reduced PIP2 affinity for the mutants. Mutating PIP2-interacting residues also reduced sensitivity for activation by the endogenous ligand pregnenolone sulfate, pointing to an allosteric interaction between PIP2 and pregnenolone sulfate. Similarly, mutating residues in the PIP2 binding site in TRPM8 resulted in increased sensitivity to PIP2 depletion and reduced sensitivity to menthol. Mutations of most PIP2-interacting residues in TRPM3 also increased sensitivity to inhibition by Gβγ, indicating allosteric interaction between Gβγ and PIP2 regulation. Disease-associated gain-of-function TRPM3 mutations on the other hand resulted in no change of PIP2 sensitivity, indicating that mutations did not increase channel activity via increasing PIP2 interactions. Our data provide insight into the mechanism of regulation of TRPM3 by PIP2, its relationship to endogenous activators and inhibitors, as well as identify similarities and differences between PIP2 regulation of TRPM3 and TRPM8.Transient receptor potential melastatin 3 (TRPM3) is a heat-activated ion channel expressed in peripheral sensory neurons and the central nervous system. TRPM3 activity depends on the membrane phospholipid phosphatidylinositol 4,5-bisphosphate P Its ches system , the nons system . TRPM3 is system , 7, 8 ans system , 8. TRPMs system , 5, 8.Activation of Gi-coupled receptors inhibits TRPM3 activity. This effect was demonstrated both by native receptors in dorsal root ganglia neurons, including μ-opioid and GABAB receptors , 10, 11,It was recently shown that mutations in TRPM3 are associated with developmental and epileptic encephalopathies manifesting as intellectual disability and seizures in children . The ori2, are common ion channel regulators Pgulators , 20. Mosgulators , 22, aregulators , but in gulators , 25, or gulators , 27, thiy member . While cis TRPM8 .2 binds to, and there is no experimentally determined structure available for TRPM3. To fill this key gap in knowledge, we generated a homology model of TRPM3, based on the experimental structure of TRPM4 in the ligand-free (apo) state P2-interacting residues in the pre-S1 segment, the S4–S5 linker, and the proximal C-terminal TRP domain. We validated our results by docking PIP2 to an apo structure of TRPM8 P2 contact residues in TRPM3, followed by ligand-binding affinity changes via molecular MM/GBSA, showed reduced PIP2 binding affinity to TRPM3. We experimentally validated the importance of these residues by demonstrating that their mutations increased sensitivity to inhibition by PIP2 depletion in electrophysiology experiments. We also showed that mutating most of these residues increased sensitivity to Gβγ inhibition, and decreased sensitivity to agonist activation, indicating allosteric interaction between PIP2 and endogenous activators and inhibitors. Furthermore, we demonstrated that gain-of-function disease–associated mutations did not change PIP2 sensitivity, indicating that the mutations do not increase channel activity via promoting PIP2 activation. Our data provide mechanistic insights into regulation of TRPM3 by its key endogenous cofactor PIP2.Currently, it is not known which residues in the TRPM3 protein PIPo) state . We thenof TRPM8 , which sructures , experim2 binding site of TRPM3. As there is currently no experimentally determined TRPM3 structure available, we generated a homology model of the human TRPM3 (hTRPM3) based on the cryo-EM structure of the mouse TRPM4 in the apo state (Protein Data Bank [PDB] ID: 6BCJ) (A and B). The template was selected as the closest homolog to TRPM3 in the TRPM family with an experimental structure available when the model was built (see the 5ZX5) as the template PD: 6BCJ)   for putative binding sites using the program SiteMap  P2. Specifically, (1) we generated a sequence alignment of TRP-domain residues, K995, R998, and R1108, in the rat TRPM8, which are conserved among TRPM family members (C), and were previously suggested to play a key role in PIP2 interactions (6BPQ) (2 at a site that includes the conserved TRP-domain residues (A and B). Comparing this complex with the apo-TRPM3 model showed that the most suitable site for lipid binding  in TRPM3 corresponded to the PIP2 site identified in TRPM8. We used this lipid-binding site to generate a model of TRPM3 in complex with a version of PIP2 with truncated tails P2, which is fully functional in experiments) by molecular docking using the program Glide PC, 2018) , 35. Sec members C, and weractions  and (2) s (6BPQ) , we builresidues , A and Bam Glide . We rank2 binding site in TRPM3 is formed by parts of the preS1 segment, the S4–S5 loop, and the proximal C-terminal TRP domain of the same subunit. The closest contact residues with PIP2 are W761 in the preS1 segment, the N991 and K992 residues in segment connecting the voltage sensor–like domain (S1–S4) to the S4–S5 linker and the R1131 in the TRP domain (B). Figure 1B also shows the location of two additional residues, K1128 and R1141 in the TRP domain, which are not in close contact with PIP2, but we experimentally characterized their mutations (see later). The numbering of these residues corresponds to the splice variant of hTRPM3 (hTRPM1325) PP domain B. FigureRPM1325) , 38, whi2 with the subsequently determined two cryo-EM structures of TRPM8 with PIP2 (6NR3), and with the menthol analog WS12 (PDB ID: 6NR2), offered a posteriori validation of our modeling P2–icilin structure, the TRPM8–PIP2–WS12 structure, and our computational model. Our model superposes very well with both structures in the transmembrane domains, but it shows a better structural alignment of the PIP2 binding site with the TRPM8–PIP2–WS12 (PDB ID: 6NR2) structure than the TRPM8–PIP2–icilin–calcium structure (PDB ID: 6NR3). In fact, the minimum RMSD values, calculated over amino-acid ranges facing the lipid-binding sites between any two aligned structures, were 1.49 and 2.34 Å, respectively PIP2 , icilin modeling . Fig. S3ectively . Intereset al. , and K605 in the neighboring N-terminal cytoplasmic Melastatin Homology Region 4 (MHR4) domain. All these residues, with the exception of R850, are in contact with, or very close to PIP2 in our TRPM8–PIP2 model. R850 is in contact with the acyl chain of PIP2 in our model, and only in contact with the PIP2 headgroup in the 6NR3, but not in the 6NR2 structure, which is consistent with the better alignment of our model with the 6NR2 PIP2-TRPM8 structure. Overall, our TRPM8–PIP2 docking model validates our computational approach to identify the TRPM3 PIP2 binding site and suggests that PIP2 likely binds to a site that is similar in TRPM3 and TRPM8.Yin et al.  listed f segment C, and K65ZX5) P2 binding site may be conserved in multiple members of the TRPM subfamily.Furthermore, superimposition of our model to the experimental structure of TRPM7 in EDTA (PDB ID: 5ZX5)  revealedmolecule . Whether2 model, residues K992 and R1131 are equivalent to the experimentally determined PIP2 contact sites R850 and R997 in TRPM8, located in the S4–S5 loop and the TRP domain (C). Specifically, N991 is adjacent to K992, and W761 in the pre-S1 segment of TRPM3 is shifted six residues from the R688 residue in TRPM8. The equivalent of W761 in TRPM8 (W682) is relatively close to PIP2 in TRPM8, and so is the equivalent of R688 in TRPM3 (M767) highlighting the generally similar importance of the pre-S1 segment in PIP2 binding in the two channels. The largest difference between the two binding sites is that the MHR4 region, which carries K605 in TRPM8, is not conserved in TRPM3, and the equivalent residue is far away from PIP2 in our TRPM3 model. Overall, the two channels bind PIP2 in a similar, yet not identical manner PP domain C. Specifl manner .2-interacting residues in TRPM3 and tested the effects of the mutations on sensitivity to inhibition by decreasing PIP2 levels. We expressed the WT and mutant channels in Xenopus oocytes and performed two-electrode voltage clamp (TEVC) experiments. We stimulated channel activity with 50 μM PregS and measured current amplitudes, then incubated the oocytes with 35 μM wortmannin for 2 h, and measured PregS-induced currents in the same oocytes (A and B). Wortmannin at this concentration inhibits phosphatidylinositol 4-kinases and has been used to inhibit the activity of PIP2-dependent ion channels  showed significantly higher inhibition after wortmannin treatment than WT TRPM3 (C–F), and their current amplitudes were also significantly lower than WT TRPM3 (H–K). We also generated two additional mutations in the TRP domain in residues that are not in contact with PIP2, K1128Q and R1141Q. Both mutants showed similar current amplitudes to WT TRPM3 (K and L). The K1128Q mutant showed similar inhibition to WT (G), but the R1141Q mutant showed a small but significant increase in wortmannin inhibition compared with WT (F). This mutation is equivalent to R1008Q in the rat TRPM8, which reduced both PIP2 and menthol sensitivity (2 in the cryo-EM structure of fcTRPM8 (R1007) P oocytes , A and Bchannels . We showchannels , indicatrtmannin . We mutaractions . The resWT TRPM3 , C–F, anWT TRPM3 , H–K. WeWT TRPM3 , K and Lon to WT G, but th with WT F. This msitivity , but it A–C shows that 100 nM rapamycin induced a significantly higher inhibition of the N993A mutant of the mouse TRPM3α2  than the WT mTRPM3α2 when the channels were stimulated with 25 μM PregS. Next, we stimulated the mTRPM3α2 with the combination of 25 μM PregS and 10 μM clotrimazole, which was shown to open an alternative pore, characterized by larger currents and less prominent outward rectification (E–G). Current amplitudes for the N993A mutant were also lower than those in the WT TRPM3 (D and H).Next, we confirmed our data in whole-cell patch-clamp experiments using the rapamycin-inducible 4′ 5′ phosphoinositide phosphatase pseudojanin . Fig. S6fication . Applica2 contact site R998Q resulted in a right shift in the diC8 PIP2 dose response in excised patches P2-interacting residue increased the relative efficiency of PI(4)P to stimulate channel activity compared with PIP2 (2 on the N991A mutant. A–C shows that the relative effect of PI(4)P compared with PIP2 did not change.Mutation of the PIP patches . TRPM3 c patches . This anIP2 . Therefo2 to the native (WT) TRPM3 versus the mutant channels that were characterized in i.e., a low-dielectric slab) and, therefore, the results should be taken as a qualitative indication.Next, we used MM/GBSA calculations  to predi2 is guided by a number of stabilizing interactions (A) established with key contact residues . Specifically, K992A had a more prominent effect than R1131Q, N991A, and W761F, respectively. This correlates well with K992A also having the most pronounced effect on inhibition by PIP2 depletion (E). Regarding the binding modes, K992 engages in multiple interactions with PIP2, including three hydrogen bonds and three salt bridges. Mutating K992 to alanine resulted in the loss of all these interactions with the exception of one hydrogen bond, the only interaction established by the amino acid backbone (C). Similar behavior was observed with the mutation R1131Q, the contact residue exerting the second largest effect on the binding affinity, which resulted in the loss of one hydrogen bond and two salt bridges (D), all established by the residue side chain. Next, mutating N991 to alanine and W761 to phenylalanine resulted in the loss of one hydrogen bond each . To further corroborate our results, we performed additional sets of calculations of the binding affinity change upon mutation P2). These calculations are clearly reproducible and agree with experimental observations. Although the trend is maintained overall (A and 2 (light blue), because of headgroup protonation states that prevent interactions via hydrogen-bond formation. Hence, it appears from our calculations, that the PIP2 protonation state featured in the proposed TRPM3 model , and therefore, it is not unexpected that mutating this residue could affect to a lesser extent the binding of a smaller ligand , R1128Q had only a very small effect on both ΔΔG and the related binding mode, which correlates well with it not being in close contact with PIP2 in our model, and the lack of effect on wortmannin inhibition. The R1141Q mutant, which is also not a PIP2 contact site, also had only a minimal effect on both ΔΔG and the related binding mode, indicating that the small, but significant, effect on wortmannin inhibition was likely because of indirect effects. Overall, all Δ affinity calculations supported our computational docking and agreed with the experimental functional characterization of the PIP2-interacting residues.Of the remaining two mutations , A and B2, it is expected that other states may exist featuring alternative networks of interactions yet compatible with the proposed phospholipids-binding site model.It is worth mentioning that, although our binding model likely captures a highly represented conformational state sampled by the TRPM3 channel when bound to PIP2-interacting residues in our TRPM3 model. The R851 residue in TRPM8 corresponds to the K992 residue in the S4–S5 linker in TRPM3 (C), and it was in direct contact with PIP2 in the cryo-EM structure of the fcTRPM8 (R850) (C), and while is not in a direct contact with PIP2 in the fcTRPM8 cryo-EM structure (R850), it is located relatively close. Since the W682A mutant was nonfunctional, we characterized the W682Q, which displayed small, yet measurable, menthol-induced currents. A–G shows that both the R851Q and the W682Q mutants showed significantly higher level of inhibition by wortmannin, with W682Q having a larger effect. Current amplitudes showed a similar pattern; both mutants were significantly lower than WT TRPM8, and the W682Q having a larger effect (H). The decrease in amplitudes was even more pronounced at negative voltages for inward currents (I), in agreement with earlier results with the R995Q PIP2 mutant , which is in contrast to TRPM3, where the deactivation kinetics after washing out PregS was not affected by wortmannin (A and B).Next, we mutated two residues in the rTRPM8 that are equivalent to PIPin TRPM3 C, and it8 (R850) . The W688 (R850) C, and whr effect H. The decurrents I, in agr2 mutant . This ismulation , A–D, whrtmannin , A and B2 P2-interacting residues in TRPM8 have an effect on agonist sensitivity. A–D shows that both the R851A and the W682Q mutant had right shifted menthol dose response. Similar to the effect on current amplitudes and wortmannin inhibition, the effect of the W682Q mutant (C and D) was more pronounced than that of R851A (B and D).Stimulation with menthol, or cold, was shown to increase the apparent affinity of TRPM8 for PIP2  indicatiQ mutant , C and Dof R851A , B and DA–D shows that both the N991A and the K992A mutant shifted the PregS dose response to the right.We also tested if a similar allosteric effect also exists in TRPM3. 2 activation is present, we expressed WT and mutant TRPM3 channels with or without Gβ1γ2 in Xenopus oocytes and measured PregS-induced currents. The N991A and K992A mutants were inhibited significantly more by Gβγ than WT TRPM3 (A–E). The K1128Q mutant, which did not affect PIP2 sensitivity, had no effect on Gβγ inhibition either (E). Interestingly, the R1131Q mutant was not inhibited, rather potentiated by coexpressing Gβγ (E). Consistently with the lack of inhibition by Gβγ, the R1131Q mutant was also not inhibited by stimulating Gi-coupled M2 muscarinic acetylcholine receptors (F–J). These data indicate that while there is an allosteric interaction between PIP2 and Gβγ, the R1131 residue in the TRP domain also plays some role in transmitting the inhibitory effect of Gβγ.TRPM3 activity is inhibited by direct binding of Gβγ to the channel . To testWT TRPM3 , A–E. Thn either E. Interesing Gβγ E. Consiseceptors , F–J. Th2. When WT (A and B) and V990M (C and D) and P1090Q mutant channels (E and F) were treated with 35 μM wortmannin for 2 h, currents evoked by 50 μM PregS were inhibited to a similar extent (G). PregS-induced average current amplitudes were not significantly different in the mutant and WT channels (not shown), similar to our earlier data (50 (H). These data indicate that the disease mutants do not increase channel activity by increasing their apparent affinity for PIP2.Gain-of-function mutations in TRPM3 have recently been shown to cause intellectual disability and seizures , 14, 15. When WT , A and Bnd V990M , C and Dchannels , E and Fr extent G. PregS-data P2-interacting residues in the channel protein. Our data indicate that residues in three regions, the pre-S1 segment, the S4–S5 loop, and the TRP domain, play important roles in forming the PIP2 binding site in TRPM3. Mutations of PIP2-interacting residues decreased the binding affinity in silico P2 and agonists as well as a physiological inhibitor. On the other hand, disease-associated gain-of-function mutations did not change PIP2 sensitivity, indicating that the mutations did not increase channel activity by enhancing PIP2 activation.Our work aims to understand the molecular mechanism of PIPeriments . Mutatin2 P2 contact residue in the fcTRPM8 structure P2 sensitivity before structures became available (2 contact residue in the fcTRPM8 structure and to the R851 residue in the rat TRPM8 that we characterized in this study (2 contact residues R688 and N692 (C). These residues are not conserved in TRPM3 (C); yet the equivalent residues in our TRPM3 model, that is, M767 and G672, are both located within 5 Å of the PIP2 headgroup (D), with M767 engaging hydrophobic interactions that stabilize the overall complex. The W761 PIP2 contact residue in the pre-S1 of TRPM3, is equivalent to the W682 residue in TRPM8, which is close to the R688 residue as well as to PIP2, but it was not close enough to designate it as a PIP2 contact site in TRPM8 P2 interactions indirectly, or both, it is difficult to tell. Finally, the K605 residue from an adjacent cytoplasmic MHR4 domain was also in contact with PIP2 in TRPM8. This residue is not conserved in TRPM3 and was not close to PIP2 in our homology model.There are currently five channels in the TRPM family for which structural data are available : TRPM2 , TRPM4 , TRPM5 , TRPM7 , and TRP2 , only TR2 . When weferences C and S5.tructure , and to vailable . The K99is study  and 6. Tand N692 C. These in TRPM3 C; yet theadgroup D, with Min TRPM8 . Interesepletion . Whether2 sensitivity (apparent affinity) for various PIP2-sensitive ion channels. For example, the apparent affinity of the G protein–activated inwardly rectifying K+ channel GIRK4 (Kir3.4) for PIP2 is increased by factors that stimulate channel activity, such as Gβγ and intracellular Na+ P2 but also induced a marked right shift in the menthol dose response P2 contact residues also decrease agonist sensitivity. Similarly, mutating most PIP2-interacting residues also made it easier for TRPM3 to be inhibited by Gβγ. This is likely to be an allosteric effect, as the Gβγ binding peptide in TRPM3 Pular Na+ . The app menthol , and theapsaicin . The oppresponse . In the ponse Psitivity  did not epletion , indicat2-interacting residues P2 structures that were determined later P2-interacting domain P2 binding site in TRPM3 is similar, yet not identical to that in TRPM8 suggests that the PIP2 binding site in TRPM channels shows substantial level of conservation.In an earlier work, well before structures became available, three residues in the TRP domain of TRPM8 were proposed to act as PIPresidues . While maffinity , only oned later . Also bein TRPM4 . Even thg domain . It was segments . The pro2-interacting residues in the epithelial Ca2+ channel TRPV6 P2 binding site in TRPV5, with the same key contact residues P2 binding site with a reasonable accuracy, even if there is no TRPV6 PIP2 costructure available currently. Similarly, in the current work, we docked PIP2 to the apo structure of TRPM8 P2 binding site with reasonable accuracy.In our earlier work, we used a homology model, based on the structure of TRPV1 combined with mutagenesis, to predict PIPel TRPV6 . Our homresidues , 56. TRPof TRPM8 , which s studies . This ma2. We identify its binding site on the channel, characterize the interaction between PIP2 and other physiological regulators of TRPM3, and compare its regulation by PIP2 to that of TRPM8.In conclusion, our data provide mechanistic insight into regulation of TRPM3 by its key physiological cofactor, PIPX. laevis oocytes were approved by the Institutional Animal Care and Use Committee at Rutgers New Jersey Medical School. Frogs were anesthetized in 0.25% ethyl 3-aminobenzoate methanesulfonate solution (pH 7.4) , then bags of ovaries were surgically collected, and rotated with 0.1 to 0.3 mg/ml type 1A collagenase (Sigma–Aldrich) at 16 °C overnight in OR2 buffer containing 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM Hepes (pH 7.4). Afterward, oocytes were washed with OR2 several times and then kept in OR2 solution supplemented with 1.8 mM CaCl2, 100 IU/ml penicillin, and 100 μg/ml streptomycin at 16 °C.All procedures of preparing in vitro transcribed from the linearized pGEMSH vectors, which contained the complementary DNA clones for hTRPM3 . RNA was r hTRPM3  rat TRPM2, and 5 mM Hepes, pH 7.4. Currents were measured with a protocol consisting a voltage step from the 0 mV holding potential to −100 mV, followed by a ramp to 100 mV once every 0.5 s with a GeneClamp 500B amplifier and analyzed with the pClamp 9.0 software (Molecular Devices). Currents were recorded by thin wall glass pipettes that contained inner filament and were filled with 1% agarose in 3 M KCl. In all TEVC experiments, different concentrations of PregS were applied to activate TRPM3 channels, and various concentrations of menthol were used to trigger responses of TRPM8 channels. The hM2 receptor was activated by 5 μM acetylcholine. For wortmannin experiments specifically, PregS, or menthol-induced currents were measured, then the same oocyte was incubated with 35 μM wortmannin for 2 h, and currents were measured again using the same protocol. In the bar graphs in Oocytes were placed in extracellular solution, which contained 97 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM Hepes, and 100 μM PregS at pH 7.4. Currents were measured by an Axopatch 200B amplifier and analyzed with the pClamp 9.0 software. Compounds were dissolved in the bath solution and delivered to the inner side of cell membrane by a custom-made gravity-driven perfusion system. Either 25 μM PIP2, 25 μM PI(4)P, or 10 μM AASt PIP2 was applied in these experiments to reactivate TRPM3. At the end of every recording, 30 μg/ml Poly-Lys (Poly-K) was applied.Oocytes were placed in a recording chamber filled with bath solution, which contained 97 mM KCl, 5 mM EGTA, 10 mM Hepes, pH 7.4. Before starting measurements, the vitelline layer was carefully removed with forceps without damaging the oocyte. Then a giga-ohm seal was formed using a borosilicate glass pipette (World Precision Instruments) with resistance from 0.8 to 1 MΩ. The pipette was filled with a solution containing 97 mM NaCl, 2 mM KCl, 1 mM MgCl2 at 37 °C. Cells were tested to confirm that they were not infected by mycoplasma. Cells were used up to 25 passages and then discarded. Cells were transiently transfected with complementary DNA encoding different TRPM3 constructs (200–400 ng) using the Effectene reagent (Qiagen). mTRPM3α2 and its mutant were cloned into the bicistronic pCAGGS/IRES-GFP vector. The components of rapamycin-inducible pseudojanin phosphatases . Human embryonic kidney 293 cells were cultured in minimum essential medium supplemented with 10% fetal bovine serum and 100 IU/ml penicillin plus 100 μg/ml streptomycin. Cells were incubated in 5% COucts 200–0 ng usind-lysine–coated 12 mm cover slips. Experiments were performed 48 to 72 h after transfection. Coverslips were placed in recording chamber filled with extracellular solution . Since mTRPM3 constructs were in the background of bicistronic pCAGGS/IRES-GFP vector and rapamycin-inducible phosphatases were labeled with red florescent protein, cells that showed both GFP and red florescent protein fluorescence were selected for the whole-cell patch-clamp experiments. Patch pipettes were prepared from borosilicate glass capillaries (Sutter Instruments) using a P-97 pipette puller (Sutter Instrument) with a resistance of 2 to 4 MΩ. Those recording pipettes were filled with intracellular solution containing 140 mM potassium gluconate, 5 mM EGTA, 1 mM MgCl2, 10 mM Hepes, and 2 mM Na-ATP, pH 7.4. After formation of gigaohm-resistance seals, the whole cell configuration was established, and currents were recorded by applying a ramp protocol once every 1 s. The holding potential was 0 mV; followed by a −100 mV step for 100 ms; plus a ramp protocol from −100 mV to +100 mV over the period of 500 ms. All recordings were made with an Axopatch 200B amplifier, filtered at 5 kHz, and digitized through a Digidata 1440A interface. Data were collected and analyzed with the pClamp10.6 (Clampex) acquisition software (Molecular Devices) and further analyzed and plotted with Prism 9 (GraphPad by Dotmatics). TRPM3 channels were activated by PregS, and 100 nM of rapamycin was applied to activate phosphatases.After 24 h of transfection, cells were plated on poly-t test, or ANOVA with Bonferroni’s post hoc test, or the Kolmogorov–Smirnov nonparametric test, using GraphPad Prism 9, as described in the figure legends. p Values are reported in figures or figure legends.Statistical analysis was performed with Origin 2021 and GraphPad Prism 9. Data were plotted as mean ± SEM and scatter plots or mean ± SD when scatter plots are not provided. Sample sizes were not predetermined by any statistical method; however, they were similar to what is generally used in the field. All recordings were performed in random order. Statistical significance was evaluated with Ficedula albicollis (PDB ID: 6BPQ) ; (2) side-chain prediction of protein residues with missing atoms, performed with no backbone sampling; (3) pKa prediction of protein residues at pH 7, followed by analysis and optimization of hydrogen-bond networks; (3) structure refinement via restrained minimization of heavy atoms (hydrogens not restrained) using the OPLS . Binding modes were ranked using the Glide standard precision scoring function. The best binding mode of PIP2 against TRPM8 is shown in 2 complex was generated and used for subsequent modeling of the TRPM3 channel as in a complex with PIP2, seven additional experimental structures of TRPM8 became available  , which cD: 6BPQ)  program D: 6BPQ)  (both dithe OPLS  force fithe OPLS . Residueam Glide  (Schrödivailable . Three o ligands .Mus musculus (PDB ID: 6BCJ) (https://swissmodel.expasy.org/), based on the human sequence UniProtKB: Q9HCF6. The choice of the template is exemplified in https://www.ebi.ac.uk/), upon performing a multiple sequence alignment (default settings) (2 binding to TRPM3 were defined by combining binding-site mapping results obtained using SiteMap (2 and the TRPM3 model (apo state) generated using Swiss-Model P2 against TRPM3 is shown in 2 lipid. Instead, starting from the PIP2 headgroup, a series of truncated versions of a growing lipid were docked successfully against the binding site on TRPM3 until a maximum tail length was reached P2 molecule, which is experimentally functional in activating TRPM3 P2. Note that in for TRPM8, the PIP2 molecule was modeled as a full-length lipid. As for the molecular docking, we used the same protocol implemented for TRPM8. Related figures were generated using the Visual Molecular Dynamics (VMD) molecular visualization program (http://www.ks.uiuc.edu/).No experimental structure of the TRPM3 channel is currently available. The cryo-EM structure of the TRPM4 channel (3.1 Å resolution) in the apo state with short coiled coil from D: 6BCJ)  was seleD: 6BCJ)  (https:/ettings) . The Swiettings) . Potenti SiteMap  with seqss-Model . Hence, ng TRPM3 ). For si program  (http://2 and that of TRPM3 in complex with (truncated) PIP2 was aligned. (2) The TRPM8/PIP2 model and the experimentally determined structure of TRPM8 in complex with the menthol analog WS-12 and PIP2 (PDB ID: 6NR2), and the complex of TRPM8 with icilin (PDB ID: 6NR3), PIP2, and calcium . All four structures were superimposed (not shown), revealing striking structural similarities. All figures related to (1) to (3) were generated using VMD.A number of structural alignments were performed to compare TRPM3 and TRPM8 structures, including models (TRPM3 and TRPM8) and experimental structures (TRPM8). Superposition of the atomic coordinates was all performed based on sequence alignments . Alignments were generated using the Match Maker tool in UCSF Chimera , version calcium . Pairwis calcium , includi calcium  included calcium  includedof TRPM4  and TRPMof TRPM4 , 32. At G in kcal/mol) of PIP2 to TRPM3 were calculated upon mutating key binding residues in the putative PIP2 binding site. These residues were also mutated experimentally. To do so, a physics-based scoring was employed P2 molecules as generated from molecular docking, that is, the native structural complexes or WT. A total of three binding modes were used as native configurations, including the TRPM3 model bound to the truncated PIP2 presented in this study, and two additional poses with different protonation states of the phospholipid headgroup and even shorter tails. Then, the “Residue-Scanning and Mutation” tool from BioLuminate  was used, and therefore, the results should be regarded as an approximation to the electrostatic energy.Changes in the binding affinity .The change in the binding affinity (the net https://www.microsoft.com/). Related figures were generated using VMD.A positive value of 2 phospholipid with short tails is also available as a supporting information file. The authors request that any published work derived from the use of such data include a reference to this publication.All data are contained in the article and supporting information. The structural model of TRPM3 in complex with a PIPThis article contains The authors declare that they have no conflicts of interest with the contents of this article.


Schedonorus arundinaceus (Sa) that efficiently converts sucrose into 1-kestotriose. In this study, Pichia pastoris PGFT6x-308 constitutively expressing nine copies of the Sa1-SST gene displayed fructosyltransferase activity in undisrupted biomass (49.8 U/ml) and culture supernatant (120.7 U/ml) in fed-batch fermentation (72 hr) with sugarcane molasses. Toluene permeabilization increased 2.3-fold the Sa1-SSTrec activity of whole cells entrapped in calcium-alginate beads. The reaction with refined or raw sugar (600 g/l) yielded 1-kestotriose and 1,1-kestotetraose in a ratio of 8:2 with their sum representing above 55% (wt/wt) of total carbohydrates. The FOSs yield decreased to 45% (wt/wt) when sugarcane syrup and molasses were used as cheaper sucrose sources. The beads retained 80% residual Sa1-SSTrec activity after a 30-day batchwise operation with refined cane sugar at 30°C and pH 5.5. The immobilized biocatalyst is attractive for the continuous production of short-chain FOSs, most particularly 1-kestotriose.Fructooligosaccharides (FOSs)—fructose-based oligosaccharides—are typical prebiotics with health-promoting effects in humans and animals. The trisaccharide 1-kestotriose is the most attractive inulin-type FOS. We previously reported a recombinant sucrose:sucrose 1-fructosyltransferase  from  Fructooligosaccharides (FOSs)—fructose-based oligosacchari-des—are prebiotic fibbers widely utilized as functional food ingredients and in the pharmaceutical industry. FOSs ingestion improves the composition of the resident intestinal microbiota by stimulating selectively the growth of probiotics (bifidobacteria and lactobacilli), which results in the competitive exclusion of pathogens. Other associated benefits are the reduction of serum cholesterol, the increase in calcium and magnesium absorption, the prevention of colon cancer, and the production of B-vitamins. Additionally, FOSs are noncariogenic, low-calorie sugars of sweet taste and exhibit antioxidant activity by free radical scavenging  and two to four fructosyl units (F) linearly connected by β-(2→1) linkages. Among inulin-type FOSs, the trisaccharide 1-kestotriose  has a superior bifidus-stimulating effect and tastes sweeter than the tetrasaccharide 1,1-kestotetraose  and the pentasaccharide 1,1,1-kestopentaose   to restrict substrate hydrolysis. Under this condition, the fungal enzymes produce a mixture of GF2, GF3, and GF4, with their sum representing around 55% (wt/wt) of total carbohydrates  and β-fructosyltransferases (EC 2.4.1.9) from few species of filamentous fungi, mainly from the genera Schedonorus arundinaceus (Sa) with catalytic properties highly attractive for the production of short-chain inulin-type FOSs  yielded 1-kestotriose and 1,1-kestotetraose in an invariable ratio of 9:1 and overall FOSs content above 55% (wt/wt) from sucrose at the wide concentration range of 100–800 g/l. Fructose release to water (sucrose hydrolysis) was negligible even at the lowest substrate concentration  and the extracellular medium (62%) in fed-batch fermentation using cane sugar for cell growth  were purchased from BDH Chemicals (England). Refined sugar, raw sugar, sugarcane syrup, and molasses were collected at the sugar mill “Melanio Hernández,” Tuinucú, Sancti Spíritus, Cuba.Sucrose, glucose, fructose, 1-kestotriose, and 1,1-kestotetraose were purchased from Sigma Aldrich (USA). Toluene (≥99.5%) and sodium alginate  with the temperature kept at 30°C and pH 5.5 controlled by the automatic addition of 40% (wt/vol) H3PO4 and 25% (wt/vol) NH3OH. The air flow rate was 3 l/min and the dissolved oxygen was maintained above 20% by increasing automatically the agitation speed from 500 rpm to 900 rpm. After 20 hr of batch cultivation, the air flow rate was adjusted to 6 l/min and the feeding phase was accomplished by the automatic addition of the carbon source at a rate range of 6–8 ml/l/hr according to the carbon demand, as monitored by the level of dissolved oxygen. The feeding solution was sugarcane molasses at 45°Bx. After 72 hr of cultivation, the cells were harvested by centrifugation .Fed-batch cultures of 2 solution. The alginate/cell mixtures were dropped through a fine needle into 1 l of 37 mM CaCl2 solution with constant stirring (100 rpm) using an impeller, type marine propeller, to avoid droplet aggregation. Gelation time was 1 hr and then the CaCl2 solution was discarded. The spherical alginate beads (diameter 2–3 mm) were hardened overnight in 67 mM CaCl2 at 4°C and stored in 1.46 M sucrose in 50 mM sodium acetate buffer (pH 5.5) at 4°C before use.Whole cells  and 2 g of sodium alginate were suspended in water to a final volume of 100 ml and stirred thoroughly (100 rpm) at 25°C to ensure a homogenous distribution before extrusion in the CaClBeads  were added to 10 ml of sodium acetate buffer  containing toluene at final concentrations of 3, 5, and 10% (vol/vol). The flasks were shaken at 100 rpm, 30°C for 1 hr to provide adequate contact between the entrapped cells and the solvent floating on the surface of the aqueous solution. After the membrane-permeabilization treatment, beads were recovered by filtration, washed twice with sodium acetate buffer  to remove residual toluene and stored in 1.46 M sucrose in 50 mM sodium acetate buffer (pH 5.5) at 4°C. Beads and solutions were assayed for sucrose-transforming activity.2 as evidence of cell viability.To determine cell viability, beads were smashed in a liquid YPG medium and aliquots (0.1 ml) of sequential dilutions were plated on YPG agar. The number of colony-forming units (cfu) per gram of wet bead was determined on plates incubated at 30°C for 48 hr. Beads  were incubated in 10 ml of sucrose solution (100 g/l) at 30°C for 12 hr to detect the release of COSa1-SSTrec is defined as the amount of enzyme releasing 1 µmol of glucose per minute at initial rates under the reaction conditions described above.Free or immobilized cells were reacted with 1.75 M sucrose in 100 mM sodium acetate buffer (pH 5.5) in a shaking water bath at 30°C with agitation (50 rpm). After 2 hr, the reaction was stopped by heating 20 min at 80°C. Glucose released to the solution was quantified by the dinitrosalicylic acid (DNSA) colorimetric method Miller, . One uniSa1-SSTrec, the enzyme recovered from the culture supernatant of sucrose-grown P. pastoris PGFT6x-308 .For evaluating the effect of toluene on the stability of free Sa1-SSTrec activity of beads collected at time intervals was measured with 1.75 M sucrose in 100 mM sodium acetate buffer (pH 5.5).For assaying pH stability, toluene-treated beads  were preincubated with 60% (wt/vol) sucrose in 100 mM sodium acetate/phosphate buffers of varying pH (4–8) at 30°C. For assaying thermal stability, the entrapped cells  were preincubated with 60% (wt/vol) sucrose in 100 mM sodium acetate buffer (pH 5.5) at temperatures of 30, 40, 50, and 60°C. The residual Beads  were incubated in a 10-ml solution of the sucrose source  adjusted to 50°Bx (around 600 g/l) in 100 mM sodium acetate buffer pH 5.5, at 30°C for 48 hr with agitation (100 rpm) in a stirred tank reactor. Samples were collected at regular intervals.Sa-1SSTrec activity. The residual activity was established by comparing with the activity of the beads stored in a sucrose solution (1.46 M) at 4°C.To evaluate the operational stability of the biocatalyst, 15 repetitive batches of 48 hr were performed under the conditions mentioned above. At the end of each cycle, the alginate beads were recovered by filtration and a fresh substrate solution was added. After 30 days, the beads were collected and assayed for 3PO4 6% (vol/vol), and absolute ethanol 4% (vol/vol) and heating for 5 min at 120°C.Qualitative FOSs analysis was performed by thin layer chromatography (TLC) on TLC Silica gel 60 F254 plates  using acetone 95% (vol/vol) as a mobile phase. Detection was achieved by spraying with a saturated solution of 1-butanol 80% (vol/vol), urea 3% (wt/vol), Hwww.chemilab.net) was used for sugars visualization.Quantitative sugar analysis was performed by HPLC using an Aminex HPX-42C column  equipped with a refractive index detector. The column temperature was kept at 85°C. Water was used as a mobile phase at a flow rate of 0.5 ml/min. Samples were appropriately diluted before injection. Solutions (20 mg/ml) of fructose, glucose, sucrose, 1-kestotriose, and 1,1-kestotetraose were used as standards. The program package ezData  15.0 was used for the data analyses. The data are presented as means ± standard deviation. The level of significance used in this study was P. pastoris PGFT6x-308 constitutively expressing nine copies of the Sa1-SST was found to secrete the recombinant enzyme (Sa1-SSTrec), a fully active glycoprotein, to the cell periplasm and the extracellular medium in fed-batch fermentations using either glycerol or cane sugar as the carbon source  and in extracellular medium (120.7 U/ml). The overall Sa1-SST activity (170.5 U/ml) was about 5.3- and 1.7-fold higher than the values reported for the carbon sources glycerol and cane sugar, respectively  and sucrose are the major carbohydrates in sugarcane molasses. Additional glucose is released from sucrose by the action of periplasmic and extracellular P. pastoris PGFT6x-308 were entrapped in calcium-alginate gel using different biomass concentrations  . The immobilization yield decreased to 98, 97, and 95% in the treatment using 200, 300, and 400 g/l of wet biomass, respectively. Similarly, the immobilization yield dropped slightly with the increase in the biomass in the Ca-alginate entrapment process of heat-killed P. pastoris cells carrying a recombinant thermostable invertase in the periplasmic space  Fig. . The higSa-1SSTrec activity (5.67 U/g) in the beads formed using the least concentrated biomass suspension (100 g/l) can be attributed to a suboptimal number of entrapped cells. The specific activity of the beads reached the maximum value (19.51 U/g wet bead) when biomass loading was 300 g/l. The treatment with the most concentrated cells (400 g/l) resulted in heavier beads of lower specific activity (13.11 U/g), suggesting a restricted internal diffusion of the substrate sucrose. Similarly, high biomass/alginate ratios failed to increase the activity of Ca-alginate entrapped cells of Saccharomyces cerevisiae  as well as to explore the 3D surface structure of the beads. SEM examinations of entrapped whole cells from different microorganisms have allowed a detailed characterization of the internal and external morphology of Ca-alginate beads  studies are necessary to confirm the homogenous distribution of whole Sa1-SSTrec is known to behave more active with the increase of sucrose concentration  at 30°C for 1 hr decreased the enzyme stability in direct proportion to the solvent concentration  with specific sucrase activity of 19.51 U/g were incubated with toluene at three different concentrations. Sucrose conversion by the whole-cell biocatalyst was increased 2.2-, 2.3-, and 1.5-fold after the treatment with 3, 5, and 10% (vol/vol) of toluene, respectively . Toluene permeabilization elicited cell death, which is technically advantageous for biocatalyst operation. The reaction of Sa1-SSTrec on sucrose releases glucose, which is metabolized by living PGFT6x-308 cells producing the contaminants ethanol and acetate in the FOSs syrup. The glucose fermentation process generates CO2. The formation of CO2 bubbles by immobilized yeast increases the internal pressure of packed-bed columns which then tend to crack  produced COSa1-SSTrec out of the Ca-alginate matrix, considering that no sucrose-transforming activity was detected in the solutions after beads recovery. We assume that the permeabilization treatment killed the yeast without causing cell disruption. Early microscopic examinations revealed that nonviable bacterial and yeast cells treated with toluene remain essentially intact  did not provoke the leakage of periplasmic Sa1-SSTrec reaction on sucrose (1.75 M) in free and immobilized P. pastoris PGFT6x-308 cells was evaluated in the range of 4–8 at 30°C  in free and immobilized cells was evaluated at pH 5.5  on the initial rates of 5.5 Fig. b. A 10-dSa1-SSTrec stability in nonviable entrapped cells was assayed at a sucrose concentration of 600 g/l to mimic the operational conditions of FOSs synthesis. No significant variations in the enzyme stability were observed for the pH range 5–7 with remaining activities of 97 and 66% after preincubation for 2 and 30 days at 30°C, respectively. The half-life values of Sa1-SSTrec in the toluene-treated beads preincubated at 30, 40, and 50°C were 37 days, 8 days, and 18 hr, respectively. The enzyme was rapidly inactivated in the beads exposed to 60°C. The half-life of Sa1-SSTrec in the beads at 30°C is comparable to the values reported for immobilized fungal fructosyltransferases, which otherwise are often operated at 50°C due to their higher thermal stability  and temperature (30–60°C) on 2) was the main product, and 1,1-kestotetraose (GF3) appeared as the reactions progressed. At the end of the four reactions, the ratio of 1-kestotriose and 1,1-kestotetraose was above 8:2. The overall FOSs yield in the reactions with refined or raw sugar represented above 55% (wt/wt) of total carbohydrates, but it decreased to 45% (wt/wt) with sugarcane syrup and molasses  in the four sucrose sources  of P. pastoris PGFT6x-308 is appropriate for sucrose conversion into 1-kestotriose in one-cycle batch reactions  during sucrose transformation. A heating treatment killed P. pastoris expressing a bacterial thermophilic invertase and enhanced sucrose hydrolysis in the cell periplasm  were entrapped in calcium-alginate beads and exposed to a membrane-permeabilizing agent. In the toluene-treated beads, the enhancement of substrate and products diffusion through the outer membrane increased the catalytic action of periplasmic Sa1-SSTrec. The immobilized whole-cell biocatalyst yielded 55% (wt/wt) FOSs, 1-kestotriose, and 1,1-kestotetraose in a ratio above 8:2, when reacted with refined or raw sugar (600 g/l) in a stirred tank reactor at 30°C and pH 5.5. The beads retained 75–80% of the initial Sa1-SSTrec activity after 15 repetitive cycles of FOSs production in a 30-day batchwise operation. The immobilized biocatalyst is suitable for reuse and cost-effective production of 1-kestotriose, the most attractive commercial FOS.kuab036_Supplemental_FileClick here for additional data file.


Clostridium botulinum neurotoxin A (BoNT/A) targets the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, by cleaving synaptosomal-associated protein of 25 kDa size (SNAP-25). Cleavage of SNAP-25 results in flaccid paralysis due to repression of synaptic transmission at the neuromuscular junction. This activity has been exploited to treat a range of diseases associated with hypersecretion of neurotransmitters, with formulations of BoNT/A commercially available as therapeutics. Generally, BoNT activity is facilitated by three essential domains within the molecule, the cell binding domain (HC), the translocation domain (HN), and the catalytic domain (LC). The HC, which consists of an N-terminal (HCN) and a C-terminal (HCC) subdomain, is responsible for BoNT’s high target specificity where it forms a dual-receptor complex with synaptic vesicle protein 2 (SV2) and a ganglioside receptor on the surface of motor neurons. In this study, we have determined the crystal structure of botulinum neurotoxin A6 cell binding domain (HC/A6) in complex with GD1a and describe the interactions involved in ganglioside binding. We also present a new crystal form of wild type HC/A6  where a large ‘hinge motion’ between the HCN and HCC subdomains is observed. These structures, along with a comparison to the previously determined wild type crystal structure of HC/A6 , reveals the degree of conformational flexibility exhibited by HC/A6. Clostridium botulinum neurotoxin (BoNT) is renowned as the most potent toxin known to humans . A detailed analysis of these two structures along with a previous HC/A6 structure [HC/A6 ]  , revealsC/A6 co-crystallisation with GD1a we identified the presence of two crystal forms in one drop. One form was identical to a previously published HC/A6 structure, that we refer to as HC/A6 ] the ‘b’ crystal lattice parameter differed by 33.47 Å with unit cell dimensions of a = 39.55 Å, b = 78.94 Å and c = 118.55 Å  required two molecular replacement (MR) search models  for correct placement of HC/A6 molecule in the electron density map. It was possible to model in the β-sandwich portion of HCN and there was clear electron density throughout the HCC subdomain, except for 12 residues (1140–1151) within the SV2 binding loop (1139–1157). Although there is variation in crystal contacts between HC/A6  and HC/A6 , both structures have the ganglioside binding site (GBS) occluded by Lys 1170 from a symmetry-related molecule  has a higher solvent content than HC/A6   and the interface between the HCN and HCC subdomain is larger .During the screening of Hode 6TWO ), which 112.41 Å . The oveto HC/A5 . However118.55 Å . HC/A6  between HC/A6:GD1a and HC/A6  (PDB code 6TWO [5 and the loop 1269–1277 appears to widen by 4 Å (measured by the difference in Cα position between Arg 1269 and Thr 1277 residues)  structusubtypes  [23,24,2C/A6    revealed a misalignment in Cα positioning across the entire molecule  when compared to HC/A6  and HC/A6:GD1a  and HC/molecule A. The pr/A6:GD1a . To datelocation .C/A6  and HC/A6:GD1a, the HCC and HCN subdomains were superimposed independently  structures form a β-sheet with residues 1003–1006. However, for the HC/A6  structure, this β-sheet could not be modelled due to disorder, possibly due to the large hinge motion between the two subdomains separating those residues. This appears to be consistent with residues 1152–1157 being rotated away from the HCN subdomain when compared to HC/A6:GD1a and HC/A6 , accompanied by a flip at Tyr 1155  and HC/A6  but is only partially modelled in HC/A6:GD1a  structure there is a continuation of electron density between the Nζ of Lys 1236 and Sγ of Cys 1280 residues  shows neither  positioned close to the GBS B. This l/A6:GD1a B, blue. /A6:GD1a B, blue. residues D, wherea neither , blue. Tnd HC/A5  and suchnd HC/A5 . It is lC/A6 we used the program BANΔIT  structures. B-factors quantify the relative motion of individual atoms within a crystal structure, where an increase in B-factor indicates an increase in motion, providing insights into the flexible regions of a protein. The raw plot shows higher B-factors overall for the HC/A6  structure, indicating more flexibility across the entire molecule compared to the HC/A6:GD1a structure. For the latter, the HCC subdomain is more flexible than the HCN  to prod the HCN B. This iC/A6) was previously cloned into the pJ401 vector [600 of 0.6. The culture was induced with 1 mM IPTG and incubated overnight at 16 °C. Cells were harvested by centrifugation and the pellet was resuspended in 50 mM Tris pH 7.4, 0.5 M NaCl. Cells were then lysed by homogenisation in a cell disrupter and the lysate was clarified by centrifugation. HC/A6 was captured using Ni2+ affinity chromatography and eluted with a 0–0.5 M imidazole linear gradient . HC/A6 was further purified by gel filtration in 50 mM Tris pH 7.2, 150 mM NaCl using a GE superdex 200 column. HC/A6 was concentrated to 1 mg/mL and flash frozen in liquid nitrogen for storage at −20 °C until required for crystallisation.The cell binding domain (residues 871–1296) of BoNT/A6  at 16 °C. Crystals were identified in the PACT premier and BCS screens supplied by Molecular Dimensions . The best HC/A6:GD1a crystals grew in 0.2 M NaCl, 0.1 M HEPES pH 7.0, 20% w/v PEG 6000. HC/A6 crystals  grew in 0.1 M magnesium acetate tetrahydrate, 0.1 M MES pH 6.5, 12% w/v of 50% PEG smear medium , 10% v/v ethylene glycol. Crystals were mounted using a cryo-loop and flash frozen in liquid nitrogen. A total of 7200 images were collected on I04 at Diamond Light Source  at 0.1° for 0.01 s per image. Indexing and integration of X-ray diffraction data were performed using DIALS [C/A6:GD1a was determined using HC/A6  as a single search model. HC/A6  was determined using the HCN and HCC subdomains as separate search models from the HC/A6  structure (PDB code 6TWO [Hng DIALS . Data reng DIALS . The iniode 6TWO ). Both sode 6TWO  and Phenode 6TWO  and moleode 6TWO , The strode 6TWO  and PDB C/A6:GD1a revealed six residues of HC/A6 which form seven hydrogen bonding interactions with the three terminal monosaccharides of GD1a. Overall, the conformation of HC/A6 upon binding GD1a does not change dramatically except for a flipping of the Phe 1278 sidechain towards Sia5 of GD1a and an accompanying widening of loop 1269–1277. However, a new crystal form of HC/A6 ) revealed a large 16.8° rotation between the HCN and HCC subdomain (hinge) that might aid membrane anchoring through both the SV2 and ganglioside binding site. Finally, a detailed comparison of the structures presented here ) with a previously reported HC/A6 structure  revealed the extent of conformational flexibility within the HCC subdomain. Two areas of particular interest are the SV2 binding loop and a polar/charged loop close to the GBS. The SV2 loop, in the HC/A6  structure, appears to have rotated away from the HCN domain protruding outward from the surface of the protein. There is a polar/charged loop that adopts a different conformation in each structure along with a dynamic bridging interaction that alternates between Cys 1235-Cys 1280, Lys 1236-O-Cys 1280, and no bridge at all. The biological implication of these structural features is yet to be established and will require further experimental investigation.The crystal structure of H


Paenibacillus sp. GKG was isolated based on its ability to produce hydrolysis zones on agar plates containing yeast cell wall substrate as the single carbon source. The extracellular enzymes secreted into the culture medium were identified by LC-MS/MS proteomics. Endo-β-1,3-glucanase PsLam81A containing GH81 catalytic and the CBM56 carbohydrate-binding modules was selected for heterologous expression in Escherichia coli. The identity of the recombinant PsLam81A was confirmed by LC-MS/MS proteomics. The PsLam81A showed the highest activity at 60 °C, and the optimal pH range was between 6.5 and 8.0. The analysis of the full-length PsLam81A and truncated PsLam81AΔCBM56 enzymes showed that the CBM56 module improved the hydrolytic activity towards linear β-1,3-glucans—curdlan and pachyman but had no effect on hydrolysis of β-1,3/β1,6-branched glucans—laminarin and yeast β-glucan. The characterization of PsLam81A enzyme broadens current knowledge on the biochemical properties and substrate specificity of family 81 glycoside hydrolases and allows prediction of the necessity of CBM56 module in the process of designing new truncated or chimeric glycosidases. A vast diversity of glycoside hydrolases (GHs) has been discovered so far. A group of GHs that catalyze the hydrolysis of β-1,3-D-glycosidic and in some cases β-1,4-D-glycosidic bonds is subdivided into three classes: exo-β-1,3-glucanases (EC 3.2.1.58), which cleave D-glucose from the nonreducing end of glucan molecule; endo-β-1,3-glucanases (EC 3.2.1.39), which require at least two β-1,3-bound glucose residues adjacent to the digested bond; and endo-β-1,3(4)-glucanases hydrolyzing both β-1,3 and β-1,4 bonds, as long as the glucose residue whose reducing group is involved in the linkage to be hydrolyzed is itself substituted at C-3 [Paenibacillus genus  and in family 81 GH ) [GHs are enzymes that participate in the breakdown of complex carbohydrates and are classified into families at the CAZy database according to amino acid sequences . Endo-β-durans)) ,7.Laminaria digitata has a heterogenous structure as it is comprised of the β-1,3-glucan backbone with a degree of polymerization (DP) ranging from 20 to 30 and with ratio of three-to-one between M-chain and G-chain molecules. M-chain molecules are terminated with nonreducing 1-linked D-mannitol, while G-chain molecules are terminated with reducing 3-linked glucose. One β-1,3 backbone can have from zero to four β-1,6 branches with an average of 1,3 branches per molecule [Laminarin is a water-soluble β-1,3-glucan with β-1,6 side chain branches majority of which are single glucosyl residues. Laminarin from molecule . Laminarmolecule . Therefomolecule ,10. The molecule . These cmolecule . This trmolecule .β-1,3-Glucanases have inspired an array of biotechnological applications including the conversion of lignocellulosic biomass into sugars, as clarification agents in winemaking, as feed additives or as biocontrol agents against pathogenic fungus in various crops ,15. EnzyPaenibacillus sp. GKG bacterium that is able to hydrolyze polysaccharides from yeast cell walls. We successfully identified and cloned the β-glucanase gene of the GH81 family responsible for this activity. The production and characterization of recombinant endo-β-1,3-glucanase revealed the substrate specificity of this enzyme and the mode of action. This work demonstrates the workflow suitable for screening new GHs using yeast cell wall polysaccharides as a substrate and broadens the current knowledge about members of the GH81 family.In this study, we have reported the isolation of Laminari-oligosaccharides, curdlan, pachyman, barley β-glucan, yeast β-glucan, sugar beet β-glucan were purchased from Megazyme . Laminarin, carboxymethyl cellulose and microcrystalline cellulose were purchased from Sigma-Aldrich . Pustulan was purchased from Biosynth-Carbosynth . Additional information about the substrates used in this study can be found in 2SO4 (96%) were purchased from Lach-Ner . Ampicilin, ethylenediaminetetraacetic acid (EDTA), KCl, K2HPO4, Na2HPO4, trizma baze was purchased from Carl-Roth . Agarose tablets, isopropyl-β-D-thiogalactopyranoside (IPTG) and proteinase K were purchased from Thermo Fisher Scientific . KH2PO4 and lysozyme were purchased from Fluka . Agar was purchased from Formedium . Ethanol (99.8%) was purchased from Honeywell . Ethidium bromide was purchased from Calbiochem . Butan-1-ol, NH4H2PO4 and MgSO4 × 7H2O were purchased from Reakhim .Ammonium bicarbonate (ABC), clotrimazole, 3,5-dinitrosalicylic acid (DNS), DL-dithiothreitol (DTT), iodoacetamide, imidazole, p-anisaldehyde, triton X-100 and urea were purchased from Sigma-Aldrich . Acetonitrile (LC-MS grade) and formic acid (LC-MS grade) were purchased from Chem-Lab NV . Glycine and ortho-phosphoric acid (85%) were purchased from Merck . Citric acid monohydrate, HCl (35–38%), NaCl and NaOH were purchased from ChemPur . Acetic acid (99.8%), potassium sodium tartrate tetrahydrate and H3 and 0.7% Na2CO3. Yeast cells were disrupted using high shear fluid processor microfluidizer M7250-10 . The operating pressure of 690 bar was used and 8 passages were performed. The temperature of the effluent did not exceed 18 °C. The resulting suspension was centrifuged at 12,200× g for 10 min. The supernatant was discarded, the yeast cell debris  layer was removed and transferred to a new centrifuge bottle, remaining undisrupted yeast cell layer was discarded. The resulting cell wall fraction was washed with water: 2 parts of water (by weight) was added to a yeast cell wall fraction. The mixture was homogenized using a kitchen blender SHB 4460WH-EUE3 . The resulting suspension was centrifuged at 12,200× g for 10 min, the supernatant was discarded, yeast cell wall layer was removed and transferred to a new centrifuge bottle. The procedure was repeated two more times. After washing with water, the yeast cell wall fraction was frozen at −80 °C and freeze-dried for 48 h at −48 °C using a freeze-dryer Modulyo D-115 . Freeze-dried yeast cell wall fraction was ground using laboratory mill M20 .Compressed baker’s yeast  suspension was prepared in reverse osmosis treated water at a concentration of 7.6 g (dry weight)/100 g of suspension. The suspension pH was 7.6 and it contained 0.7% NaHSO4H2PO4, 1 g/L K2HPO4, 2 g/L MgSO4 × 7H2O, 15 g/L agar, 3 g/L lyophilized yeast cell walls and 6 mg/L clotrimazole, pH 7.2. The plates were incubated at 30 °C for 72 h. Then the plates were visually inspected and colonies that were able to produce hydrolysis zones were isolated by repeated streaking.Soil sample was collected from the pond located in the city of Vilnius, Lithuania . The sample was enriched for yeast cell wall degrading microorganisms by adding yeast cell walls to a final concentration of 10 mg/mL and incubated at 22 °C for 120 h. Enriched samples were diluted 25-fold using 0.9% NaCl, then 50 µL aliquots were plated on a solid mineral medium containing a water-insoluble yeast cell wall substrate as a carbon source. The solid mineral medium contained 5 g/L NaCl, 1 g/L NHE. coli strain DH5α  cells were transformed with ligation mixtures and spread on LB agar plates supplemented with 50 µg/mL ampicillin. pDNA was extracted and purified using the ZR Plasmid Miniprep kit  and sequenced using pJET1.2 sequencing primers  equipped with Omega 76 mm 30K membrane discs . Concentrated supernatant was dialyzed in a 14K dialysis membrane tubing  at 4 °C against 10 mM Tris-HCl, pH 7.0, for 18 h. Afterwards, the dialyzed samples were subjected to a tryptic digest.The liquid mineral medium used for the production of extracellular enzymes contained 5 g/L NaCl, 1 g/L NH®, MWCO 10 kDa ultrafiltration centrifugal units  at 13,000× g. Sample preparation and tryptic digestion were performed according to a modified FASP method described earlier [w/w) ratio using Pierce, trypsin protease MS-grade  or Pierce, chymotrypsin protease (TLCK treated), MS-grade . Centrifugal units with digestion reaction mixture were incubated in a 37 °C water bath Sub36  for 16 h. After digestion, the flowthrough was collected into new collection tubes, the centrifugal filter units were rinsed twice with 50 µL FASP digestion buffer, the flowthrough was collected. The solvent from the recovered peptide fraction was evaporated using a vacuum dryer Speed Vac SC110 . Dried peptides were resuspended in 100 µL 50% methanol and vacuum dried two more times to remove volatile salts completely. Dried peptides were redissolved in 0.1% formic acid and Hi3 PhosB standard peptide mixture  was added to yield a final concentration of 0.2 pmol/µL. Prepared samples were analyzed by LC-MS/MS.FASP of protein samples was performed using Nanosep earlier . The proLiquid chromatography (LC) separation of trypsin cleaved peptides was performed with ACQUITY UPLC I-Class System . Peptides were separated on a reversed-phase analytical column ACQUITY UPLC Peptide BEH C18 Column 300 Å, 1.7 µm, 2.1 mm × 150 mm  at a flow rate of 40 µL/min The isocratic gradient 5% solvent B was set until 2.5 min, then a linear gradient from 5% to 35% solvent B was set until 50 min, followed by a linear gradient from 35 to 85% solvent B until 51 min . The analytical column temperature was set to 40 °C.The LC was coupled online through an ESI ionization source with Synapt G2 mass spectrometer . Data was acquired using MassLynx version 4.2 software  in positive ion mode. LC-MS data was collected using data-independent acquisition (DIA) mode MSE. The source/TOF conditions were set as follows: resolution mode, capillary voltage 2.6 kV, sampling cone voltage 40 V, extraction cone voltage 4 V, source temperature 120 °C, desolvation gas flow 800 L/h at 450 °C. During spectral data acquisition, the trap collision energy of the mass spectrometer was ramped from 18 to 35 eV for high-energy scans in MSE mode, for low-energy scans collision energy was disabled. The mass range was set to 50–2000 Da with a scan time set to 0.5 s. A reference compound  leucine enkephalin  was co-infused continuously at a 7 µL/min flow rate and scanned every 45 s as a reference for accurate mass measurements (reference mass: m/z 556.2771).For peptide and protein identification raw data files were processed using ProteinLynx Global SERVER (PLGS) version 3.0.3 . The following parameters were used to generate peak lists: low and elevated energy thresholds, 135 and 20 counts, respectively; reference mass correction window, 0.25 Da at 556.2771 Da/e. Processed data was analyzed using the following parameters: trypsin or chymotrypsin was selected as a primary digest reagent, one missed cleavage was permitted, carbamidomethylation of cysteines was set as a fixed modification, deamidation of asparagine and glutamine, oxidation of methionine were set as variable modifications. Minimal identification criteria included 2 fragment ions per peptide, 5 fragment ions per protein and a minimum of 1 peptide per protein. The false discovery rate (FDR) was set to 2%.C-terminal 6×His tag), PsLam81AΔCBM56  were cloned from genomic DNA of Paenibacillus sp. GKG using Phusion Plus PCR Master Mix (Thermo Fisher Scientific) and designed primers PsLam81A and PsLam81AΔCBM56  . E. coli strain DH5α  cells were transformed with reaction mixtures and spread on LB agar plates supplemented with 75 µg/mL ampicillin. pDNA was extracted and purified using the ZR Plasmid Miniprep kit . pDNA was sequenced using LIC sequencing primers (Gene sequence variants coding PsLam81A (native enzyme sequence with 1AΔCBM56 . The PCR primers  by the SE. coli strain BL21(DE3)  cells. Transformed bacterium were cultured in 20 mL LB medium supplemented with 75 µg/mL ampicillin until it reached OD 0.6–0.8 at 600 nm. Expression of recombinant protein was induced with 0.5 mM IPTG by incubating at 20 °C for 24 h. Bacterial cells were harvested by centrifugation at 7200× g for 30 min at 4 °C. Collected cells were resuspended in 5 mL 1 × PBS pH 7.0, sonicated and centrifuged at 30,100× g for 30 min at 4 °C. The supernatant was loaded onto a 1 mL HiTrap Chelating HP column  that had been pre-equilibrated with 10 column volumes of 1 × PBS pH 7.0. Column was washed with 5 column volumes of 28 mM imidazole in 1 × PBS pH 7.0. The recombinant protein was eluted with 400 mM imidazole in 1 × PBS pH 7.0. The collected recombinant protein fraction was desalted on a 5 mL Sephadex G25 column  using 1 × PBS pH 7.0. The molecular weight of the recombinant protein was evaluated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Prestained Protein Ladder (10–170 kDa) . Protein bands were visualized using Coomassie Brillant Blue G-250  staining. The protein concentration was estimated using Pierce Coomassie (Bradford) Protein Assay Kit .The recombinant pLATE31-PsLam81A and pLATE31-PsLam81AΔCBM56 plasmids were transformed into g for 2 min, then a sample was mixed with DNS reagent at a sample-to-DNS reagent ratio . The mixture was heated for 5 min in a boiling water bath, then cooled down for 5 min in an ice bath, then left for 5 min at 22 °C for the sample temperature to equilibrate. 100 µL of the reaction mixture were transferred to a round bottom microplate and absorbance at 540 nm was measured. The one unit of endo-1,3-β-glucanase activity is determined as the enzyme amount required to produce the amount of reducing sugar equal to 1 µmol of glucose per min of reaction time. Measurements were performed at least in triplicate and expressed as mean ± standard deviation (SD).The activity of the recombinant PsLam81A and PsLam81AΔCBM56 enzymes was determined using the DNS method by determining the reduced sugars released during the hydrolysis of polysaccharides as described previously , with mow/v) laminarin in 50 mM of various buffers: sodium citrate buffer (pH 3.0–6.5), sodium phosphate buffer (pH 6.5–8.0), Tris-HCl buffer (pH 8.0–9.0), glycine-NaOH buffer (pH 9.0–10.0).The enzymatic activity of recombinant PsLam81A was determined using laminarin as a substrate. The optimal pH was determined by measuring the activity at pH ranging between pH 4.0 and 10.0 at 40 °C for 15 min. The reaction mixture (100 µL) contained 10 µg/mL enzyme and 0.9% (w/v) laminarin in 50 mM sodium phosphate buffer pH 7.0 at different temperatures ranging from 10 °C to 80 °C for 15 min.The effect of temperature on PsLam81A and PsLam81AΔCBM56 activity was determined in a reaction mixture (100 µL) containing 5 µg/mL enzyme and 0.9% (w/v) laminarin in 50 mM sodium phosphate buffer pH 7.0 incubated at 40 °C for 15 min.The effect of temperature on the stability of PsLam81A was determined by incubating the enzyme in 1 × PBS pH 7.0 at different temperatures ranging from 4 °C to 60 °C for 15 or 60 min. After incubation activity was determined in a reaction mixture (100 µL) containing 10 µg/mL enzyme and 0.9% (w/v) laminari-oligosaccharide substrate in 50 mM sodium phosphate buffer pH 7.0 and were incubated at 40 °C for 2 h, during control reactions the enzymes were absent. The substrate specificity of PsLam81A and PsLam81AΔCBM56 was determined using various polysaccharides: laminarin, yeast β-glucan, curdlan, pachyman, barley β-glucan (w/v) polysaccharide substrate in 50 mM sodium phosphate buffer pH 7.0 and were incubated at 40 °C for 18 h, during control reactions the enzymes were absent. No temperature or chemical pre-treatment was applied to laminari-oligosaccharides or polysaccharides prior to the hydrolysis reaction. The substrates were dissolved or suspended in appropriate buffers by mixing at 1000 rpm at 22 °C for 30 min.The mode of action of PsLam81A and PsLam81AΔCBM56 was determined using laminari-oligosaccharides  solvent. TLC results were visualized by p-anisaldehyde. Developed silica plates were dipped into dye solution  for a few seconds and heated above heating plate for a few minutes until spots corresponding to hydrolysis products appeared.Samples collected at different times of hydrolysis reaction were immediately boiled for 5 min, and then analyzed by thin layer chromatography (TLC). TLC was performed as described , with slPaenibacillus sp. GKG family GH81 endo-β-1,3-glucanase gene coding sequence was deposited in GenBank under accession no. OP093985. The Paenibacillus sp. GKG 16S rRNA gene sequence was deposited in GenBank under accession no. OP081028.The Paenibacillus sp. GKG) was isolated from the water pond sample based on its ability to produce hydrolysis zones on an agar plate containing insoluble yeast cell wall substrate (Paenibacillus sp. B01 (accession no. CP045802). To identify hydrolytic enzymes, Paenibacillus sp. GKG was cultivated in a liquid mineral medium supplemented with yeast cell walls. The supernatant was collected, concentrated, dialyzed and analyzed by LC-MS/MS proteomics in order to identify the extracellular hydrolytic enzymes. The protein sequences  retrieved from UniProt using the Paenibacillus genus keyword were used as a reference database. The proteomic analysis revealed that 710 proteins from the database had one to 35 peptides matched to their amino acid sequences. The clustering of these proteins using a 90% identity allowed the identification of 163 proteins in the cell-free culture medium of Paenibacillus sp. GKG. The majority of identified proteins were not related to carbohydrate degradation. A manually curated list of 24 proteins selected as CAZymes can be found in The bacterium  has three CBM52 domains besides a catalytic domain [Aspergillus nidulans [Candida albicans [Pneumocystis carinii [Saccharomyces cerevisiae [Aspergillus fumigatus [Candida albicans [Glycine max [Rhizomucor miehei [Saccharomyces cerevisiae [Schizosaccharomyces pombe [There are 11 characterized members from the GH81 family of eukaryotic origin microorganisms, so far. Only one GH from c domain . AnEngA nidulans , CaEng1 albicans , PcEng f carinii , and ScErevisiae  has a caumigatus , CaEng2 albicans , GmGbp fcine max , RmLam81r miehei , ScEng2 revisiae , and SpEes pombe  have onlE. coli strain BL21(DE3) cells. Both enzymes were soluble independently of induction at either 20 °C or 30 °C temperatures  in the PsLam81A protein was predicted by SignalP 6.0 server [C-terminal 6 × His tags were purified successfully using immobilized metal ion affinity chromatography and desalted by gel permeation chromatography. The obtained recombinant enzymes were analyzed by SDS-PAGE (The two gene variants of selected glycoside hydrolase from family 81 (coding full PsLam81A and without carbohydrate-binding module PsLam81AΔCBM56 proteins) were cloned into a pLATE31 plasmid, expressed in eratures . Small aSDS-PAGE . The molSDS-PAGE . HoweverE. coli cells. Only a small amount of recombinant enzymes was detected in the cell-free culture medium  cells. Hence, the endoglucanase EG5B  from Paenibacillus sp. IHB B 3084 was transported to the periplasm of E. coli BL21(DE3) cells [Paenibacillus sp. DG-22 heterologously produced in E. coli M15 (pREP4) cells was also located in the periplasm when the host cells were carrying the plasmid pXA8 with a gene encoding a full-length protein including a native signal peptide, or in the cytoplasm when host cells were carrying the plasmid pQE60-XynA with a truncated gene  [For that reason the purified recombinant PsLam81A enzyme was analyzed by LC-MS/MS. The PsLam81A protein was digested with two proteases separately—trypsin and chymotrypsin. The sequence coverage obtained after tryptic and chymotryptic digestion was 86% and 78%, respectively . Overlaye medium . As the 3) cells . Moreoveremoved) .Clostridium thermocellum (Acetivibrio thermocellus) [Thermobifida fusca [The purified recombinant protein PsLam81A was active towards yeast cell walls. Therefore, the effect of pH on the activity and the effect of temperature on enzyme activity and stability were tested. The enzyme showed a broad optimum pH range exhibiting 97% of the maximum activity within the pH range of 6.5 and 8.0 and 80% of the maximum activity within the pH range of 6.0 and 9.0 a. Similaocellus)  and pH rda fusca .The highest activity of the enzyme (PsLam81A) was recorded at 60 °C b. In comLiterature research revealed that the removal of CBM modules can result in either improved ,34 or deWe have selected the temperature of 40 °C for the substrate specificity reactions . This isVarious polysaccharides  were assThe full-length enzyme variant (PsLam81A) showed the highest activity towards laminarin, followed by yeast β-glucan and curdlan with very similar specific activities and was the least active on pachyman. This data is in agreement with previously published data on the specificity of the GH81 hydrolases ,26,27,39N-terminal CBM4 module only showed a higher activity loss towards laminarin than curdlan and pachyman [The truncated variant of an enzyme (PsLam81AΔCBM56) hydrolyzed the same substrates as a full-length enzyme. The specific activity of PsLam81AΔCBM56 towards the β-1,6 branches containing polysaccharides such as laminarin and yeast β-glucan was similar or higher in comparison to a full-length counterpart. However, PsLam81AΔCBM56 had lower specific activity in the presence of the β-1,3-linear β-glucans (curdlan and pachyman). The importance of CBM modules on curdlan hydrolysis by the GH81 family enzyme was also demonstrated in a previous study . Moreovepachyman .C-terminal module CBM56 from the BH026 enzyme resulted in ∼3-fold lower activity, and the subsequent removal of the second module CBM6 resulted in ∼6-fold lower activity on curdlan relative to a native BH026 enzyme [The deletion of the 6 enzyme . In thisThe polysaccharides that were not hydrolyzed by any of the tested enzyme variants were barley β-glucan, sugar beet β-glucan, pustulan, carboxymethyl cellulose, microcrystalline cellulose . The majAnalysis of hydrolysis products of laminarin, yeast β-glucan, curdlan and pachyman by both PsLam81A and PsLam81AΔCBM56 showed similar profiles a,b and lThe analysis of hydrolysis products of laminari-oligosaccharides with PsLam81A and PsLam81AΔCBM56 showed that both enzymes were able to degrade laminarihexaose (L6), laminaripentaose (L5), laminaritetraose (L4), laminaritriose (L3) down to laminaribiose (L2) and glucose a,b.Thermobifida fusca [Clostridium thermocellum [Laminaritetraose, laminaripentaose and laminarihexaose were completely hydrolyzed after 2 h incubation while laminaritriose was not degraded completely. However, the 18 h prolonged incubation of PsLam81A and PsLam81AΔCBM56 with curdlan and pachyman resulted in glucose and laminaribiose without the accumulation of laminaritriose a,b. Thesda fusca . Also, tmocellum . Howevermocellum ,28.All this data showed that PsLam81A as several other hydrolases from the GH81 family can act on laminaritriose as a minimal substrate. In addition, the identical results for both enzymes PsLam81A and PsLam81AΔCBM56 indicated that the CBM56 module does not have an effect on the substrate specificity for oligosaccharides.Paenibacillus sp. GKG was identified, expressed heterologously and characterized as a representative of the GH81 family. PsLam81A hydrolyzed β-1,3 glycosidic bonds containing polysaccharides such as laminarin, yeast β-glucan, curdlan, and pachyman down to laminaribiose as the main hydrolysis product. The minimal laminari-oligosaccharide substrate hydrolyzed by PsLam81A was laminaritriose. PsLam81A characterized in this study has a unique domain organization compared to other characterized GHs of the family 81. PsLam81A contains a single CBM56 domain that ensures more efficient hydrolysis of water-insoluble substrates. Collected data encourage further studies focused on the application of PsLam81A for hydrolysis of the yeast wall under industrial conditions.The endo-β-1,3-glucanase PsLam81A from


OR5P2, OR5P3 and OR10A6 were associated with skin. We further examined the roles of these ORs in skin by performing weighted gene correlation network analysis (WGCNA) and c3net analysis. WGCNA suggested that the three ORs are involved in epidermal differentiation and water-impermeable barrier homeostasis, and OR10A6 showed the largest gene sub-network in the c3net network. Immunocytochemical examination of human skin keratinocytes revealed a sparse expression pattern of OR10A6, suggesting that it is not uniformly distributed among all keratinocytes. An OR10A6 agonist, 3-phenylpropyl propionate (3PPP), transiently increased intracellular Ca2+ concentration and increased cornified envelope (CE) production in cultured keratinocytes. Knock-down of OR10A6 diminished the effect of 3PPP. Overall, integration of meta-transcriptome analysis and functional analysis uncovered distinct expression patterns of ORs in various human tissues, providing basic data for future studies of the biological functions of highly expressed ORs in individual tissues. Our results further suggest that expression of OR10A6 in skin is related to epidermal differentiation, and OR10A6 may be a potential target for modulation of keratinization.Olfactory receptors (ORs) are expressed in many tissues and have multiple functions. However, most studies have focused on individual ORs. Here, we aimed to conduct a comprehensive meta-transcriptome analysis of OR gene expression in human tissues by using open-source tools to search a large, publicly available genotype-tissue expression (GTEx) data set. Analysis of RNA-seq data from GTEx revealed that OR expression patterns were tissue-dependent, and we identified distinct sets of ORs that were highly expressed in 12 tissues, involving 97 ORs in total. Among them,  For example, activation of OR2AT4 by sandalore accelerates wound healing in human epidermis  public data set  to invespidermis , while apidermis . OR51E2 pidermis . Involvepidermis . HoweverIn this work, we aimed to comprehensively define the distinct expression patterns of OR genes in different human tissues by means of GTEx meta-transcriptome analysis. Based on our research interests, we then examined the role of ORs whose expression is associated with skin by employing weight gene correlation network analysis (WGCNA) and c3net analysis. Our network analysis indicated that the three ORs highly expressed in skin compared to other tissues all belong to the same gene co-expression module, which contains genes associated with epidermal differentiation and water-impermeable barrier homeostasis. Functional and immunohistochemical studies further suggested that one of them, OR10A6, is related to keratinization.https://gtexportal.org/home/datasets). In this study, we used already-processed transcripts per million (TPM) per gene data for further data processing. The metadata was also obtained from the GTEx project. For the present purpose, we removed several tissues for which less than 100 samples were available. In total, 17,244 samples from 28 different tissues were used for the following analysis.We employed GTEx project version 8 (p” at the end (to exclude pseudogenes). This yielded 404 possible protein-coding OR genes.In the case of samples annotated as skin samples containing both skin tissue and cultured fibroblasts, we removed the fibroblast data from the skin tissue data and re-categorized it as “cultured fibroblast”. In this analysis, we employed OR gene definitions from KEGG pathway data (hsa04740) and Reactome (R-HSA-381753). These pathways contain non-OR genes, and so we used genes for which the gene symbol starts with “OR” and does not have “p-value <10^-10, fold change≥25, and area under curve for the LDA receiver operating characteristic (ROC) curve≥0.85.Before analyzing the distinct patterns of tissue expression of these OR genes, we used uniform manifold approximation and projection (UMAP) to visualize all samples  to see iTo examine possible functional interactions between OR genes and biological processes in skin tissue, we employed WGCNA according to the manual . In lineTo visualize fold gene expression levels in skin tissue, we first calculated average gene expression per tissue and then calculated the average for all tissues. Log2-transformed values were used for visualization.For further insight into the OR gene-containing network module, we employed c3net analysis . C3net up-value ≤0.05 was chosen as the threshold for significantly enriched GO terms. In addition, all GO categories have a gene count of 10 or greater.For functional insight into WGCNA network module, functional enrichment was performed in GO terms: Biological process to each gene module using clusterProfiler packages . A corre2+ medium with or without siRNA for 24 h and then used for experiments.Normal human epithelial keratinocytes were purchased from Kurabo  and cultured in EPILIFE-KG2 . Keratinocytes were seeded onto collagen-coated glass coverslips  and used within 4 days. Keratinocytes were first cultured to 100% confluency in 0.06 mM CaGAPDH: forward, gaaggtgaaggtcggagtc and reverse, gaagattggtgatgggatttc; OR5P2: forward, accttcatttatgtgatgc and reverse, aaaataacaagcatcatgag; OR5P3: forward, cagtcactctgttctatgg and reverse, taagctctctcttcagagc; OR10A6: forward, tatttacaacccaaatctg and reverse, tcagattgtgtgtaaaacc, on an LightCycler 480 System II . Results were normalized with respect to the GAPDH gene.Total RNA from human keratinocytes was isolated using a RNeasy mini kit , and complementary DNA (cDNA) synthesis was performed from 1 μg of total RNA using SuperScript VILO Master Mix . The PCR reactions were performed using LightCycler 480 Probes Master , cDNA and specific primer pairs: The cells were grown to 80% confluency, and transfected with 20 nM scramble control or OR10A6 siRNA  using the transfection reagent RNA iMAX  in OptiMem (Thermo Fisher Scientific) as described in the manual. Scramble control: ugguuuacaugucgacuaa, ugguuuacauguuguguga, ugguuuacauguuuucuga and ugguuuacauguuuuccua. OR10A6 siRNA: guucaaacaucauggguau, ccggaaaccaagaaaguga, gaaauuauggcgaaggcga and uggcuuuccugguuauuua.2 1.8, MgCl2 1.2, HEPES 25, and D-glucose 10 (pH 7.4), abbreviated as BSS(+), and incubated for a further 10 min at room temperature to allow de-esterification of the loaded dye.Changes of intracellular calcium concentration in single cells were measured with Fura-2 AM according to the manufacturer’s instructions . Briefly, cells were loaded with 5 μM Fura-2 AM at 37°C for 45 min. After loading, the cells were rinsed with balanced salt solution containing (in mM): NaCl 150, KCl 5, CaCl2+ analyzing system . The intracellular calcium concentration was measured every second with or without addition of 3-phenylpropyl propionate (3PPP) , SQ-22536 (100 μM) or L-cis-diltiazem (100 μM). Addition of 3PPP, 3PPP + SQ-22536 mixture and 3PPP + L-cis-diltiazem mixture were performed by using reflux system .The coverslip was mounted on an inverted epifluorescence microscope , equipped with a 75 W xenon lamp and band-pass filters of 340 and 380 nm. Imaging was done with a high-sensitivity CCD camera  under the control of a Cacis-diltiazem (100 μM) dissolved in medium for 15 min at 37°C. cAMP level in the cells was analyzed by using cAMP-Glo™ Max Assay  according to the manufacturer’s instructions. The luminescence was detected with a microplate reader .Keratinocytes were cultured to confluence in 96-well culture plates and then treated with 3PPP (1 mM) or ethanol (control) with or without addition of SQ-22536 (100 μM) or L-2+ medium for 3 days 3PPP (1 mM) was diluted with ethanol and added to 1.8 mM Ca2+ medium. The same amount of ethanol was added to 1.8 mM Ca2+ medium as the control. All wells were sealed with polyolefin micro sealing tape  to prevent evaporation. After 3 days, cells were washed with HBS buffer and treated with trypsin. To halt trypsinization, Trypsin Neut Solution  was added, and cells were stained with trypan blue solution . After viable cell counting, samples were centrifuged  and the supernatant was discarded. To lyse cells, lysis solution (20 mM Tris-HCl (pH 7.5), 1% β-mercaptoethanol, 1% SDS) was added to the samples and the mixtures were incubated at 95°C for 15 min. Residual CE was counted under a microscope. The CE production rate was calculated as a percentage of that of viable cells.CE count assay was conducted as described previously . Keratin2+ medium for 15 min at 37°C. After that, cell lysates were prepared according to the manufacturer’s instruction, and analyzed using a Human Phospho-Kinase Array kit . Semi-quantitation was performed by densitometry  (n = 4). The signal densities were corrected on the basis of positive reference signals.Keratinocytes were cultured to confluence in 6-well culture plates and then treated with 3PPP (1 mM) or ethanol (control) dissolved in 1.8 mM CaCultured cells were fixed with 4% paraformaldehyde in PBS for 15 min and human skin samples were fixed with acetone at −20°C for 30 min, embedded in paraffin, and sectioned at 3 µm. Immunostaining was performed with antibody to OR10A6 , KRT14 , MCSP , Ki67  and MelanA  were used as primary antibodies. Secondary antibodies were donkey anti-guinea pig Alexafluor 647, donkey anti-rabbit Alexafluor 488, anti-rat Alexafluor 594 and anti-mouse Alexafluor 594 . Nuclear staining was performed with DAPI . Samples were observed with a fluorescence microscope  using cellSens software  and Zeiss LSM 880 .Keratinocytes were cultured to confluence with or without OR10A6 siRNA in 24-well culture plates and then homogenized in RIPA buffer  containing protease inhibitor cocktail . Protein concentrations were measured with a protein assay kit . Samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis  and transferred to PVDF membranes . For the membrane staining, antibodies to OR10A6 , Phospho-Erk1/2 , Erk1/2 , Phospho-JNK1/2/3 , JNK1/2/3 , Phospho-PLC-γ1 , PLC-γ1  and α-tubulin  were used as primary antibodies and rabbit IgG HRP-linked F(ab’)2 antibody  and Goat anti-mouse IgG (H + L) Secondary Antibody HRP  were used as secondary antibodies. The protein was detected using Chemi-Lumi One Super .p < 0.05 was considered significant. Student’s t-test was used to determine the significance of differences between two groups.The statistical significance of differences among multiple groups was determined by ANOVA with Scheffé's method. First, we set out to find OR genes characteristically expressed in different tissues by means of meta-transcriptome analysis of the RNA-sequencing data in the GTEx data set. As genes relevant to olfactory signaling, we picked up 404 OR genes listed in the KEGG pathway (hsa04740) or Reactome pathway (R-HSA-381753) for further analysis. To check whether the ORs show tissue-dependent gene expression patterns, we utilized UMAP, which is an un-supervised clustering method . Most saOR5P2, OR5P3 and OR10A6 are characteristically expressed in skin tissue. To examine the functions of these genes, we employed WGCNA to identify gene network co-expression modules (Among the OR genes,  modules . We appl modules . In each modules . To infe modules . The res modules . KeratinOR5P2, OR5P3, and OR10A6 in keratinocytes and skin sections. RT-PCR analysis confirmed the expression of all three ORs (OR10A6 showed the largest gene sub-network in the c3net network (Next, we examined the expression of hree ORs . Since O network , we focu network , and a s network . The per network . They al network .cis-diltiazem (Application of 3PPP, an agonist of OR10A6 , to kerailtiazem . Elevatiiltiazem . To confiltiazem . The 3PPiltiazem .Since OR10A6 was expressed in the basal layer of human skin  and the We also examined signaling factors potentially involved in the 3PPP-induced CE production by means of human phosphokinase array analysis. 37 kinase phosphorylation sites and 2 related proteins were measured  and JNK1In accordance with previous studies, we found different expression patterns of OR genes among different human tissues , and UMAOR5P2, OR5P3, and OR10A6 were detected in RT-PCR analysis of human skin, and OR10A6 was observed at the protein level. We observed sparse expression of OR10A6 in human skin, and the OR-expressing cells were keratinocytes, but not melanocytes, because they were KRT14-positive and not MelanA-positive. Similar sparse expression of OR has been reported in human skin and human retina (n retina . In contn retina . Differen retina , and thiolf, which activates adenylyl cyclase. cAMP produced by adenylyl cyclase opens the CNG channel, resulting in an influx of Ca2+ and Na+ (cis-diltiazem significantly inhibited the 3PPP-induced calcium response in keratinocytes. Significant cAMP level elevation was also induced by 3PPP, and the elevation was inhibited by SQ-22536. OR10A6 mediation of 3PPP-induced calcium response in keratinocytes was also confirmed by OR10A6 knock-down experiments. Similar results have been reported in other non-olfactory tissues (Sparse 3PPP-induced calcium responses in keratinocytes were observed and those results did not discrepancy with the immunochemical results. The pathway of olfactory signal transduction has been elucidated, and ORs transmit information to a G protein, G and Na+ . These t and Na+ . We foun tissues .2+ sensitivity of keratinocytes and promote keratinocyte differentiation (As regards downstream signaling, human phosphokinase array analysis and western blot experiments revealed that 3PPP significantly upregulated phosphorylation of Erk1/2 and JNK1/2/3. These phosphorylation cascades are known to promote keratinocyte differentiation . Signifintiation .In line with those results, the black module genes are involved in epidermal differentiation processes, and application of 3PPP significantly stimulated CE production of keratinocytes. The CE stimulation was diminished by OR10A6 siRNA treatment. Since endogenous substances recognized by ORs include short- and medium-chain fatty acids and androstenone , OR10A6 OR5P2, OR5P3 and OR10A6 were characteristically expressed in skin. Furthermore, we confirmed that OR10A6 activation by the agonist 3PPP stimulated CE production. Our results suggest that OR10A6 might be a therapeutic target to modulate keratinization.Overall, integration of meta-transcriptome analysis and functional analysis uncovered distinct sets of ORs that were highly expressed in 12 tissues, involving 97 ORs in total. Those results provide helpful information for future studies of distinctly expressed ORs associated with various tissues. We found that