Escherichia coli O157:H7 strains possess three sodC genes encoding for periplasmic Cu, Zn superoxide dismutases: sodC, which is identical to the gene present in non-pathogenic E. coli strains, and sodC-F1 and sodC-F2, two nearly identical genes located within lambdoid prophage sequences. The significance of this apparent sodC redundancy in E. coli O157:H7 has not yet been investigated.Highly virulent enterohemorrhagic sodC genes are less resistant than the wild type strain to a challenge with hydrogen peroxide, thus confirming their involvement in the bacterial antioxidant apparatus. To understand if the different sodC genes have truly overlapping functions, we have carried out a comparison of the functional, structural and regulatory properties of the various E. coli O157:H7 SodC enzymes. We have found that the chromosomal and prophagic sodC genes are differentially regulated in vitro. sodC is exclusively expressed in aerobic cultures grown to the stationary phase. In contrast, sodC-F1 and sodC-F2 are expressed also in the logarithmic phase and in anaerobic cultures. Moreover, the abundance of SodC-F1/SodC-F2 increases with respect to that of SodC in bacteria recovered from infected Caco-2 cells, suggesting higher expression/stability of SodC-F1/SodC-F2 in intracellular environments. This observation correlates with the properties of the proteins. In fact, monomeric SodC and dimeric SodC-F1/SodC-F2 are characterized by sharp differences in catalytic activity, metal affinity, protease resistance and stability.We report that strains deleted of one or more E. coli O157:H7 sodC genes have different regulatory properties and encode for proteins with distinct structural/functional features, suggesting that they likely play distinctive roles in bacterial protection from reactive oxygen species. In particular, dimeric SodC-F1 and SodC-F2 possess physico-chemical properties which make these enzymes more suitable than SodC to resist the harsh environmental conditions which are encountered by bacteria within the infected host.Our data show that the chromosomal and bacteriophage-associated  Escherichia coli (EHEC), including strains of the highly virulent O157:H7 serotype, is responsible for a wide spectrum of diseases ranging from mild diarrhoea to hemorrhagic colitis and the potentially fatal haemolytic uremic syndrome (HUS) [E. coli O157:H7 [E. coli O157:H7 genomes [E. coli K12, it has 1.34 Mb of DNA distributed among 177 DNA segments, termed O islands, that are absent in E. coli K12 [Enterohemorrhagic me (HUS) . The sev O157:H7 ,4. Shiga genomes ,4 has resodC genes in E. coli O157:H7, encoding for periplasmic copper, zinc superoxide dismutases  [Genome analysis have also revealed the presence of three u,ZnSOD) ,4. Cu,Znu,ZnSOD) . As the u,ZnSOD) ,8. Althou,ZnSOD)  and refesodC copies of E. coli O157:H7 is localized on a chromosomal backbone sequence which is shared with E. coli K12 and it is homologous to the sodC gene from this microorganism. In contrast, the other two sodC genes, which encode nearly identical proteins differing for only one amino acid, are embedded within the sequences of two lambdoid prophages (denominated CP-933R and CP-933V in the EDL933 strain) [One of the three  strain) .sodC copies within a single bacterial genome is not unique to E. coli O157:H7 and a similar condition has been described in Salmonella enterica. In fact, many of most virulent Salmonella strains possess a bacteriophage-encoded sodC copy (sodCI) in addition to the chromosomal sodCII gene, which is present in all Salmonella strains [sodCI significantly contributes to Salmonella virulence, whereas sodCII has a modest role in pathogenesis [SodCI enzyme to Salmonella virulence [sodC genes might play a role in O157:H7 virulence.The presence of multiple  strains . A numbeogenesis ,11-13. Rirulence . Based osodC genes of E. coli O157:H7 and on the structural/functional properties of the encoded proteins. Our results point out to differences in gene regulation and in the physico-chemical properties of the enzymes, suggesting that the phage-associated sodC genes encode enzyme variants optimized to resist the harsh environmental conditions encountered by bacteria within the infected host.In this work, we have undertaken an investigation on the regulation of the different Taq DNA polymerase were obtained from EuroClone , Pfu DNA polymerase DNA polymerase from Promega. All other chemicals were purchased from BDH and were of the highest grade available. The oligonucleotides were synthesized by Primm .Antibiotics, bovine serum albumin, isopropyl β-D-thiogalactopyranoside (IPTG), pyrogallol, Triton-X114, Triton-X100 and cell culture products were purchased from Sigma-Aldrich. Restriction endonucleases, DNA-modifying enzyme and the High-Fidelity Expand DNA polymerase were obtained from Roche, Euro E. coli O157:H7 ED597 strain is a clinical human isolate connected to a HUS case . Unless otherwise specified, bacteria were grown at 37°C in Luria-Bertani (LB) medium  or in LB medium solidified with 1.5% (w/v) agar. Anaerobic growth was achieved in GasPack anaerobic jars using LB broth containing 0.2% glucose. When required, the culture media were supplemented with the appropriate antibiotics .All the bacterial strains used in this work are listed in Table sodC genes will be designed as sodC-F1 and sodC-F2. Further details about such a nomenclature are reported in the first paragraph of the results section.In this work the two prophagic E. coli ED597 strain carrying pKD46 [sodC, sodC-F1 or sodC-F2 null mutants, respectively. The mutant strains lacking the chromosomal sodC or the prophagic sodC-F genes, were designated RG101 (ΔsodC::cat), RG102 (ΔsodC-F1::cat) and RG103 (ΔsodC-F2::cat), respectively.All gene replacement experiments were carried out by the lambda-red-mediated recombination procedure as described by Datsenko and Wanner . Brieflyng pKD46 . RecombisodC-F1/ΔsodC-F2 prophagic null mutant, after deletion of the chloramphenicol resistance cassette from the strain RG102, using the FLP recombinase encoded by plasmid pCP20 [sodC-F2::cat mutant into strain RG102 carrying pKD46, recombinants were selected on chloramphenicol LB plates. Deletion of sodC-F2 was confirmed by PCR, using the oligonucleotides Clo-int and 240ATG-F2. The ΔsodC-F1/ΔsodC-F2 mutant strain was designated RG104. Subsequently, the PCR fragment amplified with H7P1 and H8P2 primers, was electroporated into the RG104 deleted of the antibiotic resistance cassette, in order to construct a triple sodC mutant strain. Recombinants were selected on chloramphenicol LB plates and confirmed by PCR using oligonucleotides 120ATGcoli-F and Clo-int. The resulting ΔsodC-F1/ΔsodC-F2/ΔsodC::cat strain was designated RG105.The same procedure was employed to obtain the double Δid pCP20 . After eE. coli O157:H7 ED597 (data not shown). Genomic DNAs were digested with ClaI, fractioned on 1% agarose gels and blotted onto nitrocellulose membrane . PCR fragments obtained with oligonucleotides specific for sodC-F1 or sodC-F2, sodC, and chloramphenicol genes  was amplified by PCR using the oligonucleotides PromEDLFor and PromEDLRev  in pMC1403, has been previously described [To analyze the transcriptional regulation of  pMC1403 , obtainiescribed .E. coli QC771 and QC1110 [pMCPromEcSOD and pMCPromO157 were introduced into d QC1110  and the d QC1110 .sodC genes was carried out according to a described procedure [XbaI-XbaI fragment from pKD3, carrying the chloramphenicol resistance cassette. These DNA fragments were independently electroporated into E. coli ED597 strain carrying pKD46; transformants were selected on LB plates containing the appropriate antibiotics. Recombination events were confirmed by PCR using the oligonucleotides 120ATGcoli-F and Kan-R or Clo-int (Table sodC::3xFLAG-kan) and RG-F107 (sodC::3xFLAG-cat).Addition of 3xFLAG epitope tails at the ends of rocedure . Brieflyrocedure  or pSUB1sodC-F1::3xFLAG-kan) and RG-F109 (sodC-F2::3xFLAG-kan) strains. In these cases, an identical PCR fragment, obtained with oligonucleotides 3xFLAG-F and 3xFLAG-R2 on pSUB11, was used to generate both the tagged strains. The same DNA fragment was also inserted into the RG-F106 (previously deleted of the kanamycin cassette) or RG-F107 strains, to obtain the doubly tagged strains RG-F110 (sodC::3xFLAG/sodC-F1::3xFLAG-kan) and RG-F111 (sodC::3xFLAG-cat/sodC-F2::3xFLAG-kan). Recombinations were confirmed by PCR using primers 240ATG-F1 or 240ATG-F2 and Kan-R on strains RG-F108, RG-F109, RG-F110 and RG-F111, while the couple of primers 120ATGcoli-F and Clo-int was used on RG-F110 and RG-F111.The same procedure was employed to construct RG-F108 , in bacteria cultivated in presence or absence of oxygen. Preparation of bacterial lysates from western-blot and immunodetection with anti-FLAG monoclonal antibodies (Sigma) was carried out as described [2O, immediately mixed with Laemmli lysis buffer [sescribed . Brieflys buffer  and boil2 at 37°C.Synthesis of epitope-tagged SodC and SodC-F proteins was analyzed both in adherent and intracellular bacteria recovered from Caco-2 cells. Caco-2 cells (from a human colonic carcinoma) were maintained in D-MEM containing glucose 1 g/l, supplemented with 4 mM L-glutamine, 1% non-essential amino acids (NEAA) and 10% foetal calf serum in a humidified atmosphere of 5% CO8CFU/ml) grown overnight a 37°C in LB broth. Monolayers were then incubated in a humidified atmosphere of 5% CO2 at 37°C for 5 h. The inoculum was removed and the monolayer was washed (3×) with HBSS to remove non-adherent bacteria. Finally, the monolayer was disrupted by incubation with a solution of 1% Triton-X100 in PBS to release bacteria. The number of adherent bacteria was determined by plating 10-fold serial dilutions onto LB agar plates. Released bacteria were prepared for immunoblotting analysis as described above. For invasion studies, bacteria were allowed to adhere as described for 2 h at which point, the monolayers were washed twice with 37°C HBSS, then overlaid with cell culture medium supplemented with 100 μg/ml gentamicin and incubated for additional 3 h. The monolayers were then disrupted to release intracellular bacteria and CFU/ml determined.Infections of Caco-2 cells were performed essentially as previously described ,21. ConfsodC-F1 and/or sodC-F2 genes of E. coli O157:H7 EDL933 were isolated by PCR amplification, using the chromosomal DNA (obtained from ATCC 700927D) as a template and the oligonucleotides O157-1For (which encompasses the ATG starting site) and O157-1Rev (downstream the translation stop codon)  of sodC-F1 was amplified with the oligonucleotides O157-2For and O157-2Rev, using pO157-F1 as a template. The amplified DNA fragment (about 470 bp) was digested with NcoI and EcoRI and cloned in pHEN-1 [pelB gene of Erwinia carotovora.To analyze the properties of the signal peptide of prophagic n pHEN-1 , obtainiE. coli 71/18 [Soluble and membrane-associated proteins from li 71/18  harbourili 71/18 , pO157-Fli 71/18 ,27.600 of 0.5 at 37°C. Then, IPTG, CuSO4 and ZnSO4 were added to a final concentration of 0.1 mM, 0.25 mM and 25 μM, respectively, and the cells were incubated for additional 3 hours at 37°C. Subsequently, bacterial cells (1.2 × 1010) were harvested by centrifugation , washed with 1 ml of ice cold 10 mM sodium phosphate buffer, pH 7.0, suspended in 0.8 ml of the same buffer and sonicated. Cell debris was removed by centrifugation at 25000 × g for 10 min (repeated twice), and the supernatant, including membranes and cytosolic proteins, was subjected to Triton-X114 partitioning [Briefly, 200 μl of overnight cultures were inoculated into 10 ml of Luria-Bertani medium containing ampicillin (100 μg/ml), and the cultures were grown to a ODitioning ,27. TritE. coli O157:H7 strains. Proteins were subsequently separated by SDS-PAGE and analyzed by western blot.The same procedure was used also to separate soluble and membrane-associated epitope-tagged proteins from E. coli QC871 [4 and 25 μM ZnSO4. After 16 h of growth, cells were harvested by centrifugation and the periplasmic proteins were extracted as described previously [Recombinant SodC was purified as described previously . Solubleli QC871  bearing eviously . Proteineviously  using boSuperoxide dismutase activity was assayed by the pyrogallol method . One uniE. coli O157:H7 ED597 was assayed incubating proteins at a concentration of 0.1 mg/ml at 37°C in 20 mM Tris-HCl, pH 6.8 or 8.0, in the presence of 0.1 mg/ml proteinase K. Aliquots were withdrawn at the indicated times and immediately assayed by the pyrogallol method to measure residual activity [Proteinase K susceptibility of Cu,ZnSODs from activity .EDTA-dependent inactivation of Cu,ZnSODs was analyzed essentially as previously described . Cu,ZnSO6 cells/ml and incubated in presence or absence of 250 μM hydrogen peroxide at 37°C. Aliquots were removed after 1 h and the number of viable cells was determined by serial dilution and plating onto LB agar. The percent survival following hydrogen peroxide exposure was calculated for each strain by the ratio of the CFU obtained upon incubation in PBS and the CFU obtained upon incubation in hydrogen peroxide. Each assays was repeated at least sixteen times, and standard deviations and Student's test were calculated.To analyze susceptibility of deletion mutants to hydrogen peroxide, overnight bacterial cultures grown on LB medium were washed in PBS, diluted to 10Heat capacity versus temperature profiles were obtained with a VP-DSC differential scanning calorimeter . Protein samples were dissolved at 0,2–0,4 mg/ml concentration, dialyzed against 0.1 M potassium phosphate buffer at the appropriate pH and degassed before the calorimetric experiment. The reference cell was filled with degassed dialysis buffer. Both cells were kept under an excess pressure of ~30 psi (~200 kPa) to avoid bubbling during the scan. A scan rate of 60°C/h was used in all the experiments. At the end of each run the solutions were cooled and subjected to a second heating cycle under the same conditions to determine the reversibility of the transitions. After each scan, samples were analyzed for integrity by SDS-PAGE and for residual activity using the pyrogallol method . ThermogE. coli O157:H7 epidemic strains EDL 933 [sodC gene typical of all E. coli strains, the presence of two sodC genes located in lambdoid prophage sequences. The two prophagic genes (sodC-F1 and sodC-F2) are identical except for a T/C mutation at nucleotide 335 downstream the ATG start codon, causing the replacement of a leucine residue (sodC-F1) with proline (sodC-F2). The mature proteins encoded by these two prophagic genes show reduced homology (61% identity), but similar size to the protein encoded by the chromosomal gene . Also the regions that surround the two coding sequences are very similar and only a few mismatches are presents. Some of these are located around the nucleotides 240 and 550 upstream the start codon, and around nucleotide 500 downstream the stop codon. These regions have been chosen to design primers able to discriminate between the sodC-F1 and sodC-F2 genes in E. coli ED597 strain.The complete genome sequencing of the two  EDL 933  has reveE. coli ED597 show a perfect identity with the sodC-F1 and sodC-F2 sequences of the EDL 933 strain, thus indicating a strict conservation of the sodC genes in these two bacterial strains.We have found that the nucleotide sequence of the DNA fragments amplified with the primers 550F1b and 500STOP1R and with the primers 550F2b and 500STOP2R . In contrast, differences in the intracellular amount of SodC and SodC-F proteins were observed under different conditions. To better appreciate the relative differences in expression between sodC and sodC-F, we constructed strains which bear the epitope-tagged copy of sodC and sodC-F1 (RG-F110 strain) or of sodC and sodC-F2 (RG-F111 strain). The relative abundance of epitope-flagged SodC and SodC-F proteins was measured in bacteria cultivated in LB broth under different conditions and in cultured epithelial cells.Expression of As shown in Figure Abundance of SodC and SodC-F1/SodC-F2 was analyzed also in anaerobic cultures and as a function of the temperature of growth (25°C or 37°C)  and rpoS mutant (QC1110) E. coli strains. We have observed that the expression of sodC is drastically reduced in the rpoS mutant strain, whereas sodC-F is not regulated by this alternative sigma factor (data not shown).As previous studies have revealed that coli K12  or in S.himurium , we haveTo examine the levels of SodC and SodC-F proteins in adherent and intracellular bacteria, Caco-2 cells were infected with strain RG-F111. Panel C of Figure sodC genes. Figure sodC and sodC-F1/sodC-F2 deletion.To investigate the role of Cu,ZnSOD in protection from exogenous oxidative stress, the effect of hydrogen peroxide on the survival of wild type and mutant strains. Experiments were carried out with bacteria grown to the stationary phase, to ensure the efficient expression of all  showed that the leader peptide sequence of the two prophage encoded sodC genes possess features compatible with processing either by SpI or by SpII  removes the leader sequence from proteins which are to be exported in the periplasmic space or secreted in the extracellular milieu, while signal peptidase II (SpII) removes the leader peptide from lipid-modified precursor of exported proteins, which are targeted to the periplasmic inner and outer membranes. The N-terminal region of most SodC proteins from Gram-negative bacteria shows the typical features of the signal peptides recognized by SpI , whereasI Figure . SpI couE. coli O157:H7 can be lipid modified and, consequently, anchored to the bacterial membrane, we have initially separated soluble and membrane-associated recombinant proteins from E. coli 71/18 cells [To verify if SodC-F1/SodC-F2 of 18 cells  harbouriE. coli O157:H7 strains. In fact, as shown in figure E. coli O157:H7 either as a soluble protein or as a lipid-modified membrane protein.This results were confirmed by analysis of soluble and lipid-associated epitope-tagged proteins extracted from cells grown for 24 h incubation at 37°C from wild type, RG-F106 and RG-F108 in vivo. In contrast, SodC is a monomeric enzyme [SodC-F1 and SodC-F2 have nearly identical amino acids sequences. Therefore we have cloned, overexpressed and characterized only one of the two proteins, namely SodC-F1. The apparent molecular weight of SodC-F1 determined by gel filtration chromatography is close to 30.6 kDa, roughly corresponding to the molecular weight expected for a dimeric enzyme (data not shown). As the only amino acidic difference between SodC-F1 and SodC-F2 does not involve a residue located at the dimer interface, it is likely that heterodimers between the two proteins may form c enzyme .S. enterica SodCI and SodCII proteins show remarkable differences in specific activity, protease resistance, metal affinity and peroxidative activity, with dimeric bacteriophage-encoded SodCI exhibiting superior stability and activity to monomeric SodCII [in vitro biochemical properties of the two Cu,ZnSODs from E. coli O157:H7.Previous studies have established that bacterial monomeric Cu,ZnSODs have quite different functional features with respect to dimeric enzymes of the same class ,35. For c SodCII . To furtWhile eukaryotic Cu,ZnSODs are highly resistant to proteolytic attack, all bacterial Cu,ZnSODs show susceptibility to proteolysis, monomeric variants showing higher sensitivity ,35,36. IE. coli O157:H7 is rapidly inactivated, and the loss of activity is greater at pH 8.0 than at pH 6.8. On the contrary, SodC-F1 activity is not affected by incubation in the presence of EDTA, independently of pH.Unlike the eukaryotic enzymes which stably bind the active site metals, bacterial Cu,ZnSODs easily lose their cofactors and are therefore irreversibly inactivated in presence of EDTA ,35,37,38As differences in the quaternary structures can influence Cu,ZnSOD activity, possibly through modulation of the active site conformational flexibility, we analyzed the catalytic activity of SodC and SodC-F1 by the pyrogallol method . The monE. coli O157:H7 Cu,ZnSODs was investigated by differential scanning calorimetry (DSC) under standardized experimental conditions . The DSC profiles of the dimeric SodC-F1 protein are characterized by a single peak, which is centered around 87°C at pH 6.0 and around 78°C at pH 7.8 , in the radioresistant bacterium Deinococcus radiodurans [Aquifex aeolicus [ we have observed that other E. coli strains, including the atypical enteropathogenic E. coli strains E110019 (serotype O111:H9) and B171 (serotype O111:NM) and the metal and antibiotic resistant environmental E. coli strain SECEC SMS-3-5 possess additional sodC copies showing high similarity with the E. coli O157:H7 sodC-F. In all the above mentioned microorganisms, Cu,ZnSODs could play a role in protecting cells from environmental reactive oxygen species, but this is not sufficient to explain the apparent redundancy of sodC. In order to better clarify this issue, we have undertaken a functional/structural characterization of the sodC genes of E. coli O157:H7 and their encoded proteins.Besides  strains ,40. Genoiodurans  and in taeolicus . MoreovesodC gene or sodC-F1 and sodC-F2 genes strongly decreases bacterial resistance to a challenge with hydrogen peroxide. This result, which is in line with previous studies carried out with different microorganisms [E. coli O157:H7 resistance against exogenous reactive oxygen species. However, the mutant strain lacking all the three sodC genes is not more susceptible to hydrogen peroxide than the strains lacking sodC or sodC-F1/sodC-F2. A similar lack of additivity of sodC mutations was previously observed in experiments carried out with S. Cholaeresuis sodC mutants [sodC mutants are not killed by exogenous superoxide but, although Cu,ZnSOD has no direct role in hydrogen peroxide scavenging, they are much more sensitive than wild type strains to treatments with hydrogen peroxide [- → O2 + OH- + OH.) [sodC mutants.Inactivation of either the chromosomal rganisms -45, indi mutants . It was  mutants . In thisperoxide ,44,47. T- + OH.) . Althoug- + OH.) . It is pE. coli O157:H7 reflects differences in the regulation and/or in properties of the enzymes encoded by chromosomal and prophagic sodC genes, we have analyzed the intracellular accumulation of the epitope-tagged SodC and SodC-F proteins. We have observed that the chromosomally encoded SodC protein is mainly expressed during the stationary phase, under control of RpoS, and that its intracellular concentration is drastically reduced under anaerobic conditions or in bacteria cultivated at 25°C. This pattern of expression is very similar to that of SodC in non-pathogenic E. coli strains or of SodCII in Salmonella [Salmonella SodCI protein [sodC genes of Salmonella was further completed by the observation that in intracellular bacteria recovered from Caco-2 cells the relative ratio between SodC and SodC-F was changed in favor of SodC-F. This could be due to differences in gene transcription and/or to a differential stability of the two enzymes in intracellular environments. To explore this possibility we have carried out a characterization of some functional/structural properties of the two proteins.To evaluate the hypothesis that the presence of multiple Cu,ZnSODs in lmonella ,10,44. I protein . The matM. tuberculosis [S. enterica [Burkholderia cenocepacia [A first difference we have identified between SodC and SodC-F concerns their exact intracellular localization. As the Cu,ZnSODs from other Gram-negative bacteria, SodC is a fully soluble periplasmic protein. In contrast, a fraction of SodC-F is found in association to the bacterial membrane, likely due to processing of the signal peptide sequence by signal peptidase II. A similar lipid-modification of a superoxide dismutase has been previously reported for the enzyme from rculosis  which, henterica  and Burkocepacia , this isPhotobacterium leiognathi [We have also compared some properties of purified SodC and SodC-F1. SodC-F1 shows higher catalytic activity than SodC. Moreover, dimeric SodC-F1 displays much higher resistance than monomeric SodC to inactivation mediated by proteolytic enzymes or by chelating agents. These properties are correlated with the higher conformational stability of SodC-F1 revealed by differential scanning calorimetry. In fact, we have observed a difference between the melting temperatures of the two enzymes of 10°C and 14°C at pH 6.0 and 7.8, respectively. It is worth nothing that the melting temperature of SodC-F1 is significantly higher than that of the dimeric bacterial enzyme from iognathi  and appriognathi , indicatE. coli O157:H7 SodC-F and those of Salmonella SodCI. In fact, both these genes have been acquired by virulent bacterial strains through bacteriophage-mediated processes, they show differences in their pattern of expression when compared to the chromosomal sodC gene and encode for dimeric proteins which are more stable and active than the monomeric SodC-enzyme.Taken together our data highlights a striking parallelism between the properties of E. coli O157:H7, this investigation supports the suggestion [E. coli genomes, has a function that is largely unrelated to pathogenesis and likely protects the cell from endogenous superoxide produced during the aerobic metabolism. In contrast, the sodC genes carried by prophages, which can be identified only in a subset of bacterial strains, codify for proteins which have structural and regulatory features which likely favour bacterial survival within the hostile environments encountered during the infection.Although further studies are required to clarify the exact role of the Cu,ZnSODs in ggestion  that thesodC genes of E. coli O157:H7 are differently regulated and encode for proteins with distinct structural and functional properties, thus indicating that their functions are not truly redundant. These findings contribute to establish that the acquisition of additional sodC copies within bacterial genomes helps bacteria to expand their ability to withstand the reactive oxygen species generated within the host or in other hostile environments.This work demonstrates that the E. coli O157:H7 mutants construction; performed hydrogen peroxide resistance tests and in vitro expression studies, RG also participated in the design and writing of the manuscript; LN and GR participated in the design of the study and in the draft of the manuscript; LC carried out the DSC experiments; AB coordinated the study, participated in its design and in the writing of the manuscript. All authors read and approved the final manuscript.MDO carried out protein characterization and expression studies in Caco-2 cells and participated in the design and writing of the manuscript; RS and RG carried out


M) or valine (V) at codon 129. All of the mutants lost their ability to prevent Bax-mediated chromatin condensation or DNA fragmentation in primary human neurons. In the breast carcinoma MCF-7 cells, the F198SV, D202NV, P102LV and Q217RV retained, whereas the P102LM, P105LV, Y145stopM and Q212PM PrP mutants lost their ability to inhibit Bax-mediated condensed chromatin. The inhibition of Bax-mediated condensed chromatin depended on the ability of the mutants to generate cytosolic PrP. However, except for the P102LV, none of the mutants significantly inhibited Bax-mediated caspase activation. These results show that the cytosolic PrP generated from the GSS mutants is not as efficient as wild type PrP in inhibiting Bax-mediated cell death. Furthermore, these results indicate that the anti-Bax function is also disrupted in GSS-associated PrP mutants and is not associated with the difference between CJD and GSS.Previously, we have shown the loss of anti-Bax function in Creutzfeldt Jakob disease (CJD)-associated prion protein (PrP) mutants that are unable to generate cytosolic PrP (CyPrP). To determine if the anti-Bax function of PrP modulates the manifestation of prion diseases, we further investigated the anti-Bax function of eight familial Gerstmann-Sträussler-Scheinker Syndrome (GSS)-associated PrP mutants. These PrP mutants contained their respective methionine ( Autosomal dominant inherited prion protein (PrP) gene mutations cause approximately 10% of all prion diseases The underlying molecular mechanism of prion diseases associated with each of these mutations is unclear. Gain of function is suspected as the cause of the disease. However, while the mutant proteins are expressed throughout the lifespan of the individual, disease rarely develops before the fourth or fifth decade of life, indicating that misfolding of PrP caused by the mutation is not sufficient to initiate the neurodegeneration. This prompted us to investigate if loss of function could be involved in the length and severity of disease progression.While the normal function of PrP is still not entirely clear, there is growing evidence for the neuroprotective roles of PrP We previously showed that the GSS-associated A117V PrP mutant with a valine at codon 129 retained the anti-Bax function while all of the 11 CJD-associated PrP mutations carrying a methionine at codon 129 or the FFI-associated D178N PrP mutant lost the ability to prevent Bax-mediated condensed chromatin in human neurons Cultures of human primary neurons were done as previously described under the ethical approval from the McGill University Institutional Review Board V) or a methionine (WT PrPM) at the polymorphic codon 129 M, P102LV, P105LV, Y145stopM, F198SV, D202NV, Q212PM, and Q217RV are the following: F198S, 5′-AGGGGGAGAACTCCACCGAGACCGAC-3′; D202N, 5′-GAACTTCACCGAGACCAACGTTAAGATGATGGAG-3′; Q217R, 5′-CAGATGTGTAT CACCCGGTACGAGAGGGAATCTC-3′; P102L, 5′-GTCAGTGGAACAAGCTGAGTAAGC CAAAAACC-3′; P105L, 5′-GGAACAAGCCGAGTAAGCTAAAAACCAACATGAAGCAC-3′; Y145stop, 5′-CGGCAGTGACTAGGAGGATCGTTACTATCG-3′; and Q212P, GCGTGGTTGAGCC GATGTGTATCACCC. All PrP mutations were confirmed by sequencing using the Sequenase Version 2.0 DNA Sequencing kit  and the polymorphism at codon 129 was confirmed by PCR Sca I and Xba I sites of pBud-EGFP or pBud-EGFP-Bax generating pBud-EGFP/PrP mutants or pBud-EGFP-Bax/PrP mutants.GSS-associated mutations were produced by QuikChange Site-Directed Mutagenesis  from the pcDNA3.1(+)-PrP wild type carrying a valine (WT PrP5 cells) plated onto poly-L-lysine-coated plastic coverslips and MCF-7 cells (2.5×105 cells) plated onto glass coverslips were transfected with the Helios Gene Gun system  at a shooting pressure of 100 psi for human neurons and 220 psi for MCF-7 cells. Cartridges contained 1 µg DNA and 0.125 mg gold per shot 6 cells) plated in six-well plates were transfected with 4 µg of pBud-EGFP/PrP or/PrP mutant DNAs using the Lipofectamine2000 reagent (Invitrogen). To generate SP-CyPrP, N2a cells were transfected with 10 and 20 µg of pBud-EGFP/PrP DNA, as previously described Human neurons , fixed 20 min at room temperature in 4% paraformaldehyde and 4% sucrose in PBS, and the chromatin stained 20 min with 1 µg/mL Hoechst 33342  in PBS. The percentage of cell death was calculated by counting the number of EGFP-positive cells with condensed chromatin over the total number of EGFP-positive cells using a Nikon Eclipse TE2000-U microscope.Twenty hours after transfection, MCF-7 cells and human neurons were washed with phosphate-buffered saline . The percentage of TUNEL-positive cells was obtained by counting the TUNEL- and EGFP-positive cells over the total number of EGFP-positive cells.Human neurons were fixed 20 hours after transfection, as described above, and Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was performed with the TM Red MCF-7 cells were labeled 20 hours after transfection for active caspases with the SR Fluorochrome-labeled inhibitors of caspases (FLICA) poly caspase kit . The percentage of FLICA-positive cells was calculated by counting the FLICA- and EGFP-positive cells over the number of total EGFP-positive cells.g 15 min at 4°C, dried, and solubilized in the Laemmli's sample buffer (2% SDS (w/v), 5% β-mercaptoethanol (v/v), 10% glycerol, 0.01% bromophenol blue (w/v), and 62.5 mM Tris-HCl, pH 6.8).N2a cells were treated 24 hours after transfection with 0.25 µM epoxomycin  and 5 µg/mL Brefeldin A (BioMol) for 18 hours. Subcellular fractionation was performed as described previously g for 15 min at 4°C to separate detergent-soluble proteins (supernatant) from the detergent-insoluble proteins (pellet). Detergent-soluble proteins were concentrated by methanol precipitation and these as well as the detergent insoluble proteins solubilized in the Laemmli's sample buffer for western blotting.Cytosolic proteins obtained from the subcellular fractions were solubilized 20 min on ice in lysis buffer , 0.5% sodium deoxycholate (NaDOC) (w/v), 50 mM Tris-HCl, pH 7.5) and centrifuged at 11 500×Membrane fractions from N2a cells were treated with Peptide-N-glycosidase F (PNGase F) according to manufacturer's instructions . Briefly, proteins from membrane fractions were adjusted to 0.5% SDS and 1% β-mercaptoethanol, denatured 10 min at 100°C and incubated overnight at 37°C with 2 units of PNGase F in the presence of protease inhibitors. The proteins were methanol precipitated as described above.The proteins were separated on 15% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes. The membranes were probed with the anti-PrP 3F4 antibody The intensity of PrP detected by western blot analyses was resolved with the Image Quant TL software . The ratio of cytosolic PrP over membrane PrP or the ratio of nuclear PrP over membrane PrP was calculated.post hoc Scheffé test. A p value≤0.05 was considered a significant difference. To determine the correlation coefficient and its significance, we used the Fisher's r to z transformation.Statistical analyses were performed with StatView software . To determine the statistically significant difference, we used ANOVA followed by a M) or valine (PrPV) at codon 129 prevented Bax-mediated condensed chromatin. Similar to our previous observations with CJD mutants, the P102LM, P105LV, Y145stopM and Q212PM PrP mutants completely lost their ability to prevent Bax-mediated condensed chromatin. However, F198SV, D202NV, and Q217RV completely while the P102LV partially retained the anti-Bax function. None of the GSS-associated PrP mutants co-expressed with EGFP induced chromatin condensation in MCF-7 cells, except for the Y145stopM which, consistent with previous observations GSS-associated PrP mutations P105L, A117V, F198S, D202N, and Q217R have been reported with a valine at codon 129, whereas P102L, Y145stop, and Q212P carry a methionine at codon 129 V, D202NV, and Q217RV PrP mutants completely inhibited Bax-mediated apoptosis or simply delayed condensed chromatin formation, we measured caspase activity with the FLICA pan-caspase substrate, SR-VAD-fmk  expression because of a deletion in the Casp3 gene and thus cannot undergo DNA fragmentation in apoptotic conditions -VAD-fmk Fig. 2A.To determine if the loss of anti-Bax function from the GSS-associated PrP mutants is due to lower levels of CyPrP as previously shown in CJD mutants M, WT PrPV, F198SV, D202NV, Q217RV and Q212PM PrP mutants generated detectable levels of CyPrP while the P102LM, P102LV, P105LV, and Y145stopM did not  SP-CyPrP Fig. 4C.To determine the anti-Bax function of GSS-associated PrP mutants in a neuronal cell type, we assessed the ability of the GSS-associated mutants against Bax-mediated cell death in primary human neurons. None of the GSS-associated PrP mutants induced cell death assessed by condensed chromatin in primary human neurons Fig. 5A.M, P105LV, Y145stopM, and Q212PM could not inhibit Bax-mediated condensed chromatin. However, four of the GSS-associated mutants, F198SV, D202NV, P102LV and Q217RV, prevented Bax-mediated condensed chromatin. However, except for the P102LV, these mutants did not inhibit caspase activation, a step that is upstream of chromatin condensation in the Bax-activated death pathway. The results indicated that these mutants simply delayed the cell death pathway because, in addition to the caspase activation in condensed chromatin positive cells, some transfected cells showed caspase activation in non-condensed chromatin positive cells. In contrast to human neurons, the MCF-7 cells lack the Casp3 gene and thus do not undergo DNA fragmentation although condensation of chromatin occurs through Casp6 and Casp7 activation In this manuscript, we examined the anti-Bax function of GSS-associated PrP mutants. WT PrP inhibits the first step of Bax activation in human primary neurons and in MCF-7 cells V/M, P105LV, Y145stopM. The results with PrP Y145stop are consistent with others that show that PrP Y145stop has a short half-life and accumulates in the nucleus, ER, and Golgi but not significantly in the cytosol M mostly in the nuclear fraction. In those mutants that did generate detectable CyPrP, most of the CyPrP likely resulted from retrotranslocation because CyPrP migrated at the expected size for deglycosylated PrP lacking the N-terminal or C-terminal signal peptides. Retention of the signal peptides would show a protein migrating at 28 to 30 kDa rather than at 23–25 kDa as we observe. Furthermore, western blots of CyPrP with anti-signal peptide antiserum gave only a weak signal compared to that obtained with the anti-PrP 3F4 antibody. The anti-Bax function could have been influenced also by the aggregation of CyPrP, a condition detected in GSS mutants expressed in other cell types in vitro and in vivo studies We also show in this paper that the loss of GSS mutants in inhibiting Bax-mediated condensed chromatin results from the inability of these mutants to generate CyPrP in MCF-7 cells. As shown previously with the eleven CJD-associated PrP mutants V, Q217RV, and D202NV mutants generated as much CyPrP as WT PrPV and while this level was sufficient in preventing Bax-mediated caspase activation in WT PrP, it was not in mutant PrPs. Others have shown that PrP Q217RV escapes the ER quality control and accumulates as an abnormally folded form in the post-Golgi compartment of neuroblastoma cells V accumulates in the cytosol V, Q217RV, and D202NV location in helix 3 is consistent with the observed loss of anti-Bax function in these mutants, despite their ability to generate CyPrP. Therefore, the loss of anti-Bax function in PrP mutants can occur through two mechanisms: low levels of CyPrP through decreased retrotranslocation or pre-emptive quality control and loss in anti-Bax activity for those mutants that can generate CyPrP.Most importantly, we observed that the GSS-associated mutants that generated significant levels of CyPrP and prevented condensed chromatin could not prevent caspase activation. Therefore, the CyPrP generated from these GSS-associated PrP mutants did not have normal anti-Bax function. Indeed, the F198SWe found it interesting to observe significant amounts of PrP in the nuclear fraction from Wt PrP and most mutant PrP-transfected cells. We can exclude the possibility of contaminating endoplasmic reticulum membranes in the nuclear fraction since BiP is not detected in the nuclear proteins. Possibly, nuclear PrP was translocated from the cytosol to the nucleus. Others have reported fluorescent-tagged PrP and C-terminally truncated forms of PrP in the nuclear and perinuclear compartments In conclusion, we have excluded the possibility that the retention of PrP's anti-Bax function in GSS-associated mutants could be responsible for the longer duration of GSS disease relative to CJD. However, the fact that nineteen different single point mutations of the prion protein associated with CJD, GSS and FFI significantly inhibit PrP's anti-Bax function suggests that both trafficking and structural problems arise with mutations in the PrP.


Mycobacterium smegmatis, we studied the drug susceptibility pattern of various mutants with genetic alterations in the 16S rRNA decoding A-site in the context of wild-type and mutant protein S12. RpsL K42R substitution was found not to affect the drug resistance pattern associated with mutational alterations in 16S rRNA H44.Recent studies have suggested that ribosomal protein S12 modulates 16S rRNA function and susceptibility to 2-deoxystreptamine aminoglycosides. To study whether the non-restrictive K42R mutation in RpsL affects 2-deoxystreptamine susceptibility in  Representatives for each of the two disubstituted 2-deoxystreptamine subclasses were included: the 4,5-disubstituted 2-deoxystreptamines paromomycin and neomycin and the 4,6-disubstituted 2-deoxystreptamines gentamicin, tobramycin and kanamycin. The results are presented in M. smegmatis, the A1408G mutation confers moderate resistance to paromomycin (6′ OH) but high level resistance to 2-deoxystreptamine aminoglycosides with an amino group at the 6′ position of ring I. A key element in drug binding is the pseudo base-pair interaction between the ring of the aminoglycosides I and A 1408 Among all A-site mutations that confer aminoglycoside resistance, the A1408G mutation is the most significant. In The Watson–Crick base pair C1409–G1491 forms the base of the drug binding pocket. In the crystal structures vs K42R, see M. smegmatis H44 mutants to 2-deoxystreptamines. Notably, this finding is not fully congruent with a previous finding in E. coli where K42R  reportedly modulated the level of paromomycin resistance of a G1491U mutant Strains with mutations in 16S rRNA residues 1408, 1409 and 1491 show a mutation-specific drug susceptibility pattern that is independent of the amino acid residue 42 in ribosomal protein S12  versus A-U (E. coli), 1411–1489 U-A (M. smegmatis) versus C-G (E. coli). We found that the various H44 mutations  resulted in identical drug susceptibility patterns regardless of a mycobacterial and proteobacterial H44 sequence context at residues 1410–1490 and 1411–1489 (data not shown).To study whether the different findings reported for helix 44 . M. smegT. thermophilusCrystal structures of streptomycin bound to the small ribosomal subunit of In contrast to K42R, mutations K42A or K42T disrupt the salt bridge to H27 . This moS. cerevisiae 18S rRNA (homologous to E. coli residue A1408/G1491) reversed the natural resistance of yeast cytoplasmic ribosomes, i.e, increased the susceptibility to aminoglycosides from completely resistant (MIC>5000 µg/ml) up to highly susceptible (MIC about 3 µg/ml). These levels of drug susceptibility are similar to those found in drug susceptible E. coli (MIC about 2.5–5 µg/ml) S. cerevisiae or E. coli), susceptibility to 2-deoxystreptamines is apparently determined by the nucleotide residues in drug binding pocket of 16S rRNA. This finding contrasts with the view of a universal interrelation between S12, base pair 1409–1491 and 2-deoxystreptamine susceptibility. Instead, it suggests that functional interactions between S12 and the 2-deoxystreptamine binding site are limited to certain bacterial genera and/or specific rpsL mutations. Interaction of S12 and 2-deoxystreptamine aminoglycosides apparently is not at the level of drug binding, but provoked by interference of two opposite effects on translation fidelity induced by error-restrictive rpsL mutations and misreading-inducing aminoglycoside antibiotics. As a result, this interplay is limited to S12 amino acid substitutions which confer a hyperaccurate phenotype and thus are functional antagonists of drug-induced misreading.Our data demonstrate that K42R in ribosomal protein S12 does not affect resistance to 2-deoxystreptamine aminoglycosides as conferred by 16S rRNA mutations in H44, and that S12 most likely plays little role in the species-specific pattern of susceptibility to 2-deoxystreptamines. This conclusion is supported by the observation that mutations G1645A/A1754G in M. smegmatis ΔrrnB (SZ380) was generated by unmarked deletion mutagenesis of the rrnB operon in M. smegmatis mc2155. A suicide vector pH022 containing two DNA fragments flanking the rrnB operon (generated by PCR), a selectable marker (GmR) and counter-selectable marker sacB (both outside the rrnB DNA fragments), was transformed into M. smegmatis mc2155. A single rRNA allelic derivative was obtained by a two-step selection procedure: selection of transformants on agar plates containing gentamicin followed by a counter-selection step on agar plates with sucrose. Deletion of rrnB was confirmed by Southern blot analysis and partial sequencing.A single rRNA allelic strain M. smegmatis ΔrrnB (SZ380) and in strain M. smegmatis ΔrrnB rpsL K42R (SZ004) Mutagenesis of the H44 decoding-site RNA was performed in strain M. smegmatis cultures were resuspended in LB broth supplemented with 0.05% of Tween 80, diluted to an absorbance at 600 nm of 0.025 and incubated in the presence of 2-fold serial dilutions of 2-deoxystreptamine aminoglycosides. After incubation at 37°C for 72 h, the MIC was recorded as the lowest concentration of drug inhibiting visible growth.Drug susceptibility was studied by determining Minimal Inhibitory Concentrations (MIC). MIC tests were performed in a microtiter plate format as described T. thermophilus  PyMol (DeLano Scientific) was used to render the structure of the A-site of 30S ribosomal subunit from Figure S1Chemical structures of disubstituted 2-deoxystreptamine antibiotics used in this study.(0.48 MB DOC)Click here for additional data file.Table S1Strains used in this study.(0.04 MB DOC)Click here for additional data file.Table S2Plasmids used in this study.(0.03 MB DOC)Click here for additional data file.


All-trans retinoic acid (RA) was previously shown to regulate the growth of gastric cancer cells derived from the cell line SC-M1. This study was designed to investigate the effect of RA on the sensitivity of SC-M1 cells to lymphokine-activated killer (LAK) activity. RA at the concentration range of 0.001-10 microM was shown to induce SC-M1 cells to exhibit resistance to LAK activity in a dose-dependent manner. A kinetics study indicated that a significantly increased resistance was detected after 2 days of co-culturing SC-M1 cells with RA and reached a maximum after 6 days of culture. Similar results were obtained from two other cancer cell lines: promyelocytic leukaemia HL-60 and hepatic cancer Hep 3B. A binding assay demonstrated that the binding efficacy between target SC-M1 cells and effector LAK cells was not altered by RA. Flow cytometric analyses revealed that RA exhibited no effect on the expression of cell surface molecules, including HLA class I and class II antigens, intercellular adhesion molecule-1 and -2, and lymphocyte function antigen-3. Cell cycle analysis revealed that culture of SC-M1 cells with RA resulted in an increase in G0/G1 phase and a decrease in S phase, accompanied by a decrease in cyclin A and cyclin B1 mRNA as determined by Northern blot analysis. Additionally, RA was shown to enhance the expression of retinoic acid receptor alpha  in SC-M1 cells, and to have no effect on the expression of RARbeta or RARgamma. Taken together, these results indicate that RA can significantly increase gastric cancer cells SC-M1 to resist LAK cytotoxicity by means of a cytostatic effect through a mechanism relating to cell cycle regulation. The prevailing ideas, such as a decrease in effector to target cell binding, a reduced MHC class I antigen expression or an altered RARbeta expression, are not involved.


Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the binding event is coupled to a hinge-bending motion that brings together two domains in a closed form. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has also been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Understanding the mechanistic details of PBP conformational changes, both global  and local , therefore is not only important for understanding their biological function but also for protein engineering experiments.Thermotoga maritima in its ribose-bound and unliganded state. The T. maritima RBP (tmRBP) has 39% sequence identity and is considerably more resistant to thermal denaturation  than the mesophilic Escherichia coli homolog (ecRBP) . Polar ligand interactions and ligand-induced global conformational changes are conserved among ecRBP and tmRBP; however local structural rearrangements involving side-chain motions in the ligand-binding site are not conserved.Here we present biochemical characterization and crystal structure determination of the periplasmic ribose-binding protein (RBP) from the hyperthermophile Although the large-scale ligand-induced changes are mediated through similar regions, and are produced by similar backbone movements in tmRBP and ecRBP, the small-scale ligand-induced structural rearrangements differentiate the mesophile and thermophile. This suggests there are mechanistic differences in the manner by which these two proteins bind their ligands and are an example of how two structurally similar proteins utilize different mechanisms to form a ligand-bound state. Bacterial periplasmic binding proteins (PBP) are receptors for extracellular solutes in metabolite uptake , chemotaLigand-free PBPs adopt an open conformation in which the inter-domain interface is exposed to solvent. Solute binding induces a conformational change to form a closed state in which the ligand is bound at the domain interface and buried by the surrounding protein -8. This E. coli ribose-binding protein [E. coli ribose-binding protein (RBP) [A collection of PBP structures determined in both apo and ligand-bound states (Table  protein  to as li protein . PBPs suin (RBP)  and alloin (RBP)  have beeE. coli RBP (ecRBP) small-scale backbone movements are restricted to the hinge region, whereas the secondary structure elements in the two domains and the amino acids in the binding pocket adopt essentially the same conformations in both the apo and ribose-bound forms [E. coli leucine-binding protein, not only the hinge region, but also loops and amino acid side-chains in the binding pocket show ligand-induced changes [In nd forms . However changes , many of changes ,13,14.Thermotoga maritima (tmRBP), the mesophilic homolog, ecRBP, of which has been studied in detail [The ligand-induced conformational changes have not been described previously in a thermophilic PBP. We have characterized the stability, determined the ligand-binding properties, and solved the X-ray crystal structures of the apo and ligand-bound forms of a thermophilic periplasmic ribose-binding protein from the hyperthermophile n detail ,15,16. TT. maritima genome sequence [tm0958, based on sequence similarity to the E. coli RBP, and genetic linkage of this ORF within a putative operon that contains sequences for ABC transporters characteristic of a ribose transport system [tm0958 was amplified from T. maritima genomic DNA using the polymerase chain reaction. The resulting DNA fragment was cloned into a pET21a vector with a C-terminal hexa-histidine tag preceded by a glycine-serine linker. The nucleotide sequence of the recombinant was confirmed by DNA sequencing. Over-expression of this ORF in E. coli produced ~50 mg of pure protein per liter of growth medium, which was purified by immobilized metal affinity chromatography [The RBP gene was identified in the sequence  as open t system . ORF tm0tography  followedtm0958 DNA sequence suggests that this truncation may result from translation initiation at methionine 142 (numbering according to NCBI NP 228766), which is preceded by a ribosome binding site , the other consistent with a ~55 kDa protein Figure . SDS-PAGappTm) of full-length monomeric wild-type tmRBP was determined by thermal denaturation using circular dichroism (CD) [appTm in the absence of GdCl was determined by linear extrapolation of a series of melting point determinations carried out at different GdCl concentrations [appTm value is 56°C .The apparent thermal stability (ism (CD) . In the ism (CD) ,22. An atrations  . AdditiappTm of full-length wild-type monomeric tmRBP in the presence of 5.5 M GdCl. Under these conditions the appTm is 71°C in the absence of sugar and 97°C in the presence of 1 mM ribose, indicating that tmRBP is a ribose-binding protein, as predicted from sequence homology , containing a M142A mutation to prevent expression of the in-frame ORF. We were unable to obtain crystals of the heterodimeric form. The apo-protein and ribose-complex diffract to 1.4 Å and 2.15 Å resolution and were refined to Rcryst/Rfree values of 18.0/20.3 and 19.3/22.3 respectively. The X-ray crystal structure of ribose-bound tmRBP was solved by molecular replacement using ecRBP as the search model [Crystals of ribose-complexed tmRBP were grown using a full-length wild-type construct (residues 30–323) that lacks the periplasmic signal sequence (residues 1–29). The apo-protein was crystallized using a construct that consisted of residues 30–310 . It is possible that the absence of this region could in some way influence the observed conformation of apo form of tmRBP. However, superimposition of the tmRBP ribose complex C-terminal domain onto the C-terminal region of the apo protein suggests that these this region does not form interdomain interactions in the absence of ligand.Thermoanaerobacter tengcongensis [α atoms s Figure . The strs Figure . One of E. coli protein [Although ribose is commonly found as a furanose carbohydrate in biological molecules (e.g. nucleic acids), all periplasmic RBPs, including tmRBP, bind the β-anomer of D-pyranose ribose ,26  which make extensive van der Waals interactions with the sugar ring. In ecRBP the equivalent aromatic binding pocket residues are all phenylalanines. Alignment of tmRBP and ecRBP structures indicates that the six-membered ring of W15 in tmRBP is equivalent to F15 in ecRBP  that interacts with the ribose  and buried surface area  it has been postulated that ordered binding occurs and ribose initially interacts with N-terminal domain of ecRBP [We have characterized the ligand-binding properties of a putative ribose-binding protein identified in the genomic sequence of the extremophilic bacterium onserved ,24. Locaof ecRBP . If an oWater molecules have been suggested to play an important mechanistic role in the evolution and adaptation of the PBP hinge . In partLigand-induced hinge bending motion is a key characteristic of the periplasmic binding protein superfamily. Analysis of PBP structures has provided a detailed description of this class of conformational change ,12-14. Ttm0958 gene was amplified from T. maritima genomic DNA (American Type Culture Collection) by the sticky-end PCR method [NP 228766: PO4--TATGAAAGGAA AGATGGCTATTGTGATCTCC and for the 5'-TGAAAGGAA AGATGGCTAT TGTGATCTCC end of the genes; PO4--AATTCTA ATGGTGATGGTGATGGTGACTGCCTTCTTCTTTTCTGCCGTAAGCAGTG and CTAATGGTGATGGTGATGGTGACTGCCTTCTTCTTTTCTGCCGTAAGCAGTG for the 3'end of the full-length tmRBP gene, PO4-AATTCTAATGGTGATGGTGATGGTGACTGCCTTCTCTTGTCACCAGCTCAACAGTGAC and CTAATGGTGATGGTGATGGTGACTGCCTTCTCTTGTCACCAGCTCAACAGTGA C for the 3' end of the tmRBP-apo gene [E. coli. This ORF lacks the periplasmic signal sequence. The coding sequence starting at lysine 30 was cloned in-frame with an ATG start codon. A hexa-histidine affinity tag, preceded by a glycine-serine linker, was fused in-frame at the carboxy terminus to facilitate purification by immobilized metal affinity chromatography (IMAC). Protein concentration was determined spectrophotometrically  [The R method  using thapo gene . The 30–M-1cm-1) . The resM-1cm-1) . Pooled Proteins were excised from a 12% Tris-HCl SDS-PAGE gel and were digested in-gel using the Pierce In-gel Tryptic Digest Kit. Mass spectra were acquired on an Applied Biosystems Voyager DE MALDI-TOF mass spectrometer using an α-cyano-4-hydroxycinnamic acid matrix with a 300 ns delay time.Circular dichroism (CD) measurements were carried out on an Aviv Model 202 CD spectrophotometer. Thermal denaturations were determined by measuring the CD signal at 222 nm (1 cm path length) as a function of temperature, using 1.0 μM of full-length wild-type monomeric tmRBP  in the presence or absence of 1 mM ribose at several GdCl concentrations extrapolated to 0 M GdCl . Proteina = 72.1 Å, b = 98.2 Å, c = 131.1 Å)   mixed with 2 μl of 0.1 M MES pH 6.0, 20% (w/v) PEG 8000 and 0.1 M RbCl. Crystals of the C-terminally truncated M142A apoprotein were grown in micro-batch drops containing 2 μl of the protein solution  mixed with 2 μl of 0.1 M Bis-Tris pH 5.9, 25% (w/v) PEG 3350, 0.2 M NaCl. Diffraction quality crystals typically grew within two weeks at 17.0°C. The ribose-complexed crystals diffract to 2.15 Å resolution and belong to the I222 space group , one ribose molecule, and 142 water molecules. The final model for the apoprotein includes two intact monomers (residues 30–310) and 627 water molecules. The models exhibit good stereochemistry as determined by PROCHECK  and MolPLarge-scale hinge bending motions were analyzed with the DynDom web server . Local CMJC purified, crystallized, solved the structure of tmRBP, and carried out circular dichroism experiments. MJC, LSB and HWH undertook sequence and structural analysis of the tmRBP and ecRBP structures. MJC and HWH wrote the manuscript. All authors have read and approved the final manuscript.


A and B haplotypes that are under balancing selection and differentially function in defense and reproduction. Our study shows how the qualitative differences that distinguish the human and chimpanzee systems of KIR and MHC class I predominantly derive from adaptations on the human line in response to selective pressures placed on human NK cells by the competing needs of defense and reproduction.Natural killer (NK) cells serve essential functions in immunity and reproduction. Diversifying these functions within individuals and populations are rapidly-evolving interactions between highly polymorphic major histocompatibility complex (MHC) class I ligands and variable NK cell receptors. Specific to simian primates is the family of Killer cell Immunoglobulin-like Receptors (KIR), which recognize MHC class I and associate with a range of human diseases. Because KIR have considerable species-specificity and are lacking from common animal models, we performed extensive comparison of the systems of KIR and MHC class I interaction in humans and chimpanzees. Although of similar complexity, they differ in genomic organization, gene content, and diversification mechanisms, mainly because of human-specific specialization in the KIR that recognizes the C1 and C2 epitopes of MHC-B and -C. Humans uniquely focused KIR recognition on MHC-C, while losing C1-bearing MHC-B. Reversing this trend, C1-bearing HLA-B46 was recently driven to unprecedented high frequency in Southeast Asia. Chimpanzees have a variety of ancient, avid, and predominantly inhibitory receptors, whereas human receptors are fewer, recently evolved, and combine avid inhibitory receptors with attenuated activating receptors. These differences accompany human-specific evolution of the  Natural killer (NK) cells are versatile lymphocytes that make essential contributions to immune defense and placental reproduction. Essential to NK cell development, diversification and function are variable families of surface receptors that recognize equally variable determinants of polymorphic major histocompatibility complex (MHC) class I molecules, better known as the tissue types matched in clinical organ transplantation. These ligand-receptor interactions evolve rapidly, exhibiting much species specificity and convergent evolution. Consequently, mice represent a poor model, because their receptors are so disparate from the independently evolved human counterparts that are restricted to simian primates. To identify unique and shared aspects of human NK cell biology, we have defined the genomics, population biology, and immunology of variable chimpanzee NK cell receptors and ligands to a level permitting accurate, informed comparison with the well-characterized human system. In both receptors and ligands there are dramatic, qualitative differences between humans and chimpanzees. We show these differences arose during human evolution from the last common human–chimpanzee ancestor, while the chimpanzee system remained relatively stable. That two so closely related species exhibit major differences in NK cell receptors and ligands testifies to the strong and varying selection imposed by the different demands and competing needs of defense and reproduction. Natural killer (NK) cells are lymphocytes that contribute to both the immune and reproductive systems. NK cells provide first-line, innate immune defense against infection KIR locus combines gene content variability with allelic polymorphism KIR3DL gene during the last ∼40–58 million years KIR genes are expressed in the brain, but not by NK cells Ly49 gene family independently evolved as a variable family of NK cell receptors for MHC class I KIR3DL gene, but a diversified system of CD94 and NKG2 genes KIR3DL gene, cattle expanded and diversified the distantly related KIR3DX gene The human HLA class I genes KIR cDNA and one KIR haplotype KIR genes is shared by humans and chimpanzees; and second, that the organization of KIR genes into haplotypes is qualitatively different in the two species. To test these hypotheses we performed extensive analysis of chimpanzee KIR haplotype structure and variation, permitting definitive genetic and functional comparison with the human KIR system.HLA-A, B, C and G serve as ligands for human KIR KIR haplotypes . Restricted to the centromeric region, chimpanzee 2DL5 has absolute LD with inhibitory KIR2DL8, whereas human 2DL5 has absolute LD with activating KIR2DS3/S5 and is alternatively found in the centromeric region, the telomeric region, or both  lack both of them.Although human and chimpanzee each have ten variable plotypes . Whereasc region  and presc region . Also va or both . Thus huKIR form five pairs within the centromeric region , being replaced by the human-specific sublineage that includes both C1- and C2-specific receptors. The shared sublineage includes additional chimpanzee inhibitory C2 receptors and 2DS4. The differences in the MHC-C system of receptors in human and chimpanzee are seen to be mainly the result of human-specific evolution. These differences alter basic functional characteristics such as the number and avidity of receptors  . PositioLineage III KIR recognize the C1 and C2 epitopes of MHC class I. C2 depends upon valine 76 (V76) and lysine 80 (K80), a motif restricted to a subset of MHC-C allotypes. C1 depends upon V76 and asparagine 80 (N80), a motif present in subsets of MHC-C and -B allotypes; 22% of chimpanzee MHC-B allotypes have C1, compared to only 2.5% for HLA-B . AncestrOf 12 distinct V76-containing HLA-B , 11 emerB*46 frequencies of 25.4% and 22.9% in the Yunnan Dai and Guizhou Miao, respectively, are the highest for any HLA-B allele in populations exceeding one million individuals, being of a magnitude typical for small, isolated or bottlenecked populations  as well as more limited type II functional divergence  . Of eighType II functional divergence was more limited and equally distributed between MHC-B and -C (two positions each). Notably, however, this divergence included the valine to glutamate change at position 76 in the α1 domain of MHC-B . OverallKIR haplotype structure and gene content. These differences arose from specializations evolved on the human line. For chimpanzee KIR haplotypes, variable gene content is confined to the centromeric region, where ten KIR genes are variably found, leaving the telomeric region empty. During human evolution the telomeric region was ‘colonized’ to create a second region of gene content variability. As a consequence of this reorganization, symmetrical recombination between the centromeric and telomeric regions has evolved to be a major mechanism for diversifying KIR haplotypes in humans Since their ancestors separated 6.5–10 million years ago, human and chimpanzee acquired major differences in KIR with no human equivalents, all of which encode high-avidity activating or inhibitory receptors for C1 or C2 KIR with no chimpanzee counterparts encode high-avidity inhibitory receptors for C1 and C2 and four activating receptors, which acquired mutations that caused three to lose avidity for HLA-C completely and one to have it reduced A second major human-specific specialization has been to fix mutations reducing the avidity of activating KIR for HLA class I, while retaining high-avidity inhibitory KIR. This process is most striking for the lineage III KIR that comprise receptors for MHC-C, the form of MHC class I molecule that became the major source of KIR ligands (the C1 and C2 epitopes) in the course of hominid evolution. Chimpanzees have eight lineage III KIR haplotype groups, A and B, that are under balancing selection, present in all populations, and differently associated with disease KIR haplotypes are variations on a theme emphasizing multiple and variable high-avidity C1 and C2 receptors. The character of the A haplotypes is closer to that of the chimpanzee KIR haplotypes: they have genes encoding high-avidity inhibitory C1, C2 and Bw4 receptors and lack their attenuated activating counterparts. In contrast, B haplotypes carry genes for the attenuated activating receptors and distinctive variants of the inhibitory receptors. Disease associations suggest a basis for the balancing selection, in that A haplotypes favor successful anti-viral defense, whereas maternal B haplotypes favor successful reproduction A haplotype KIR genes are more polymorphic than their B haplotype counterparts KIR haplotypes. In this context, human-specific evolution of group B KIR haplotypes can be seen as an adaptation to life-threatening complications of pregnancy, such as pre-eclampsia and eclampsia, which have not affected the chimpanzee. For example, these hypertensive conditions could have arisen from imbalance between the supply and increased demands on maternal blood caused by selection to increase the size of the neonatal human brain, to double that of the chimpanzee The major consequence of these two specializations is that humans evolved two distinctive HLA-B allele. Resolution of human hepatitis C virus infection was associated with homozygosity for KIR2DL3 and its C1 ligand A third human-specific specialization has been the decreasing use of MHC-B allotypes as ligands for lineage III KIR. C1 originated in a molecule resembling MHC-B, which with duplication and differentiation led to the modern MHC-B and -C A panel of 39 individuals was studied; 35 western chimpanzees, two eastern chimpanzees, one central chimpanzee, and one individual of unknown geographical origin. This study was approved by the Stanford University administrative panels on human subjects in medical research and laboratory animal care.Haplotypes H13 and H2 originate from the RPCI-43 BAC library  while H8 belongs to the CHORI-251 BAC library . The final sequence of the H13 haplotype (clone RP43-84K19) has ‘finished sequencing’ quality , and the third character is either a ‘L’, ‘S’ or ‘P’ to indicate ‘Long’ (inhibitory) or ‘Short’ (activating) cytoplasmic tails, or a pseudogene, respectively. To simplify the text in this manuscript, the acronym KIR is sometimes omitted.Based on phylogenetics, the Ig-like domains form three groups: D0 , D1, and D2 . Based on domain structure and phylogenetic comparison, human KIR are seen to form four distinct lineages: KIR of the lineages III (3DL1-2) and V (3DL3) have three Ig-like domains, while KIR of the lineages I (2DL4-5) and III  have two, with D0-D2 and D1-D2 configuration, respectively.KIR haplotypes sequenced was performed using peripheral blood mononuclear cells from the two individuals whose genomic DNA was used to construct the BAC libraries RPCI-43 and CHORI-251 , KIR content was determined as previously described KIR a new typing system was developed. KIR haplotype structures were predicted from genotype data using the HAPLO-IHP program For five of the 14 KIR of To compare genotype diversity in human and chimpanzee, data from human populations from Africa KIR, linkage disequilibrium (LD) was investigated from the haplotype data of KIR haplotype structures using KIR2DL5 as a reference. In these analyses, gene linkage to 2DL5+/− was estimated for each KIR as a ratio: for example KIR3DS1 is seven times more common on 2DL5A+ haplotypes than on 2DL5A− haplotypes. All ratios were normalized to account for the difference in frequency between +2DL5 and −2DL5 haplotypes. In the human locus, KIR2DS3/5 can occupy two different genomic locations 2DL5 (A or B) and 2DS3/5 was assumed to be absolute, as supported by currently available haplotype sequences. Gene linkage to 2DL5 in human was assessed from Caucasian individuals For chimpanzee dN/dS (ω) ratios were estimated by maximum-likelihood using PAML4 MHC-B and -C sequences was gathered and phylogenetic analyses conducted using the approach described for the KIR genomic analysis (see A representative set of ysis see . Ancestrhttp://www.ncbi.nlm.nih.gov/Traces/home/), and generated by the Sanger Center as part of the gorilla genome project.Data for the distribution of the MHC-B and -C residues affecting interaction with lineage III KIR in hominoids were compiled using IMGT-HLA and IPD-MHC sequences Allele frequencies for C1-bearing HLA-B allotypes are from the Allele Frequency Net Database The B46 distribution map was generated using the GMT software package Type I (site-specific shift of evolutionary rate) and type II (site-specific shift of amino acid property) functional divergence analyses were performed with DIVERGE2 KIR and HLA phenotypic frequencies (PF) in 33 human populations (see PF*C2PF)+(2DL2PF*C2PF)+(2DL2PF*C1PF)+(2DL3PF*C1PF). For East Asian populations we also added the interaction between HLA-B*46 and KIR2DL2-3: (2DL2PF*B46PF)+(2DL3PF*B46PF). An alternative model where the interaction between KIR2DL2 and HLA-C2 was not taken into account was also considered but the results were similar  Intron-exon structure of a typical KIR gene showing the 14 datasets used in phylogenetic analyses. (B–F) Pairwise comparison of haplotypes: H13 (top) - H2 (left) (B); H8 (top) - H2 (left) (C); H13 (top) - H8 (left) (D); H13 (top) - human A (E) and H13 (top) - human B (F). Phylogenetic analyses were performed individually for the 14 segments defined in (A) using Bayesian, maximum-likelihood (ML), neighbor-joining (NJ) and parsimony methods (KIR gene names indicate the lineages: I (green), II (orange), III (purple) and V (red). (E–F) Orange: human and chimpanzee sequences are mixed with orthologs from other species.Phylogenetic comparison of human and chimpanzee  methods . Colored(0.05 MB PDF)Click here for additional data file.Figure S2KIR haplotypes. The phylogenetic reconstruction was performed on each of the 14 segments described in Phylogenetic analysis of the 14 genomic segments used to compare (0.11 MB PDF)Click here for additional data file.Figure S3Emergence of MHC-B allotypes with V76. This phylogenetic tree represents the MHC-B subtree of the tree presented in (0.03 MB PDF)Click here for additional data file.Figure S4B*4601 is the only HLA-B allele with an allele frequency >25% in a large population. The two left-most columns list the 24 most common HLA-B alleles worldwide and their respective worldwide allele frequency (AF) in a study of 146 worldwide population samples HLA-B AF. Two alleles initially included (HLA-B*0704 and B*3705) were subsequently excluded as no data were available for them in the Allele Frequency Net Database HLA-B alleles the name of the large population with the highest AF, and the AF in this population. While we set a minimum of ∼200,000 individuals for a population to be included in column 5, all the populations listed in this column have modern population sizes well in excess of 1,000,000 individuals. AF>25% are shaded in orange.(0.02 MB PDF)Click here for additional data file.Figure S5PF*C1PF quantity  in 33 human populations. Area between the gapped lines is the 25–75 percentile range; area between the dotted lines (top part only) is the non-outlier range (Whisker plot with 1.5 coefficient). Colors in the top part are as defined in (A). Populations in purple (bottom part) have HLA-B*46PF of 8.7–27.5%. SWA, Southwest Asia; OCE, Oceania; NEA, Northeast Asia; NAM, North America; SAM, South America.MHC-B allotypes that reacquire binding to lineage III KIR restore or increase NK cell effector capacity. (A) Summary of the KIR2DL/HLA-B (magenta) and KIR2DL/HLA-C (blue) interactions. (B) Average number of distinct KIR2DL-HLA interactions (ANDI) (top) and 2DL3(0.03 MB PDF)Click here for additional data file.Figure S6PF*C2PF) quantity in 33 populations. Dark gray area represents the 25-75 percentile range. (C-D) Pearson product-moment correlations between KIR2DL2PF and HLA-C2PF in 33 (C) or 32 (D) populations. The red point in (C) is an outlier (Nasioi population) and was removed for the analysis in (D). (E) Pearson product-moment correlations between HLA-C1/C2 and KIR2DL1-3 phenotypic frequencies. The Nasioi population was consistently an outlier, indicating that the HLA-C1/C2 and KIR2DL1-3 frequencies in this population have evolved differently than in the other 32 populations.Variability of KIR2DL2 and HLA-C2 frequencies in human populations. Amongst the components of the KIR2DL/HLA-C interactions HLA-C2 and KIR2DL2 display the widest range of phenotypic frequencies in populations . HLA-C2 (0.12 MB PDF)Click here for additional data file.Figure S7ML: coefficient of type I functional divergence. θ ΙΙ: coefficient of type II functional divergence. Significance of the type I analysis was assessed using a likelihood ratio test (LRT): twice the difference in likelihood (‘LRT’) was compared to a χ 2 distribution with one degree of freedom. For the type II analysis a two-tailed Z-test was used to assess the significance. S.E., Standard Error. (B) Identification of the type I and type II functional divergence-related residues  >θ ML/ΙΙ+0.3 [Gu]). Group-specific residues are functional divergence-related in all three comparisons while MHC-B or -C specific positions are related to functional divergence in two of the three comparisons and display an average, or lower than average, functional divergence in the third comparison. For the type I divergence, MHC-B specific positions were defined as related to functional divergence in the MHC-B/MHC-C and MHC-B/OWM comparisons  but not in the MHC-C/OWM comparison . For the type II divergence, MHC-B specific positions were defined as related to functional divergence in the MHC-B/MHC-C and MHC-B/OWM comparisons  but not in the MHC-C/OWM comparison . The same approach was used for the MHC-C specific positions. OWM, Old World monkey. (C) Identification of residues with a low type I functional divergence (defined as having p<θ ML-0.3). (D-F) Results of the type I  and type II (E) functional divergence analyses. Functionally-divergent sites are listed at the bottom of each graph. MHC-B specific sites are colored blue, MHC-C specific sites are colored red. Sites that are functionally-divergent in all three comparisons are green, those with a low type-I divergence are orange. Underlined residues have a better support (G2 in panel B). Boxed residues are in the recombinant region of HLA-B*46. Y axis, posterior probability of a site to be functional divergence-related. [Gu X (2006) A simple statistical method for estimating type-II (cluster-specific) functional divergence of protein sequences. Mol Biol Evol 23: 1937-1945.]Functional divergence between the α 1 and α 2 domains of MHC-B and MHC-C. (A) Summary of the type I and type II functional divergence analyses. θ (0.04 MB PDF)Click here for additional data file.Figure S8HLA-B*46 (‘c’ indicates that this probability was corrected for multiple comparisons while ‘nc’ indicates no correction for multiple comparisons). §, the significance is for a subgroup of HLA-B*46 haplotypes (listed in ‘Other HLA factors’). The column ‘Other HLA factors’ lists genetic factors also found to be associated with the disease in the same study, and genetic factors in bold displayed a more significant association than B*46. Brackets in the ‘Other HLA factors’ column designate haplotypes. #, only in males. References are: Disease associations with HLA-B*46. The ‘Disease’ column on the left lists the name of the disease investigated, and for infectious diseases the name of the causal pathogen. The country where the study was conducted, the type of association (resistance ‘R’ or susceptibility ‘S’) and the reference to the study are given in the ‘Association’ column. The column ‘Significance’ gives the significance of the association between the disease and (0.05 MB PDF)Click here for additional data file.Figure S9KIR cDNA and gene sequences. Results of the expression study in peripheral blood mononuclear cells are given for the ten KIR gene sequences with no prior cDNA equivalent. *, Pt-KIR3DL3 is from the same lineage as KIR3DL3, a gene expressed at low or undetectable levels in peripheral blood NK cells.Relationships between chimpanzee (0.01 MB PDF)Click here for additional data file.Figure S10Pt-KIR3DS6. (A) Sequence of Pt-KIR3DL6 and 3DS6 at the end of exon 7 and at the beginning of exon 8. Pt-KIR3DL6 has a stretch of seven adenosines at the end of exon 7 that maintains the typical KIR reading frame for exons 8 and 9, resulting in a protein with two ITIM in the cytoplasmic tail. Pt-KIR3DS6 has eight adenosines at the end of exon 7: this changes the typical KIR reading frame for exons 8 and 9, resulting in a protein with a short cytoplasmic tail with no ITIM. Nucleotides in blue represent splice sites. The adenosine in red represents the extra base pair of Pt-KIR3DS6 comparing to 3DL6. (B) Sequencing of exon 7 of Pt-KIR3DL6/S6 in six individuals that typed positives for Pt-KIR3DL6, including Alex, an individual used in the study where Pt-KIR3DL6 was characterized Pt-KIR3DL6 and 3DS6, respectively. For each amplification and individual, several clones were sequenced, and the number of clones in each category is mentioned . (C) Schematic representation of the gene-to-gene PCR amplification used to characterize the position of Pt-KIR3DS6. F, forward primer; R, reverse primer. TM, exon encoding the transmembrane domain; CYT1-2, exons encoding the cytoplasmic tail. L1-2, exons encoding the leader peptide.Sequence and position of (0.41 MB PDF)Click here for additional data file.Figure S11KIR and HLA frequencies used to establish the average number of distinct KIR2DL-HLA-B/C interactions (ANDI) in human populations. AFR, Africa; EUR, Europe; SWA, Southwest Asia; EAS, East Asia; OCE, Oceania; NEA, Northeast Asia; NAM, North America; SAM, South America. Numbers in parenthesis indicate the number of populations in each group. References are: (0.02 MB PDF)Click here for additional data file.Figure S12Alternative model for the interactions between HLA-C and KIR2DL. Unlike the analyses presented in (0.03 MB PDF)Click here for additional data file.Figure S13KIR sequences. The NJ method was used for the reconstructions and phylogenetic trees were rooted at the midpoint. For nodes, the bootstrap proportion support is given when >50. The eleven cDNA sequences described in the first study of chimpanzee KIR KIR lineages are delimited by boxes and are indicated with white roman letters. (A) D0 domain (or pseudoexon 3). (B) D1 domain. (C) D2 domain. (D) Stem, transmembrane and cytoplasmic domains (S/TM/CYT). (E) Full-length coding sequences (including the pseudoexon 3). White brackets indicate non-recombinant alleles. Allelic relationships were established based on the overall distance in the full-length sequence analysis and consistency in the domain-by-domain analysis (panels A-D).Domain-by-domain phylogenetic analysis of chimpanzee (0.03 MB PDF)Click here for additional data file.Figure S14KIR polymorphism in chimpanzee. Amino acid variation is listed for 12 of the 13 chimpanzee KIR for which such data are available (A-L). For each KIR, the positions of variability are displayed. The origin of each sequence is given in parenthesis: Hxx and Tx (KIR haplotypes), cDNA (first cDNA study Summary of (0.03 MB PDF)Click here for additional data file.Text S1Supporting text.(0.19 MB PDF)Click here for additional data file.


Four cell lines, the mouse L1210 leukaemia, the human W1L2 lymphoblastoid and two human ovarian (CH1 and 41M) cell lines, were made resistant to ZD1694 (Tomudex) by continual exposure to incremental doses of the drug. A 500-fold increase in thymidylate synthase (TS) activity is the primary mechanism of resistance to ZD1694 in the W1L2:RD1694 cell line, which is consequently highly cross-resistant to other folate-based TS inhibitors, including BW1843U89, LY231514 and AG337, but sensitive to antifolates with other enzyme targets. The CH1:RD1694 cell line is 14-fold resistant to ZD1694, largely accounted for by the 4.2-fold increase in TS activity. Cross-resistance was observed to other TS inhibitors, including 5-fluorodeoxyuridine (FdUrd). 41M:RD1694 cells, when exposed to 0.1 microM [3H]ZD1694, accumulated approximately 20-fold less 3H-labelled material over 24 h than the parental line. Data are consistent with this being the result of impaired transport of the drug via the reduced folate/methotrexate carrier. Resistance was therefore observed to methotrexate but not to CB3717, a compound known to use this transport mechanism poorly. The mouse L1210:RD1694 cell line does not accumulate ZD1694 or Methotrexate (MTX) polyglutamates. Folylpolyglutamate synthetase substrate activity (using ZD1694 as the substrate) was decreased to approximately 13% of that observed in the parental line. Cross-resistance was found to those compounds known to be active through polyglutamation.


The present study mainly aimed to investigate the direct effects of Endostar (ES) on the proliferation and radiosensitivity of human lung squamous cancer cell line H-520.0/G1 arrest in H-520 cells, whereas irradiation induced G2/M arrest. The phospho-p38-MAPK and p-Akt protein levels were decreased in H-520 cells after ES treatment. Furthermore, activated caspase protein level increased and Bcl-2 protein levels decreased after treatment with ES and irradiation.ES significantly inhibited H-520 cell proliferation in a time- and dose-dependent manner. According to the colony-forming assays, ES could increase the H-520 cell radiosensitivity. ES induced cell apoptosis, the apoptosis rate increased with the raise of ES concentration. Irradiation induced significantly higher apoptosis rate in ES-treated H-520 cells than non-treated H-520 cells. ES induced cell cycle distribution and GES significantly enhanced the sensitivity of H-520 cells to irradiation by inhibition of cellular proliferation, promotion of cell apoptosis and redistribution of cell cycle, possibly via deactivation of Akt pathway. The present study supports the possibility to use the combination of ES and ionizing irradiation to treat patients with lung squamous cell cancer in clinics. About 40% of patients with stage III or IV non-small cell lung carcinoma (NSCLC) cannot be resected at present . Radiothν, α5, β1 and cell surface glypicans and nucleolin have been identified as receptors interact with ED [et al reported that ED inhibited migration and invasion of head and neck squamous cell carcinoma cells [et al reported that ED directly modulated lung cancer cell function in vitro [et al demonstrated that ED exhibited a direct anti-tumor effect in addition to its anti-angiogenic activity in colon cancer cells [Endostatin (ED) is a 22 kDa polypeptide derived from the C-terminal fragment of type XVIII collogen. Both recombinant human and murine ED have been reported to inhibit endothelial cell proliferation but not smooth muscle cells or fibroblast proliferation in vitro, which suggested that the anti-proliferation effect was endothelial-specific ,9. The a with ED -16. Recema cells . Cui et in vitro . Dkhissier cells . All theE. coli, and was approved by the State Food and Drug Administration (SFDA) for the treatment of NSCLC in 2005 [Endostar (ES) is a novel recombinant human ED which expressed and purified in  in 2005 . ES is t2 at 37°C. ES-treated H-520 cells were obtained by culturing cells with 200 μg/mL ES  for 24 h before irradiation. Phospho-P38 mitogen-activated protein kinases (MAPK) mAb (Alexa Fluor) was provided by Cell Signaling Technology, US.The human lung squamous cancer cell line H-520 was purchased from the Institute of Basic Medical Sciences Chinese Academy of Medical Sciences and cultured in DMEM supplemented with 100 IU/mL penicillin, 100 mg/mL streptomycin, 4 mM glutamine and 10% heat-inactivated fetal bovine serum  in a humidified atmosphere of 95% air and 5% CO5 cells were plated onto 25 cm2 culture flasks one day before drug treatment. Cultures were between 50% and 70% confluence at the time of harvest.For all in vitro experiments, cells were released from flasks using phosphate- buffered saline containing 0.01% trypsin and 0.20 mmol/L EDTA, and 1 × 102 atmosphere. The studies were completed with 3 × l03 cells/well plated into 96-well flat bottom microplates (Costar), treated with ES of desired concentration when cells began to grow exponentially. After incubation for 24-96 h, 20 μL of 3--2,5-diphenyl tetrazolium bromide (MTT 5 mg/mL) was added to each well, and the cells were further incubated at 37°C for 4 h. The medium was then removed and 200 μL of DMSO was added to dissolve the reduced formazan product. The plate was then read on a microplate reader  at 590 nm.Cells were maintained as mono-layer cultures in DMEM with 10% FBS and antibiotics (100 U/ml of penicillin and l00 mg/ml streptomycin). The cells were incubated in 5% CO60Co γ ray source at 1.953 Gy/min dose rate. Radiation was performed in the Radiation Department of Peking University.Exponentially growing H-520 cells were irradiated using -D/D0)N was applied to calculate the cellular radiosensitivity , the capacity for sublethal damage repair , and the extrapolation number (N). Those values were used to calculate the SF after irradiation at a dose of 2Gy (SF2) and the sensitization enhancement ratio (SER).H-520 cells of the control and ES groups were exposed to different radiation doses  and incubated for 21 d, then cells were fixed with methanol and stained with Giemsa. Cell colonies which contained more than 50 cells were manually counted. Radiation survival data from ES-treated cells were corrected for plating efficiency (PE) using an unirradiated plate treated with ES under the same conditions. The PE and survival fraction (SF) were calculated as follows: PE = (colony number/inoculating cell number) × 100%. SF = PE (tested group)/PE (0Gy group) × 100%. A dose-survival curve was obtained for each experiment and used for calculating the radiobiological parameters. Three replicates were set at each radiation dose. The cell-survival curve was fitted using Origin 7.5 software according to the multi-target single-hit model after normalizing for the cytotoxicity induced by Endostar alone. The equation of SF = 1- double staining were used for evaluation of cellular apoptosis rate. The H-520 cells were treated with 200 μg/mL ES for 24 h and then irradiated with different doses of 0, 2, 4 and 8Gy. After a 72 h post- irradiation incubation period in 200 μg/mL, all cells were collected, stained with annexin V-FITC for 30 min at 4°C, then stained with PI and analyzed by flow cytometry (FCM) immediately.5 H-520 cells were harvested after treatment with irradiation for 24 h. Then, washed once in 1 ml PBS, and re-suspended in 200 μl staining solution containing FITC-VAD-fmk . After incubation for 20 min at 30°C, cells were washed in 1 ml PBS and re-suspended in 200 μl PBS [5 × 100 μl PBS . SamplesH-520 cells from the control and ES groups were treated with irradiation (0 and 4Gy) and 8, 24, and 48 h later, cells were trypsinized, counted, washed, and re-suspended from all flasks. Cells were then fixed by dropwise addition of 70% ice-cold ethanol and stored at 4°C until the day of analysis. Cells were then washed in phosphate-buffered saline, and re-suspended in PI solution at 50 μg/mL and analyzed by FCM using Coulter EPICS and ModFit software .Bcl-2 and phosphor-p38-MAPK were quantified in control and ES-treated H-520 cells at 24 h after irradiation. Cells were stained with p38-MAPK mAb (Alexa Fluor) or Bcl-2 mAb  and analyzed by FACScan using Coulter EPICS and ModFit software.H-520 cells from the control and ES groups were treated with irradiation (0 and 4Gy), harvested and total cell lysates were resolved on 10% SDS-PAGE, and processed according to standard protocols. The antibodies (Abs) used for western blotting included: monoclonal anti-β Actin , monoclonal antibody against phosphor-Akt . Optimal dilutions of primary Abs were 1:1000 to 1:5000. The secondary Abs anti-rabbit were conjugated to horseradish peroxidase (dilution 1: 5000 to 1: 10000); signals were detected using the ECL system.p < 0.05 was used in this study.Results are presented as mean ± SE of at least three experiments. Student's t test was used to assess the statistical significance of differences. A significance level threshold of The effect of ES on the proliferation of human lung squamous cancer cells H-520 was first determined using MTT assay. As shown in Fig. 2) was 0.56 vs. 0.37 and mean inactivation dose (D0) was 2Gy vs. 1.33Gy in the absence and presence of ES pre-treatment respectively. The SER of ES in H-520 cells was 1.51. The other radiobiological parameters of H-520 cells were: Dq = 0.41 Gy, N = 1.60 and D10 = 4.60Gy, whereas these parameters of ES-treated H-520 cells were: Dq = 0.34Gy, N = 1.79 and D10 = 3.05Gy, respectively. These data indicated that ES had efficient radiosensitization effects on H-520 cells in vitro.In the coming assays, we treated H-520 cells with ES in a dose of 200 μg/mL for 24 h before those cells received different doses of irradiation. The cell survival was detected by a clonogenic assay at 21d after irradiation. As shown in Fig. To evaluate the induction of apoptosis after the combination of ES and irradiation, H-520 cells were treated with 200 μg/mL ES for 24 h immediately before irradiation. Apoptosis cell death was determined by fluorescence microscopy using cells stained with Hoechst 33258. H-520 cells showed typical morphologic changes of apoptosis as cytoplasmic and nuclear shrinkage and chromatin condensation or fragmentation morphology Fig. .The amount of apoptotic cells was determined by annexin V-FITC and PI double staining. As expected, more apoptotic cells were observed when the radiation doses increased Fig. . Radiati0/G1 peak was increased and the G2/M peak decreased in a time-dependent style. There was no significant change in the S-phase after treated with ES for 12 h, but a marked decrease was observed from 24 to 48 h after treatment with ES  for 12 to 48 h, the GProteins involved in apoptosis including Bcl-2 and activated caspase which were tested by flow cytometry. The Bcl-2 expression was decreased in H-520 cells after the treatment with irradiation or ES, but less expression of Bcl-2 in H-520 cells was observed after the treatment with both ES and irradiation Fig. . On the ES influenced signaling pathway, we examined the expression of the phosphorylation status of Akt by Western blotting and p38-MAPK by FCM, which mainly associated with mitogenicity and cell proliferation. As shown in Fig. S) and the invasion of MDA-MB-435 human breast cancer cells [It is known that ED inhibits the angiogenesis of malignant diseases, but it does not inhibit the functions of cancer cells. However, recent studies showed that ED possess direct antitumor activity on cancer cell migration, metastasis, and proliferation ,17,21-23er cells ,24. But q) and mean lethal doses (D0) respectively. It indicates that the repair of sublethal damages and sensitivity to lethal radiation dose was decreased. A significant radiosensitivity effect with the sensitization enhancement ratio (SER) is 1.51. So our findings showed that ES could enhance the sensitivity of H-520 cells to irradiation and had the potential to be a radiosensitizer.It has been reported that ED enhanced the anti-tumor effects of ionizing irradiation in vivo -29. ES s2/M phase, less sensitive in the G0/G1 phase, and least sensitive during the latter part of the S phase [0/G1 phase and a decrease in the S phase [0/G1 phase and a reduction in the H-520 cells of S phase in ES-treated H-520 cells with a time-dependent style. H-520 cells progressing past the G1/S block might accumulate proapoptotic signals caused by both radiation and ES, resulting in increased H-520 cell death [The mechanisms of ES improved H-520 cell response to radiation were multiple, especially in the in vivo models -32. ES c S phase . It was  S phase -39. In tll death . RadiatiA fundamental view on apoptosis was that apoptotic signaling transduction act mostly upon pathways initiating from mitochondria and they eventually met at the level of Bcl-2 family members and the caspases, an ultimate executioners of cell death . It has Previous studies showed that the PI3K-Akt pathway and MAPKs have a significant effect on the cell survival of cancer cells and play an important role in the anti-apoptotic effects of growth factors ,45. TherIn conclusion, ES significantly inhibited cell proliferation and induced apoptosis in human lung squamous cancer cell line H-520 and has the potential to be a radiosensitizer, which may due to the cell cycle redistribution, increase of cell apoptosis and influences on the PI3K-Akt pathway and MAPKs. The present data offer basic information of the combining ES and irradiation on tumor treatment, which may have clinical significance.ED: recombinant human endostatin; ES: Endostar; PE: plating efficiency; SF: survival fraction; SER: sensitization enhancement ratio; D0: mean lethal dose; mean inactivation dose, Dq: quasithreshold dose; N: extrapolation number; SF2: surviving fraction after 2Gy irradiation; MTT: methyl thiazolyl tetrazolium; FCM: flow cytometry; NSCLC: non-small cell lung cancer; HUVECS: human umbilical vein endothelial cells.The authors declare that they have no competing interests.ZYY carried out cell colony-forming assay, fluorescence-activated cell sorting, flow cytometric analysis, and drafted the manuscript. JJW participated in its design and revised the manuscript. FL performed the statistical analysis. YDZ carried out the irradiation experiment. YZ supervised experimental work and revised the manuscript. All authors read and approved the final manuscript.