It is widely accepted that FAM20C functions as a Golgi casein kinase and has large numbers of kinase substrates within the secretory pathway. It has been previously reported that FAM20C is required for maintenance of healthy periodontal tissues. However, there has been no report that any extracellular matrix molecules expressed in periodontal tissues are indeed substrates of FAM20C. In this study, we sought to identify the binding partner(s) of FAM20C. FAM20C wild-type (WT) and its kinase inactive form D478A proteins were generated. These proteins were electrophoresed and the Coomassie Brilliant Blue (CBB)-positive bands were analyzed to identify FAM20C-binding protein(s) by Mass Spectrometry (MS) analysis. Periostin was found by the analysis and the binding between FAM20C and Periostin was investigated in cell cultures and in vitro. We further determined the binding region(s) within Periostin responsible for FAM20C-binding. Immunolocalization of FAM20C and Periostin was examined using mouse periodontium tissues by immunohistochemical analysis. In vitro kinase assay was performed using Periostin and FAM20C proteins to see whether FAM20C phosphorylates Periostin in vitro. We identified Periostin as one of FAM20C-binding proteins by MS analysis. Periostin interacted with FAM20C in a kinase-activity independent manner and the binding was direct in vitro. We further identified the binding domain of FAM20C in Periostin, which was mapped within Fasciclin (Fas) I domain 1–4 of Periostin. Immunolocalization of FAM20C was observed in periodontal ligament (PDL) extracellular matrix where that of Periostin was also immunostained in murine periodontal tissues. FAM20C WT, but not D478A, phosphorylated Periostin in vitro. Consistent with the overlapped expression pattern of FAM20C and Periostin, our data demonstrate for the first time that Periostin is a direct FAM20C-binding partner and that FAM20C phosphorylates Periostin in vitro. PDL is a soft, unmineralized connective tissue between the cementum and the alveolar bone, which functions to support teeth in their sockets as a shock absorber against mastication impact. PDL also functions as a sensory receptor and a cell reservoir for tissue homeostasis, repair and regeneration1. It is well known that fibroblast is the major cell type observed in PDL. PDL fibroblasts produce type I collagen, which is the major constituent of PDL extracellular matrices to support the ligament, while other non-collagenous proteins are found in the PDL region. However, the role(s) of these non-collagenous gene products for PDL tissue integrity and maintenance is still not fully clear.Periodontium consists of root cementum, periodontal ligament (PDL), alveolar bone, gingiva and alveolar mucosa2, later renamed based on the expression patterns in periosteum and PDL3. Periostin is highly expressed in collagen-rich connective tissues subjected to constant mechanical loading in vivo and associated with various tissue homeostasis and pathology including bone, skin, PDL, cardiac valves, muscle injury, vascular injury, myocardial infarction, epithelial ovarian cancer, colorectal cancer, pulmonary vascular remodeling, bronchial asthma and oral cancer6. Periostin is a 90-kDa secretory protein that has several domains composed of an amino-terminal small cysteine-rich domain in the EMILIN family (EMI domain), tandem repeats of 4 Fasciclin (Fas) I domains ), and a carboxyl-terminal region (CTR)7. Periostin is reported to bind to several extracellular matrix (ECM) molecules and growth factors such as type I collagen and Notch19. It has been reported that Periostin directly interacts with Fibronectin10 through its EMI domain, while Tenascin-C7, CCN311 and BMP112 through Fas I domain. These findings suggest that Periostin is a multi-functional protein possibly through orchestrating these binding proteins inside and outside of the cell13.Periostin was first identified as osteoblast-specific factor 2 (OSF-2) from mouse osteoblastic MC3T3-E1 cell cDNA library14, is highly expressed in chondrocytes, osteoblasts, osteocytes, odontoblasts, ameloblasts and cementoblasts as well as dentin, enamel and bone matrices15. The expression pattern of FAM20C suggests that it has an important role in the formation of these mineralized tissues and subsequent mineralization process. Recently, FAM20C was identified as an intracellular kinase, namely golgi casein kinase (GCK), which was first described in lactating mammary glands that GCK enzymatically phosphorylates endogenous casein16. GCK/FAM20C phosphorylates secretory pathway proteins within Ser-X-Glu/phospho-Ser (SXE/pS) motif, which many of small integrin-binding ligand, N-linked glycoprotein (SIBLING) family members possess17. SIBLING proteins are known to function as nucleaters or inhibitors of biomineralization. It has been previously reported that acidic serine- and aspartate-rich motif (ASARM) peptide derived from osteopontin inhibits mineralization by binding to hydroxyapatite in a phosphorylation-dependent manner18. FAM20C is also known as a causative gene for Raine syndrome (OMIM#259775)19. Raine syndrome is an autosomal recessive rare disorder characterized by generalized osteosclerosis with periosteal bone formation, manifesting distinctive facial phenotype20. Oral/craniofacial phenotypes have been reported that the non-lethal type of Raine syndrome patients have small teeth with enamel dysplasia20, enlarged gingiva21 and amelogenesis imperfecta with significant gingivitis22. A previous report suggested that FAM20C is required for maintenance of healthy periodontal tissues23. As FAM20C functions as an intracellular protein kinase in the secretory pathway, it is reasonable to speculate that there might be some secretory proteins working together with FAM20C in PDL tissues.FAM20C is a member of “Family with sequence similarity 20”, consisting of three members; FAM20A, FAM20B and FAM20C. FAM20C, also known as DMP4 in miceHere we show that Periostin was identified during the course of FAM20C protein purification by Mass Spectrometry analysis. FAM20C interacted with Periostin in cell cultures. Using recombinant proteins, FAM20C directly bound to Periostin in vitro. We further narrowed down the binding domain of FAM20C and found that Fas I domain in Periostin is necessary for the binding to FAM20C. Immunohistochemical analysis demonstrated that immunolocalization of FAM20C was observed in murine PDL, which was overlapped with that of Periostin. Periostin was phosphorylated by FAM20C in vitro.We intended to obtain FAM20C proteins, thus FAM20C-stablely transfected clones were first generated by transfecting FAM20C-WT-V5/His or FAM20C-D478A-V5/His expression vector into HEK 293 cells. HEK 293 cells were selected for their higher transfection efficiency.17 likely due to the presence of V5/His tag (~ 5 kDa). Interestingly another band appeared at a higher molecular weight than the FAM20C-WT-V5/His or FAM20C-D478A-V5/His protein when the amount of FAM20C-WT-V5/His  and 59.25%  . The purified FAM20C-WT-V5/His and FAM20C-D478A-V5/His proteins were prepared, electrophoresed, and the gel was stained with CBB. A single CBB-positive band was then found at a molecular weight corresponding to the size of FAM20C-WT-V5/His or -D478A-V5/His protein  and a Raine-syndrome mutation form (P328S) in addition to FAM20C-WT were used for the binding assay. HEK 293 cells were transiently transfected with POSTN-WT-HA and FAM20C-WT-V5/His, FAM20C-D478A-V5/His, or FAM20C-P328S-V5/His to investigate the interaction. The results demonstrated that POSTN-WT interacted with FAM20C-WT -Western blot analysis was performed. The results showed that the binding between POSTN and FAM20C-WT  within Periostin responsible for FAM20C binding, several deletion constructs of Periostin were used and the binding assay was performed in the similar manner as described above  or anti-Periostin  antibody. Non-immune goat immunoglobulin (IgG) was also used as a negative control for anti-FAM20C  and non-immune rabbit IgG for anti-Periostin . Distal root of maxillary first molar was selected as a region of interest. Panels  show higher magnification views of the open boxed areas in (a), (c), (e) or (g), respectively. Immunolocalization for FAM20C (brown color) was present in PDL , with preferential distribution at the bone surface region  and bone embedded Sharpey’s fibres . Periostin was present (brown color) in PDL  with strong positive signals along the thick PDL collagen, both at the bone surface and cementum surface regions of PDL , and bone embedded Sharpey’s fibres . No immunoreactivities were detected when non-immune IgG was used (e–h).25 and phosphorylated substances were enriched with Phos-tag agarose. Isolated phosphorylated proteins were separated by SDS-PAGE and detected by anti-Periostin antibody. Our results demonstrated that Periostin was detected when FAM20C WT  mice were generated and the homozygous mice showed periodontal tissue phenotypes28. In Periostin KO mice, PDL fibroblasts were irregularly distributed among collagen fibrils and collagen fibrils were disorganized28. While periodontium of WT and KO mice appeared intact when teeth were unerupted, after tooth eruption, dramatic periodontal defects were observed including enlarged gingival tissue, attachment loss, irregular PDL width, alveolar bone loss and external root resorption. Removal of masticatory forces partially rescued the PDL phenotype, suggesting the potential role of Periostin via transcription level26. It has also been reported that Periostin deficiency leads to decrease in collagen fibril diameter, which may explain why KO mice’s PDL collagen fibrils were susceptible to occlusal force and damaged8. Besides the PDL defect, Periostin KO mice exhibited dwarfism and enamel defects, suggesting that Periostin plays crucial roles in bone and tooth development27. Despite its possible critical role of Periostin reported in certain tissues including PDL, there has been so far no gene mutations found in humans.There has been increasing evidence that Periostin plays an important role in periodontal tissue development. 29. Some reports showed the presence of periodontitis in non-lethal type of Raine syndrome, where FAM20C is mutated, but genotype–phenotype correlation was not established. Although FAM20A is considered to be an allosteric activator of FAM20C kinase30 and some clinical phenotypes in patients with FAM20A mutations are overlapped with those who with FAM20C mutations, it has not been ruled out that periodontitis in Raine syndrome patients is just coincidence. On the contrary, in mice, the involvement of FAM20C in periodontal tissues has been more extensively studied and established. The expression of Fam20C mRNA is detected in PDL fibroblasts at 5 and 7 weeks and FAM20C protein is expressed in PDL matrix in the incisor at 7 weeks15. Consistent with this, our data demonstrated that FAM20C is expressed in PDL tissues by immunohistochemistry  was generated and showed periodontal disease phenotype. Since collagen fibers appeared thinner and unevenly distributed in these mice, this disrupts PDL structure, which likely allows direct infiltration of bacteria from periodontal pocket leading to periodontal disease. Additionally, in Fam20C cKO mice, the expression level of Periostin in PDL was dramatically reduced23. Fam20C global KO mouse (Fam20C −/− mouse) was generated by another group and demonstrated approximately 20% of mortality rate. Surviving Fam20C −/− mice showed decreased body weight and length, bone abnormalities and lack of tooth enamel31, manifesting similar clinical phenotypes to Raine syndrome patients. Therefore, these findings in mouse model studies and observations in human case reports highly suggest the possible relationship between FAM20C and Periostin in PDL, bone and enamel, and these two genes are genetically associated.Among the FAM20 family, it has been reported that periodontitis may be a part of clinical phenotypic spectrum in FAM20A mutationstry Fig. . More re32. Despite the critical biological importance of phosphorylation in non-collagenous matrix proteins, kinase(s) responsible for phosphorylation have not been identified until recently. FAM20C kinase favors phosphorylation of an S-X-E/phosphorylated-S motif33. As illustrated in Fig. 140-N-E, S548-E-E, S611-K-E, S805-R-E; based on NCBI Reference Sequence; NP_056599.1), of which three of them are conserved between mouse and human Periostin . The conserved three S-X-E motifs are located within Fas I domains, i.e. S140-N-E in mouse RD1, S548-E-E and S611-K-E in mouse RD4. A possible reason why we could not retrieve any S-X-E motif peptides from our MS analysis is likely due to the detection limitation. As trypsin was used for peptide digestion and due to the presence of K or R residues near S-X-E motif, the length of peptide is too short to detect by the analysis. For example, S546-E-E containing peptide is supposed to be digested as “GMTSEEK”, and S609-K-E containing peptide as “SK”. At this point, it is still unclear why we could not detect any S138-N-E containing motif, however the peptide detection setting is normally 7–20 peptides and this S138-N-E containing peptide may be too long to detect. Further studies are required to verify the location of phosphorylation site(s) in Periostin.It has been known for almost 30 years that non-collagenous proteins in bone, dentin and enamel contain phosphoproteins with O-phosphoserine34. More recently, functional domain of this Periostin’s biological effect has been identified and reported by two independent groups; one is that a peptide sequence (amino acids 142–151) of Periostin stimulates chemotactic migration, adhesion, proliferation and endothelial tube formation of human endothelial colony forming cells in vitro35 and another that monoclonal antibodies that recognize amino acids 136–151 of Periostin inhibit Periostin-induced migration of human endothelial colony forming cells36. Interestingly, the functional domain reported in both studies is located within Fas I domain of Periostin, more specifically in RD1 which contains S138-N-E motif  at Boston University Medical Campus (approved protocol number: AN-15050), and all efforts were made to minimize suffering animals. This study was performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals.38 and used in this study.The human embryonic kidney (HEK) 293 cells were maintained as previously described39 was used in the immunohistochemical analysis.X-tremeGENE 9 DNA transfection reagent was obtained from Roche Life Science. Recombinant mouse Periostin protein (2955-F2) was obtained from R&D Systems. The antibodies used in this study were as follows; anti-V5 (Life Technologies), anti-HA , anti-HA high affinity , goat polyclonal anti-FAM20C (Santa Cruz Biotechnology), and rabbit polyclonal anti-Periostin  antibody. Rabbit polyclonal anti-Periostin antibody against RD1 domain of Periostin previously generated40. The plasmids harboring FAM20C-WT, FAM20C-D478A, and FAM20C-P328S cDNAs followed by V5—6XHis-tag  were used in this study. Mouse Periostin expression vectors previously generated in pCAGI-puro vector7 were used in this study.Human FAM20C expression vector constructs, including wild-type (WT), Raine syndrome-mutant form (P328S), and a mutant form lacking its kinase activity (D478A) were generated by PCR methods as previously reported41. Briefly, cells were transiently transfected using X-tremeGENE 9 DNA transfection reagent with pcDNA3.1-FAM20C-WT-V5/His and pcDNA3.1-FAM20C-D478A-V5/His according to the manufacturer’s protocol. The transfected cells were treated with 400 μg/ml of G418 neomycin analogue and further cultured. Ten of single colony-derived clones transfected with either FAM20C-WT or FAM20C-D478A were isolated, further cultured and the expression of FAM20C proteins was verified by Western blotting with anti-V5 antibody. The cell clone that expressed the strongest band intensity for FAM20C-WT-V5/His or FAM20C-D478A-V5/His was chosen, further cultured in a larger scale for protein production, and the conditioned media of FAM20C-WT-V5/His and FAM20C-D478A-V5/His were collected. The conditioned media were centrifuged at 1500 rpm for 5 min, the supernatant was incubated with Ni–NTA agarose beads (Qiagen), and FAM20C-WT-V5/His and FAM20C-D478A-V5/His proteins were purified as previously described41. The purified proteins were dialyzed against distilled water, lyophilized and resuspended in distilled water. The protein concentration was measured and the purified proteins were kept at -20 °C until use.The stable transfected HEK 293 cell clones that overexpress FAM20C-WT-V5/His or FAM20C-D478A-V5/His were generated and FAM20C-V5/His proteins were purified in the same manner as previously describedVarious amounts of purified FAM20C-WT-V5/His  and FAM20C-D478A-V5/His (2.5 and 5.0 µg) proteins were prepared, applied to 4–12% SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue R-250 (CBB). The CBB staining positive, selected bands were excised and the protein identification was analyzed at Taplin Mass Spectrometry Facility . Peptide sequences were determined by matching protein databases with the acquired fragmentation pattern.5 cells/well and transfected with mouse Periostin with C-terminal HA tag (pCAGI-puro delta Periostin (b-)-WT-HA; POSTN-WT-HA) and FAM20C-WT-V5/His, FAM20C-D478A-V5/His, or FAM20C-P328S-V5/His. Total amount of cDNA was kept constant by supplementation with empty vector. After 24 h of transfection, cell lysates were collected, immunoprecipitated with anti-V5 antibody and subjected to Western blotting (WB) analysis using anti-HA antibody to identify the binding. The same membrane was stripped by stripping buffer, and WB with anti-V5 antibody was performed to verify the expression of FAM20C-V5/His proteins. An aliquot of the same cell lysates was subjected to WB analysis with anti-HA antibody to verify the expression of POSTN-WT-HA.HEK 293 cells were plated onto 6 well culture plates at a density of 3 × 10The mouse recombinant Periostin and FAM20C-WT-V5/His or FAM20C-D478A-V5/His proteins were prepared in FAM20C protein’s dose dependent manner and incubated in PBS where the total amount of proteins per sample was kept constant by adding bovine serum albumin (BSA). Samples were then immunoprecipitated with anti-V5 antibody followed by Western blot (WB) analysis with anti-Periostin (Abcam) antibody. The same membrane was stripped, and WB with anti-V5 antibody was performed to verify the expression of FAM20C-V5/His proteins.7 were used to identify the interaction domain of Periostin with FAM20C. The following was the deletion forms; Periostin (POSTN)-WT-HA, Periostin delta CTR-HA (POSTN-dCTR-HA) lacking its C-terminal region, Periostin delta EMI-HA (POSTN-dEMI-HA) lacking EMI domain, Periostin delta EMI-CTR-HA (POSTN-dEMI-CTR-HA) lacking both EMI and CTR domains, and Periostin EMI-HA (EMI domain only). HEK 293 cells were transiently transfected with POSTN-WT-HA, POSTN-dCTR-HA, POSTN-dEMI-HA, POSTN-dEMI-CTR-HA, or POSTN-EMI-HA and FAM20C-WT-V5/His. Total amount of cDNA was kept constant by supplementation with empty vector. After 24 h of transfection, cell lysates were collected and the binding domain was identified in the same manner as described above.Various deletion mutant forms of pCAGI-puro delta Periostin (b-)-HA generated previously37 with anti-FAM20C antibody (1:100 dilution), anti-Periostin antibody (1:400 dilution)39 or a non-immune goat or rabbit immunoglobulin (IgG) (at the same concentration as primary antibodies) as negative controls. The immuno-reactivity was amplified using VECTASTAIN Elite ABC HRP kit (Vector Laboratories Inc.). The sections were counterstained with hematoxylin (Sigma).The maxillary periodontal tissue including molars were dissected from mouse neonates , fixed, decalcified and embedded in paraffin. Immunohistochemical staining was performed as previously described2, 250 ng of recombinant POSTN and 500 ng of FAM20C-WT-V5/His or -D478A-V5/His protein. The total protein amount was kept constant supplemented by BSA. The mixtures were incubated for 30 min at 30 °C, Phos-tag agarose (Wako USA) was added, further incubated for 1 h at 4 °C, washed with lysis buffer and SDS sample buffer was added. Reaction products were separated by 4–12% SDS-PAGE and detected by Western blotting using anti-Periostin antibody.Kinase assays were performed in 20 μl reaction mixture containing wit 1 mM cold ATP (Thermo Fisher), 50 mM Tris–HCl pH 7.0, 10 mM MnClSupplementary Information 1


Haementeria ghilianii, the 66mer three-disulfide-bonded peptide tridegin, specifically inhibits the blood coagulation factor FXIIIa. Tridegin represents a potential tool for antithrombotic and thrombolytic therapy. We recently synthesized two-disulfide-bonded tridegin variants, which retained their inhibitory potential. For further lead optimization, however, structure information is required. We thus analyzed the structure of a two-disulfide-bonded tridegin isomer by solution 2D NMR spectroscopy in a combinatory approach with subsequent MD simulations. The isomer was studied using two fragments, i.e., the disulfide-bonded N-terminal (Lys1–Cys37) and the flexible C-terminal part (Arg38–Glu66), which allowed for a simplified, label-free NMR-structure elucidation of the 66mer peptide. The structural information was subsequently used in molecular modeling and docking studies to provide insights into the structure–activity relationships. The present study will prospectively support the development of anticoagulant-therapy-relevant compounds targeting FXIIIa.The saliva of blood-sucking leeches contains a plethora of anticoagulant substances. One of these compounds derived from  Hirudo medicinalis, have been used as a biomedical tool from ancient times up to the present day. They were mainly used to remove blood, which should help to detoxify the patient  with the disulfide bonds Cys5–Cys37 and Cys17–Cys31, are used as a lead structure for further research on tridegin and its influence on FXIIIa  was determined by a combination of NMR (nuclear magnetic resonance) spectroscopy and molecular dynamics (MD) simulation studies. This two-disulfide-bonded isomer, B, was subsequently applied for molecular docking studies on FXIIIa. The information received from this study was used to refine structure–activity relationships of the tridegin–FXIIIa interaction. Our results may pave the way for further investigations on FXIIIa as well as the optimization/development of lead structures for antithrombotic and thrombolytic therapy.The aim of the current study is to gain further insight into tridegin’s structure, folding, and interaction with FXIIIa. In order to obtain a more precise understanding of the mode of action against FXIIIa, a structure of the tridegin isomer B  spectra and calculated by Cyana   was deteby Cyana  based onN–Hα region of the NOESY and TOCSY spectra , which were successfully attributed to the amino acid positions by assigning the amino acid spin system and subsequent sequential walk within the HHα range b, as it Hα range . Especia structure  structure     a massiv9S,C25S]  is only  structure  structures  structure  structure  structure    . The mod9S,C25S]  is very 9S,C25S]  also pot9S,C25S] . In this9S,C25S]  was obse] models , whose r N-terminal part      and FXII9S,C25S] , when we9S,C25S]  model, tFmoc-amino acids were purchased from Orpegen Peptide Chemicals  and Novabiochem . HBTU, resins, and further chemicals for solid-phase peptide synthesis, including reagent-grade N-methylmorpholine, piperidine, trifluoroacetic acid (TFA), and N,N-dimethylformamide (DMF) were purchased from IRIS Biotech , Sigma-Aldrich Chemie GmbH , Alfa Aesar , Abcr GmbH , VWR International , and FLUKA Chemika . Solvents  and chemicals  used for peptide purification and oxidation were obtained from VWR International  and Fisher Scientific .The synthesis of the linear peptide precursors according to an automated standard Fmoc SPPS protocol using a ResPep SL peptide synthesizer from Intavis Bioanalytical Instruments GmbH  as well as the subsequent peptide cleavage from the resin and the purification step via RP-HPLC were carried out as previously described .The purity of the linear peptides was >95%, which was assessed by analytical RP-HPLC on a Shimadzu LC-20AD system equipped with a Vydac 218TP column . The gradient elution system contained 0.1% TFA in water (eluent A) and 0.1% TFA in acetonitrile (eluent B). Peptide elution was achieved with a gradient of 20% to 50% eluent B in 30 min and a flow rate of 1 mL/min. The peptides were detected at λ = 220 nm.The selective oxidation of the two-disulfide-bonded N-terminal peptide Lys1–Cys37 proceeded in a one-pot reaction slightly modified to the already described protocol . In briePeptide characterization was achieved by means of analytical RP-HPLC (see above for the linear precursor), mass spectrometry, and amino acid analysis.The peptide concentration as well as the amino acid composition were analyzed using an LC 3000 system from Eppendorf-Biotronik . Peptide hydrolysis was carried out in 6 N HCl at 110 °C in sealed tubes for 24 h. Subsequently, the hydrolyzed peptides were dried in a vacuum concentrator and redissolved. The respective amino acid concentrations were determined by comparison with an amino acid standard solution .Peptide masses were determined using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. As matrix 2,5-dihydroxyacetophenone  was used according to the manufacture’s guideline . MALDI mass spectra were produced on an UltrafleXtreme instrument .The disulfide connectivity of the N-terminal fragment was determined as described recently ,30. Ther2)-Glu(NH-(CH2)4-NH-Abz)-Val-Lys-Val-Ile-NH2 as described previously -TOCSY, -NOESY, -COSY, and -HSQC spectra using water suppression. The spectra were processed with TOP SPIN 4.0.6 (Bruker) and analyzed using CcpNmr Analysis (Collaborative Computing Project for NMR). Distance constraints were extracted form -NOESY spectra acquired with a mixing time of 150 ms and a recycle delay of 1.5 s. Upper-limit-distance constraints were calibrated according to their intensities in the NOESY spectra. To calculate the structure of the fragments based on the chemical shift data the program Cyana    was used9S,C25S]  with add9S,C25S]  force fi9S,C25S] . Subsequ9S,C25S] ,43. Duri9S,C25S] . The pro9S,C25S] ,46. Peri9S,C25S] , which w   was used9S,C25S] . The PDB9S,C25S] . The fule server . The Hdoe server . Only th complex . The modnet.in/) . Similar  .      generate9S,C25S]  binds in9S,C25S] . The iso9S,C25S] , especia9S,C25S]  and thatIn conclusion, the deeper insight into the structure–activity relationships of the FXIIIa inhibitor tridegin from the docking studies will prospectively support the future development of anticoagulant-therapy-relevant compounds targeting FXIIIa.


In vitro growth kinetics of these viruses were comparable to the virus containing the wild-type M41-S1. Furthermore, Neu5Ac binding by the recombinant viruses containing single N-glycosylation site knock-out mutations matched the Neu5Ac binding observed with the recombinant RBDs. Five N-glycosylation mutants lost the ability to bind Neu5Ac and gained binding to a different, yet unknown, sialylated glycan receptor on host cells. These results demonstrate that N-glycosylation of IBV is a determinant for receptor specificity.Infection of chicken coronavirus infectious bronchitis virus (IBV) is initiated by binding of the viral heavily N-glycosylated attachment protein spike to the alpha-2,3-linked sialic acid receptor Neu5Ac. Previously, we have shown that N-glycosylation of recombinantly expressed receptor binding domain (RBD) of the spike of IBV-M41 is of critical importance for binding to chicken trachea tissue. Here we investigated the role of N-glycosylation of the RBD on receptor specificity and virus replication in the context of the virus particle. Using our reverse genetics system we were able to generate recombinant IBVs for nine-out-of-ten individual N-glycosylation mutants.  Gammacoronavirus in the order of Nidovirales. IBV is endemic in most countries around the world and causes huge economical losses in the poultry industry. Many different IBV strains circulate worldwide, which were recently classified into 32 phylogenetic lineages  [Infectious bronchitis virus (IBV) is an enveloped, positive-strand RNA virus, belonging to the genus The virion of IBV is composed of four structural proteins, including the nucleocapsid (N) protein, encapsidating the genomic RNA, and the envelope proteins membrane (M), envelope (E) and spike (S). The S protein of IBV is a homo-trimer, which is translated as a single polypeptide and subsequently cleaved by host proteases to generate two subunits, S1 and S2. These subunits remain non-covalently bound in the virion, where S2, the stalk of the spike, is anchored in the membrane and essential for virion–host membrane fusion. The S1 subunit forms the head of the spike protein, which is essential for receptor binding. IBV strains use  sialic acids  for viraSpikes of coronaviruses are heavily decorated with glycans on asparagine (N) residues , and conserved across different genotypes . GlycosyS1 of vaccine strain H52 was replaced by that of IBV-M41 (r-M41-S1) or by individual N-glycosylation mutants thereof (r-M41-N-#-A). While nine-out-of-ten recombinant viruses could be rescued, showing similar replication kinetics in chicken embryo kidney (CEK) cells compared to r-M41-S1, the receptor specificity of five mutants was changed, based on glycan ELISA and haemagglutination assays using chicken erythrocytes. This reveals that N-glycosylation of S1 in the virion has implications on receptor binding and specificity.To assess the role of N-glycosylation of the spike of pathogenic IBV-M41, we set out to rescue recombinant viruses using our previously published targeted recombination system , where tM41-S1 gene, RNA was isolated from a virus stock of IBV-M41  using the QIAamp viral RNA Mini Kit . Reverse transcription was performed using the Transcriptor First Strand cDNA Synthesis Kit  with random hexamers according to the manufacturer’s protocol. PCR was performed with Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific) using the primers listed in S1 domain of IBV-H52 for that of M41 in the plasmid used for targeted recombination, the M41-S1 PCR product and the previously generated plasmid containing the H52-S gene [PacI and SnaBI , ligated and subsequently transformed in HB101 E. coli. Sequences were confirmed by automated nucleotide sequencing . A step-wise ligation approach was used to obtain the H52 M41-S1 donor plasmid. To introduce mutations leading to individual N-to-A substitutions in N-glycosylation sites in the receptor binding domain (RBD) sequence, site-directed mutagenesis was performed using the primers listed in S1 gene with the introduced mutations were confirmed by automated nucleotide sequencing .To obtain the fragment coding for the 2-S gene  were digM41-S1 sequence  and r-M41-N-#-A representing N-glycosylation mutants, all in the IBV-H52 genetic background . The rescued r-M41 viruses were passaged twice in 8-day-embryonated chicken eggs (ECE) until embryo death was observed (maximum of 7 days post inoculation). Allantoic fluid was harvested and the sequence of the S1 gene was assessed by automated sequencing . Virus stocks were subsequently produced by inoculation of passage 2 (P2) virus (diluted 1 : 10000) in 8-day-ECE, and allantoic fluid was harvested 24 h later to use for subsequent experiments. Viral quantity was calculated based on RT-qPCR, as described previously [Targeted recombination was performed as described before . For eaceviously . PrimersAnti-IBV immunohistochemistry on chorioallantoic membranes (CAM), collected in P0, was performed as described previously  using a 5 cells per well onto glass coverslips. CEK cells were infected in triplicates with 1.5*106 virus particles based on the calculated RT-qPCR titre. Culture media samples were collected at 8, 24, 48 and 72 h post infection (p.i.), after which RNA was isolated and RT-qPCR was performed in duplicates as previously reported [P<0.05 between r-M41-S1 and IBV-M41wt.CEK cells were isolated from 18-day-ECE . Single-reported  using a CEK cells grown on coverslips were analysed by immunofluorescent staining as described previously . Antibod6 genome equivalents) were pre-treated with 50 mU ml−1 of Arthrobacter ureafaciens neuraminidase (AUNA)  for 1 h at 37 °C, before application to the coated plates and incubated for 2 h at room temperature. After washing with PBS, primary monoclonal mouse anti IBV-S1  was applied in phosphate buffered Normal Antibody Diluent (NAD)  for 1 h at room temperature. Dako Envision HRPO labelled polymer anti-mouse  at a 1 : 1 ratio (v/v) in NAD was added to the wells, and after 30 min the wells were washed with PBS. TMB buffer  substrate was used as peroxidase substrate to visualize binding, after which the reaction was terminated using 2 N H2SO4. FLUOstar Omega (BMG Labtech) was used to measure optical densities (OD450nm) and MARS Data Analysis Software for analysis of the data.Synthetic Neu5Acα2-3Galβ1-3GlcNAc-polyacrylamide  (0.5 µg per well) was coated in a 96-well maxisorp plate  overnight at 4 ˚C. This was followed by a blocking step using 3 % (v/v) BSA  in PBS/0.1 % (v/v) Tween. Virus dilutions were made in triplicate and added to the plates, followed by incubation for 2 h at room temperature. Where indicated, virus stocks  and/or chRBC’s were pre-treated with 5 mU ml−1 of AUNA  for 1 h at 37 °C before twofold serial dilutions of the virus were made in in PBS-/-. chRBC were applied to the (pre-treated) viruses in a 1 : 1 vol ratio. Haemagglutination titres were determined after 15 min incubation at room temperature.Chicken erythrocytes (chRBC) were collected from the brachial vein and collected in 3.2 % (v/v) sodium citrate (NaC) . chRBC were washed four times (1500 r.p.m. for 10 min) in PBS without calcium chloride and magnesium chloride (PBS-/-)  and diluted in PBS-/- to a concentration of 2 % (v/v). Virus stocks  to alanine (A) substitution . To this end, in the plasmids used to generate recombinant IBV-H52, the ies ml−1 , and use2 throughout the course of infection, whereas titres of r-M41-S1 were between 1×104 and 1×105 from 8 h p.i. onwards for both clones. IBV-M41wt was included as a positive control, which showed a significant higher viral load at 48 and 72 h p.i., potentially explained by the chimeric nature of the genetic background of the recombinant viruses and IBV-M41wt. These results show that all rescued recombinant viruses were able to enter CEK cells, and replication in CEK cells appears not to be affected significantly by N-glycosylation of the RBD of the IBV spike.To compare the growth kinetics of the rescued recombinant viruses, CEK cells were inoculated with r-M41-S1 and each of the rescued N-glycosylation mutants. After 8 h, infection of CEK cells was confirmed by observing viral nucleocapsid protein expression for all viruses in immunofluorescent staining , with thTo assess whether the rescue of nine-out-of-ten recombinant viruses was based on infection through binding of Neu5Ac, we investigated whether N-glycosylation mutants in the viral context retained affinity for this receptor. r-M41-N145A, N219A, N229A and N246A, were still able to bind Neu5Ac in a concentration-dependent manner, whereas for r-M41-N33A, -N59A, -N85A, -N160A and -N194A no binding to Neu5Ac was observed . InteracTo demonstrate and confirm the N-glycosylation mutants unable to bind Neu5Ac are dependent on a different (glycan) receptor, haemagglutination assays were performed using chicken erythrocytes (chRBC). These host cells display a variety of glycans on their surface , and areTaken together, we observed for r-M41-S1, as well as for r-M41-N145A, -N219A, -N229A and -N246A binding to Neu5Ac in ELISA , indepenHere we reveal that N-glycosylation of at least five sites in the spike protein of IBV-M41 affects the binding specificity for sialylated host glycans. The ability of recombinant viruses to recognize Neu5Ac is completely in line with binding of recombinant RBD proteins in ELISA , indicatNine-out-of-ten single substitutions in N-glycosylation sites in the spike protein of IBV allowed rescue of recombinant viruses. We cannot draw any definitive conclusion for the reason why r-M41-N126A could not be grown in embryonated eggs, but some speculations can be addressed. Previously, we observed that recombinantly expressed RBD-N126A lost its binding to chicken trachea tissue; however, this cannot be the sole explanation, as the same was observed for five other mutants  , which ain silico ligand binding predictions previously point towards involvement of amino acids S87, N144 and T162 of the M41-RBD [in vivo, which remains to be revealed.Recently the cryo-EM structure of IBV-M41 was resolved, revealing the complete spike of IBV-M41 is decorated with N-glycans to (at least) 20 asparagine residues . In our  M41-RBD . These aThe infectivity and growth kinetics of the recombinant viruses were not affected by N-to-A mutations in the RBD. Whereas wild-type IBV-M41 showed significantly higher titres than r-M41-S1, possibly due to its slightly different genetic background, the mutants showed titres comparable to r-M41-S1 at all timepoints . These rMany viruses are decorated with N-glycans on the virion surface, including the flaviviruses Dengue and Zika virus, influenza A virus, Ebola virus, HIV, Nipah virus and human coronaviruses . The gly


Furthermore, three rounds of directed molecular evolution demonstrated that known cefotaximase-enhancing mutations were accessible regardless of the differences in dynamics. Interestingly, specific sequence differences between the related ß-lactamases were shown to have a higher effect in evolutionary outcomes than did differences in dynamics. Overall, these ß-lactamase models show tolerance to protein dynamics at the timescale of catalytic turnover in the evolution of a new function.The evolution of new protein functions is dependent upon inherent biophysical features of proteins. Whereas, it has been shown that changes in protein dynamics can occur in the course of directed molecular evolution trajectories and contribute to new function, it is not known whether varying protein dynamics modify the course of evolution. We investigate this question using three related ß-lactamases displaying dynamics that differ broadly at the slow timescale that corresponds to catalytic turnover yet have similar fast dynamics, thermal stability, catalytic, and substrate recognition profiles. Introduction of substitutions E104K and G238S, that are known to have a synergistic effect on function in the parent ß-lactamase, showed similar increases in catalytic efficiency toward cefotaxime in the related ß-lactamases. Molecular simulations using Protein Energy Landscape Exploration reveal that this results from stabilizing the catalytically-productive conformations, demonstrating the dominance of the synergistic effect of the E014K and G238S substitutions  Therefore, focused combinatorial libraries and directed molecular evolution experiments are currently the most useful approach to uncover epistatic interactions  or can be deleterious when combined (negative sign epistasis); on the other hand, deleterious mutations can be beneficial when combined , E104K-R (5′-CTGTGACTGGTGAGTACTTAACCAAGTCATTCTGAGAATA-3′), G238S-F (5′- GATAAATCTGGAGCCAGTGAGCGTGGGTCTC-3′), and G238S-R (5′- GAGACCCACGCTCACTGGCTCCAGATTTATC-3′). Pfu polymerase (Agilent) was used, with reaction conditions according to the manufacturer's recommendations and an extension time of 6 min (~1 min/kb). The reaction product was digested with DpnI (NEB) for 1 h at 37°C to eliminate the template and one-tenth of the reaction was transformed into CaCl2-competent E. coli XL1-Blue prepared following the Inoue method .Mutations E104K and G238S were introduced individually and jointly into ß-lactamase constructs TEM-1, cTEM-2m, and cTEM-17m. The ß-lactamase genes were fused to an OmpA signal peptide in 3′ for periplasmic export and cloned into the plasmid pET-24 as previously reported  and spread onto Luria-Bertani agar plates containing 50 μg/mL kanamycin. After overnight incubation at 37°C, the DNA of 10–20 colonies was amplified using 2x PCR Precision Master mix (ABM) and sequenced to verify the mutation rate. DNA sequencing was done with by the Genomic Platform of IRIC or the Genome Quebec Innovation Center at McGill University, Canada. Library size was estimated by counting the resulting colony forming units after overnight incubation at 37°C in Luria-Bertani agar plates containing 50 μg/mL kanamycin. Subsequently, the libraries were pooled, miniprepped using the Monarch Plasmid miniprep kit (NEB) and transformed into E. coli BL21(DE3) for expression and screening.Libraries were generated according to the protocol of Copp et al. . BrieflyLibraries were screened on agar plates containing the antibiotic cefotaxime (Sigma-Aldrich) at concentrations 0.08–0.032 μg/mL. Several dilutions of the library were plated on non-selective media to ensure that colony forming units were 10–20-fold higher than the estimated library size. To favor genetic diversity and avoid early evolutionary dead-ends, between 25 and 70 colonies were pooled and selected at each round. Plasmid DNA was extracted with Monarch plasmid miniprep kit (NEB), amplified and used as a template for random mutagenesis to produce the next generation. This process was repeated for a total of three generations. The selection process was tracked by sequencing between 5 and 14 colonies after each selection .Expression and purification of the ß-lactamase variants were performed as in Gobeil et al. . BrieflyE. coli BL21(DE3) cells expressing the ß-lactamase variants were propagated overnight in Luria-Bertani media containing 50 μg/mL kanamycin. An inoculum of 104 cfu was spotted onto Luria-Bertani agar plates, containing 0.25 mM IPTG and 0.004–1048 μg/mL cefotaxime in 2-fold dilutions. After overnight incubation at 37°C, the plate which had the lowest cefotaxime concentration at which there was no visible growth was considered as the minimal inhibitory concentration. Each construct was assayed in triplicate.Minimal inhibitory concentration (MIC) assays were performed according to Wiegand et al.  using th264nm = 7,250 M−1cm−1  spectrophotometer, where εexc = 483 nm and λem = 568 nm. Melting temperature values were calculated from the negative derivative of the fluorescence value vs. temperature where the minimum represents the melting temperature . Fluorescence was measured with λThe crystal structures 1XPB for TEM-1, 4MEZ for cTEM-2m, and 4ID4 for cTEM-17m were prepared with Schrodinger's Preparation Wizard. Active-site waters blocking substrate docking were manually removed, taking care to keep the water molecule bridging S70 and E166. Missing side chains were added and sampled through Prime. Hydrogens were added and the protonation states manually checked with the interactive optimizer. For TEM-1, this resulted in H26 and H96 in epsilon protonation, the remaining histidines being delta-protonated. In cTEM-2m, H96 was additionally delta-protonated; it is quite solvent exposed and far from the active site. For cTEM-17m, H26, and H29 were epsilon-protonated and H112 was doubly protonated (thus having a +1 charge). Although the Wizard suggested that K234, immediately next to the nucleophilic S70, should be neutral, we kept it positive as it might be crucial for substrate binding and for stabilizing deprotonation S70  inner box diameter to 16 Å to guarantee substrate accommodation. We attempted docking before and after computing quantum charges for the ligands, at the B3LYP DFT and 6/31Gcomplex - Ereceptor - Esubstrate.Using the cefotaxime-docked structures as input, local PELE explorations were run with the recent AdaptivePELE version  are within one order of magnitude of the native TEM-1 [ see large conformational rearrangements associated with ligand-binding and substrate turnover. We previously reported two variants of TEM-1 ß-lactamase where functionality is maintained despite sequence differences marked differences the magnitude and location of slow protein motions  Gobeil , 2019. Vin vitro kinetics of cefotaxime hydrolysis  was strongly improved by inclusion of mutations E104K/G238S in all three settings: a ~180-fold increase in cTEM-2m and cTEM-17m, significantly greater than the ~30-fold increase in TEM-1. This was accompanied by a modest increase in productive binding to cefotaxime: KM was reduced by ~2-3-fold in cTEM-2m and cTEM-17m, which both had higher affinity at the outset, compared to ~10-fold in TEM-1. Although mutations E104K/G238S have been found to be destabilizing in TEM-1 and are often accompanied with the stabilizing mutation M182T  and differs most in the range and extent of its slow dynamics, yet inclusion of mutations E104K/G238S had essentially the same effect on catalytic efficiency (190 mMkcat due to G238S (30 to 160-fold). In contrast, the effects of the individual mutations on KM were modest, whether in improving KM in TEM-1 (0.2 to 0.5-fold) or weakening it in cTEM-2m and cTEM-17m (1.2 to 4-fold)  . Indeed,kcat of the three host ß-lactamases; although the inclusion of E104K/G238S increased its kcat 200-fold, reflected in its 600-fold improvement in catalytic efficiency, MIC increased only ~4-fold  interaction energies at the catalytic distance monitored  than TEM-1  and cTEM-2m  (3), bringing it in the range of TEM-1 E104K/G238S  and cTEM-2m E104K/G238S  which showed smaller variation in cavity size with their respective hosts. The observations made with cTEM-17m demonstrate that variation in the active site cavity volume is tolerated in TEM-1 variants.It is interesting to note that cTEM-17m has a larger active site pocket  . The actE. coli were variable. Importantly, the individual E104K or G238S substitutions had significantly different effects on MIC in the three hosts, potentially because low catalytic turnover (kcat) is a limiting factor upon selection for cefotaxime resistance in bacteria. This led us to question whether the evolutionary paths to cefotaxime resistance available to these three hosts could differ, should epistatic interactions differ significantly due to initial slow motions.Overall, the combined E104K/G238S substitutions consistently led to important improvements in catalytic efficiency in the three hosts, as reflected in the more favorable energies predicted for their cefotaxime complexes. Nonetheless, the effects of the E104K/G238S substitutions on MIC in Having demonstrated that the prevalent E104K/G238S evolutionary solution to cefotaxime resistance in TEM-1 ß-lactamase is valid in variants cTEM-2m and cTEM-17m, we investigated whether other known evolutionary paths toward cefotaxime resistance in TEM-1  was observed in 7 out of 9 colonies sequenced and was the sole variant identified after selection of the third generation (Fixation of one variant was identified following two rounds of evolution for cTEM-2m: variant cTEM-2m (neration . PCR bianeration .K158N/R164R/K192N/K234K) was identified in 10 out of 14 colonies sequenced. Of special interest is that mutations E104K and G238S were individually observed following the selection of the second generation, accompanied by one or more non-synonymous mutations, but neither became dominant following selection of the third generation  variant includes two non-synonymous mutations in the Ω-loop that had previously been reported in clinical isolates or directed molecular evolution experiments in TEM-1  after two rounds of evolution, it conferred no observable increase in resistance in vivo . This is consistent with the slight increases in MIC that result from introduction of the synergistic mutations E104K/G238S into cTEM-2m. Overall, cTEM-2m did not evolve cefotaxime resistance in E. coli as readily as TEM-1 and cTEM-17m.The predominant cTEM-2m (K158N/R164R/K192N/K234K) variant conferred a 4-fold increase in MIC compared to cTEM-17m (MIC = 0.11 μg/mL). It includes two non-synonymous mutations, K158N and K192N, that have both been previously identified. K158N defines the clinical isolate TEM-127 (H158N in wild-type TEM-1)  NCBI, , whereas1) NCBI, .Overall, the most prevalent mutations in TEM-1 laboratory experiments and clinical isolates such as E104K, R164S, A237T, G238S, or E240K were not fixated. However, evolutionary path sampling did reveal previously reported mutations in cefotaximase variants. And even though it was not fixated, variants containing E104K and G238S were obtained in cTEM-17m evolution, the variant with higher slow dynamics.Evolution of a new protein function often requires the initial protein to possess at least some promiscuous activity  displays the highest occurrence of the salt bridge  and on binding energy predicted upon docking CTX was most similar upon introduction into TEM-1 and cTEM-17m. This was unexpected since the latter includes 17 mutations in the active-site region relative to TEM-1 and displays vastly increased slow dynamics , did not suffice to procure CTX resistance in E. coli.The synergistic impact of mutations E104K/G238S on catalytic efficiency, on CTX resistance in dynamics . DespiteE. coli for TEM-1 and for host cTEM-17m that displays extensive slow dynamics, despite its possessing greater sequence divergence than cTEM-2m. Furthermore, the strongest positive epistasis observed in vitro for cTEM-2m E104K/G238S demonstrates that its differences in slow dynamics do not prevent evolvability; instead, the poor evolutionary outcome of cTEM-2m in E. coli appears to result from the deleterious effect of its initial mutations M68L and M69T on kinetics of cefotaxime hydrolysis.Similar parallels in evolutionary outcome were observed when sampling evolutionary paths in directed evolution. Mutations E104K, G238S and further mutations reported in clinical isolates of TEM-1 were selected in cTEM-17m libraries (Barlow and Hall, Overall, three vastly differing patterns of protein dynamics at the timescale of catalytic turnover did not impede the high evolvability of TEM-1 ß-lactamase. Its tolerance to extensive protein dynamics at slow timescales is consistent with the robustness of TEM-1 ß-lactamases and has been hypothesized to facilitate evolution toward the recognition of new substrates (Gobeil et al., The original contributions presented in the study are included in the article/LA and JP designed experiments and analyzed data. LA and CL-S-D performed protein expression, purification, and kinetic experiments. LA performed directed molecular evolution. FS-J designed preliminary computational simulations. VG performed computational simulations. CP provided Python scripts. LA, JP, and VG wrote the manuscript. All authors revised and approved the final version of the manuscript.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.


S22A and S22B on rice chromosome 2 inducing F1 pollen sterility in hybrids between Oryza sativa japonica-type cultivar Taichung 65 (T65) and a wild relative of rice species Oryza glumaepatula. The causal gene of S22B in T65 encodes a protein containing DUF1668 and gametophytically expressed in the anthers, designated S22B_j. The O. glumaepatula allele S22B-g, allelic to S22B_j, possesses three non-synonymous substitutions and a 2-bp deletion, leading to a frameshifted translation at the S22B C-terminal region. Transcription level of S22B-j and/or S22B_g did not solely determine the fertility of pollen grains by genotypes at S22B. Western blotting of S22B found that one major band with approximately 46 kDa appeared only at the mature stage and was reduced on semi-sterile heterozygotes at S22B, implying that the 46 kDa band may associated in hybrid sterility. In addition, causal genes of S22A in T65 were found to be S22A_j1 and S22A_j3 encoding DUF1668-containing protein. The allele of a wild rice species Oryza meridionalis Ng at S22B, designated S22B_m, is a loss-of-function allele probably due to large deletion of the gene lacking DUF1668 domain and evolved from the different lineage of O. glumaepatula. Phylogenetic analysis of DUF1668 suggested that many gene duplications occurred before the divergence of current crops in Poaceae, and loss-of-function mutations of DUF1668-containing genes represent the candidate causal genetic events contributing to hybrid incompatibilities. The duplicated DUF1668-domain gene may provide genetic potential to induce hybrid incompatibility by consequent mutations after divergence.Postzygotic reproductive isolation maintains species integrity and uniformity and contributes to speciation by restricting the free gene flow between divergent species. In this study we identify causal genes of two Mendelian factors  In planArabidopsis  incompatibilities are a classical genetic model , which pbidopsis , cotton bidopsis , monkey bidopsis , and whebidopsis . Gene clbidopsis . Gene dulization . Recipro S27/S28 , DPL1/DPPL1/DPL2 , and DGSGS1/DGS2  loci, anGS1/DGS2 .Sa locus to induce pollen sterility  at a single Mendelian locus in heterozygotes in plants. For example, in rice, intra-subspecific hybrids of cultivated rice carry two adjacent genes encoding F-Box protein and SUMO E3 ligase-like protein at the terility , and endlity  genes in plants  and O. glumaepatula accession IRGC105668 in the genetic background of T65, the F1 pollen sterility gene S22 was identified as a Mendelian genetic factor on the short-arm end of chromosome 2 (S22 responsible for pollen sterility were dissected into the two independent genetic loci: S22A and S22B (+S22A-T65/sS22A-glum|+S22B-T65/+S22B-T65 genotype (S22A_SS plants) showed approximately 50% pollen fertility (semi-sterility) due to sterility of pollen grains carrying the “sterile allele” sS22A-glum. Similarly, plants carrying the +S22A-T65/+S22A-T65|+S22B-T65/sS22B-glum genotype (S22B_SS plants) showed pollen semi-sterility because of sterility of pollen grains carrying the “sterile allele” sS22B-glum. The coupling phase linkage of sS22A-glum and sS22B-glum on O. glumaepatula-derived chromosomal segments could explain the initial identification of S22 as a single Mendelian factor  markers RM12317 and RM7451 for S22A_SS plants, RM7033 and RM279 for S22B_SS plants, and RM12317 and RM279 for S22A+B_SS plants. Phenotypes of S22A_SS, S22A+B_SS, and S22B_SS plants were discriminated according to the morphology of the sterile pollen grains stained with a 1% iodine-potassium iodide solution. Semi-sterile plants, in which almost all of the sterile pollen grains show no staining, stain slightly, and stain dark brown in color and are smaller than normal grains, were classified as S22A+B_SS, S22A_SS, and S22B_SS , and 10 ng genomic DNA template. PCR was performed in a GeneAmp PCR System 9,700 . The cycling profile was an initial denaturation step at 95°C for 5 min; 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 40 s; and a final elongation step at 72°C for 7 min. Amplified products were electrophoresed on a 4% agarose gel in 0.5× TBE buffer. Agrobacterium-mediated transformation was conducted as described by S22A_SS and S22B_SS plants. The copy numbers of transgenes were analyzed by quantitative PCR (qPCR) in an MX3000P QPCR system  using QuantiTect SYBR Green PCR Kits  according to our previous analysis (http://rice.plantbiology.msu.edu/) in O. sativa cv. Nipponbare Os-Nipponbare-Reference-IRGSP-1.0 and O. glumaepatula W1183 accession ALNU02000000  from the ground anther using a Multibeads shocker . Protein fractions of the Trizol extract were reserved for subsequent western blotting analysis. Extracted RNA was treated with DNase I  to degrade contaminated genomic DNA. The first-strand cDNA was synthesized from approximately 100 ng of the extracted RNA and then reverse-transcribed using Revertra Ace . Reverse transcription-quantitative PCR (RT-qPCR) was conducted in a MX3000P QPCR system (Agilent Technologies) using QuantiTect SYBR Green PCR Kits (Qiagen). For RT-qPCR of S22B, a pair of primers, 5'-CTC TGC CAA CTT CTG CAT CGC CAG G-3'/5'-GCT GAT AAG CTT GTA CAT CTC CGA C-3' and 5'-TGG AGG ATC CAT CTT GGC ATC AT-3'/5'-ACA GCT CCT CTT GGC TTA GCA-3' were used for amplification of S22B and the actin 1 gene (Os03g0718100) as an internal control, respectively.For temporal expression analysis of β-glucuronidase (GUS) assay, genomic sequences of the 1,572-bp region upstream of the initiation codon (ATG) of +S22B-T65 were cloned into the Gateway-entry vector pENTR/TOPO using pENTR Directional TOPO Cloning Kits (Life Technologies Japan). The cloned insert was transferred into the destination vector pGWB3 (GUS gene derived from pGWB3. The construct was transformed to T65 by Agrobacterium-mediated transformation.For the promoter-or pGWB3  using LRS22B was amplified from the Nipponbare full-length cDNA clone J023058D10 (AK070727) provided by the National Agriculture and Food Research Organization (NARO), Japan, and was cloned into the Gateway-entry vector pENTR/TOPO (Life Technologies Japan). The GFP-fused gene at N-terminal under a control of 35S CaMV promoter was constructed using LR clonase (Life Technologies Japan) into the destination binary vector pGWB6  after 500 nM of mitochondrial fluorescent dye MitoTracker Red CMXRos staining.The coding sequence at or pGWB6 . The conS22B product, we prepared a rabbit polyclonal antibody against two synthesized 14-amino acid peptide sequences: N-KLATPLDAGAHDG-C and N-ISGGRKPEQHSLLP-C . The antibody specificity was confirmed by western blotting of a 6×His + S22B-T65 fused recombinant protein expressed in Escherichia coli strain BL21. The protein fractions extracted by Trizol were mixed with 2× Laemmli sample buffer and 2-mercaptoethanol, and then run on a 12% TGX gel . The proteins were transferred onto a polyvinylidene difluoride membrane with a 0.2-μm pore size . Goat anti-rabbit IgG (H + L) antibody and horseradish peroxidase-conjugated antibody (Bio-Rad) were used for the secondary antibody reactions, and detection was performed using chemiluminescence detection with Western BLoT Hyper HRP Substrate .For western blotting of the Setaria viridis, Setaria italica, Panicum virgatum, Botryococcus distachyon, Sorghum bicolor, Zea mays, Botryococcus stacei, and Oryza sativa were downloaded from the Phytozome 12 database  were the most informative plants to map S22B within 18.4 kb of the genomic region in the reference sequence Nipponbare between DNA markers M48 and M46 , 13.7-kb SalI, and 10.7-kb XmnI (XmnI_b) fragments of the Nipponbare genomic fragment derived from BAC clone OSJNBa008C13 were introduced into S22B_SS plants by Agrobacterium-mediated transformation  of sS22-glum alleles (k = 0.11) via pollen from the theoretical transmission (k = 0.5) was observed, which was attributed to the sterility of pollen grains carrying sS22-glum  BAC clone GL47D11 showed that the O. glumaepatula allele S22B-g, allelic to S22B_j, possesses three non-synonymous substitutions CCG(P) > GCG(A), TTC(F) > GTC (V), and CCC(P) > GCC(A) and a 2-bp deletion, leading to a frameshifted translation at the S22B C-terminal region .To narrow down the candidate region of nd RM279 , 308 rec and M46 . For theormation . In the 22-glums . Similar22-glums . Meanwhineration , suggestobserved . The twofragment . In the  control . LOC_Os0d S22B_j ,D. The gl region . Many nul region . The S22S22B from the anthers at the unicellular, bicellular, and mature stages was investigated by RT-qPCR among +S22B-T65 homozygotes, sS22B-glum heterozygotes, and sS22B-glum homozygotes. Transcript levels gradually decreased during the progression of post-meiotic male gametogenesis  gradually increased during the pollen stage. Another major band at a molecular mass probably 46 kDa (46 kDa band) appeared only at the mature stage, and was reduced in the sS22B-glum heterozygotes as compared with those of homozygotes for +S22B-T65 or sS22B-glum alleles. Although the levels of S22B transcripts were reduced in homozygotes for the sS22B-glum allele  transformed by the construct of 35SCaMV prom::N-GFP-S22B protein  derived from the BC4F6 plants heterozygous at the S22A genomic region between the SSR markers RM12317 and RM12350, and homozygous for T65 at S22B in the genetic background of T65  in the genetic background of T65 .During the process of development of introgression lines of d of T65 , pollen pulation . Linkage protein . Howeveraepatula  except tS22B_j as a query in the proteome of angiosperm species in the Phytozome 12 database  harboring other members from Setaria, Panicum, Botryococcus, and Oryza species , ancestral DUF1668-containing genes likely originated before divergence of the BOP and PACMAD clades and are thus shared among these species. Since S22A_j1, S22A_j2, and S22A_j3 also formed a monophyletic clade within Clade 1  and S22B may have at least an ancestral population of the divergence of the BOP and PACMAD clades. Although this study demonstrated that genetic variants at both S22A and S22B induce HI, it will be necessary to further examine whether the DUF1668 domain has evolved as an HI factor in other Poaceae species.Here, we revealed that the genes  sterile . The tra alleles  and tranncreased . Althouga pollen , demonst mapping . Althoug Clade 1 . Since C Clade 1 , they liS22B transcript levels were reduced in the order of homozygotes for +S22B-T65, heterozygotes, and homozygotes for sS22B-glum in the anthers at each of the unicellular, bicellular, and mature stages using forward and reverse primers targeting the same sequences at S22B_j and S22B_g  based on the results of the promoter-GUS assay , homozygotes for sS22A-glum were not obtained (S22A_SS plants was fertile, and self-pollinated seeds of S22A_SS showed normal seed germination. These results demonstrate that the sS22A-glum allele is insufficient for male gametogenesis in heterozygotes. Alternatively, the sS22B-glum allele is transmitted via male gametophytes in heterozygotes and homozygotes, which segregates at low frequency. The sS22A-glum allele encodes a truncated protein with loss of the complete DUF1668 domain (sS22B-glum allele has a few single nucleotide substitutions and a 2-bp frameshift mutation at the C-terminal, but the DUF1668 domain was predicted to exist in the Pfam search (S22B_m is likely a loss-of-function allele, and a homozygous plant for the S22-mer allele segregated and showed normal pollen fertility (O. glumaepatula may inactivate gene function, although the DUF1668 domain was predicted in silico.In the linkage analysis of obtained . The tot8 domain . In contm search . S22B_m ertility . TherefoSa, S5, and S1. The incompatible gene complex includes sporophytic genetic factors and gametophytic genetic factors that determine pollen fertility of its own gametophytes in heterozygotes. The BLAST similarity search using the cloned genes at the known HI as a query did not find homologous sequences within the S22A and S22B mapping regions. If HI systems conferred by S22A and S22B also evolve incompatible haplotypes to induce HI in hybrids, the genes cloned in this study only represent a portion of gametophytic members acting as a protector and were not sufficient to induce HI. To further identify other possible genes including a killer factor involved in HI at S22A or S22B, it is necessary to conduct defective mutant experiments using genome-editing methods such as CRISPR/Cas9 in heterozygotes showing HI. As suggested in the BDM model, nucleotide variants causing HI are heterozygous only at birth, which is necessary to escape from negative selection due to their own maladaptive phenotypes owing to this incompatibility. When S22A_j1, S22A_j2, and S22A_j3 are considered as the ancestral types and the truncated genes at sS22A-glum are considered as the variant types, nucleotide variants of sS22A-glum occurring only at birth need to escape from natural selection via BDM partners such as duplicated genes or interacting genes in other genomic regions of O. glumaepatula or ancestral species. Since near-isogenic lines of S22A and S22B in the T65 genetic background were used in this study, the genetic phenomena may appear as monogenic events, and other BDM partners have not yet been identified.Diverged haplotypes, including multiple tightly linked genes, are known to induce HI in intra-specific and interspecific hybrids of rice, such as Sc locus results from the feedback-mediated regulation of gene expression between differentiated alleles in inter-subspecific hybrids between japonica and indica rice (S22B_SS, transcription level on heterozygote would show half of the homozygotes for +S22B-T65 because transcription of sS22B-glum is suppressed on heterozygote. However, expression level on the S22B_SS was closed to average of homozygotes of +S22B-T65 and sS22B-glum, suggesting that allelic suppression has not occurred on transcription level. Instead, we suggest that alternative splicing of S22B_j and/or S22B_g or production of the 46 kDa band resulted from post-translational modification is suppressed in pollen grains harboring sS22B-glum in allele-specific manner. The further studies may reveal this opinion. Another possibility may be that copy number variation of functional copies of DUF1668-containing genes in pollen grains results in competitive transmission efficiencies between fertile and sterile alleles. The allelic differences in development and starch absorption capacity may also result in the biased allocation of nutrient resources between gametophytes, leading to the distorted transmission efficiency of alleles.In contrast, a single gene can also cause an HI system. Allelic suppression of the japonica allele at the ica rice . If the S22A and S22B could be caused by gene complexes within the candidate region, or by structural changes in a single gene. Further genetic analysis to identify other genetic loci interacting with S22A or S22B, and biochemical and genetic characterization of DUF1668-containing genes may reveal the HI mechanism via DUF1668.In summary, HI at The original contributions presented in the study are included in the article/MS: investigation, development of genetic materials, review, writing draft manuscript, and editing of the manuscript. NS and YM: investigation and development of plant materials. HK, JW, and TM: investigation and sequencing. HY and AY: project administration, funding acquisition, and supervision. YY: conceptualization, methodology, investigation, data curation, and writing draft manuscript. All authors contributed to the article and approved the submitted version.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.


We found that RNase G determines the half-life of hns mRNA by cleaving its 5′ untranslated region and that altering its cleavage sites by genome editing stabilizes hns mRNA, thus decreasing S. Typhimurium virulence in mice. Under anaerobic conditions, the FNR-mediated transcriptional repression of rnc encoding RNase III, which degrades rng mRNA, and simultaneous induction of rng transcription resulted in rapid hns mRNA degradation, leading to the derepression of genes involved in the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS). Together, our findings show that RNase III and RNase G levels-mediated control of hns mRNA abundance acts as a regulatory pathway upstream of a complex feed-forward loop for SPI-1 expression.Bacteria utilize endoribonuclease-mediated RNA processing and decay to rapidly adapt to environmental changes. Here, we report that the modulation of  Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS). In addition, we showed that this regulatory pathway plays a key role in the virulence of S. Typhimurium in the host. Thus, our study improves the understanding of the mechanisms through which bacterial pathogens sense the host environment and respond precisely by expressing gene products required for adaptation to that particular niche.Recent studies have shown that pathogenic bacteria with ribonuclease mutations display attenuated virulence, impaired mobility, and reduced proliferation in host cells. However, the molecular mechanisms underlying ribonuclease-associated pathogenesis have not yet been characterised. Here, we provide strong experimental evidence that the coordinated modulation of endoribonuclease activity constitutes an additional regulatory layer upstream of a complex feed-forward loop controlling global regulatory systems in the  Bacterial ribonucleases regulate gene expression via RNA processing and decay. They are essential for processing RNA precursors into mature, functional RNAs, and initiate the reactions that cleave and differentially regulate certain polycistronic mRNAs –5. BacteEscherichia coli, mRNA decay is heavily dependent on endoribonuclease E (RNase E) -1-propanesulfonate (CHAPS), and 10 mM dithiothreitol (DTT), and disrupted using 0.5 mm glass beads and a MINI BEADBEATER (BioSpec). Next, whole cell lysates were obtained by centrifugation at 4°C and 13,200 rpm for 15 min and protein concentration was determined using a Quick Start 1 × Dye Bradford reagent and bovine serum albumin standard (Bio-Rad Laboratories). Cellular enolase (Eno) levels in the whole cell lysates of WT and Δrng cells were assessed by western blotting to evaluate whether eno mRNA levels were controlled by RNase G, as in E. coli ATP  and T4 polynucleotide kinase  and purified using 12% polyacrylamide gel containing 8 M urea. RNase G cleavage assays were performed as described previously [Synthetic 600 of 0.6. To stop RNA transcription, rifampicin (Sigma-Aldrich) was added to the cultures at a final concentration of 1 mg ml-1. For the zero-time point, culture samples were taken before rifampicin addition. To measure the half-life of cat mRNA, culture samples were taken 1, 2, 4, and 8 min after rifampicin addition and qRT-PCR analysis was performed as described above. To measure the half-life of rng mRNA, overnight cultures of WT or Δrnc cells grown in LB medium at 37°C with aeration were diluted 1:100 in same medium and incubated at 37°C to an OD600 of 0.6 and 0.3, respectively, in the presence or absence of oxygen. Total RNA preparation and qRT-PCR were carried out as described above.WT cells harbouring pSt-hns (WT)-cat, pSt-hns (A-31C)-cat, pSt-hns (A-9G)-cat, and pSt-hns (U-8C)-cat plasmids were cultured overnight in LB medium at 37°C with aeration, diluted 1:100 in same medium, and incubated at 37°C to an OD4 CFUs of the WT, hns A-31C, hns A-9G, or hns U-8C strains in 100 μl of PBS. Mice were euthanised after 2 days and their spleens, livers, and mesenteric lymph nodes were removed aseptically and homogenised in 500 μl of ice-cold TRIzol (Thermo Fisher Scientific). Total RNA samples were purified by phenol extraction and ethanol precipitation.Three 6-week-old female mice were intraperitoneally infected with 10t-tests were used for control comparisons in SAS v. 9.2 and SigmaPlot 10.0 (Systat Software). Data represent the mean ± standard error of the mean (s. e. m) and P < 0.05 was considered to indicate statistical significance. See The numerical data used in all main figures are included in S1 DataExcel spreadsheet containing, in separate sheets, the underlying numerical data and statistical analysis for Figure panels: 1A, 1B, 1C, 1D, 1E, 1F, 2B, 3A, 3B, 3C, 4A, 5A, 5C, 5D, 5E, 6A, 6B, 6C, 6D, 6F, and 6G.(XLSX)Click here for additional data file.S1 Fig2, 103, 104, and 105 CFUs per mice (CFUs contained in 100 μl) of WT cells and incubated at 22 ± 1°C, and mortality was monitored daily. Kaplan-Meier survival curves were determined from three independent experiments. The control corresponds to the injection of PBS alone. Data are representative of three independent experiments, and similar results were obtained. ** P < 0.01 and **** P < 0.0001 for 102-, 103-, 104-, or 105 CFUs-injected mice versus PBS-treated mice (two-sided unpaired Student’s t-test). NS; not significant.Mice were injected with 10(TIF)Click here for additional data file.S2 FigS. Typhimurium strains  for 60 h. The serum was analyzed for the expression of cytokines IL-1β, IL-12p70, IL-17A, IL-23, IL-27, IFN-β, and GM-CSF by multiplex cytokine analysis. The data are presented as the mean ± s. e. m. of at least two independent experiments, and statistically significant differences are indicated with different letters . n. d.; not detectable.Serum was collected from mice that were left uninfected or infected with (TIF)Click here for additional data file.S3 Fig(A) Growth and protein production during growth of the cells in LB with aeration at 37°C. The incubation periods for the samples of exponential (3 h), early-stationary (9 h), stationary (24 h), and death phase (48 h) are denoted on the graph. The data are presented as the mean ± s. e. m. of three independent experiments. (B) Protein expression profiles in concentrated supernatants and whole-cell lysates of S. Typhimurium strains (WT and Δrng), showing abundance changes in the secretion of type III secretion system (T3SS) effector proteins and flagellar proteins during different growth phases. Separated protein bands in four lanes  of the WT and Δrng strains were sliced into three gel pieces (red dot line boxes) between 30 and 90 kDa for identification of proteins in the pooled data sets generated by tandem mass spectrometry, as shown in (C) Western blot intensities of Enolase (Eno) in S. Typhimurium strains (WT and Δrng) at the indicated culture periods. WT Eno levels were set to 1. For (B) and (C), M; size marker.(TIF)Click here for additional data file.S4 Fig(A) RNase G cleavage sites-dependent expression of CAT on the hns::cat fusion. Top: for each construct, a DNA fragment containing trpc promoter, the 5′ UTR from TSS and the hns CDS for the first 10 amino acids was cloned in-frame with the CDS of the chloramphenicol acetyl transferase (cat) gene in the pCAT924 vector. RNase G cleavage sites are indicated in blue bold characters and nucleotide substitutions are indicated in red bold characters. Red arrows indicate primers used for qRT-PCR in (B). Bottom: WT strain harboring hns::cat fusion constructs were grown in LB at 37°C to an OD600 of 0.6, and were collected for western blot analysis of CAT using protein-specific polyclonal antibodies. WT CAT levels were set to 1. The data are presented as the mean ± s. e. m. of at least three independent experiments, and statistically significant differences are indicated with different letters . (B) RNase G cleavage sites-dependent stability of cat mRNA on the hns::cat fusion. Total RNA samples of S. Typhimurium cultures used in (A), were prepared from the cultures 0, 1, 2, 4, and 8 min after the addition of rifampicin (1 mg ml-1) and cDNA synthesis was performed using random hexamer, and analyzed of cat mRNA levels using qRT-PCR. The expression levels of cat mRNA were normalized using 16S rRNA mRNA levels. Gene expression levels were quantified using the ΔΔCt method and represented semi-logarithmic plot. The data are presented as the mean ± s. e. m. of three independent experiments. Statistically significant values from two-sided unpaired Student’s t-tests are indicated. For (A), ribosomal protein S1 was used as an internal standard to evaluate the amounts of cell extract in each lane.(TIF)Click here for additional data file.S5 Fighns 5′ UTR. TSS; Transcription start site.Sequencing shows successful subsequent introduction of a point mutation in the (TIF)Click here for additional data file.S6 Fig(A)In vitro RNase G cleavage of the 5′-32P-end-labeled synthetic full-length hns transcripts containing the wild-type or the mutated sequences  generated cleavage products. Bold, blue characters indicate hydrolysis products. Black arrows indicate the cleavage sites identified in (B) The relative amount of RNase G cleavage product from the wild-type or the mutated hns transcripts was assessed by measuring the radioactivity of each cleavage product and plotted. (C) The secondary structures of the full-length hns transcripts containing the wild-type or mutated sequences . The secondary structures were deduced using the M-Fold program. Arrows indicate RNase G cleavage sites. The start codon is indicated by bold, green characters.(TIF)Click here for additional data file.S7 FigS. Typhimurium strains -infected mice (n = 3 mice per group), and cDNA was synthesized from the total RNA purified from each organ. The relative abundance of each group of hns and SPI-1-related genes mRNAs was quantified and is shown right side the gel images. The expression levels of hns and SPI-1-related genes mRNAs were normalized using ribE mRNA levels. The data are presented as the mean ± s. e. m. of at least three independent experiments, and statistically significant differences are indicated with different letters .Total RNA was isolated from the spleens and livers of (TIF)Click here for additional data file.S8 Fig(A) Predicted FNR and ArcA binding sites in the promoter region of rnc using the online software Prodoric Virtual Footprint Promoter Analysis Version 3.0 online tool to predict E. coli K12 FNR and ArcA binding sites. (B) Predicted FNR and ArcA binding sites in the promoter region of rng using the online software Prodoric Virtual Footprint Promoter Analysis Version 3.0 online tool to predict E. coli K12 FNR and ArcA binding sites. (C) Predicted FNR and ArcA binding sites in the promoter region of hns using the online software Prodoric Virtual Footprint Promoter Analysis Version 3.0 online tool to predict E. coli K12 FNR and ArcA binding sites. For (A), (B), and (C), FNR and ArcA binding sites are highlighted in black box, and a score indicating how closely the binding site match FNR and ArcA binding sites consensus logo are given. A perfect match to the consensus sequence scores 8.93 and 7.44, respectively, as determined by Prodoric.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table1See METHOD DETAILS for detailed description scheme for plasmids. Abbreviation: Ampr, ampicillin resistance; Kmr, kanamycin resistance; Tcr, tetracycline resistance; Cmr, chloramphenicol resistance; Sptr, spectinomycin resistance; Strr, streptomycin resistance.(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 TableP values, and significance for all graphs in this manuscript.All statistical tests, biological replicates, exact (PDF)Click here for additional data file.