Conversely, shRNA-mediated knockdown of IL13RA2 successfully overcame the sunitinib-resistance in Caki-1 cells. Histopathological analyses revealed that IL13RA2 repressed sunitinib-induced apoptosis without increasing tumor vasculature in vivo. To our knowledge, this is a novel mechanism of developing resistance to sunitinib in a certain population of ccRCC, and these results indicate that IL13RA2 could be one of potential target to overcome sunitinib resistance.Vascular endothelial growth factor (VEGF) and mammalian target of rapamycin are well-known therapeutic targets for renal cell carcinoma (RCC). Sunitinib is an agent that targets VEGF receptors and is considered to be a standard treatment for metastatic or unresectable clear cell RCC (ccRCC). However, ccRCC eventually develops resistance to sunitinib in most cases, and the mechanisms underlying this resistance are not fully elucidated. In the present study, we established unique primary xenograft models, KURC1  and KURC2, from freshly isolated ccRCC specimens. The KURC1 xenograft initially responded to sunitinib treatment, however finally acquired resistance. KURC2 retained sensitivity to sunitinib for over 6 months. Comparing gene expression profiles between the two xenograft models with different sensitivity to sunitinib, we identified interleukin 13 receptor alpha 2 (IL13RA2) as a candidate molecule associated with the acquired sunitinib-resistance in ccRCC. And patients with high IL13RA2 expression in immunohistochemistry in primary ccRCC tumor tends to have sunitinib-resistant metastatic site. Next, we showed that sunitinib-sensitive 786-O cells acquired resistance  Renal cell carcinoma (RCC) accounts for 2–3% of all malignancies in adults and causes approximately 102,000 deaths worldwide each year .Recent increased understanding of the underlying molecular biology of RCC has established the vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTOR) pathways as relevant therapeutic targets . Agents Sunitinib is a small molecule inhibitor of multiple receptor tyrosine kinases (RTKs), including VEGF receptors , platelet-derived growth factor receptors (PDGFR-α and PDGFR-β), fms-related tyrosine kinase 3 (FLT3), stem cell growth factor receptor KIT, and RET , 8, and Previous reports indicated that primary xenograft models directly transplanted from patient surgical specimens can recapitulate the original clinical course because these implanted tumor grafts contain both epithelial and stromal cells that may co-proliferate to form histologically complex tumors in host mice , 15. TheRCC tumor specimens were obtained at the Department of Urology, Kyoto University Hospital with appropriate written informed consent. This study was approved by the Kyoto University’s institutional review board .786-O and Caki-1 cell lines were purchased from the American Type Culture Collection . Cells were cultured routinely in Dulbecco's Modified Eagle Medium  containing 10% fetal bovine serum supplemented with 1% penicillin/streptomycin. Caki-1 cells stably infected with shRNA lentivirus and 786-O cells stably infected with pBABE-puro retroviruses were selected in the presence of 1.5 μg/ml puromycin.3 fragments, and subcutaneously transplanted into 5-week-old CB-17/Icr-crj SCID mice  on the day of surgery. KURC1  and KURC2 xenografts were established within 3–4 months after the first inoculation. Xenograft tumors were extracted and transplanted into several SCID mice.All experiments involving laboratory animals were performed in accordance with the Guidelines for Animal Experiments of Kyoto University . To establish primary xenograft models, local tumors of the kidney were resected by radical nephrectomy, minced into 20–30 mm7 cells were subcutaneously injected into both flanks of 5–6-week-old female BALB/cA Jcl nude(nu/nu) mice  and tumor volumes were measured twice a week. All experiments were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Following the experimental procedures, all animals were euthanized by carbon dioxide and tumors were excised.To establish cell line xenograft models, a total of 1.0×10Sunitinib provided by Pfizer Global Pharmaceuticals was diluted in dimethyl sulfoxide (DMSO). Sunitinib treatment for primary or cell line xenograft tumors was commenced from the fourth week after transplantation. Forty mg/kg sunitinib was orally administered once a day for the treatment group, and vehicle only was given to the control group. Affected tumors were considered resistant when the tumor growth rate was comparable to that of the control tumor. Tumors were resected 24 hours later from the last sunitinib or vehicle administration. Because of the difference of growth rate and sunitinib response between 786-O and Caki-1 xenograft tumors, 17, we Antibodies were purchased commercially as follows: human IL13RA2 , human STAT6, human phospho-STAT6 Y641 , human ssDNA , human β-actin, human N-cadherin and mouse CD31 .Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded 17 clinical ccRCC samples or xenograft tissues as described previously . The degWhole cell proteins were isolated from snap-frozen specimens or cultured cells and analyzed by immunoblotting as previously described , 21.GAPDH). The primer sequences were as follows: GAPDH, 5′-GAAGGTGAAGGTCGGAGTC-3′ (sense) and 5′-GAAGATGGTGATGGGATTTC-3′ (antisense); and IL13RA2, 5′-TTGCTTGGCTATCGGATGCT-3′ (sense) and 5′-GGGTTAACTTTTATCTCGGTGTCTGA-3′ (antisense).Total RNA was extracted from vehicle-treated control, sunitinib-treated sensitive or resistant tumors of KURC1, KURC2, and cell line subclones, as previously described . cDNA waFor microarray experiments, we used total RNA extracted from two resistant, one sensitive, and two control KURC1 tumors, and one sensitive and one control KURC2 tumor. Microarray analyses were performed at Genetic Lab Co., Ltd.  using GeneChip Human Gene 1.0 ST Arrays . The results were analyzed using GeneSpring GX  by Genetic Lab Co., Ltd. Our microarray data was registered at the NCBI GEO (GSE66346).Lentivirus-based plasmids containing pLKO.1 shRNA sets to human IL13RA2  were purchased from Open Biosystems . Non-silencing shRNAs (Open Biosystems) were used as negative controls. The experimental procedure for shRNA transfection was performed according to the Open Biosystems technical manual.IL13RA2, 5′-CACCATGTATCCATATGATGTTCCAGATTATGCTGGATCCGCTTTCGTTTGCTTGGCTATCGGATGCTTATATAC-3′ and 5′-ACACCCTAACTGACACACATTCCCAGGGTCGACTCATGTATCACAGAAAAATTCTGGAATCATTTTTG-3′. The PCR products were inserted into pBABE-puro vector at BamH1/Xho1 sites using an Infusion-HD Cloning Kit . G3T-hi packaging cells were infected with retroviral plasmids using a Retrovirus Packaging Kit . These experiments were performed according to the manufacturer’s instructions.Full-length IL13RA2 cDNA was amplified by PCR from Caki-1 cell cDNA using PrimeSTAR HS DNA polymerase  and cloned into the pBABE-puro retroviral vector. The oligonucleotide sequences used in the construction of the expression construct were as follows: Oncomine data was obtained from Vasselli et al.  and Bittt-test. In vitro cell proliferation and tumor growth in vivo were analyzed by two-way repeated ANOVA. P values of < 0.05 were considered statistically significant.Data are expressed as the mean ± SE. The significance of differences between means was assessed using the Student’s in vivo. We named them as primary xenograft KURC1 and KURC2. Histopathological diagnosis of primary tumor was clear cell type RCC of Grade 2 > 3, pT2N0M0 for KURC1 and clear cell type RCC, G3, pT3aN0M1 for KURC2. Each xenograft mostly recaptured the histopathological features of original tumors in tumor grade and architecture (Two cohorts of primary xenografts were established and stably engrafted following three or more passages itecture . We haveitecture .th) was shorter than vehicle group (vehicle 5th), and the xenograft of sunitinib 5th acquired a complete resistance to sunitinib with maintaining clear-cell histological appearance and exhibited a similar tumor growth curve to the xenograft treated with vehicle 5th . KURC1 and KURC2 sensitive tumors treated with sunitinib were harvested at day 30 and 60, respectively . SunitinThree genes, IL13RA2, MTMR7 and FAM5B, were significantly highly expressed in sunitinib-resistant KURC1 xenografts (day 50) compared with those treated with vehicle . Conversely, the expression levels of these genes were not significantly changed in sunitinib-sensitive KURC1 (day 30) and sunitinib-sensitive KURC2 compared with those in vehicle-treated KURC1 and KURC2 .Among the three genes, qPCR analysis confirmed that IL13RA2 mRNA was significantly upregulated in sunitinib-resistant KURC1 compared with sunitinib-sensitive KURC1 or those treated with vehicles . The othImmunohistochemical analysis confirmed that the protein expression of IL13RA2 was upregulated in sunitinib-resistant KURC1 tumors compared with sunitinib-sensitive KURC1 tumors or those treated with vehicle . KURC1 wNext, we examined IL13RA2 expression in human metastatic ccRCC tumors. Tumor samples were collected from primary lesion of metastatic ccRCC patients. These patients were subsequently treated with sunitinib and the response was evaluated by Response Evaluation Criteria in Solid Tumors guidelines. Five patients showed progressive disease within 3 month under sunitinib treatment (non-responder), whereas 12 patients showed partial response or stable disease over 3 month (responder). Although 3 patients were responder to sunitinib even high expression of IL13RA2 in their primary tumors, most patients with primary ccRCC tumors of higher IL13RA2 expression were non-responder group when they were treated with sunitinib for metastatic sites compared with those of lower IL13RA2 expression . CollectTo clarify the effect of IL13RA2 on sunitinib sensitivity, sunitinib-sensitive 786-O cells  were infin vivo was significantly suppressed by shRNA-mediated knockdown of IL13RA2  activation and/or upregulation of other pro-angiogenic signaling pathways, for example fibroblast growth factor 1 and 2 (FGF1/2), ephrin A1 and A2 (Efna1/2), and angiopoietin 1 (Ang1) ; 2) incrro model .However, these proposed mechanisms do not fully address the process of acquired resistance to sunitinib in human RCC, because these mechanisms have not been validated in models that are well recapitulated for the clinical course of human RCCs. Furthermore, it is also difficult to compare human clinical tumor tissues before treatment with those after treatment.Although human cancer cell lines are extremely useful for cancer research , 38, manIn our primary xenograft models, KURC1 and KURC2 tumors displayed different characteristics regarding sunitinib sensitivity. KURC1 developed resistance to sunitinib after 4 weeks of treatment but KURC2 remained sensitive for more than 6 months. Although there have been some reports that primary xenograft tumors acquire resistance to sunitinib, previous studies showed that re-transplanted tumors in the next cohort of other mice recovered sensitivity to sunitinib . SurprisTo explore the underlying mechanisms of acquired resistance, we performed a microarray analysis comparing sunitinib-resistant and-sensitive KURC1 with controls, and sunitinib-sensitive KURC2 with controls. Additionally, we performed whole exome sequencing to compare completely sunitinib-resistant KURC1 with controls. Some gene expression changes were identified by microarray analysis, but acquired somatic mutations were not found in the exon regions within these genes of interest (unpublished data). Therefore, we compared gene expression changes in our data set with those in previous reports and evaluated whether these gene expression changes were related to sunitinib resistance.The pro-angiogenic cytokine interleukin 8 (IL-8) was increased in the plasma of sunitinib-resistant xenograft mice, and neutralization of IL-8 blocked tumor angiogenesis and caused tumor re-sensitization to sunitinib , 42. RedIL13RA2 was identified as a candidate molecule associated with sunitinib resistance in our model KURC1, and IL13RA2 expression was significantly higher in human primary ccRCC tumor of patients with sunitinib-resistant metastatic sites. Even though 3 patients were responder to sunitinib whose primary RCC tumors were higher expression of IL13RA2, this molecule may be one candidate of sunitinib-resistance and other mechanism or molecules have been reported. Albeit, it would be better that we evaluated IL13RA2 expression level in the affected tumors after sunitinib treatment or after developing sunitinib-resistance, it is ethically difficult to obtain such samples. Therefore we evaluated that IL13RA2 expression in human primary tumors and their systemic response to sunitinib.Besides, gene expression data derived from Oncomine reported by Vasselli indicated that ccRCC specimens of Furhman Grade 4 showed a significantly higher expression of IL13RA2 compared with those of Grade 3. Moreover, higher expression of this gene was also significantly associated with poor prognosis (dead at 1 year). In general, patients with high grade ccRCC tumors or patients of poor prognosis developed resistance to sunitinib earlier. These data might support our results. However other available dataset in ccRCC reported by Bittner showed IL13RA2 expression was not correlated with tumor grade, albeit it was slightly elevated in the cases of tumor Grade 4 . ConsideIL13RA2 was originally cloned from the human renal cell carcinoma Caki-1 cell line . This prGenerally, in 786-O tumor xenograft model, MVD in sensitive status was significantly decreased by sunitinib treatment, but after long term of sunitinib treatment MVD in resistant status was slightly increased in compared with sensitive status. This mechanism was suggested that other angiogenic factors  were elevated or induced by anti-angiogenic treatment .in vivo. Additionally, growth of xenograft tumors was promoted by upregulation of IL13RA2 and suppressed by silencing of IL13RA2. And moreover, MVD was also decreased by sunitinib treatment independent of IL13RA2 expression level. These data suggested that the mechanisms of acquired resistance related with IL13RA2 expression were not associated with re-angiogenesis in tumors despite the observations in previous reports [In addition to these theories, our data suggested that IL13RA2 expression was correlated with tumor growth and sensitivity to sunitinib. Sunitinib failed to suppress tumor growth of 786-O xenograft tumors when IL13RA2 was overexpressed, and sunitinib inhibited tumor growth of Caki-1 xenograft tumors knocked down for IL13RA2  reports , 49.It was reported that tumor apoptosis was induced by upregulation of IL13RA2 in glioblastoma cells . AnotherRecent studies showed that targeting IL13RA2 may be a new therapy for glioblastoma and head and neck squamous cell carcinoma , 52. We S1 FigN-cadherin staining of xenograft tumors derived from KURC1 and KURC2. Scale bar, 50 μm.(PDF)Click here for additional data file.S2 FigP < 0.01; Students’ t-test). There was no significant difference in KURC2 groups.Evaluation of FAM5B and MTMR7 mRNA expression in KURC1 and KURC2 xenograft tumors treated with sunitinib or vehicle by qPCR. All samples were prepared in triplicate and data are presented as the mean ± SE from indicated number of samples. Columns, mean; bar, SE. The difference in the mRNA expression levels between the sunitinib-treated group and control or sensitive group in KURC1 was statistically significant (*(PDF)Click here for additional data file.S3 Figth by qPCR. Columns, mean; bar, SE. The difference in the mRNA expression levels between the sunitinib-treated group and vehicle group in KURC1 was statistically significant . (B) Immunohistochemical staining of IL13RA2 in KURC1 xenograft tumors repeatedly treated with sunitinib 5th or vehicle 5th. Scale bar, 50 μm. (C) Immunohistochemical staining of IL13RA2 in KURC2 xenograft tumors. Scale bar, 50 μm.(A) Evaluation of IL13RA2 mRNA expression in KURC1 xenograft tumors repeatedly treated with sunitinib or vehicle 5(PDF)Click here for additional data file.S4 Figin vivo. (B) Xenograft tumor growth of 786-O subclone overexpressing IL13RA2 was increased compared with mock control. Day 0 is the day of transplantation. The difference was statistically significant .(A) IL13RA2 staining of xenograft tumors derived from 786-O subclone overexpressing IL13RA2 or mock control. The difference of IL13RA2 expression level was maintained (PDF)Click here for additional data file.S5 Figin vivo. (B) Xenograft tumor growth of Caki-1 subclone was suppressed by shRNA-mediated knockdown of IL13RA2 (#525 and #526) compared with sh-scrambled subclones. Day 0 is the day of transplantation. The difference was statistically significant .(A) IL13RA2 staining of xenograft tumors derived from Caki-1 subclone suppressed by shRNA-mediated knockdown of IL13RA2 and sh-scrambled subclones. The difference of IL13RA2 expression level was maintained (PDF)Click here for additional data file.S6 FigCD31 staining of xenograft tumors derived from (A) 786-O subclones and (B) Caki-1 subclones treated with sunitinib or control. Scale bar, 50 μm.(PDF)Click here for additional data file.S7 FigssDNA staining of xenograft tumors derived from (A) 786-O subclones and (B) Caki-1 subclones treated with sunitinib or control. Scale bar, 50 μm.(PDF)Click here for additional data file.S8 FigData originated from Vasselli et al. (21). Left: IL13RA2 mRNA expression of Grade 3 ccRCC versus Grade 4. Right: IL13RA2 mRNA expression of ccRCC patients alive at 1 year versus those dead at 1 year. Lower: Data originated from Bittner IL13RA2 mRNA expression of ccRCC grouped by tumor grade.(PDF)Click here for additional data file.S1 TableMicroarray analysis were performed using Affymetrix GeneChip Human Gene 1.0 ST Arrays. The 8 genes were previous report related with resistance to sunitinib or sorafenib in RCC.(XLSX)Click here for additional data file.


Purified cFANCD2 L234R did not bind to chicken FANCI, and its monoubiquitination was significantly deficient, probably due to the abnormal ID complex formation. In addition, the histone chaperone activity of cFANCD2 L234R was also defective. These findings may explain some aspects of Fanconi anemia pathogenesis by a FANCD2 missense mutation.FANCD2 is a product of one of the genes associated with Fanconi anemia (FA), a rare recessive disease characterized by bone marrow failure, skeletal malformations, developmental defects, and cancer predisposition. FANCD2 forms a complex with FANCI (ID complex) and is monoubiquitinated, which facilitates the downstream interstrand crosslink (ICL) repair steps, such as ICL unhooking and nucleolytic end resection. In the present study, we focused on the chicken FANCD2 (cFANCD2) mutant harboring the Leu234 to Arg (L234R) substitution. cFANCD2 L234R corresponds to the human FANCD2 L231R mutation identified in an FA patient. We found that cFANCD2 L234R did not complement the defective ICL repair in FANCD2 Fanconi anemia (FA) is a rare recessive disease, with symptoms including bone marrow failure, skeletal malformations, developmental defects, and cancer predisposition Sixteen genes have been identified as FA causative genes, and all are conserved in higher eukaryotes except for FANCM and FANCP, which are found even in yeast species in vitro, and the monoubiquitination of FANCD2 in the ID complex is robustly enhanced in the presence of DNA The FA core complex, which contains a ubiquitin E3 ligase subunit (FANCL), then monoubiquitinates both the FANCI and FANCD2 subunits of the ID complex in vitro and in vivo, and complements the ICL repair-defective phenotype of the FANCD2−/− DT40 cells with a significantly reduced rate In addition, FANCD2 possesses histone chaperone activity, which modifies the chromatin structure by promoting histone deposition/eviction around the ICL sites A number of FANCD2 mutations, which are generally considered to reduce protein stability, have been identified in FA patients −/− DT40 cells For the construction of the cFANCD2 fusions, the cDNA encoding FANCD2 was inserted into the GFP or the histone H2B-GFP expression vector, using the Gateway system (Invitrogen). To establish stable cell lines expressing the cFANCD2 fusions, the plasmids were transfected into FANCD2The cisplatin sensitivity of the DT40 cell lines was assessed by colony formation, in medium containing the indicated amount of cisplatin (Nihon-Kayaku) and 1.4% methylcellulose, as previously described The chicken DT40 cells were treated with MMC (500 ng/ml for 6 h) or without MMC, and the chromatin fraction was obtained by the method described previously The chicken DT40 cells producing the GFP-cFANCD2 proteins were lysed in lysis buffer, containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, 5 mM NaF, and protease inhibitor cocktail (Roche). Afterward, the lysates were treated with 50 unit/mL of Benzonase, and were mixed with anti-GFP beads (MBL). After three washes with lysis buffer, the immunoprecipitates were analyzed by western blotting using anti-chicken FANCD2 and cFANCI antibodies.6-tag fused proteins in the Escherichia coli BL21(DE3) codon(+)RIL strain (Stratagene), and were purified as previously described Wild type cFANCD2 and chicken FANCI (cFANCI) were expressed as HisEscherichia coli BL21(DE3) codon(+)RIL strain. The GST-tag was removed from hUBE2T by PreScission protease digestion during the purification steps. The concentrations of the purified proteins were assayed by the Bradford method The purification of chicken FANCL (cFANCL) and human UBE2T (hUBE2T) was performed by the previously described methods Escherichia coli BL21(DE3) cells, and human histone H4 was overexpressed in Escherichia coli JM109(DE3) cells. All core histones were expressed as His6-tagged proteins. The histone H3/H4 and H2A/H2B complexes were each formed by the salt dialysis method, and were purified as described previously 6-tag was removed during the purification, using thrombin protease.Human core histones were purified by the same method described previously The purified cFANCD2, cFANCI, and equimolar mixture of cFANCD2 and cFANCI were subjected to Superdex 200 10/300 GL gel filtration column chromatography , in buffer containing 20 mM Tris-HCl (pH 8.0), 10% glycerol, 200 mM NaCl, and 5 mM 2-mercaptoethanol. The elution profiles of the purified proteins were monitored by UV absorption at 280 nm. The fractions at 7.5–14.5 mL of elution volume were separated by 7% SDS-PAGE, and the proteins were detected by silver staining.2, and 0.5 mM dithiothreitol. The reaction mixtures were incubated for 90 min at 30°C. The reactions were then stopped by the addition of 10 µL of 2-fold SDS buffer, containing 125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.01% bromophenol blue, and 0.2 M dithiothreitol. The samples were separated by 7% SDS-PAGE, and the ubiquitinated products were visualized by western blotting using an anti-HA antibody  and by Coomassie Brilliant Blue staining. The amounts of monoubiquitinated FANCD2 were quantitated by LAS4000 using Multi Gauge software (Fujifilm).The monoubiquitination assay was performed as previously described 2, 2.8 mM dithiothreitol, and 3.3% glycerol. To start the nucleosome assembly reaction, 100 ng of relaxed circular φX174 dsDNA, which was preincubated with 2 units of wheat germ topoisomerase I (Promega) at 37°C for 150 min, in buffer containing 18 mM Tris-HCl (pH 8.0), 8% glycerol, 84 mM NaCl, 2 mM MgCl2, 0.2 mM EDTA, and 2.7 mM dithiothreitol, was added to the reaction. The reaction was continued at 37°C for 60 min. Afterwards, the reaction mixture was deproteinized with 60 µL of a proteinase K solution, containing 20 mM Tris-HCl (pH 8.0), 20 mM EDTA, 0.5% SDS, and 0.5 mg/mL proteinase K. The DNA was then extracted with phenol-chloroform, and subsequently precipitated with ethanol. Topoisomers were separated by 1% agarose electrophoresis in TAE buffer (40 mM Tris-acetate and 1 mM EDTA), and were visualized by SYBR Gold (Invitrogen) staining.The supercoiling assay for nucleosome assembly was performed as previously described −/− chicken DT40 cells producing the cFANCD2 L234R mutant, in which the Leu234 residue was replaced by Arg. We found that the cFANCD2 L234R mutant was not monoubiquitinated in the whole cell extracts, unlike wild-type cFANCD2 , which corresponds to the R302W mutant of human FANCD2, exhibited substantially decreased histone chaperone activity isplatin . On the S1 FigureThe full-length gel images for .  Cells expressing the GFP-fused wild type (WT) cFANCD2 or cFANCD2 L234R were treated with or without mitomycin C (MMC), and the WCEs were analyzed by western blotting using anti-FANCD2 (left panel) and anti-FANCI antibodies (right panel). S-forms and L-forms indicate non-ubiquitinated forms and monoubiquitinated forms of cFANCD2 and cFANCI, respectively. Non-specific bands are marked by the asterisks.  Cells expressing GFP-fused WT cFANCD2 or cFANCD2 L234R were treated with or without MMC, and the chromatin fractions were analyzed by western blotting using anti-FANCD2 (left panel) and anti-FANCI antibodies (right panel). “S” indicates non-ubiquitinated forms of cFANCD2 and cFANCI, and “L” indicates monoubiquitinated forms of cFANCD2 and cFANCI. Asterisks indicate the degradation products of GFP-cFANCD2 generated during the chromatin preparation.(TIF)Click here for additional data file.S2 FigureThe full-length gel images for .  Gel filtration analysis of the ID complex formation. Fraction numbers are shown in the bottom of gel-filtration profiles, and fractions indicated above the gel images were analyzed by 7% SDS-PAGE, and proteins were visualized by silver staining. Markers are indicated as ‘M’. Experiments with wild type (WT) cFANCD2 and WT cFANCI (A), cFANCD2 L234R and WT cFANCI (B).  Immunoprecipitation analysis of the cFANCD2 and cFANCI interaction. The cell extracts from chicken DT40 cells expressing GFP-WT cFANCD2 or cFANCD2 L234R were immunoprecipitated with an anti-GFP antibody. The immunoprecipitates were analyzed by western blotting using anti-chicken FANCD2 (C) and FANCI antibodies (D).(TIF)Click here for additional data file.


In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors , kinases , proteases , signal transduction proteins  and transcription factors . Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study.Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY), or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx) and maltose binding protein (MBP) were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions.E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases proteins may be truncated to minimise molecular weight and the numbers of contiguous hydrophobic amino acids and low complexity regions to aid soluble expression in E. coli.By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in  To achieve this on a whole proteome scale a generic approach must be taken to the expression of protein families, unlike the traditional approach of protein chemistry in optimising the isolation of individual proteins on a case by case basis. E. coli has been the expression system of choice for the majority of laboratories engaged in high-throughput, multi-plexed cloning, expression and purification of proteins for structural genomics [E. coli as an expression host include well studied physiology, genetics and availability of advanced genetic tools [C. elegans full-length genes were cloned and expressed in E. coli only 11 % yielded soluble protein [in vitro refolding has been used in a number of cases, but refolding conditions are highly protein specific and so unlikely to be useful for high-throughput protein expression.The production of purified proteins is important for several experimental approaches aimed to assign gene function including antibody generation for immunocytochemistry and immunoprecipitation studies -3, in viractions ,5 and stractions . The avagenomics . The advic tools -10, rapiic tools  and protic tools . There a protein . Similar protein . With th protein ,16 or en protein  assays. E. coli one of which is to truncate long multi-domain proteins into separate domains, as has been performed for the Ephb2 receptor [There are several fall-back strategies for expression of correctly folded eukaryotic proteins in receptor ,18,19. Rreceptor  or inducreceptor . Alternareceptor ) or chapreceptor ; the indreceptor  or chemireceptor ; or the receptor ,26, overreceptor  or lipidreceptor . Perhapsreceptor -31.The aim of this work was to ask if it is possible to derive some general conclusions regarding which expression strategy would most likely result in the expression of soluble, functionally active mammalian protein on a family-by-family or domain-by-domain basis. A deep-mining approach was taken to maximise the chances of successful expression by examining the soluble expression of 30 different proteins using 14 different expression vectors. This study allowed us to make several conclusions regarding the best strategies to adopt for the soluble expression of different mammalian proteins in bacteria. The conclusions were tested by the expression of an additional 95 mammalian proteins. and sequence confirmed. A recombinational cloning strategy was employed termed "GATEWAY" cloning [The 30 proteins chosen for this expression study are listed in Table  cloning ,36 based cloning . Primers cloning  and openFor this study a set of destination vectors were constructed by modifying pET-DEST42 . The T7 promoter was chosen over other promoters commonly used for bacterial expression because of the high specificity and processivity of T7 RNA polymerase and the wide choice of expression strains currently available. Briefly, multicloning sites were created either 5' of the attR1 or 3' of the attR2 recombination sites for insertion of DNA inserts encoding N or C-terminal tags respectively. The expression vectors contained a T7lac promoter  for imprE. coli BL21(DE3), an expression strain containing chromosomally integrated T7 RNA polymerase gene (λDE3 lysogen) under the control of the lacUV5 promoter. To handle a large number of expression experiments  and associated manipulations to screen for total and soluble expression in E. coli, the recombinational cloning, transformation, growth of expression cultures and cell lysis and filtration separation of insoluble protein were performed in 96-well plate format. Figure Expression plasmids generated by recombination reactions were used to transform eriments 0 total aLooking first at the results for total (soluble and insoluble) expression, no clear patterns emerge for the various expression vectors used. With the exception of CASP2, CDKN2A, Trp53, EGFR(TK), FOS and CD44 most proteins expressed well across all expression vectors. Interesting differences are apparent however when one looks at the production of soluble protein. Using decahistidine green fluorescent protein (H10-GFP) or decahistidine glutathione-S-transferase (H10-GST) as fusion partners at the N-terminus gave poor yields of soluble intact product. This may not be because they were poor at promoting soluble expression but because they were prone to proteolysis during cell lysis reducing the yield of full-length soluble protein. A set of proteins , HRAS, mdm2(p53-bd), Ephb2(TK) and CCND2) gave high soluble expression levels in the baseline N-terminal decahistidine vector, which was not improved when expressed as decahistidine thioredoxin (H10-Trx) or decahistidine maltose binding protein (H10-MBP) fusions. The molecular weight of these proteins ranged from 9 – 35 Kda and averaged 22.8 Kda. These proteins are all expressed in the cytoplasm, have an average of 1 low-complexity region, 3.8 contiguous hydrophobic amino acids (hp_aa), pI of 6.6, grand average of hydropathicity index  was dramatically poorer at expressing soluble protein compared with the vector pN110 (which encodes a H10 tag), with average soluble expression yields of 0.8 and 3.3 mg per litre of culture respectively. Both vectors contain T7 RNA polymerase promoters, but pN110 also contains a lac operator (lacO) downstream of the promoter and the gene encoding the lac repressor (lacI) for tighter control of gene expression. This may result in a faster rate of transcript synthesis, after induction with IPTG, and hence translation rates (due to an increased concentration of mRNA) for pDEST17 compared with pN110. If translation rate exceeds the rate of protein folding, then increased production of insoluble protein would occur.A similar study was performed where the 30 ORFs were cloned into 8 different C-terminal tag expression vectors shown in Figure MBP-H10 was the most effective tag at the C-terminus to promote protein solubility with an average construct full-length soluble yield of 5.0 mg/l, which compares well with an average of 5.8 mg/l when this tag is fused at the N-terminus. The order of C-terminal tags to promote soluble expression was similar for total expression: MBP-H10 > GST-H10 > V5-H6 > Dhfr-H10 ~ GFP-H10 ~ Trx-H10 > H10 ~ DHFR-H10. Thioredoxin was not as effective a solubility enhancing tag when fused at the C-terminus with an average soluble yield of only 0.7 mg/l compared with 6.0 mg/l when fused to the N-terminus.Several correlations with protein features are seen when one groups the MPB fusions according to soluble protein expression levels. For the first group, where soluble expression levels were in the range of 5 – 50 mg/l, the average molecular weight, pI and GRAVY score were 20.6 KDa, 5.9 and -0.58 respectively. The average numbers of contiguous hydrophobic amino acids, low complexity and coiled-coil regions were 3.1, 0.56 and 0.22 respectively. The second group displayed soluble expression levels between 1 – 5 mg/l. Here, the average molecular weight, pI and GRAVY score were 25.1 KDa, 7.9 and -0.39 respectively and the average numbers of contiguous hydrophobic amino acids, low complexity and coiled-coil regions were 4.3, 0.71 and 0 respectively. The last group displayed soluble expression levels between 0 – 1 mg / l. Here the average molecular weight, pI and GRAVY score were 41.1 KDa, 6.2 and -0.51 respectively and the average numbers of contiguous hydrophobic amino acids, low complexity and coiled-coil regions were 5, 2.43 and 0.21 respectively. There were representatives of nuclear, cytoplasmic and extra-cellular proteins in all three groupings.E. coli previously (Nfkb1), to those that contain no PfamA domains and have not been expressed in E. coli previously . They included 24 proteins not annotated as PfamA domains, with unknown function. All cDNAs were amplified from a primary cDNA library, cloned into pDONR221 and sequence confirmed prior to transfer to pDEST-N112-MBP  , sub-celet al.  tested tet al. found a similar relationship with their set of 32 human proteins with 4 different N-terminal fusions [There was a strong correlation between successful soluble expression and molecular weight of the protein. Small proteins with an average molecular weight of 22.8 KDa did not require to be fused with solubility enhancing proteins for soluble expression whereas proteins that required to be fused with N-terminal MBP or Trx for soluble expression had an average molecular weight of 40.4 KDa and those where the addition of a N-terminal fusion could not rescue soluble expression had an average size of 51.4 KDa. The same pattern also emerged in the C-terminal fusion study. The decreasing probability of successful soluble expression of mammalian proteins with increasing molecular weight is likely due to increasing protein complexity, perhaps requiring specialised eukaryotic chaperones for folding or stabilising binding partners. The majority of proteins solubly expressed in this study contained single domains and as fusion proteins were either capable of self-folding or were folded with the aid of prokaryotic chaperones. Braun  fusions .in vivo.A correlation in this study was observed between increasing numbers of contiguous hydrophobic amino (hp_aa) acids (AILFWV) and soluble expression. This ranged from an average of 3.8 hp_aa for those proteins not requiring a N-terminal fusion for high level soluble expression to 5 hp_aa for proteins requiring a N-terminal fusion for successful expression and 5.6 hp_aa where expression failed under the conditions described here. This pattern was also repeated in the C-terminal fusion study where good expression proteins had an average of 3.1 hp_aa whereas poor expression proteins had an average of 5 hp_aa. In a study of the sequences of 2753 non-membrane proteins it was found that the sequences of three or more consecutive hydrophobic residues are suppressed in globular proteins . Low comSome interesting conclusions were drawn when soluble expression was measured for an additional set of 95 mammalian proteins expressed as H10-MBP fusions Table . In seveet al. [There have been three comparative studies recently where sets of proteins were cloned into several expression vectors and the effects of the fusion partner on total and soluble expression yield were examined. Hammarstrom et al.  cloned 2et al.  Here GSTet al. . HoweverIn this study it was found that, on average, N-terminal fusion partners are preferable for optimal protein expression. When proteins are expressed with their native N-terminus, as in our C-terminal fusion proteins, total expression levels can be more variable than when expressed with a constant N-terminal tag. This may be because of variable RNA secondary structures in the region around the start codon which could interfere with ribosome binding. An additional explanation is that during translation the expressed protein emerges from the ribosome first and initiates an incorrect, irreversible, folding pathway before the soluble fusion partner has been translated and folded. The mis-folded protein would be ubiquitin labelled and targeted to the proteasome for degradation resulting in lower total expression levels. This scenario is more likely when expressing mammalian proteins in a bacterial system which lacks specific eukaryotic chaperone proteins. It has been shown previously that proteins prone to mis-folding and aggregation can arrest GFP folding when fused at the C-terminus . HoweverIt was found that Trx and MBP were the best N-terminal protein fusions to promote protein solubility. The best C-terminal fusion to promote protein solubility was MBP and this may be acting as a true intra-molecular chaperone , able toIt must be stressed here that although protein solubility is a useful indicator of correct folding, additional measurements need to be performed to give supporting evidence for correct folding. These may include removing the protein fusion with a protease and analysis of the cleaved protein of interest by a variety of biophysical and functional assays such as analysis of monodispersity by light scattering , NMR 5656,57, CDGFP did not significantly enhance soluble protein expression when fused to the C-terminus of the proteins in this study, supporting the use of this tag as an indicator of soluble protein expression of fused ORFs.,41 The oE. coli? If the protein has a molecular weight of less than 30 KDa and contains 1 or less low complexity regions and less than 4 contiguous hydrophobic amino acids expression of the full-length protein in E. coli should give good levels of soluble protein. As a generic strategy we would recommend expressing the protein with a fusion partner and found MBP and Trx to be the best fusions to enhance protein solubility as N-terminal tags with MBP being superior as a C-terminal fusion. C-terminal fusions are desirable for proteins such as the P450s where N-terminal tags can inhibit functional activity. When fused to an optimal fusion partner, nuclear, cytoplasmic and extra-cellular domains were equally likely to be expressed solubly. For larger proteins over 50 KDa, truncations should be considered to express specific protein domains and to minimise the molecular weight, number of low complexity regions and contiguous hydrophobic amino acids. In conclusion, this study will help enable a systematic expansion in the number mammalian proteins and domains that can be successfully expressed in E. coli as soluble product, and also predict which are best targeted for a eukaryotic expression system.What guidelines have emerged from this study in developing a strategy for the production of soluble mammalian proteins in E. coli DB3.1 and BL21(DE3)Star pLysS, Gateway BP and LR clonase enzyme mix, pre-cast 4–12 % NuPAGE Bis-Tris gels and PVDF membranes (0.45 μm pore size) were all from Invitrogen . Entry plasmids in both open (minus stop codon) or closed format (plus stop codon) containing the full-length genes for GRB2, HRAS, JUN, FOS, MAD, MAX, CDK2, CDK4, CDKN1B, CASP2, MMP1, CDKN2A and CD44 were provided by Pascal Braun and Josh LaBaer . A full length clone containing the full-length human EGFR ORF was provided by the RIKEN BioResource Center  and Efna1 from the Mammalian Gene Collection (MGC) archived at the Wellcome Trust Sanger Institute . First strand synthesis human and mouse cDNA was from BD Biosciences . Plasmid, gel extraction and PCR purification kits and 6xHis protein ladder were purchased from Qiagen . The expression strain BL21(DE3), BugBuster protein extraction reagent and His tag monoclonal antibody was from Merck Biosciences . The 96-well multiscreen-DV durapore filter plate with 0.65 μm pore size was from Millipore  and Cy5-labelled goat anti-mouse IgG from Amersham Biosciences . Europium labelled antibodies and DELFIA reagents were from Perkin Elmer  and all other chemicals unless otherwise stated were from Sigma-Aldrich .Oligonucleotides were synthesised by Qiagen-Operon  or Sigma-Genosys . All restriction enzymes were from New England Biolabs . The vectors pET-DEST42, pDEST17 and pDONR201 and 4, and 0.5 unit KOD hot start DNA polymerase (Novagen). PCR cycling conditions were: 94°C – 2 mins followed by 15 cycles of 94°C – 15 s, 59°C – 30 s, 68°C – 9 mins. The 7468 bp PCR product was purified using a PCR purification spin column (Qiagen) and eluted with 30 μl of 10 mM Tris-HCl (pH8.5), digested with 20 units of DpnI enzyme at 37°C for 4 hrs, to remove methylated plasmid DNA, purified by spin column and an intramolecular ligation reaction performed using 16 ng of linear PCR product and 5 units T4 DNA ligase and the buffers from the rapid ligation kit (Roche). The ligated PCR product was used to transform E. coli DB3.1 and the resultant pET-DEST42-MCS plasmid DNA prepared and sequence confirmed. Insert 1, encoding a decahistidine tag with a 5'-NdeI site and blunt 3' end, was prepared by PCR with primer pairs 22 (5' GGAATTCCATATGAAUCAC 3') and 24 (5' pGTGATGGTGATGGTGATGGTGATGGTGATTCATATGGAATTCC) and insert 2 encoding a decahistidine tag flanked by a 5'-NdeI site and 3'-KpnI site was prepared with primer pairs 22 and 26 (5' CGGGGTACCATGGTGATGGTGATGGTGATGGTGATGGTGATTCATATGGAATTCC 3'). PCR reactions were as above except the annealing temperature dropped to 44°C, extension time to 10 s and 12 cycles employed. Insert size was checked by 10 % TBE-PAGE and purified by a nucleotide removal kit (Qiagen). Expression vectors (b) pDEST-N110 and pDEST-N112 , pET41a or pET32 (Novagen) respectively The primer pairs for MBP were 78 (5' TTATTACACGAAGTGAAAATCGAAGAAGGTAAACTGGTAATC 3') and 79 (5' pGTTCGAGCTCGAATTAGTCTGCGCGTCTTTC), for GFP 84 (5' TTATTACACGAAGTGGCTAGCAAAGGAGAAGAACTTTTCACTGGAG 3') and 85 (5' pTTTGTAGAGCTCATCCATGCCATGTGTAATC 3'), for GST 86 (5' TTATTACACGAAGTGTCCCCTATACTAGGTTATTGGAAAATTAAGGG 3') and 87 (5' pATCCGATTTTGGAGGATGGTCGCCACC 3') and for Trx 88 (5' TTATTACACGAAGTGAGCGATAAAATTATTCACCTGACTGAC 3') and 89 (5' p CAGGTTAGCGTCGAGGAACTCTTTC 3'). The inserts were digested with DraIII and ligated with DraIII, BfrBI cut pDEST-N112 vector to create the GATEWAY destination vectors pDEST-112-MBP, pDEST-112-GFP, pDEST-112-GST, pDEST-112-Trx.To prepare pET-DEST42-MCS, a multi-cloning site was inserted into pET-DEST42 (Invitrogen) at nt396, between the shine-dalgarno sequence and the attR1 recombination site, encoding the recognition sequences for NdeI, KpnI, DraIII and BfrBI. Inverse or whole plasmid PCR was performed on pET-DEST42 with 5'-phosphorylated PAGE purified primer pairs 20 (5' TACCCACGAAGTGATGCATACAAGTTTGTACAAAAAAGCTGAACG 3') and 21 (5' CCCATATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAG 3') in a 20 μl reaction containing 10 ng pET-DEST42, 0.3 μM primers 20 and 21, 20 mM Tris-HCl (pH 7.5), 0.5 mM DTT, 200 μM each of dATP, dCTP, dGTP and dTTP, 1 mM MgSOpDEST-C101 was designed to insert a decahistidine encoded sequence between the attR2 recombination site and T7 transcription termination region. pDEST-C102 is as C101 except a DraIII, BfrBI site was inserted downstream of the attR2 recombination site. Inverse PCR was performed as described above with primer pairs 1 (5' pCACCATCACCATCATCACCATCACCATTGAGTTTGATCCGGC) and 2 (5' pATGCACCACTTTGTACAAGAAAGCTGAAC) to generate pDEST-C101 and primer pairs 1 and 3 (5' pATGCATACCACTCACTTCGTGCACCACTTTGTACAAGAAAGCTGAAC) to prepare pDEST-C102. Murine and human dihydrofolate reductase (Dhfr and DHFR respectively) inserts flanked by a 5' DraIII site and blunt end at the 3' were amplified from MGC clones using the primer pairs 82 (5' TTATTACACGAAGTGCGACCATTGAACTGCATCGTCGCCGTG) and 83 (5' pGTCTTTCTTCTCGTAGACTTCAAACTTATAC 3') for Dhfr and 80 (5' TTATTACACGAAGTGGGTTCGCTAAACTGCATCGTCGCTGTG) and 81 (5' pATCATTCTTCTCATATACTTCAAATTTG) for DHFR. The DraIII digested inserts were ligated with DraIII, BfrBI digested pDEST-C102 vector to create pDEST-C102-MBP, GFP, GST, Trx, Dhfr and DHFR as shown in Figure et al. [4, and 0.5 unit KOD hot start DNA polymerase (Novagen) in a total volume of 20 μl. The PCR reaction consisted of 94°C – 2 mins, and 30 cycles of 94°C – 15 s, 55°C – 30 s, 68°C – 2.5 mins followed by 68°C – 5 mins. A 50-fold dilution of the PCR 1 reaction was made for the second 30 cycle PCR containing the ORF specific primers. Linkers were added to these primers encoding half the attB1 and attB2 sites for forward and reverse primers respectively. For entry clone generation to be transferred to N-terminal tag expression vectors the 5'-linkers for the forward and reverse primers were 5' AAAAAGCAGGCTCT 3' and 5' AGAAAGCTGGGTTCTA 3' respectively with the reverse primer adding a stop codon. For inserts destined to the C-terminal tag expression vectors the forward and reverse primers were 5' AAAAAGCAGGCTTCGAAGGAGATAGAACCATGG 3' and 5' AGAAAGCTGGGTT 3' respectively with the forward primer encoding the shine-dalgarno and kozak sequences and start codon. PCR 2 products were analysed by 1 % TBE-agarose electrophoresis[A nested PCR strategy was used to isolate protein encoding ORFs directly from cDNA adapted for GATEWAY cloning from the method described by J. E. Collins et al. . Brieflyophoresis and corrophoresis except tophoresis were use600 had reached 1 (3 hrs for BL21(DE3)), 60 μl was transferred to 1.2 ml 2xYT media in a 96-deep-well block containing the appropriate antibiotics, placed on a shaking incubator at 37°C and when the OD600 reached 0.5 (2 hrs for BL21(DE3)) IPTG added to a final concentration of 1 mM and shaking continued at 25°C for 12 hours. Total protein was analysed by transferring a 20 μl aliquot of the induced culture to a 96-well PCR plate containing 20 μl of 2 × NuPage LDS loading buffer (Invitrogen), 0.1 M DTT, heated to 95°C for 10 mins and cooled on ice prior to loading 10 μl on a 17-well 4–12 % NuPAGE Bis-Tris gels with a multi-channel gel loading syringe (Hamilton). Soluble protein was extracted by transferring 290 μl of induced culture to a shallow well plate, centrifugation at 3000 g for 5 mins, supernatant removed and cells were resuspended in 58 μl BugBuster containing 1.4 units of benzonase and 58 units of recombinant lysozyme (Novagen). For the C-terminal tag and expression strain comparison this buffer was also supplemented with 0.58 μl protease inhibitor cocktail set III 10-fold diluted in DMSO (Novagen). The cell-pellets were resuspended with a multi-channel pipette and incubated with slow shaking for 20 mins at room temperature prior to transfer to 96-well multiscreen-DV durapore filter plates with 0.65 μm pore size (Millipore). The filter plate was placed on top of a shallow 96-well plate and centrifuged at 1000 g for 2 mins. 4 μl of the filtrate was then added to a 96-well plate containing 5 μl of 4 × NuPage LDS loading buffer (Invitrogen), 11 μl of 182 mM DTT, the plate heated at 95°C for 5 mins and loaded onto a 17-well 4–12 % NuPAGE Bis-Tris gel. A His-tagged molecular weight ladder (Qiagen) was also loaded onto each gel. Gel electrophoresis and electro-transfer to PVDF membrane was as described.[All BL21(DE3) transformants were selected and propagated in the presence of 100 μg/ml ampicillin. A single antibiotic resistant colony was used to inoculate 0.5 ml 2xYT media in a 96-deep well block containing the appropriate antibiotics and shaken at 210 rpm at 37°C. When the average ODMRD performed the molecular biology, participated in the bioinformatics, expression screening, quantitation, experimental design and drafted the manuscript. SPS and RLP participated in the expression screening and quantitation. KJV helped with the bioinformatics . JM participated in the experimental design, coordination and helped to draft the manuscript. All authors approved the final manuscript.


Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies.Serotyping of O-(lipopolysaccharide) and H-(flagellar) antigens is a wideley used method for identification of pathogenic strains and clones of fliC genes present in E. coli reference strains U9-41 (O2:K1:H4) and P12b (O15:H17) was determined and both were found 99.3% (1043 of 1050 nucleotides) identical in their coding sequence. A PCR/RFLP protocol was developed for typing of fliC-H4 strains and 88 E. coli strains reacting with H4 antiserum were investigated. Nucleotide sequencing of complete fliC genes of six E. coli strains which were selected based on serum agglutination titers, fliC-PCR genotyping and reference data revealed 96.6 to 100% identity on the amino acid level. The functional expression of flagellin encoded by fliC-H4 from strain U9-41 and from our strain P12b which is an H4 expressing variant type was investigated in the E. coli K-12 strain JM109 which encodes flagellar type H48. The fliC recombinant plasmid carrying JM109 strains reacted with both H4 and H48 specific antisera whereas JM109 reacted only with the H48 antiserum. By immunoelectron microscopy, we could show that the flagella made by the fliC-H4 recombinant plasmid carrying strain are constituted of H48 and H4 flagellins which are co-assembled into functional flagella.The complete nucleotide sequence of fliC genes present in serologically different types of E. coli strainswhich were isolated at different time periods and geographical locations. Our expression studies show for the first time, that flagellins of different molecular weigth are functionally expressed and coassembled in the same flagellar filament in E. coli.The flagellar serotype H4 is encoded by closely related  Enterobacteriaceae species Escherichia coli are common as commensals in the intestinal flora of humans and warm-blooded animals [E. coli strains were developed in the early 1940ies when it became evident that certain E. coli strains were agents of infantile gastroenteritis [E. coli O- (lipopolysaccharide) and H-(flagellar) antigens which allowed the grouping of E. coli strains according to their O:H-types (serotypes) [E. coli (EPEC) strains from stools of diarrhoeic infants [E. coli strains from both humans and animals [Bacterial strains belonging to the  animals . Typing nteritis . In 1944rotypes) . Serotyp infants  and is s animals ,3.E. coli populations by multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (MLST) allowed the detection of clonal types of strains which carry specific virulence markers and are associated with disease in humans [E. coli [E. coli which can occur in different combinations in wildtype isolates of strains [E. coli serotyping and the laborious typing procedure restricts its usage to a few reference laboratories. Therefore, alternative typing methods were developed including molecular characterization of genes coding for the O- and H-antigens in E. coli [fliC genes by PCR was successfully employed for characterization of human pathogenic O157:H7 and O26:H11 strains [fliC genes coding for flagellar antigens H7 and H6 revealed large sequence similarities between strains sharing the same H-type but different O-types [fliC gene allows H-typing of non-flagellated (non-motile) E. coli isolates which cannot be analyzed for their flagella with H-specific antisera [The genetic analysis of n humans ,7. It wa[E. coli ,7. At pr strains ,3,5,8. H E. coli -12. Typi strains ,14. Anal O-types . Moreoveantisera .E. coli belonging to many different O-groups including strains of shiga toxin-producing E. coli (STEC) and extraintestinal pathogenic serotypes [). Moreover, a cryptic fliC-H4 gene was described to be present and to be spontaneously expressed in E. coli strain P12b (O15:H17) which carries another type of flagella called H17 which is not encoded by the fliC gene [E. coli strains belonging to different O-serogroups and pathotypes. We have used the published nucleotide sequence of the fliC gene present in the E. coli H4 reference strain U9-41 (accession AB028472) to develop a PCR which allows discrimination between the fliC-H4 gene variants. Nucleotide sequence analysis of the fliC gene was performed on other E. coli H4 strains that either showed deviations in the PCR analysis or were reported to harbour allelic types of the H4-fliC gene [fliC genes and have introduced them into the laboratory E. coli K-12 strain JM109 [The flagellar type H4 is frequently occurring in erotypes -20,  and P12b (O15:H17) [E. coli K-12 strain JM109 (O-rough:H48) (this study), which was used for expression studies were investigated for inhibition of motility in swarm-agar containing 1:600 dilutions of either H4, or H48-antiserum (see Methods). U9-41 and P12b were fully inhibited for their motility in the presence of H4 antiserum derived from strains U9-41 or from P12b but not in the presence of H48 specific antiserum. The E. coli K-12 strain JM109 was not inhibited for motility by H4 antisera but by H48 antiserum. These findings indicate that H4 antisera specifically inhibited the motility of E. coli H4 strains and that the antigenically different flagellar type H17 was not expressed or lost in our P12b isolate, similar as previously described with spontaneously arising variants of P12b [E. coli H17 strains would also carry the genes for expression of H4 type flagella as it was described for P12b [E. coli H17 strains  for their serological reaction with different H4 specific antisera and all strains showed specific positive reactions . Differences in agglutinating titers between H4U9-41 and H4P12b antisera were not more than twofold with either strain indicating that both sera were similar for their specificity (Table E. coli H-types for polymorphisms in the fliC gene by HhaI digestion of fliC-PCR products as described in the Method section.O15:H17)  and the  of P12b . We becafor P12b ,22,24. FE. coli H-serotypes [E. coli fliC genes coding for flagellar types H1 to H56 revealed individual patterns corresponding to the different H-serotypes  in the corresponding E. coli strains and the fliC HhaI patterns obtained from these strains do therefore not correspond to their H-serotypes [fliC gene in strain P12b codes for flagella of serotype H4. The relationship between fliC genes present in the different E. coli strains was further investigated by nucleotide sequencing.PCR products obtained with primers fliC-1 and fliC-2 were digested with HhaI to obtain H-serotype specific RFLP patterns from reference strains for the 53 different erotypes . HhaI-RFerotypes ,25-27. Herotypes  , P12b (O15:H17), U1-41 (O5:K4:H4), P7d (O68:H4), C107-74 (O15:H17) and E1541-68 (O154:H4) as listed in Table fliC genes present in these strains revealed in all cases a length of 1050 bp. As a control, the fliC gene of the strain U9-41 was sequenced and found to be identical to the previously deposited fliC sequence (accession AB028472). The identity of the fliC-H4 sequences ranges from 97,6 % to 100 %. The greatest sequence difference to the fliC gene coding sequence of strain U9-41 was found in the fliC gene of strain P7d (exchange of 20 nucleotides), whereas the fliC sequences of strains U1-41 and U9-41 were identical to each other. All deduced flagellins have a length of 349 amino acids. The greatest deviation in the primary structure to the FliC protein of the reference strain U9-41 was again observed for the FliC protein of strain P7d (exchange of 9 aa in a stretch of 349 aa). Fig. To obtain the entire coding region of the s U9-41 O:K1:H4, Ps U9-41 O:K1:H4, PfliC genes present in E. coli strains U9-41 and P12b with primers fliC-1 and fliC-2 resulted in the generation of a 953 bp internal PCR product with both strains  from 88 E. coli strains which showed agglutination reactions with H4 antisera (Table MboI restriction profiles which corresponded to the patterns obtained with strain U9-41 (O2:K1:H4) (Table Amplification of the fliC-H4 genes in a different genetic background we cloned the corresponding PCR products of strains U9-41 and P12b into the vector pLITMUS38 as described in the Methods section. The fliC coding regions were inserted downstream of the lacZ promoter of pLITMUS38 and the fliC recombinant plasmids were transformed into the laboratory E. coli K-12 strain JM109 [fliC-pattern as the E. coli reference strain P4 (O16:H48) [fliC-H4 genes in the JM109 derivative strains TPE1976 (fliC-H4U9-41 clone) and TPE1978 (fliC-H4P12b clone) was analyzed by tube agglutination with H4 and H48 antisera, respectively. The strains TPE1976 and TPE1978 showed agglutination with H48 and H4 sera whereas the parental JM109 strain reacted only with H48 serum  and H17 (P12b) flagellar antigen [fliC-H4 genes from different E. coli strains with primers which were generated on the basis of the previously published fliC sequence (accession AB028472) of the E. coli H4 reference strain U9-41. The comparison of the fliC gene sequence encoding H4 flagella in strain P12b with the fliC sequences of five other E. coli H4 or H17 strains revealed a high similarity on the DNA and on the amino acid level  is a 51.3 kDa protein consisting of 498 amino acids and thus much larger than the 36.3 kDa H4 flagellin which is composed of 349 amino acids. However, both flagellins share conserved N- and C-terminal sequences which are known to be involved in the structural assembly of flagella [The functional expression of the cloned ace Fig. . Single ace Fig.  and doubace Fig.  experimeflagella . H48 andflagella .fliC gene in E. coli can change the antigenic properties of the flagella made by this strain. Horizontal transfer of fliC genes may contribute to the diversity of flagellar serotypes by recombination within E. coli recipient strains. The mammalian host immune system is the driving force for continuous selection of new flagellar antigens in E. coli. Published data indicate that both mutation and recombination events in the fliC gene have taken place in the evolution of E. coli flagellar antigens [We were able to show that the introduction of an isolated antigens ,16.fliC sequence data have shown that the flagellar type H4 which is present in E. coli strains of clinical importance covers several genetic variants which are closely related to each other. We have shown for the first time that flagellins of different molecular size are expressed and coassembled into functional flagella in a laboratory E. coli K-12 strain.Our E. coli were obtained from the International Escherichia and Klebsiella Centre, Statens Seruminstitut, Copenhagen, Denmark and are described elsewhere [E. coli isolates originating from humans and animals was investigated by PCR/RFLP typing for fliC-H4 genes. These strains were previously investigated for the O-types and for production of Shiga-toxins (Stx) and were isolated in different countries between 1941 to 2002 [E. coli K-12 strain JM109 is described elsewhere [The reference strains used for production of antisera for O- and H-typing of lsewhere . Origin  to 2002 ,19,30 (Tlsewhere .E. coli were prepared according to ∅rskov and ∅rskov [fliC encoded H4 antigen and produced flagellar type H4 (this work).Rabbit antisera against the different O- and H-antigens of d ∅rskov . Antiserd ∅rskov . Our strE. coli strains was tested by inoculating bacteria in tubes containing 10 ml portions of swarm-agar (L-broth + 0.3% agar) as described [E. coli strains in the presence of flagellar-specific antiserum (H4 and H48) was tested in swarm-agar containing a 1:600 dilution of the respective antiserum. Cultures which were inhibited for motility were observed over two weeks for possible switch to motility by phase variation.Expression of flagella and swarming of H-serotyping was performed as described . In briefliC genes present in the E. coli reference strains as described [E. coli reference strains for 53 different H-types [The oligonucleotide primers fliC-1 (5' CAA GTC ATT AAT AC(A/C) AAC AGC C 3') and fliC-2 (5' GAC AT(A/G) TT(A/G) GA(G/A/C) ACT TC(G/C) GT 3') were used for amplification of internal parts of escribed . The PCRescribed . PCR profliC-H4 sequence (AB028472) were used for the amplification of the entire fliC-H4 coding regions. The PCR was performed for 30 cycles: 30 sec at 94°C, 60 sec at 58.1°C and 90 sec at 72°C with primers fliC-5 (5'-TGA GTG ACC AGA CGA TAA CAG GG-3') and fliC-6 (5'-GGA CGA TTA GTG GGT GAA ATG AGG-3') and yielded a 1243 bp product. PCR products were purified with the QIAquick™ PCR Purification Kit  and used for sequencing. Sequencing reactions were carried out using the dye terminator chemistry  and separated on an automated DNA sequencer . The sequences were analysed using the Lasergene software  and the Mac Vector software  to assemblings and alignings.Two primers deduced from the published E. coli strain P12b (O15:H17) with a size of 1234 bp containing the fliC gene for flagellin has been submitted to EMBL data library under accession number AJ515904. The coding sequences of the different fliC genes from the following strains have been assigned the following accession numbers: AJ605764 for strain U1-41 (O5:K4:H4), AJ605765 for strain P7d (O68:H4), AJ605766 for strain C107-74 (O15:H17) and AJ536600 for strain E1541-68 (O154:H4). The origin of the strains is listed in Table The nucleotide sequence of the genomic region of fliC-H4 genes were obtained from genomic DNA prepared as described [E. coli strains U9-41 and P12b using primers fliC-5 and fliC-6. The amplification products were inserted into the vector pLITMUS38  digested with EcoRV. The orientation of the the insert PCR products was determined by using commercially available sequencing primers LITMUS forward 28/38 and LITMUS Reverse 28/38 (New England Biolabs).PCR products encompassing the complete coding sequences of the escribed  of E. coE. coli strains were produced by repeated passage on semi-solid agar followed by growth in L-Broth as described above. Cultures carrying recombinant pLITMUS38 plasmids were grown in the presence of 100 μg/ml ampicillin. IEM was performed using fresh, non-formalized cultures of motile bacteria. For IEM, aliquots of respective bacterial cultures were diluted 1:2 in PBS pH 7.2 and adsorbed onto glow-discharge treated 400 mesh grids coated with Pioloform and carbon  [E. coli strains were incubated with rabbit anti-H4 (1:1000) followed by anti-rabbit-IgG-5 nm gold and two washing steps on droplets of blocking buffer for 10 min. The samples were subsequently incubated with anti H48 antibody (1:1000) followed by incubation with anti-rabbit-IgG- 10 nm gold. Removal of surplus conjugate and negative staining were performed as detailed above.Motile cultures of Germany) . Grids wfliC recombinant plasmid carrying strains. AM and HG developed the IEM methodology and HG contributed to the data analysis. All authors participated in review and preparation of the final manuscript.LB conceived of the study and carried out PCR genotyping and coexpression studies. ES carried out sequence determination and alignments and construction of recombinant plasmids. SZ and SK performed serological assays and PCR genotyping. CS performed analysis of


We also determined whether subchronic treatment with pramipexole regulates dopamine transporter function, thereby reducing intracellular transport of the active metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP+).Our aim was to determine if pramipexole, a Dh). Mice were pretreated for 3 days and during the 2-day MPTP regimen with pramipexole (0.1 mg/kg/day) or saline. Stereological quantification of dopamine neuron number and optical density measurement of dopamine fiber loss were carried out at 1 week after treatment, using immunostaining for dopamine transporter (DAT) and tyrosine hydroxylase (TH). Additional wild-type (WT) and D3 receptor knockout (KO) mice were treated for 5 days with pramipexole (0.1 mg/kg/day) or vehicle. The kinetics of [3H]MPP+ and [3H]DA uptake (Vmax and Km) were determined 24 h later; and at 24 h and 14 days dopamine transporter density was measured by quantitative autoradiography.Ten 12-month old C57BL/6 mice were treated with MPTP  twice per day at 20 mg/kg s.c. (4 injections over 48 Vmax for [3H]DA and [3H]MPP+ uptake into striatal synaptosomes of WT mice. Pramipexole treatment lowered Vmax in WT but not D3 KO mice; however, D3 KO mice had lower Vmax for [3H]DA uptake. There was no change in DAT number in WT with pramipexole treatment or D3 KO mice at 24 h post-treatment, but there was a reduction in WT-pramipexole treated and not in D3 KO mice at 14 days post-treatment.Pramipexole treatment completely antagonized the neurotoxic effects of MPTP, as measured by substantia nigra and ventral tegmental area TH-immunoreactive cell counts. MPTP- induced loss of striatal innervation, as measured by DAT-immunoreactivity, was partially prevented by pramipexole, but not with regard to TH-IR. Pramipexole also reduced DAT- immunoreactivity in non-MPTP treated mice. Subchronic treatment with pramipexole lowered the + taken up into DA terminals via DAT. D3 receptor plays an important role in this regulation of transporter uptake and availability.These results suggest that protection occurs at clinically suitable doses of pramipexole. Protection could be due to a reduced amount of MPP An interesting development in the use of dopamine (DA) agonists for treatment of Parkinson's disease (PD) is that some of them have proven to be neuroprotective in animal models of PD. Antiparkinsonian agents that are direct DA agonists, such as apomorphine , bromocr2 receptor subtype DA and [3H]MPP+ uptake through the DAT. Kinetics of [3H]DA uptake in mouse striata demonstrated a higher Vmax but a similar Km to that reported for rat fresh striatal postnuclear preparation [Km was similar due to inclusion of COMT inhibitor in the preparations [Vmax (P = 0.0008) and the Km (P = 0.016) for [3H]DA uptake was significantly decreased in pramipexole treated mice. The Vmax (P < 0.001) and the Km (P = 0.001) for [3H]MPP+ uptake were also significantly decreased in pramipexole treated mice.Since pramipexole treatment in non-MPTP treated mice altered levels of DAT-IR, it was important to identify if pramipexole treatment altered the kinetics of [paration . Uptake arations . C57BL/6Vmax and the Km for [3H]DA uptake by subchronic treatment with pramipexole was due to the D3 receptor, the values for Vmax and the Km for [3H]DA uptake was measured in WT (C57BL/6) and D3 KO littermate mice  and for Km values . Synaptosomes prepared from D3 KO mice had significantly lower Vmax  and Km , values for [3H]DA uptake than WT mice  and Km  values for [3H]DA uptake compared with WT mice treated with saline. D3 KO mice treated with pramipexole had significantly lower Vmax  and Km  values for [3H]DA uptake than WT mice treated with saline, but D3 KO mice treated with pramipexole were not different from D3 KO mice treated with vehicle. WT mice treated with pramipexole were also not different from D3 KO mice treated with pramipexole.To determine if the reduction in  methods ), treateVmax and the Km for [3H]DA uptake by subchronic treatment with pramipexole at 24 h post-treatment was due to the D3 receptor regulation of the number of DAT sites, the density of DAT sites was measured in WT and D3 KO mice treated with vehicle or pramipexole for 5 days. ANOVA showed that there were no group differences -PD 128907 does interact with D3 autoreceptors to regulate extracellular DA levels. Those data are consistent with acute treatment with D2/D3 agonists leading to increased Vmax DA and [3H]MPP+ uptake in mice at 24 h post-treatment. Interestingly, D3 receptor KO mice exhibited substantially lower Vmax and Km values of [3H]DA uptake than WT mice, and pramipexole did not further reduce Vmax and Km values in D3 receptor KO mice. However, the actual density of sites on the DA terminals was not reduced 24 h after pramipexole treatment in WT mice (or in D3 receptor KO mice), as determined by DAT autoradiography. The inability to modulate the number of DAT binding sites 12 h after termination of subchronic treatment with D2/D3 receptor agonists has previously been reported RTI binding to DAT sites in pramipexole treated WT mice 14 days after pramipexole treatment, but not in D3 receptor KO mice. Thus, there might be both rapid and slower modifications in DAT function produced by D3/D2 agonist treatment, ultimately resulting in lower DAT number, and mediated by the D3 receptor.We observed that subchronic treatment with pramipexole reduced the reported , suggest2/D3 receptor agonists, and increased by the dopamine D2/D3 receptor antagonist, but not by D1 agonists and antagonists 1-methyl-4-phenylpyridinium (MPP+) and [3H]DA uptake in synaptosomes derived from the striatum were deterimined by the method of Eshleman et al [Km and Vmax of [3H]MPP+ and DA uptake were made by t-test (0.05 level of significance).Fourteen C57/Bl6 mice  were divided into 2 groups: one group received pramipexole (0.1 mg/kg) once a day for 5 days and the other group received the vehicle . An additional 10 WT and 10 Dan et al , with mi4 1.2 mM, pargyline 10 uM, glucose 0.2%, ascorbic acid 0.02%, pH7.4). To the 8 ml sample, butaclamol was added at a final concentration of 100 nM, 4 ml of sample was than removed and placed in a separate 15 ml centrifuge tube  and Mazindol  was added at a final concentration of 40 uM. 50μl of the samples were added to borosilicate tubes  and placed in a 25°C water bath with the drugs for a 10 min preincubation. The assay was initiated by adding 50μl concentrations of unlabeled MPP+ or DA ranging from 0–300 nM with [3H]MPP+ or [3H]DA at a final concentration of 20 nM. The samples were incubated at 25°C for 10 min. Specific uptake was defined as the difference in uptake observed in the absence and presence of mazindol (40μM). Uptake was terminated after 5 min by filtration through Whatman GF/C filters presoaked in HEPES buffer. Scintillation fluid was added to each filtered spot and radioactivity remaining on the filters was determined using a Wallac β-scintillation spectrometer. Each experiment involved triplicate determinations, and 6 independent experiments for each drug competition curve were performed.Mouse striata were dissected and homogenized with a glass-Teflon homogenizer in ice-cold modified HEPES (1 ml). The sample was centrifuged at 1000 g, for 10 min at 4°C. The supernatant was collected and centrifuged at 14,000 g for 10 min at 4°C. The pellet was resuspended in 8 ml of HEPES buffer  were divided into 2 groups: one group received pramipexole (0.1 mg/kg) once a day for 5 days and the other group received the vehicle . Twenty-four h after the last injection of pramipexole or vehicle, the mice were overdosed as above and the brains rapidly removed and frozen on dry- ice. An additional group of 10 WT and 10 D3 receptor knockout mice  were similarly treated but their brains were processed 14 days after the last treatment. Autoradiography of DAT sites were quantified following labeling with [125I]RTI-55 (3ß-(4-iodophenyl)tropan-2 ß-carboxylic acid methyl ester)  in the presence of 100 nM paroxetine (Smith Klein Beecham BRL 29060A) to block the serotonin transporter, according to published methodology [3H-Hyperfilm for 18 h for DAT. Autoradiographs were analyzed using a computer-based image analysis system  that converts transmitted optical density to the amount of radioligand bound in pmol per microgram of protein.10 WT and 10 D3 KO = D3 receptor knockoutDDA = dopamineDAT = dopamine transporterMPP+ = 1-methyl-4-phenylpyridiniumMPTP = 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinePD = Parkinson's diseaseSN = substantia nigraTH = tyrosine hydroxylaseVTA = ventral tegmental areaWT = wild typeThe author of this manuscript received a grant from Pharmacia Corporation to partially support the costs of the research. This company is no longer in existence.CW and HR carried out the MPTP treatment, performed the immunocytochemistry and participated in the statistical analysis. SB and DH carried out the uptake assays, performed the DAT autoradiography and participated in the statistical analysis. JNJ conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.