The bacterial flagellum is a motile organelle driven by a rotary motor, and its axial portions function as a drive shaft (rod), a universal joint (hook) and a helical propeller (filament). The rod and hook are directly connected to each other, with their subunit proteins FlgG and FlgE having 39% sequence identity, but show distinct mechanical properties; the rod is straight and rigid as a drive shaft whereas the hook is flexible in bending as a universal joint. Here we report the structure of the rod and comparison with that of the hook. While these two structures have the same helical symmetry and repeat distance and nearly identical folds of corresponding domains, the domain orientations differ by ∼7°, resulting in tight and loose axial subunit packing in the rod and hook, respectively, conferring the rigidity on the rod and flexibility on the hook. This provides a good example of versatile use of a protein structure in biological organisms. The bacterial flagellum is a motile organelle that enables bacterial movement. Here the authors explain how the structurally similar flagellum components FlgG and FlgE can give rise to distinct macrostructures—the rod and hook—through subtle differences in domain orientation attributable to a short N-terminal insertion in FlgG. Salmonella enterica and Escherichia coli, the basal body can be divided into five parts according to their functions: the C ring is the cytoplasmic ring with a diameter of 45 nm and is involved in torque generation as well as regulating the directional switching of motor rotation; the MS ring is the cytoplasmic membrane ring with the M ring in the membrane and the S ring in the periplasm and works as the rotor as well as the base for entire flagellar assembly; the LP ring is the bushing to support fast rotation of the rod through the peptidoglycan layer and the outer membrane; the flagellar type III protein export apparatus forms the central gate that selectively translocates flagellar axial proteins from the cytoplasm into the central channel of the flagellum; and the rod is the most proximal portion of the flagellar axial structure that works as a drive shaft  with the N- and C-terminal regions of FlgEThe hook-basal body (HBB) structure has been revealed by electron cryomicroscopy (cryoEM) and image analysis  but the 825et al.16In the axial tubular structure, protein domains are arranged radially. The hook protein FlgE has three domains, D0, D1 and D2. Domain D2 lies around 7.5 nm from the centre and forms the surface of the hook with an outer diameter of 18 nm. Domain D1 is just inside domain D2 and lies between 5 and 6 nm from the centre. Domain D0 forms a tube with a 1-nm thick wall and the axial central channel of about 2 nm in diameter. The crystal structure of a 31 kDa FlgE fragment containing domains D1 and D2 has been solved by Samatey The diameter of the polyrod is 13 nm, much smaller than those of the flagellar filament (23 nm) and the hook (18 nm) . In addiWe determined the structure of the polyrod by cryoEM image analysis using the iterative helical real space reconstruction (IHRSR) algorithm91–222 into the cryoEM density map by using the real-space correlations in ChimeraWe built an atomic model of the polyrod by docking the homology model of FlgGThe density map also resolved two rod-like densities in the inner core of the polyrod just as those in domain D0 of the hook  and 5. O16de novo model building of these two gap regions.We thus built an atomic model of the FlgG rod structure with residues 1–23 (D0), 91–222 (D1) and 227–260 (D0), but we could not complete the model by filling the chains in the two gaps (24–90 and 223–226) into domain Dc , which cAlthough the rod (13 nm) is thinner than the hook (18 nm) by the absence of domain D2 of the hook, which forms the right-handed six-stranded continuous helical densities on the surface to stabilize the hook structure, EM observations of the polyrods and polyhooks on negatively stained specimen grids indicate that the polyrod is quite rigid against bending, much more rigid than the polyhook. The atomic model of the rod clearly indicate that the rod is rigid because the D0 domains and the D1 domains are both highly packed in all the three main helical directions: the left-handed 5-start, the right-handed 6-start and the 11-start (protofilament) helix, in each of the inner and outer radial regions of the rod, respectively . In contIt is puzzling how the FlgG and FlgE subunits can form such significantly distinct packing interactions in the rod and hook, respectively, to give rise to their markedly different mechanical properties, despite their nearly identical folds, helical symmetries and repeat distances. What we found in the structural comparison are the following two factors. The first is a slightly longer N-terminal α-helix of FlgG than that of FlgE (26 residues for FlgG and 24 residues for FlgE), and the presence of an extra density in the rod structure in the yet un-modelled region of the rod and hook density maps (domain Dc), which are formed mainly by their N-terminal regions connecting the N-terminal α-helix and domain D1. This extra density is likely to be formed by the 18 residues insertion in FlgG . The extTo examine our hypothesis that the rod is straight and more rigid as a drive shaft than the hook as a universal joint by FlgG specific insertion of 18 residues not present in FlgE , we inseBiological systems and their dynamic activities are all supported by complex interaction networks of macromolecules, such as protein, DNA and RNA, exchanging and converting small molecules, information and energy to exert specific functions for specific cellular activities. For each specific function, the amino acid sequence and 3D structure of a protein is designed and optimized by the evolutionary process over millions of years. Therefore, in general, proteins with distinct sequences and 3D structures are used for distinct functions. However, occasionally, biological systems take advantage of the versatility of protein design, where proteins of largely distinct functions are produced by small modifications in the amino acid sequence. In the present study, we have reported an interesting example of such kind. We show that two axial component proteins of the bacterial flagellum, FlgG and FlgE, which form the rod and the hook, respectively, share a high homology in their sequences and tertiary folds and assemble into very similar quaternary structures to form tubular structures, but produce distinct mechanical functions of the rod being rigid and the hook being flexible by a small insertion of amino acid residues.We obtained structural evidence explaining how these two proteins of a nearly identical fold can produce different quaternary structures with distinct functions. Both FlgG and FlgE are considered to originate from a common ancestor, as they share 230 amino acids with 39% identity, and their tertiary structures are predicted to be nearly identical for the corresponding domains D0 and D1 . The helThe structure of the rod also revealed that domain D0, the innermost domain of the tubular structure, shows a very similar structure but distinct axial subunit packing in comparison with that of the hook. Domain D0 is composed of the terminal α-helices in both FlgG and FlgE. These terminal α-helices form a coiled coil in a similar manner to those of FliC in the flagellar filament, but they are significantly more tilted than those of FliC, with the longer C-terminal helix more tilted than the N-terminal one . In the The structural and functional differences between the hook and the filament require a structural and mechanical adaptor to join them. FlgK and FlgL are the adaptor proteins that form the junction between these two tubular structures. Despite the functional differences between the rod and the hook, however, there are no junction proteins connecting them. Despite the ∼7° difference in the orientation of the D1 domains in the rod and hook structures, the entire subunit structures and domain arrangements of FlgG and FlgE are probably similar enough to allow the intersubunit interactions between FlgG and FlgE to be maintained at the rod-hook junction . That isThe C-terminal α-helix faces the inner lumen while the N-terminal α-helix is located outside, facing domain Dc. This relative placement is the same as that in the atomic model of the filamentYersinia YscP, a FliK homolog, stays inside the central channel of the needle complex during needle assembly, partly in an α-helical conformation, and monitors the needle length as a ruler31The hook length control is one of the intriguing questions in the regulatory mechanisms of flagellar assembly. It has been suggested that the extended N-terminal chain of FliK monitors the hook length during hook assembly, and the interaction of its C-terminal domain with the cytoplasmic domain of FlhB is responsible for the substrate specificity switch of the protein export apparatus to stop rod/hook protein export and start filament protein export upon the hook grows to 55 nm. How does FliK measure the hook length? One of the hypotheses proposed for needle length control in the assembly mechanism of the type III secretion system of pathogenic bacteria, which is homologous to the flagellar hook basal body, is that the N-terminal chain of 35S-labelled HBB complex, normalizing for the number of sulfur-containing residues of each protein, and converting the relative stoichiometry values into absolute ones based on the number of FlgE subunits in the hook (∼130) obtained from the hook length and its helical latticeThe stoichiometries of the rod proteins FlgB, FlgC, FlgF and FlgG have been estimated to be about 6, 6, 6 and 26, respectively, as part of the study to determine the stoichiometries of the HBB component proteins by analyzing et al.Chevance The outer surface of the polyrod has an electrostatic potential distribution strongly biased to negative . Three nSalmonella enterica serovar Typhimurium, TH9709 [ΔfliK::tetRA ΔflgHI flgG*5667(G65V)]For preparation of the polyrod specimen, we used a polyrod strain of tetRA genes were amplified by PCR using 5′-TGGCTTTAAGTCCGGTACGGCATCATTTGCCGATATGTTCTTAAGACCCACTTTCACATT-3′ and 5′-TCCCCGCCACTTTTACGCCCAGCCCCACTTTGGAACCGGCCTAAGCACTTGTCTCCTG-3′ as DNA primers. The reaction mixture was purified using a QIAquick PCR purification kit (QIAGEN). Salmonella SJW3076 strain harboring pKD46  per liter of LB) containing tetracycline to generate the flgE::tetRA strain. Then, to remove the tetRA genes from the chromosome, the DNA encoding the RSS was amplified by PCR using 5′- GGCTTTAAGTCCGGTACGGCATCATTTGCCGATATGTTCTATCAGACCATCCGCCAGCC-3′ and 5′- TCCCCGCCACTTTTACGCCCAGCCCCACTTTGGACCGGCAGGCAGCGTCGTCTGCTCGG-3′ as DNA primers. The flgE::tetRA strain carrying pKD46 was electroporated with purified PCR products. After 1-hour incubation at 37 °C, cells were spread onto tetracycline-sensitive plates and incubated at 42 °C overnight to generate a Salmonella flgE::RSS strain (MME1001). The flgE::RSS allele was confirmed by DNA sequencing. DNA sequencing reactions were carried out using BigDye v3.1 as described in the manufacturer's instructions (Applied Biosystems), and then the reaction mixtures were analysed by a 3,130 Genetic Analyzer (Applied Biosystems). The ΔfliK::tetRA allele derived from the Salmonella ΔflgE ΔfliK::tetRA strain (NMEK001)Salmonella MME1001 and SJW1103 (wild type)strains by P22-mediated transduction to create the flgE+RSSΔfliK::tetRA (MMEK1001) and ΔfliK::tetRA (MMK001) strains, respectively, which produce polyhooks composed of FlgE with and without RSS insertion, respectively.To insert the FlgG specific 18 residues sequence (YQTIRQPGAQSSEQTTLP), hereafter called ‘RSS' (Rod Specific Sequence), between Phe 42 and Ala 43 of FlgE, we used the λ Red homologous recombination systemD46 ref.  was grow2. The magnification of × 91,463 and the pixel size of 1.64 Å per pixel were determined by measuring the 23.0 Å layer line spacing from images of tobacco mosaic virus (TMV) recorded in the same CCD frames with polyrods and polyhooks.A 4 μl sample solution was applied onto a Quantifoil grid , and the grid was plunge-frozen into liquid ethane using a fully automated vitrification device . The specimen was observed at a specimen temperature of around 50 K using a JEOL JEM-3200FSC electron microscope, equipped with a liquid-helium cooled specimen stage, an Ω-type energy filter and a field-emission electron gun operated at 200 kV. Zero-loss images (10 eV) were recorded on a 4 k × 4 k 15 μm-per-pixel slow scan CCD camera . The electron dose was ∼20 electrons per ÅImage analysis was performed as previously described2 was applied to the final 3D image reconstruction, and the 3D image was filtered at 7.4 Å resolution. Image collection and processing statistics are summarized in 91–222 and model fitting into the cryoEM density were carried out as described in Results.Further image processing was carried out with the SPIDER package600 had reached 0.6. The culture solution was centrifuged at 7,000 r.p.m. for 10 min at 4 °C, and the cells in the pellet were suspended in a solution containing 0.75 M sucrose, 150 mM Tris base and 10 mM EDTA and incubated at 37 °C for 20 min. The cell solution was centrifuged at 14,000 r.p.m. for 10 min at 4 °C, and the cells in the pellet were suspended in 20 ml of ice cold water for osmotic shock. Then, 1 μl (5 units) of DNAase-I (Takara) and 1 ml of 0.1 M MgSO4 were added, and after incubation at 30 °C for 20 min, it was centrifuged again at 14,000 r.p.m. for 20 min at 4 °C. The cells in the pellet was suspended in 200 μl of water and used for EM observation of polyhooks. A 4 μl solution of the osmotically shocked cells was applied on an EM grid, and the cells were negatively stained with 0.1% phosphotungstic acid solution (pH 6.0) by repeating blotting and staining three times. A JEOL JEM-1011 electron microscope was operated at 100 kV to observe the cells on the EM grid at a magnification of × 7,500.A 50 μl overnight culture solution was inoculated to 5 ml of LB and cultured at 37 °C until ODThe reconstructed density was deposited to Electron Microscopy Data Bank with accession code EMD-6683 and the atomic coordinate to Protein Data Bank with PDB ID code 5WRH. The data that support the findings of this study are available from the corresponding author upon request.How to cite this article: Fujii, T. et al. Identical folds used for distinct mechanical functions of the bacterial flagellar rod and hook. Nat. Commun.8, 14276 doi: 10.1038/ncomms14276 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Arabidopsis thaliana both genes show high level of expression in transcriptionally active cells. Here, we focus on two important differences between A. thaliana fibrillarins. First and most relevant is the enzymatic activity by AtFib2. The AtFib2 shows a novel ribonuclease activity that is not seen with AtFib1. Second is a difference in the ability to interact with phosphoinositides and phosphatidic acid between both proteins. We also show that the novel ribonuclease activity as well as the phospholipid binding region of fibrillarin is confine to the GAR domain. The ribonuclease activity of fibrillarin reveals in this study represents a new role for this protein in rRNA processing.Fibrillarin is one of the most important nucleolar proteins that have been shown as essential for life. Fibrillarin localizes primarily at the periphery between fibrillar center and dense fibrillar component as well as in Cajal bodies. In most plants there are at least two different genes for fibrillarin. In  S-adenosyl-L-methionine-dependent methyltransferase which is found in all eukaryotic cells and a shorter version exists in the Archaea kingdom as well  from A. thaliana as a model plant. In most eukaryotic cells, fibrillarin localizes primarily in the FC and DFC regions of the nucleolus, where active ribosomal DNA (rDNA) transcription and rRNA processing takes place. Both proteins contain three domains; glycine-arginine rich domain (GAR domain), methyltransferase domain and alpha region. The domains are very well conserved with the exception of the GAR domain that does not exist in the Archaea. The GAR domain has been shown to be required for nucleolar localization of fibrillarin . The heatmap in Figure 1B was generated by using the ComplexHeatmap package . Sequences of AtFib1 and AtFib2 were obtained with SuperScriptTM III One-Step RT-PCR System with PlatinumTM Taq DNA Polymerase (Thermo Fischer Scientific). The specific primers to amplify AtFib1 were: forward 5′ – 3′ CATATGATGAGACCCCCAGTTACAGGA and reverse 5′ – 3′ GGATCCCTATGA GGCTGGGGTCTTTTG. To amplify Atfib2 the specific primers were: forward 5′ – 3′ CATATGATGAGACCTCCTCTAACTGGAAG and reverse 5′ – 3′ GGATCCTCTAAG CAGCAGTAGCAGCCTTTG. Forward primers have NdeI restriction enzyme sequence, reverse primers have BamHI restriction enzyme sequence for pET15b expression plasmid cloning. Same strategy was used for GAR (AtGAR2) and alpha helix (Atα2) domain cloning of AtFib2. AtGAR2 primers: forward: 5′ – 3′ CATATGATGAGACCTCCTCTA ACTGGAAG, reverse 5′ – 3′: GGATCCCACAATCACTTTGCTTCCTCC. Atα2 primers: forward 5′ – 3′: CATATGCTTGTAGGCATGGTTGATGT, reverse 5′ – 3′: GGATCC CAAAGGCTGCTACTGCTGCTTAG.∗at 20°C . RNA extArabidopsis thaliana fibrillarins were expressed in E. coli Artic competent cells induced with 1 mM isopropyl-D-1-thiogalactopyranoside at 11°C for 24 h. Harvested cells were suspended in protein extraction buffer  and sonicated. After clarification by centrifugation (17400 × g × 15 min), the supernatant was subjected to further purification steps. The clarified supernatant was loaded onto a Ni-NTA agarose column (Thermo Fisher Scientific) and washed three times with the extraction buffer. Fibrillarins were eluted  in a linear gradient from 20 to 200 mM of imidazole. Fibrillarins containing fractions were further purified by Q sepharose chromatography leading to single band detection of fibrillarins. Same strategy was used for AtGAR2 and Atα2 domains.A. thaliana. After electrophoresis, gel was washed for 10 min with buffer I  and consequent incubation for 30 min in buffer II  and buffer III . Gel was stained with 0.2% of toluidine blue and washed with water . Once cloned, vector was linearized with NdeI enzyme for 1h at 37°C. Transcription was made with T7 RNA polymerase (New England Biolabs Inc.) for 2 h at 37°C. Specific primers for AtsnoU3 used are: forward 5′–3′ ACGACCTTACTTGAACAGGA, reverse 5′–3′ CCTGTCAGACCGCCGTGC GAC.A. thaliana was mixed with each fibrillarin on BC200 buffer , incubated for 1 h at 37°C and then loaded in a 1% agarose gel.Total RNA extracted from PIP strip with spotted phosphoinositides  was probed with anti-Fib antibody. For this, the membrane was blocked with 3% BSA in PBS for 1 h followed by 3 h at room temperature of 1% BSA in PBS and 0.4 μg of each protein. After that, PIP strip was washed three times, 10-min each, with PBS-T and incubated with primary antibody for 1 h. Again washed with PBS-T and incubated with the appropriate IRDye secondary antibody for 1 h. The immunoblotting signals were analyzed by Odyssey Infrared Imager 9120 .Fifty nanograms of each fibrillarin was loaded in a 12% acrylamide gel to perform a SDS-PAGE. Subsequently we transfer the protein to a nitrocellulose membrane and blocked with 3% of BSA in PBS at room temperature. Later was made incubation with anti-Fib rabbit antibody (1/5000) for 2 h at room temperature and a third with secondary antibody (1/4000) 1 h at room temperature, with three washes between incubations and revealed. The immunoblotting signals were analyzed by Odyssey Infrared Imager 9120 .Arabidopsis thaliana callus were made according to A. thaliana instead of leaves. Images were acquired in confocal microscope (Leica TCS SP5 AOBS TANDEM) and a laser-scanning microscope FV100 Olympus with 60X (NA 1.4) oil immersion objective lens.A. thaliana shows the greater amino acid difference in the GAR domain represented by a dotted blue contour . Considering that this part of the protein resides the main difference we check if their expression would be tissue or developmental stage specific. Transcriptional patterns of AtFib genes  demonstrate high expression levels in the different tissues and on different developmental stages in wild-type Arabidopsis Col-0 . Sequence differences between AtFib proteins (GAR domain + fibrillarin domain) could have major implications on protein activity. At transcriptional level, we found differences between AtFib genes. AtFib1 shows the highest expression levels but also little variations between treatments, while AtFib2 shows the most variation between treatments and seems to be more affected by development stages but remains expressed in all stages. AtFib3 shows the lowest level of expression values and almost no variation between tissues and development stages. We focused on AtFib1 and AtFib2 as they are expressed in almost all conditions. The in silico structure prediction between them  shows that the main structural difference is due to an angle changed for the exposure of the GAR domain as can be seen in the overlay of the structures in Figure 2B. As in other crystal structure of fibrillarin . Different eluates were loaded in the ribonuclease activity gel assay and show that both fibrillarins (AtFib1 and AtFib2) have ribonuclease activity. Western blot of the bands confirmed their correspondence to fibrillarin . AtFib2 is more susceptible to degradation as showed by Western blot and as in gel activity assay.Our initial studies where to test gel mobility alterations by fibrillarin with RNA resulted in degradation of the RNA when a short incubation was carried out at room temperature. We therefore tested the purified fibrillarin with an Figure 3A) in the exact same procedure and tested their activity under native conditions. Both fibrillarins were incubated with rRNA to test their ability to cleave rRNA. The reactions were carried out using the same amounts of fibrillarins as what is shown in the silver stained gel . The results shown in Figure 3B demonstrate that AtFib2 has a potent ribonuclease activity in a dose dependent manner while AtFib1 can only show activity under the greatest amount. This correlates well with the in gel activity assay which shows both proteins to have activity but AtFib2 show significant rRNA cleavage. We tested if A. thaliana fibrillarins are activated by calcium, as other ribonucleases . Interestingly, the activation of the ribonuclease activity by calcium shown for AtFib differs from that of human fibrillarin that we tested (data not shown).We decided to characterize this novel ribonuclease activity and purified both proteins to homogeneity P, while AtFib2 interacts with all phosphoinositides, as well as with phosphatidic acid (PA). This is similar to what we have detected with the unique human fibrillarin .Our previous studies with human fibrillarin had shown its interaction with phosphoinositides . In Figu2 antibody. We carried out confocal microscopy of Arabidopsis callus which had membrane bound PtdInsP2 removed by Triton X-100 as it was done in other publications . We show that nuclear PtdInsP2 has a partial colocalization with fibrillarin. Since the antibodies against fibrillarin detect both forms of fibrillarin it is impossible to discern between the two forms at this stage. We have unsuccessfully tried to raise antibodies, which would distinguish between these two fibrillarins that may lead to a better colocalization of one of them with phosphoinositides. The PtdInsP2 exhibits a dotted pattern in nucleoli regions and a diffuse pattern in other nuclear regions. Fibrillarin colocalizes with PtdInsP2 in the nucleolus but not in other regions like Cajal bodies.Nuclear phosphoinositides have been extensively studied in plant membranes but studies are lacking on the nuclear forms. To provide more information on nuclear phosphoinositides, we took advantage of the PtdInsPications  . The N terminus contains the GAR domain and the C terminus the α domain. We purified both domains  and tested them for activity. Only AtGAR2 domain showed high ribonuclease activity both in an in gel activity assay with RNA as substrate, as well as under native conditions . The ribonuclease activity of AtGAR2 domain is less selective than the full fibrillarin protein as it degrades both 28S and 18S simultaneously and gives a less selective pattern of bands as well; Figure 5D, lanes 4 and 5. The AtGAR2 domain is also the interacting domain for phospholipid binding, including all phosphoinositides species as well as PA and phosphatidylserine (PS) and resemble the full protein binding, while the alpha region of AtFib2 had no ribonuclease activity and only binds to PtdIns(5)P . Finally, we compared AtFib1 and AtFib2 for their ribonuclease activity on U3 guide RNA and overall rRNA. We found that AtFib2 was able to cut RNA as compared between Figures 6A,B. AtFib1 showed only a minor reduction in the amount of rRNA but maintained the exact same pattern, while AtFib2 showed a different pattern of rRNA and U3 after interacting with this fibrillarin as seen in Figure 6B, lane 6 compared to lanes 7 and 8.In order to define the domain that has ribonuclease activity, we overexpressed two domains of the protein, which were shown to have an enzymatic activity assigned  known as paleopolyploidy, events that occurred throughout plant evolution. These events have played a major role in Brassicaceae evolution. One of the main features of fibrillarin is the N-terminal GAR domain. It is the least evolutionary conserved domain of the protein; however, this sequence was added in the transition between Archaea to eukaryotic cells as it is absent in all archaebacteria. This domain is also responsible for the phosphoinositide binding, which well correlates with the lack of it in Archaea kingdom . Furtherin vitro. The authors could not discern which protein had the ribonuclease activity, but they identified nucleolin and fibrillarin as important proteins in this fraction ositides . Amino aositides . The intositides . The comA. thaliana fibrillarins. Taken into account that phosphoinositides–protein interaction affects the protein ability to form new complexes it is therefore likely that both fibrillarins in A. thaliana bind to different partners. This may also explain why confocal microscopy of both fibrillarins does not colocalize 100% with the PtdInsP2 signal as it does in human cells  affect ribonuclease activity?UR-C: Experiments design and conception and wrote the article. AP-S: Bioinformatics and wrote the article. MS: Microscopy related experiments. LR-Z: Experiments design and article discussions. PH: Article discussions and review. EC: Experiment design and original set of experiments that led to the research, article writing, and revisions.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.


An equiatomic FeCoCrNi high-entropy alloy with a face-centered cubic structure was fabricated by a powder metallurgy route, and then processed by high-pressure torsion. Detailed microscopy investigations revealed that grain refinement from coarse grains to nanocrystalline grains occurred mainly via concurrent nanoband (NB) subdivision and deformation twinning. NB–NB, twin–NB and twin–twin interactions contributed to the deformation process. The twin–twin interactions resulted in severe lattice distortion and accumulation of high densities of dislocations in the interaction areas. With increasing strain, NB subdivision and interactions between primary twins and inclined secondary stacking faults (SFs)/nanotwins occurred. Secondary nanotwins divided the primary twins into many equiaxed parts, leading to further grain refinement. The interactions between secondary SFs/nanotwins associated with the presence of Shockley partials and primary twins also transformed the primary twin boundaries into incoherent high-angle grain boundaries. High-entropy alloys (HEAs) have received considerable attention, and become one of the most promising structural materials since their discovery in 20041370.1CoCrFeNi HEA18−2)Severe plastic deformation (SPD) induced grain refinement processes involve complicated microstructural evolutions, which could significantly affect the mechanical behavior of the materials9et al. reported that a nanostructure of Al0.3CoCrFeNi alloy processed by HPT processing has a high incremental hardness, and subsequent annealing at appropriate temperatures gave an ordered BCC secondary phase with an additional increase in hardnesset al. showed that the plastic deformation mechanisms of an Al0.1CoCrFeNi FCC HEA induced by HPT at room temperature include dislocation slip at low strains and twinning at high strainsThe plastic deformation mechanisms of HEAs could be different from that of conventional alloys, since HEAs have highly distorted lattice structures due to the different atomic sizes and chemical bonds of their constituent elementset al. reported that the FeCoCrNi HEA is metastable at intermediate temperatures1523xxFeCoCrNi HEA is a stable single FCC solid solution with high toughness even at cryogenic temperatures25During the unconfined HPT processing, the equivalent von Mises strain (ε) can be calculated from the following equation0 and h are the initial and final thicknesses, respectively. where r is the radius of the disk, N is the number of the revolution, and hIn deformation stage 1, grains mainly maintain the original size (~25 μm) and present a microstructure with significant numbers of NBs and deformation twins. Deformation twins with TB spacing ranging from ~5 nm to ~40 nm were also observed at this deformation stage, as shown in Although the morphologies of NBs and deformation twins were very similar in this deformation stage, the boundaries of NBs are not as straight as those of the twins. They can also be easily differentiated by electron diffraction along a <110> direction. Further, while TBs were exactly parallel to each other at this deformation stage see , NBs in Three interaction characteristics can be observed in this deformation stage. 3133With increasing strain, the misorientation between NBs and the matrix increased, and some NBs were divided into several segments due to NBs interactions. The HRTEM image  shows th38T) and the matrix (marked as (111)M). The originally coherent TBs lost their coherency and transformed into incoherent high-angle GBs after accumulating a large number of dislocations at and near TBset al.After dislocation–TB interactions, a very high density of dislocations accumulated at and near primary TBs, as shown in arked as 1M). The T2) of T2 interaction area still form a twin relationship with the T1) of T1, whereas the (111) plane of T2 and T1 has a misorientation about 43°. It can thus be concluded that with further deformation, secondary nanotwins could divide primary twins into many equiaxed parts and transform the primary equiaxed parts into an equiaxed NC structure, and the distorted secondary TB , low SFE and large lattice friction stresset al.It’s worth noting that NBs were observed in the initial deformation stages with extremely low strain , while sBecause of the low SFE of the HEA, twinning is an important deformation mode in the coarse-grained HEA. Therefore, deformation twins with thicknesses of only a few tens nanometers were frequently observed at the very early stages of the deformation process. Twinning at these early stages of deformation should occur via a mechanism that operates in coarse-grained materialsWith increasing strain, abundant thin secondary SFs/nanotwins inclined to the primary TBs formed . Activatet al.The formation mechanism of steps along TBs  during d5456During the HPT process, the hardness increased continuously with increasing the equivalent strain before reaching a plateau see in , indicatThe microstructural evolution of FeCoCrNi HEA is divided into four deformation stages. The grain refinement process can also be described in four steps, as illustrated schematically in Different from the SPD-induced grain refinement mechanisms in conventional materials, the SPD-induced grain refinement mechanism in the HEA includes concurrent NB subdivision and deformation twinning. The concurrent deformation mechanism causes significant grain refinement from an average grain size of ~25 μm in the as-received sample to ~68 nm.The misorientation between a NB and its neighboring matrix increases with increasing strain because of dislocation accumulation at the boundaries of the NB with increasing strain. The interaction between two sets of NBs on different slip planes causes boundary distortion and segmentation of NBs.The interaction between the tips of secondary twins and the boundaries of primary twins leads to the emission of new secondary twins on the other side of the primary twins to release local stress concentration. High densities of dislocations accumulated in the interaction areas result in local heavy lattice distortion.Significant dislocation–TB reactions produce atomic steps at primary twin boundaries that gradually transform the originally coherent TBs into incoherent high-angle GBs.The microhardness and microstructural evolution of an FeCoCrNi HEA processed by HPT was investigated in detail. The following conclusions are drawn:The FeCoCrNi HEA used in this investigation was fabricated by a powder metallurgy route. The mixed high purity Fe, Co, Cr and Ni raw materials with an equiatomic ratio were induction melted in vacuum, and then, the melt were disintegrated by high pressure argon into fine liquid particles. The droplets were then cooled down in a stainless steel chamber. The pre-alloyed FeCoCrNi powder was collected and sieved, and filled in a steel can. The canned powder was hot extruded at 1200 °C with an extrusion ratio of 6:1 to obtain a billet with a high density. Details of the fabrication technique and the characterizations of the powder have been reported in our previous studyThe as-received alloy was firstly cut into several disks along the transverse direction with a diameter of 10 mm and thickness of about 1.2 mm. The disks were ground carefully on each side with 1000 grit and 2000 grit SiC paper sequentially, resulting in a smooth surface and a final specimen thickness of ~1 mm for HPT processing. An unconstrained HPT die set was used in our experiments. The disks were subjected to pressure along the normal direction in a non-hydrostatic environmentThe microhardness measurements were taken along the radii from the center to edge at different radial directions (distance between two adjacent indentations is 0.5 mm) using a load of 100 g for 10 s. The structures of both the as-received and the deformed samples were characterized using an X-ray diffractometer  with Cu-Kα radiation and a TEM (Tecnai G2 F20). XRD results showed that both the as-received and deformed samples have an FCC single phase. Samples for TEM observations were cut along a radius direction of the disks. The location of each sample center relative to its disk center was recorded in order to calculate the HPT strain. These samples were thinned to a thickness of ~70 μm, and then electropolished using a solution of 5% perchloric acid and 95% alcohol at −40 °C with an applied voltage of 27 V. Grain sizes were measured directly from the TEM images by measuring the width of the individual grains along linear traverses. The average grain sizes of samples after deformation were estimated from at least 200 grains. For each deformation stage, the average twin/NB thicknesses were estimated from up to 20 TEM and HRTEM images.How to cite this article: Wu, W. et al. Dual mechanisms of grain refinement in a FeCoCrNi high-entropy alloy processed by high-pressure torsion. Sci. Rep.7, 46720; doi: 10.1038/srep46720 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The mechanical properties of the high-strength low-alloy pipeline steels were mainly controlled by the subsequent phase transformations after rolling. The influence of hot uniaxial compression on the phase transformation of acicular ferrite was explored by viewing of the deformation degree, the deformation temperature, and the strain rate. The results show that the increase of deformation amounts raises the transformation starting and finishing temperature during the subsequent cooling and also promotes the polygonal ferrite transformation, which leads to the decrease of Vickers hardness accordingly. With the increasing of the deformation temperature, the achieved microstructure becomes coarsened and thus decreases the hardness. As the strain rate increases, the microstructure is refined and thus the hardness increases gradually; increasing the strain rate appropriately is beneficial to the refinement of the microstructure. Since the high-strength low-alloy (HSLA) pipeline steels were developed in the early 1970s, they have been widely used in the construction of long-distance oil and gas transportation systems, due to their good combination of strength, toughness and weldability ,3. In orThe mechanical properties of pipeline steels depend on the alloy chemistry and thermo-mechanical control processing (TMCP) ,7,8. TheThis project focuses on the thermo-mechanical process in the metastable austenite field. The effects of hot uniaxial compression on the microstructure, phase transformation behaviors and micro-hardness of HSLA pipeline steel were investigated in view of the deformation degree, the deformation temperature, and the strain rate.The chemical composition of the employed material used in this project, an HSLA pipeline steel, is given in −1, 1 s−1 and 10 s−1) followed by cooling to room temperature  equipped with a dilatometer. The simulation procedures are illustrated in perature c. Due toAfter the heat treatments, the samples were cut in half to ensure the middle of the samples can be shown for microstructural observation. Then they were mounted, polished, and etched in solution of nitric acid (4%) and ethyl alcohol. The microstructures were studied by C-35A OLYMPUS Optical Microscope (OM) (Japan). The phase transformation points were determined by the accessory dilatometer of the Gleeble 1500 thermal simulation testing system . The starting and finishing points of transformation were determined by 5% and 95% for transformation fraction respectively. Vickers micro-hardness tests were performed using a load of 2 N.s, and finishing temperature, Tf, of the samples during continuous cooling after deformation with various deformation reductions are shown in s and Tf are both raised as the deformation amount increases, suggesting that the process of transformation is facilitated. When the deformation reduction is higher than 50%, the increase tendency of Ts and Tf is more obvious. This is because the increase of the deformation amount results in the multiplication of dislocations and deformation bands in the metastable austenitic grain, and in the increase of cumulative strain energy, thus facilitating phase transformation during cooling after hot uniaxial compression. The transformation starting temperature, Ts and Tf presented in The optical micrographs of the samples with various deformation reductions  are shown in s, and finishing temperature, Tf, of the samples during continuous cooling after hot uniaxial compression at various temperatures. With the increase of the deformation temperature, Ts decreases, whereas Tf remains keeps unchanged. On one hand, the rate of dynamic recovery and recrystallization is higher at the high deformation temperature, which results in the decrease of the density of dislocations and amount of deformation bands, and thus hinders the decomposition of metastable austenite during continuous cooling by decreasing the nucleation sites. On the other hand, higher deformation temperature implies that cooling to the transformation starting temperature requires more time, since the cooling rate is constant for all samples. Hence, the sample deformed at high temperature has more time for recovery and recrystallization, resulting in the decrease of nucleation sites for the subsequent phase transformation during cooling, and thus the decrease of Ts.r3 temperature results in the formation of the ultra-fine ferrite grains, namely DSIT. The strain-induced ferrite grains form on the prior austenite grain boundaries at first, and then grow into the austenite grains. The increase of the deformation reduction can promote DSIT. The starting temperature for transformation without deformation is determined by the dilatometer measurement of 767 °C, which is closed to that for the sample deformed at 800 °C. According to The optical micrographs of the samples deformed at various temperatures are shown in −1 has evident fluctuations (see Stress-strain curves of the samples during hot uniaxial compression with various strain rates are presented in ions see . This maThe Vickers micro-hardness results of the samples with various strain rates are shown in In this paper, the effects of hot uniaxial compression on the microstructure, phase transformation behaviors and mechanical properties in view of the deformation degree, deformation temperature and strain rate were investigated. The conclusions are as follows:i. The increase of the deformation reduction results in the increase of the transformation starting and finishing temperature for polygonal ferrite and acicular ferrite, due to the multiplication of dislocations and deformation bands providing more nucleation sites. The increase of the deformation reduction promotes the formation of polygonal ferrite and hinders that of acicular ferrite, and thus decreases the Vickers hardness.ii. The increase of the deformation temperature leads to the decrease of the transformation starting temperature, due to dynamic recovery at high temperature and enough recovery time during cooling. The increase of the deformation temperature results in coarse grains, and thus decreases the Vickers hardness. Deformation at 800 °C leads to dynamic strain-induced transformation.iii. A higher strain rate results in a finer microstructure and higher Vickers hardness. The relatively low strain rate provides enough time for the recovery of dislocations and deformation bands, and thus decreases the nucleation sites, resulting in the formation of coarse grains.


The F-type lectin (FTL) family is one of the most recent to be identified and structurally characterized. Members of the FTL family are characterized by a fucose recognition domain [F-type lectin domain (FTLD)] that displays a novel jellyroll fold (“F-type” fold) and unique carbohydrate- and calcium-binding sequence motifs. This novel lectin family comprises widely distributed proteins exhibiting single, double, or greater multiples of the FTLD, either tandemly arrayed or combined with other structurally and functionally distinct domains, yielding lectin subunits of pleiotropic properties even within a single species. Furthermore, the extraordinary variability of FTL sequences (isoforms) that are expressed in a single individual has revealed genetic mechanisms of diversification in ligand recognition that are unique to FTLs. Functions of FTLs in self/non-self-recognition include innate immunity, fertilization, microbial adhesion, and pathogenesis, among others. In addition, although the F-type fold is distinctive for FTLs, a structure-based search revealed apparently unrelated proteins with minor sequence similarity to FTLs that displayed the FTLD fold. In general, the phylogenetic analysis of FTLD sequences from viruses to mammals reveals clades that are consistent with the currently accepted taxonomy of extant species. However, the surprisingly discontinuous distribution of FTLDs within each taxonomic category suggests not only an extensive structural/functional diversification of the FTLs along evolutionary lineages but also that this intriguing lectin family has been subject to frequent gene duplication, secondary loss, lateral transfer, and functional co-option. Recognition of glycans exposed on the surface of microbial pathogens and parasites by the host’s cell-associated and soluble lectins is considered the initial key step in the innate immune response of both invertebrates and vertebrates –5. MembeF-type lectins are fucose-binding proteins of wide taxonomic distribution from viruses to vertebrates and constitute the most recently identified lectin family –14. TheyMorone saxatilis) (l-fucose-Sepharose yielded a 32 kDa protein (MsaFBP32) that did not require calcium or other divalent cations for binding to cells. Partial Edman sequencing of the protein enabled cDNA and genomic cloning and revealed the presence of two 140-amino acid tandemly arrayed domains. Analysis of the deduced polypeptide sequence of MsaFBP32 failed to identify the signature motif of the CTLs or any of the known lectin families described at the time and suggested that MsaFBP32 represented a novel lectin type. Although no matches to known lectins were initially identified, the search of sequence databases revealed a stretch of N-terminus sequence from a single protein named PXN1-XENLA (Xenopus laevis), consists of an MsaFBP32-like domain linked to a PXN domain that also exhibits lectin activity  characterized by proteins present in both prokaryotes and eukaryotes, which displayed the newly identified lectin domain (FTLD), either tandemly arrayed or in mosaic combinations with other structurally and functionally distinct domains (The identification of the FTL family was a fortuitous discovery that resulted from the search for fucose-binding CTLs in serum and liver extracts from the striped bass (xatilis) , 12. AffN1-XENLA  that shaactivity . Furtherphibians , 12. Sureumoniae , 12. As  domains , 12.Anguilla anguilla agglutinin (AAA)]  that carries two tandemly arrayed FTLDs ] . These wed FTLDs . The resed FTLDs ] revealeed FTLDs . This inStreptococcus spp. FTL sequences as virulence factors (lectinolysins) (Crassostrea gigas as extremely diversified FTLs with role(s) in fertilization  –22, and lization –26. In rlization . FinallyM. saxatilis FTL (MsaFBP32) and X. laevis PXN-fusion protein (PXN1-XENLA) led to the identification of an approximately 140-amino acid long lectin domain and a tentative amino acid sequence motif common to a number of lectins, as well as selected domains present in sequences that had been described in other contexts such as the Drosophila furrowed gene and the Streptococcus fucose regulon. In turn, this resulted in the identification of a novel fucose-binding lectin family (FTL family) that included both prokaryotes (S. pneumoniae TIGR4) and eukaryotes   , 12  . In turn others) .l-fucose (α-Fuc) complex  tightly coordinates a cation  via seven oxygen atoms of six residues  both from the peptide backbone and side chains in a pentagonal bipyramidal geometry. The distance between the cation binding site and the sugar binding pocket indicates that the divalent cation does not directly interact with the carbohydrate as in CTLs, but that together with two disulfide bridges (Cys50-Cys146 and Cys108-Cys124) and two salt bridges (Arg41-Glu149 and Asp64-Arg131) that clamp the structure together, rather stabilizes the fold and shape the key CDR1 and CDR2 loops (2+) per domain and several Cl− placed on the three-fold axis, and two trimers can form hexamers with opposing carbohydrate-binding surfaces  situated in a shallow cleft and the axial 4-OH of the sugar. Interactions are also established between this basic triad and the ring O5 and equatorial 3-OH of the sugar (82 and Cys83) establishes a van der Waals contact with the bond between C1 and C2 of the α-Fuc ring, and the C6, which fits into a hydrophobic pocket formed by His27 and Phe45, together with Leu23 and Tyr46 , it becomes clear that as for most animal lectins, the specificity of AAA for α-Fuc is nominal rather than absolute.The AAA/α-Fuc structure revealed that the protein binds to α-Fuc through hydrogen bonds established between the side chains of three basic amino acid residues  oligosaccharides via additional interactions established between amino acid residues in CDRs 1–5 that encircle the binding cleft, with the subterminal units of the H1 and Lea trisaccharides. Specifically, Glu26 and His27 on CDR1 can interact with hydroxyls (3-OH and 2-OH) in Gal and oxygen of the GlcNAc 2-N-Acetyl group in Lea, or GlcNAc 6-OH and 4-OH groups in H. The OH group of Tyr46 in CDR2 can interact with the glycosidic bond oxygen between Gal and GlcNAc moieties. Furthermore, Asp81 and Arg79 in CDR4 interact with the GlcNAc 6-OH group in Lea, and a water molecule can bridge the Gal 4-OH group with Asp81 in H1. The rigidity of the CDR1 loop prevents recognition of Lex, in which the 2-N-acetyl is pointed toward the Fuc side of the oligosaccharide , where X indicates any amino acid residue]. Loops participating in the hydrophobic pocket for the fucose methyl group, such as CDR2, are also conserved in both hydropathic profile and length. The cation binding sequence motif is h2DGx, where h indicates a small hydrophobic amino acid residue  and x stands for a small hydrophilic residue . Three of the seven oxygens that bind the cation are contributed by this motif, which in AAA is located just after the 310-helix h3 . Similarly, replacements of metal-coordinating residues are frequent, and those that occur at Ser49 are of special interest, since as a bidentate ligand, it is central to the coordination geometry. In most cases, this position is substituted by residues that are able to form similar coordination bonds, such as Asp, Gln, Glu, Thr, and Tyr, but in some sequences this is not the case. In the latter, a water molecule may substitute in cation coordination, or it is possible that the coordination geometry is modified  and would not be subject to junctional diversity during splicing  are permissible as long as any potentially disruptive effect on the core fold is minimal, as in the FTLD CDRs, where most indels are present. Interestingly, the CDR1 loop, which interacts with subterminal sugar units, shows considerable divergence suggesting that it might determine the fine specificity for a wide diversity of glycoconjugates. Coincidentally, in the MsaFBP32 gene, the exons coding the two FTLDs are split by introns localized at the lower side of the barrel close to a turn that is also variable in length  and C-terminal FTLD (C-FTLD) of MsaFBP32 revealed that they are similar but not identical , 12. SeqA Figure . In addig pocket    and A. japonica conserve the basic amino acid triad that interacts with the C4 hydroxyl in fucose, showing strict conservation of His52 and the CDR4 sequence  Figure  may expasequence , 15. CDR6 in AAA ] that fothe 6-OH .C. gigas, the genetic mechanisms for generating diversity in ligand recognition by lectin isoforms have been characterized in detail  that in the AAA structure interact with the hydroxyl on C4 of L-Fuc  of the Pacific oyster n detail –26. Oystn detail . First,  Figures A,B. It ic Figure A, while c Figure B. Secondc Figure . Interesc Figure , 26. In c Figure , 26. In c Figure –26.The identification of a large number of proteins exhibiting the FTL sequence motif as multiple tandemly arrayed FTLDs enabled the establishment of the FTLs as a novel lectin family , 12 Fig, 12. In Drosophila (CG9095 and furrowed) , a CTLD, and a predicted transmembrane domain  , sea urcurrowed) , S. pneuae TIGR4 , 12, Strus mitis , 21, andfloridae  represene domain . It is ne domain , 12  of the triad of basic residues that interact with the axial hydroxyl of fucose in AAA are conserved. CSIase, the galactose-binding domain of the bacterial sialidase, has been shown to bind carbohydrate (40 and Arg73) homologous to those involved in carbohydrate recognition by AAA (His52 and Arg79) and in CSIase (His539 and Arg572) are conserved, suggesting that it may bind carbohydrate. In FVa-C2, all residues of the basic triad related to carbohydrate binding are absent, making this pocket the most hydrophobic and the deepest. Interestingly, FVa-C2 has affinity for phospholipids instead of carbohydrates   , 12. Howamander) , 12. Theamander) , 12, 30.The advent of innovative sequencing technologies during the last decade has enabled comprehensive genomic and transcriptomic studies on a large variety of organisms and significantly expanded our knowledge about the taxonomic distribution of the FTLD from viruses to prokaryotes and eukaryotes. In this regard, a rigorous and exhaustive computational study on publically available databases by Bishnoi et al. has recently provided significant insight and greatly expanded the range of taxa in which the FTLD is found . Using aOstreococcus sp., O. tauri, and O. lucimarinus , and one from the coccolithophore Emiliania huxleyi (E. huxleyi virus 203), which are microalgal species abundant in photosynthetic phytoplankton. Except for a viral FTLD joined by a PTX domain found in the E. huxleyi virus 203, all other viral FTLD sequences are single. It is noteworthy that although E. huxleyi and Ostreococcus spp. also display FTLDs, some with high similarity to the viral FTLDs, the microalgal host’s FTLDs are associated with other distinct non-FTLD domains  that in AAA interacts with the bond between ring atoms C1 and C2 of α-Fuc. Second, two strands of the AAA fold (AAA residues 126–136 and residues 145–155), of which the former strand  and with several oyster (Crassostrea spp.) and mussel (Mytilus sp.) species (Phytophthora spp.) also possess singly or tandemly arrayed FTLDs, mostly associated with other structurally and functionally distinct domains, but no FTLDs were identified in higher plants (From over 400 FTLD sequence clusters (at 80% sequence identity) tentatively identified in available databases by Bishnoy et al. , six FTL domains . The str domains  revealed domains A–D. Firse Figure C. This se Figure D. Intere species . The facr plants .M. degradans, a Gram-negative bacterium with broad polysaccharide substrate degrading capability , a large family of pore-forming and platelet-aggregating toxins (S. pneumoniae (GA41301 1.2 and 1.1) and S. mitis FTLDs with the structure of AAA revealed the conserved triad of basic residues (His-Arg-Arg) that in the AAA structure  were shown to interact with the hydroxyl on C4 to provide α-Fuc specificity. The other residues in the primary and extended carbohydrate-binding sites of AAA are not conserved among the three streptococcal FTLDs and could reflect recognition of different fucose-containing oligosaccharides  that encircle the binding cleft  from pability  and a ge regulon  in the Gg toxins –23. CompBishnoi et al.  recentlyDictyostelium discoideum) are trimers of protein subunits that carry two distinct lectin domains: an N-terminal “discoidin” domain that displays the FTL fold  H-type domains  from the slime mold  (B. floridae)  . In moll1008811) –26, as w1008811) –52. Similoridae) , 30.Xenopus spp. FTLs are organized from single FTLDs to combinations of two, three, or four FTLDs and as chimeric proteins containing five tandemly arrayed FTLDs adjacent to a PXN domain  of DiscI and DiscII remains to be rigorously established  or l-Rha  as non-reducing terminal residues on the microbial surface (Dicentrarchus labrax) and gilt head bream  , followed by viral  challenge, Cho et al. , the FTL JspFL was only upregulated in spleen, while it was also constitutively expressed in liver and gills  and Garnderella vaginalis, among others, produce CDCs  that bind to and disrupt the host cell membrane  and Lewis b (Leb) moieties], and it has been controversial whether the fucose-binding site remains masked in the CDC monomer and is only exposed following contact with the cell surface , discussed in a previous section, are highly polymorphic proteins stored in the acrosomal rings of sperm cells that bind to the surface of the egg perivitelline envelope during fertilization (Pinctada martensii) is highly expressed in hemocytes and gill and significantly upregulated (13-fold) by infectious challenge , suggesting that PmF-lectin is involved in the innate immune response (Littorina littorea) has been hypothesized as an immune defense system  and with several oyster (Crassostrea spp.) and mussel (Mytilus sp.) species , enzymes, or other proteins. Thus, FTLs are essentially pleiotropic and can orchestrate a vast array of functions based on “self” and “non-self”-recognition that encompass not only innate immunity but also fertilization, cell adhesion, and microbial virulence, among others yet to be unraveled Figure . In rece species . In thisbona fide FTL homologs are detectable in genomes of eutherian mammals  and immunoglobulin- and B/T cell-mediated immune responses and solely rely on innate immunity for defense against infection. Thus, the increased FTLD diversification in hemichordates and cephalochordates, together with the lack of FTLDs in urochordates could be rather attributed to compensatory effects among multiple lectin families, depending on selective advantage(s) that each can provide to any given taxa as more or less effective pattern recognition receptors in innate immunity. In support of this view, it is noteworthy that the urochordata ascidian Clavelina picta, which lacks FTLs, expresses a highly diversified repertoire of fucose-binding CTLs, suggesting that the expansion of the CTL repertoire probably reflects the selective advantage that fucose-binding CTLs provides over FTLs to the ascidian’s innate immune responses  the different specificity of their binding sites, (b) the distinct architecture of the presentation and multivalency of the carbohydrate ligands on the microbial cell surface or the host, and (c) the biophysical properties of the microenvironment where the interactions occur –14. Bishesponses , 67. In  complex , 67. TheThe rapidly expanding genomic databases and their increasing availability for numerous animal species have provided further insight into the structural and functional diversification of lectin repertoires from prokaryotes, invertebrates, protochordates, and vertebrates. In this context, the recent identification of novel lectin families such as the FTLs –14, undeGV designed, drafted, and edited the final manuscript; LA and MB developed and analyzed structural models, evaluated and edited the draft manuscript; and MC, CF, and KS evaluated and edited the draft 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.


Self-nonself discrimination is a common theme for all of the organisms in different evolutionary branches, which is also the most fundamental step for host immune protection. Plenty of pattern recognition receptors (PRRs) with great diversity have been identified from different organisms to recognize various pathogen-associated molecular patterns (PAMPs) in the last two decades, depicting a complicated scene of host-pathogen interaction. However, the detailed mechanism of the complicate PAMPs–PRRs interactions at the contacting interface between pathogens and hosts is still not well understood. All of the cells are coated by glycosylation complex and thick carbohydrates layer. The different polysaccharides in extracellular matrix of pathogen-host are important for nonself recognition of most organisms. Coincidentally, massive expansion of PRRs, majority of which contain recognition domains of Ig, leucine-rich repeat (LRR), C-type lectin (CTL), C1q and scavenger receptor (SR), have been annotated and identified in invertebrates by screening the available genomic sequence. The phylum Mollusca is one of the largest groups in the animal kingdom with abundant biodiversity providing plenty of solutions about pathogen recognition and immune protection, which might offer a suitable model to figure out the common rules of immune recognition mechanism. The present review summarizes the diverse PRRs and common elements of various PAMPs, especially focusing on the structural and functional characteristics of canonical carbohydrate recognition proteins and some novel proteins functioning in molluscan immune defense system, with the objective to provide new ideas about the immune recognition mechanisms. All of the organisms, including prokaryote, plant, and animal are facing potentially life-threatening infections by a variety of microbial pathogens. The survival of the host depends on its ability to recognize infectious microbes and induce appropriate defense responses ,2. The iIn the long-term evolution, the immune protection first starts with non-professional immune cells in plant, cnidarians, and nematode, and the epithelial cells are able to mediate innate immune responses . The truThere are four major classes of biological macromolecules that are required for all the living organisms, including carbohydrates, proteins, nucleic acids, and lipids. They are composed of different types of subunit  and exhibit different profile in component, structure, and distribution. For example, the polysaccharides in the extracellular matrix (ECM) of algae, plants, bacteria, fungi and animals are different, such as cellulose in plants, peptidoglycan (PGN) in bacteria, chitin and glucans in fungi, and sulfated polysaccharides in animals . The fouCarbohydrates (glycans), as the most stable organic macromolecule with complex structure, have long been known to play fundamental roles in antigen formation and innate immune recognition ,11. GlycWith the long evolution, all organisms have developed an effective immune recognition system, in which several families of germline-encoded PRRs can detect the conserved molecular patterns of microbes. Recently, a picture of the evolutionarily conserved sensing system has emerged by comparing the structural homologies of these pathogen sensors across species . Most PRIn terms of structure, almost all of the PRRs contain specific domains classified into different types. Immunoglobulin (Ig) domain, leucine-rich repeat (LRR) domain, C-type lectin (CTL) domain, C1q domain, and scavenger receptor (SR) cysteine-rich domain are identified as the most prominent motifs, which are also rearranged with various combinations to build immune recognition rhyme . For exaArabidopsis [Nematostella vectensis [Crassostrea gigas, there is significant expansion of C1q, CTL, and Ig recognition domains, and 321 C1qDCs, 266 CTLs, and 190 FREPs [Strongylocentrotus purpuratus [Recently, species-specific expansion of recognition domains has been significantly characterized in plants and invertebrates by screening the available genomic sequence. There is a big family of LRR containing recognition proteins reported in plant bidopsis , and 187ectensis . In oyst90 FREPs  are annorpuratus . These rThe sophisticated repertoire of PRRs in molluscs could be mainly classified into several families, depending on their recognition domains, such as Ig domain containing PRRs , LRR domain containing PRRs (TLRs and LRRs), lectin domain containing PRRs , C1q domain containing PRRs (C1qDC), SR domain containing PRRs (SRs), and other domain containing PRRs  . These mIg domain is an evolutionarily ancient domain, and it can tandemly build a functional protein or assemble complexes with other domains. An individual Ig domain is approximately 100 amino acids in length and forms an “Ig-fold” structure, consisting of two anti-parallel β-sheets packed face to face . Four seC. gigas [Biomphalaria glabrata [Aplysia californica [C. gigas [Lottia gigantea [B. glabrata, a surprising diversity of FREP genes were generated through point mutation and recombination [FREP is a kind of Ig and fibrinogen-like (FBG) domain containing protein with high levels of sequence diversity, and it is involved in innate defense responses of invertebrates . FREPs hC. gigas ,23, snaiglabrata ,25, and ifornica . There agigantea , respectbination , and thebination ,29. But,M. galloprovincialis was significantly up-regulated after LPS, LTA, poly I:C, and zymosan stimulations [AiFREP-1 from scallop Argopecten irradians could agglutinate Gram-negative bacteria E. coli JM109, V. anguillarum and Gram-positive bacteria M. luteus in the presence of calcium ions [AiFREP-2 could bind different PAMP ligands including LPS, PGN and GLU, and various microbes, including Gram-negative bacteria (V. anguillarum), Gram-positive bacteria (Staphylococcus aureus), and fungi (Pichia pastoris and Yarrowia lipolytica) [B. glabrata are also able to bind trematode Echinostoma paraensei sporocysts, as well as a variety of microbes (Gram-positive and Gram-negative bacteria and yeast) with certain specificity with respect to pathogen type [E. paraensei, whereas FREPs with higher molecular-weight bound to Gram-positive, Gram-negative bacteria, and yeast [M. galloprovincialis were reported to promote the phagocytosis of fluorescent beads [Molluscan FREPs have been reported to be involved in immune response as an important kind of PRRs, mainly with binding and opsonic activities . The mRNulations . AiFREP-ium ions . AiFREP-olytica) . FREPs fgen type . FREP4 ond yeast . Except nt beads .However, the mechanisms of binding specificity of molluscan FREP and the polymorphism of Ig domain still need further investigation. The identification of more FREPs and their polymorphisms, and the comparison of structure and function will further clarify the taxonomic diversification of FREPs and their underlying recombination mechanisms in molluscs , providiC. gigas (CgSiglec-1) with two I-set Ig domains [CgSiglec-1 protein (rCgSiglec-1) could bind poly sialic acid (pSIAS), LPS, and PGN in a dose-dependent manner, which was different from other Siglecs characterized by binding ligands only in a sialic acid-dependent manner [CgSiglec-1 by specific polyclonal antibodies enhanced the LPS-induced cell apoptosis and phagocytosis towards V. splendidus. The broader PAMP binding spectrum and the induction of cellular immunity might endow CgSiglec-1 with important roles in the immune response of oyster against a variety of foreign invaders [CgJAM-A-L) was identified from oyster C. gigas with typical JAM-A domain, which functioned as an adhesion molecule [CgJAM-A-L could bind to LPS, PGN, LTA, MAN, GLU, and poly I:C, as well as various microorganisms including M. luteus, S. aureus, E. coli, V. anguillarum, V. splendidus, P. pastoris, and Y. lipolytica. The phagocytic rates of oyster hemocytes towards Gram-negative bacteria V. anguillarum and yeast P. pastoris were significantly enhanced after the incubation of rCgJAM-A-L [CgSiglec and CgJAM-A) in mollusc would provide useful clews for the evolutionary study of Ig domains as well as the adaptive immunity.Except for FREPs, several Ig domain only proteins have also been identified as vital PRRs in the mollusc immune system. Siglec is a member of IgSF, which involves in the host-pathogen recognition, cell–cell interactions and subsequent signaling pathways in the immune and nervous systems in vertebrates. The orthologs of mammalian Siglec are composed of one N-terminal V-set Ig domain, C2-set Ig domains, and cytosolic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) ,35. The  domains . The rect manner . The siginvaders . Junctioinvaders . A strucmolecule . The recgJAM-A-L . These rLRR domain is a most widespread protein domain, which has a conserved core consensus of L-x-x-L-x-L-x-x-N to form a β-strand followed by a more variable sequence . PDB strDrosophila [Cg26493-D2 elevated after challenges with LPS and all Vibrio strains, while Cg13671 was only induced by V. anguillarum. About ten bivalve TLRs have been identified to participate in immune recognition of various microbes and PAMPs. A newly identified non-phagocytic receptor CgTLR-6 from C. gigas exhibited affinity to LPS and PGN, and broader recognition spectrum for bacteria and fungi [CgTLR1–4 from C. gigas lack the activities to recognize specific PAMPs, but they can activate NF-κB responsive reporter in HEK293 cells with a dose-dependent manner [HcToll1, HcToll-2, and HcToll3) from the pearl mussel H. cumingii could respond to bacterial and viral challenge and regulate the expression of antimicrobial peptides (AMPs) [Chlamys farreri, including SARM (ORF08051) and TRAM/TICAM2 (ORF08726), indicating the possibility of MyD88-independent TLR signaling pathway in mollusc [The LRR-containing proteins could be divided into two groups, proteins containing LRR motif and other motifs or domains, and proteins merely containing LRR motif termed LRR-only proteins . The TLRosophila  and V-tyosophila , suggestosophila . For exand fungi . Astoniss (AMPs) ,51,52. As (AMPs) . Recentl mollusc . All of C. farreri. Six CfLRRops (CfLRRop 1-6) containing varied number (1–4) of LRR domains have been identified [CfLRRop-1, -2, and -3 in hemocytes could be induced by the stimulations of LPS, PGN, GLU, and poly I:C, while the mRNA transcripts of CfLRRop-4, -5, and -6 in hemocytes could respond to the stimulations of different microbes, including V. anguillarum, M. luteus, and P. pastoris. The recombinant CfLRRop-1, -3, -4, -5, -6 proteins, but not rCfLRRop-2 protein, could directly bind LPS, PGN, GLU, and poly I:C with varied affinity, indicating LRR-only proteins represented a new type of PRR in bivalve with similar recognition characters as TLR [CfLRRop-1 and -2, but not rCfLRRop-3, could induce the release of TNF-α in primary cultured scallop hemocytes. rCfLRRop-4, -5, and -6 could induce varied immune effectors, including tumor necrosis factor α, superoxide dismutase, catalase, and lysozyme. The versatile CfLRRops could function in immune response not only as PRRs, but also as an immune effector or pro-inflammatory factor, suggesting a functional differentiation among the diverse CfLRRops [The versatile functions of proteins merely containing LRR motif  in immune response have also been recently reported in scallop entified ,55,56 wis as TLR ,55,56. ACfLRRops ,55,56. TCfLRRops ,55,56.www.imperial.ac.uk/research/animallectins/), and at least seven groups of lectins have been identified in mollusc, including C-type, P-type, F-type, I-type, galectins, ficolins, and chitinase-like lectins [Lectins are the most classical carbohydrate-binding proteins to bind the glycans of glycoproteins and glycolipids with high affinity. They play crucial roles in the innate immune responses of invertebrates by recognizing and eliminating pathogens . Based o lectins ,59. Two C. gigas [Venerupis philippinarum (VpCTL) [Haliotis discus hannai (CLHd and HdhCTL1) [Hyriopsis cumingii (Hcperlucin and HcLec4) [Ruditapes philippinarum (MCL-3 and MCL-4) [C. farreri  [C. gigas (CgCLec-1-CgCLec-7) [A. irradians (AiCTL-1-AiCTL-10) [CTLs are structurally characterized by double-loop composed of two highly conserved disulfide bridges located at the bases of the loops, and they can recognize and bind to terminal sugars on glycoproteins and glycolipids in a calcium-dependent manner ,60. GeneC. gigas . More th (VpCTL) , two froHdhCTL1) ,63, Hyri HcLec4) ,65, and d MCL-4) ,67,68, rgCLec-7) , and ten2+-binding site 2 in CRDs. Most molluscan CTLs contain single CRD, while there are three or four CRDs in CfLec-3 and CfLec-4 from C. farreri [AiCTL-9 from A. irradians [2+-dependent carbohydrate binding. There are four Ca2+-binding sites in this structure, among which the site 2 is known to be involved in the carbohydrate binding [2+-binding sites 2, there are two conserved motifs determining the CRD binding ability. In vertebrates, the first motif is always EPN (Glu-Pro-Asn) or QPD (Gln-Pro-Asp), and the second one always WND (Trp-Asn-Asp). While in invertebrates, much more diversities are reported, and there are at least seven types in the first motif and more than ten types in the second motif presented in different molluscan CTLs [The diversity of CRD structure is mainly embodied in the number of CRD as well as variety of Ca farreri ,71 and Arradians , respectrradians . The CRD binding ,73,74. Fcan CTLs . The fircan CTLs . HoweverCfLec-3 from C. farreri with three CRDs can bind more PAMPs than CfLec-1 and CfLec-2 with single CRD [AiCTL-9, a multi-CRD lectin from scallop A. irradians, can bind LPS, PGN, glucan, and mannan [CfLec-4 from C. farreri , rCRD3 and rCRD4 could bind to all four PAMPs tested, while rCRD1 and rCRD2 could only bind to LPS and mannan [2+-binding site 2 displayed different PAMPs binding spectrums. In C. farreri, CfLec-1 containing the motif EPD could bind LPS, PGN and mannan in vitro, while CfLec-2 with the same motif could also bind zymosan besides the other three PAMPs [HcLec4 in H. cumingii with a “LND” motif could bind to LPS and PGN, while CRD3 of HcLec4 with “LND and EPN” motifs could not bind to LPS and PGN [R. philippinarum not only markedly suppressed the growth of Alteromonas haloplanktis, but also significantly enhanced the hemocyte phagocytic ability toward the bacteria [C. farreri [C. gigas [H. cumingii, the bacteria clearance efficiency and the expression level of AMPs were both increased after HcLec4 was knocked down, revealing that HcLec4 exerted its antibacterial effect by regulating the expression of AMPs at the early stage of bacterial infection [Diverse immunological functions of C-type lectins in molluscs have been demonstrated in the past decades. The mRNA transcripts of almost all the CTLs are increased after stimulation with pathogens or PAMPs, implying that they are involved in innate immune response ,76. As angle CRD . AiCTL-9d mannan . Among td mannan . It is nee PAMPs ,80. Simi and PGN . Besidesbacteria ,81. Simi farreri ,80 and CC. gigas  also inhnfection .A large number of CTLs identified from molluscs shared similar structural features and displayed versatile immunocompetence. Since molluscs lack of antibody mediated adaptive immunity, abundant CTLs with diverse expression profiles and bioactivities might function as nonclonal effectors in the molluscan immune system.CvGal identified from the hemocytes of C. virginica [PoGal-1 and PoGal-2 from P. fucata [AiGal-1 and AiGal-2 from A. irradians [R. philippinarum [Galectins are a family of most conserved and ubiquitous lectin, which is defined by a conserved CRD with a canonical amino acid sequence and affinity primary for β-galactoside . Similarirginica , PoGal-1. fucata ,85, AiGarradians ,87, and ppinarum . The cluppinarum ,85. P. fucata, PoGal-1 and PoGal-2, were significantly up-regulated after V. alginolyticus stimulation, suggesting that PoGals were involved in the immune response against bacteria [CvGal in C. virginica facilitated particularly the recognition of a variety of potential microbial pathogens, unicellular algae, and preferentially Perkinsus trophozoites [CvGal and P. trophozoites suggests that CvGal functions as a hemocyte surface receptor for the parasite and mediates phagocytosis [Perkinsus olseni and agglutinate V. tapetis in vitro [AiGal-2 from A. irradians displayed high binding and agglutinating activities toward a variety of microbes, including V. anguillarum, M. luteus, E. coli, V. fluvialis, and Edwardsiella tarda, and could also recruit hemocytes and promote hemocytes encapsulation. These results suggested that AiGal2 functioned as a pattern recognition receptor in immune defense and contributed to the nonself recognition and elimination in cellular immune response of bay scallop [It has been demonstrated that galectin of molluscs can target glycans on the surfaces of bacteria and parasites, and plays a crucial role in innate immune responses . Two galbacteria ,85. CvGahozoites . The strocytosis . MCGal ein vitro . AiGal-2 scallop .Pinctada martensii (PmF-lectin) and C. gigas. The mRNA transcript of PmF-lectin is abundant in hemocytes and gill, and its expression level is dramatically up-regulated after a challenge with V. alginolyticus, suggesting that PmF-lectin is involved in the innate immune response [F-type lectins (fucolectins) are fucose-binding proteins, which have also been identified in oyster response . CgClp1 and CgClp2) were identified from C. gigas, and their mRNA expressions in hemocytes could be induced by LPS stimulation, strongly suggesting that these two close paralogous genes were involved in oyster immunity [ChFCN) was identified from C. hongkongensis. It contained a typical signal peptide and a fibrinogen-related domain at the N- and C-terminus, respectively, but no additional ficolin collagen-like domain. ChFCN mRNA transcripts increased under microbial infections and the recombinant ChFCN protein could bind and agglutinate microorganisms, and enhance the phagocytosis [The chitinase-like lectins and ficolin-like proteins are also found in molluscs. Two chitinase-like proteins  was isolated by affinity chromatography with the activities to agglutinate and inhibit biofilm formation of Staphylococcus aureus, suggesting that this lectin might be a potential alternative to the conventional use of antimicrobial agents in the treatment of infections caused by Staphylococcal biofilms [Recently, some new family lectins were proposed in molluscs. For instance, a glycosylated galactose-binding lectin (MCL) from analysis . A new lbiofilms .M. galloprovincialis (MgC1q) [Hyriopsis cumingii (HcC1qDC5) [C. farreri (CfC1qDC and CfC1qDC-2), A. irradians (AiC1qDC-1 and AiC1qDC-2) [C. gigas (CgC1qDC-1–4) [MgC1q in M. galloprovincialis hemocytes was drastically increased after the stimulations of Gram-positive bacteria and Gram-negative bacteria [CfC1qDC (rCfC1qDC) and AiC1qDC-2 (rAiC1qDC-2) proteins both bound LPS, PGN, β-glucan, and poly I:C, while the latter could also bind mannan and yeast-glucan [AiC1qDC-2 contributed to its broader bacterial agglutinating spectrum to fungus P. pastoris, Gram-positive bacteria B. subtilis, and Gram-negative bacteria E. coli and V. anguillarum [CgC1qDCs in oyster (CgC1qDC-2–4) could enhance the phagocytosis of oyster hemocytes, and the enhancements towards Gram-negative bacteria were significantly higher than that towards Gram-positive bacteria, indicating the important roles of molluscan C1qDC as an opsonin in the clearance of invading microbes [CgC1qDC-3 exhibited higher binding affinity to LPS and stronger opsonization, and its mRNA expression could be up-regulated rapidly and persisted upon the secondary challenge with homologous Vibrios. It was proposed to exert efficient functions in the immune response against invading pathogens [CfC1qDC could interact with human heat-aggregated IgG, which gave a hint for the conservative property of C1qDC proteins to functionally interact with immunoglobulins in evolution [C1q domain containing proteins (C1qDCs) are a family of proteins containing a globular head C1q domain (ghC1q) in C-terminus, which recognize a broad range of ligands and trigger a serial of immune responses, such as the classical pathway of complement . There w (MgC1q) , pearl mcC1qDC5) , C. farrC1qDC-2) , and C. qDC-1–4) . All of bacteria . The rect-glucan ,98. The uillarum . Moreovemicrobes . Comparaathogens . Most inAn expanded set of 321 and 168 C1qDCs are annotated in oyster  and mussP. martensii and scallop C. farreri [CfSR was significantly up-regulated by the stimulations of LPS, PGN, and β-glucan. The recombined protein CfSR displayed unique broad ligand-binding properties not only for acetylated low density lipoprotein (Ac-LDL) and dextran sulfate, but also for various PAMPs, such as LPS, PGN, mannan, and zymosan [C. gigas are highly up-regulated in response to summer mortality syndrome, hypoxia, and bacterial challenge [SRs represent a large family of endocytic receptors with multifunction to bind and internalize a variety of microbial pathogens, as well as modified or endogenous molecules derived from the host, and contribute to a range of physiological or pathological processes . Accordi farreri . The mRN zymosan . Moreove zymosan . The mRNhallenge , suggestC. gigas genome, and six of them  have been characterized [CgPGRP family [A. irradians (AiPGRP) [C. farreri (CfPGRP S1) [CgPGRP-L is close to that of long PGRP groups, it is homologous to short PGRPs and possesses an additional goose-type (g-type) lysozyme domain, suggesting that CgPGRP-L may have both binding and lytic functions against the bacterial cell wall [CfPGRP-S1 displayed strong activities to agglutinate Gram-positive bacteria M. luteus and Bacillus subtilis, while weak activities to agglutinate Gram-negative bacteria E. coli [E. coli and S. aureus in virtue of the conserved Zn2+ binding sites in amidase-2/PGRP domain. The recombinant PGRP-S1S (rPGRP-S1S) from oyster C. gigas did not show any bactericidal activity, but agglutinated E. coli and induced secretion of granular contents by hemocyte degranulation [PGRPs are a kind of highly conserved PRRs that specifically bind to PGN, a unique cell wall component of all virtual bacteria, but is not present in eukaryotic cells . Nine PGcterized ,69. A poP family . Mollusc(AiPGRP)  and ZhikPGRP S1) . The ideell wall . CfPGRP- E. coli . It alsonulation . AlthougP. fucata (poLGBP) [C. farreri (CfLGBP) [CfLGBP exhibited obvious binding and agglutination activity toward various PAMPs and microorganisms, and the polymorphism of 7679 G/G in CfLGBP enhanced its binding activity to LPS and β-glucan, which was associated with the disease resistance of scallop against V. anguillarum [CgβGBP-1 and CgβGBP-2) reported in oyster. Although domain structures of both CgβGBPs are similar to other invertebrate βGBPs, they seem to have evolved with different immunological functions. The recombinant CgβGBP-2 enhanced the phenoloxidase (PO) activity of hemocyte suspensions under the presence of laminarin, but rCgβGBP-1 did not show this enhancement. Because there are integrin recognition sites that areidentified in CgβGBP-1, but not in CgβGBP-2, it is suggested that the hemocyte-related functions of CgβGBP-1 are possibly evolved from integrin [Perna viridis with an inherent serine protease activity [The GNBP family, including the LPS and β-1,3-glucan binding protein (LGBP), and β-1,3-Glucan binding proteins (βGBP), can bind Gram-negative bacteria, LPS, and GLU. The information about molluscan GNBP is still limited and there are only a few GNBP genes identified from (poLGBP)  and C. f(CfLGBP) , respect(CfLGBP) ,111. Theuillarum . There aintegrin . Additioactivity . It agglExcept for the canonical carbohydrates recognition proteins, some novel molecules were discovered in molluscs to play roles in PAMPs recognition. Concomitantly, some unconventional immune defense strategies or novel pathopoiesis mechanisms are prompted with great potentiality. These un-traditional recognition domains or proteins are under revealing with increasing attention. C. gigas (CgDM9CP-1) by two laboratories. DM9 domain was originally identified in Drosophila melanogaster with no defined functions. The DM9 containing protein (CgDM9CP-1) was significantly enriched from oyster hemolymph by affinity chromatography through a mannose-conjugated cellulose column. CgDM9CP-1 exhibited high binding specificity and avidity toward d-mannose residue [CgDM9CP-1 demonstrated that Asp22 and Lys43 were the critical residues for ligand recognition [CgDM9CP-1 protein was found to mainly distribute on the surface of C. gigas hemocytes, and it could be translocated into cytoplasm and colocalized with the engulfed microbes during hemocytes phagocytosis, which collectively indicated its important role as classical PRR. It is worth noting that DM9 domain has been found to exist in various proteins from a number of species, and its existence in prokaryotic cells indicates that the universal DM9 domain is an ancient protein domain that probably evolved from prokaryotes [Recently, a novel PRR with two DM9 domains was identified from oyster  residue . It servognition ,115. Morkaryotes . But, thC. gigas sooner after the first publication. The oyster CgCaspase-3 was confirmed to have the specific binding activity to LPS, but not to LTA, mannan, and β-1,3-glucan by ELISA-based LPS binding assay and surface plasmon resonance (SPR) analysis. The in vivo interaction of CgCaspase-3 with LPS specifically inhibited the cell apoptosis, just opposite to pyroptosis promoting effect of LPS-caspase binding in mammals [CgCaspase-3 was dependent on CASc domain, which was totally different from mammalian caspase-4/5/11 relying on the CARD domain. In addition, CgCaspase-1, only containing one CASc domain, also displayed direct LPS binding activity, which was similar to CgCaspase-3 [Caspases is well-known as a family of cysteine-dependent aspartate-directed proteases for their key roles in apoptosis and inflammatory responses. Recently, they were reported as the PRRs to play important role in immune recognition. Following an unexpected result that cytoplasmic LPS could activate caspase-11 “non-canonical inflammasome” in a TLR4-independent manner in mice , human c mammals . Interesaspase-3 . The greC. gigas (rCgIL17-5) was proved to directly bind to PGN, LPS, poly (I:C), and β-1,3-glucan, with the highest affinity to PGN, which had never been reported in vertebrate interleukins [CgIL17-5 was strongly up-regulated after PGN stimulation, suggesting that its involvement in the recognition of diverse pathogens as a PRR and preference to recognize Gram-positive bacteria [Eisenia foetida, the coelomic cytolytic factor (CCF), displayed both lectin activity to bind LPS, PGN, and GLU, as well as TNF-like lytic activity [CgIL17-5 could significantly inhibit the growth of both Gram-positive bacteria M. luteus and Gram-negative bacteria E. coli, indicating its direct anti-bacteria effect by inhibiting their proliferation [In mammals, cytokines are the major regulators of the host immune response by the interaction with specific receptors. An unexpected carbohydrate-binding (lectin) property of cytokines was discovered in the study of cytokine-receptor interaction. Some cytokines could recognize and bind to the specific glycosylation sites of their individual receptors and induce immune modulation ,121. Forrleukins . The mRNbacteria . Anotheractivity . The lecactivity . Moreoveferation . The endferation .CgPEPCK) in oyster C. gigas, which is well known as a key enzyme that is involved in the metabolic pathway of gluconeogenesis, also displays the LPS and PGN binding activities [CgAK) from the hemolymph of C. gigas by LPS affinity chromatography. CgAK could directly bind to LPS in a concentration-dependent manner. The interaction with LPS significantly decreased the ATP hydrolytic activity of CgAK and this in turn led to the accumulation of ATP in vitro, which is of great importance for the detection of pathogens and the induction of host immune responses [Metabolic enzymes have long been known accustomedly for their function of catalyzing substrate to produce new products. Recently, the unexpected non-metabolic functions of metabolic enzymes were observed in the context of inflammation in both vertebrates and invertebrates. For example, the metabolic enzyme hexokinase could unexpectedly act as a pattern recognition receptor to recognize bacterial peptidoglycan and trigger the activation of inflammasome in mice ,128. Simtivities . Besidesesponses . These nRecognition is the fundamental step for the interaction of hosts–pathogens. Many glycans show remarkably discontinuous distribution across various microbes, and certain lineage-specific glycans have become important signals for nonself recognition of hosts by a toolkit of innate recognizing molecules . Coincid


The Tribbles (TRIB) pseudokinases control multiple aspects of eukaryotic cell biology and evolved unique features distinguishing them from all other protein kinases. The atypical pseudokinase domain retains a regulated binding platform for substrates, which are ubiquitinated by context-specific E3 ligases. This plastic configuration has also been exploited as a scaffold to support the modulation of canonical MAPK and AKT modules. In this review, we discuss the evolution of TRIBs and their roles in vertebrate cell biology. TRIB2 is the most ancestral member of the family, whereas the emergence of TRIB3 homologs in mammals supports additional biological roles, many of which are currently being dissected. Given their pleiotropic role in diseases, the unusual TRIB pseudokinase conformation provides a highly attractive opportunity for drug design. Pseudoenzymes are inactive counterparts of classical enzymes and have evolved in all kingdoms of life, where they regulate a vast array of biological processes. The pseudokinases are one of the best-studied families of human pseudoenzymes.Eukaryotic TRIB pseudokinases evolved from a common ancestor (the human TRIB2 homolog), and contain a highly atypical pseudokinase domain fused to a unique docking site in an extended C tail that binds to ubiquitin E3 ligases.TRIB evolution has led to the appearance of three mammalian TRIB pseudokinases, termed TRIB1, TRIB2, and TRIB3, which contain both unique and shared features.In cells, TRIB pseudokinases act as modulators of substrate ubiquitination and as molecular scaffolds for the assembly and regulation of signaling modules, including the C/EBPα transcription factor and AKT and ERK networks.TRIB1 and TRIB2 have potent oncogenic activities in vertebrate cells, and recent evidence also suggests that TRIB2 acts as a tumour suppressor, consistent with the requirement for balanced TRIB signaling in the regulation of transcription, differentiation, proliferation, and apoptosis. Protein kinases and phosphorylation modulate all aspects of eukaryotic cell biology and, together with members of the Ubiquitin system, have become highly significant for mechanistic drug targeting TRIB pseudokinases represent a subbranch of the CAMK subfamily in the human kinome in vitroin cis with the TRIB regulatory pseudokinase domain In mammalian cells, the three related TRIB family members (TRIB1–3) are classed as serine/threonine pseudokinases that either lack (TRIB1), or have low, (TRIB2 and TRIB3) vestigial ATP affinity and phosphotransferase capacity when analyzed Trbl), which encodes a pseudokinase with developmental roles in this model genetic organism. Trbl was discovered through three independent Drosophila screens, which revealed genes and mutations that affect either oogenesis via Slbo, the alpha ortholog of the mammalian CCAAT enhancer-binding protein (C/EBPα) transcription factor Drosophila ortholog of the CDC25 dual-specificity phosphatases, which initiate eukaryotic S-phase and mitosis and are themselves regulated at various cell cycle checkpoints. Recent data confirmed that TRIB2 protein expression is also cell cycle regulated in human cells, positioning it as a potential modulator of CDC25 phosphatases, which it degrades through a ubiquitin- and proteasome-dependent mechanism TRIB proteins derive their name from the single metazoan fly gene ‘Tribbles’ (Nematostella vectensis) and sponges (Amphimedon queenslandica), whereas TRIB1 and TRIB3 orthologs are restricted to specific later metazoan lineages that led to the vertebrates, where all three TRIBs pseudokinases are consistently preserved. It is likely that the emergence of TRIB1 and TRIB3 at later stages of evolution was driven by unique regulatory requirements associated with signaling integration by these proteins in higher organisms. TRIB1 likely appeared through TRIB2 gene duplication during the diversification of vertebrates from invertebrates, since multiple copies of TRIB2 and a single copy of TRIB1 are observed in non-mammalian chordates, such as fish (Actinopterygii) and reptiles. Remarkably, the TRIB3-specific sequence signature appears in all mammalian lineages, and from the extensive data analyzed here, emerged relatively recently during the diversification of mammals from non-mammalian chordates  and a C-terminal COP1-binding peptide region, which interacts e domain . Recent e domain , the avae domain A and flae domain A. In pasWithin the pseudokinase domain, TRIB-specific constraints are imposed on residues and/or motifs that are dispersed in primary sequence, but that spatially cluster in two critical regions of the pseudokinase domain A, namelyIn addition to characteristic residues and/or motifs in the pseudokinase domain, the critical C-terminal tail involved in protein–protein interactions is readily identified as a distinguishing feature of TRIB kinases; this segment is highly conserved in TRIB kinases, but strikingly different in kinases outside of the TRIB subfamily A. No detInterestingly, the HPW[FL] motif is tethered to the C-lobe of the pseudokinase domain in the recently solved TRIB1 crystal structure, while the DQXVP[D/E] motif is tethered to the N-lobe. Why would such tethering of the C-tail to the pseudokinase domain be important for TRIB functions? One possibility is that this interaction allows efficient coupling of COP1 binding (or other regulatory binding proteins) in the C-tail with coopted substrate-binding functions associated with the degenerate pseudokinase domain. Such a view may also explain why the C-helix and activation loop conformation in the TRIB1 (and presumably TRIB2 and TRIB3) pseudokinase domain are stabilized in a unique conformation In humans, the TRIB gene family has been implicated in many different cancers, but especially in melanoma, lung, liver, and acute leukemias Trib2 gene was first identified as a murine myeloid oncogene, since its overexpression in a bone marrow transplant model leads to the development of a potent transplantable AML with 100% penetrance and short latency TRIB1 gene is located at the same chromosomal locus (8q24.13) as the MYC oncogene and, therefore, MYC  cancer susceptibility genes are co-amplified in a significant percentage of human tumours, where the 8q24 amplicon is the most commonly amplified region across multiple cancer types cis-acting flanking regulatory segments in TRIB itself. Of related interest, TRIB1 gene polymorphisms are also associated with nonalcoholic liver and metabolic syndromes The al locus q24.13 asin vivoIn the hematopoietic system, C/EBPα is essential for granulopoiesis TRIB2 gene promoter to drive expression of the TRIB2 protein, which is then positively reinforced via p30 C/EBPα expression of E2F1 Through their interaction with ubiquitylated transcription factors, or regulation of signaling pathways that culminate in cell type-specific reprogramming, TRIB pseudokinases are regulated by, and fundamental regulators of, gene transcription networks. An analysis of TRIB1, 2, and 3 promoters confirms that TRIB1 and 2 both have putative E2F and C/EBPα transcription factor-binding sites among a large number of conserved canonical transcription factor sequence determinants . ConsistHOXA9, MEIS1, NUP98-HOXD13, and C/EBPAp30TRIB2 expression levels, while generally low in AML, are higher in PML-RARA-positive leukemia than in PML-RARA-negative leukemia TRIB1 gene is located on chromosome 8, the most common chromosomal gain in human AML and APL (distinguished by the fusion oncogene PML/RARA). The hypothesis that TRIB1 and PML/RARA cooperate has been tested and, while they do not cooperate to drive a shorter latency leukemia in vivo, TRIB1 and PML/RARA have functionally redundant inhibitory effects on C/EBPα, which also impacts responses Trans Retinoic Acid  of the vestigial ATP binding detected in human TRIB2 and TRIB3 Leading on from this, is the atypical nucleotide-binding site suitable for targeting with small molecules? Do known protein kinase inhibitors have unappreciated ‘off-target’ effects on TRIB pseudokinases in cells? Can small molecules be designed to probe TRIB structural dynamics and cell biology? In particular, can compounds be identified that promote changes in TRIB stability in different cell types? These classes of chemical might be useful leads for new types of drug.How are the expression levels of TRIB pseudokinases regulated under physiological conditions, and before and during pathology? Related to this, what are the transcriptional networks in operation that fulfill the obligations of the TRIB proteins in eukaryotes, and how have these changed during evolution?The appearance and retention of pseudoenzymes during evolution in essentially all enzyme families suggests that these domains are malleable templates that can be coopted for new biological functions when required