TARDBP, which encodes TAR DNA-binding protein 43 (TDP-43), are a rare cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). While the majority of mutations are found in the C-terminal glycine-rich domain, an alanine to valine amino acid change at position 90 (A90V) in the bipartite nuclear localization signal (NLS) of TDP-43 has been described. This sequence variant has previously been shown to cause cytoplasmic mislocalization of TDP-43 and decrease protein solubility, leading to the formation of insoluble aggregates. Since the A90V mutation has been described both in patients as well as healthy controls, its pathogenic potential in ALS and FTD remains unclear. Here we compare properties of overexpressed A90V to the highly pathogenic M337V mutation. Though both mutations drive mislocalization of the protein to the cytoplasm to the same extent, M337V produces more significant damage in terms of protein solubility, levels of pathogenic phosphorylation, and formation of C-terminal truncated protein species. Furthermore, the M337V, but not the A90V mutant, leads to a downregulation of histone deacetylase 6 and Ras GTPase-activating protein-binding protein. We conclude that in the absence of another genetic or environmental ‘hit’ the A90V variant is not sufficient to cause the deleterious phenotypes associated with ALS and FTD, despite prominent cytoplasmic protein relocalization of TDP-43.Mutations in the gene  Amyotrophic lateral sclerosis (ALS) is a fast progressing neurodegenerative disorder associated with atrophy of upper and/or lower motor neurons leading to muscle wasting due to denervation. It is the third most frequently occurring neurodegenerative disease after Alzheimer’s disease and Parkinson’s disease . Death uFigures 1A,C)  . Mutatio in 2008 . The fre in 2008 .TARDBP mutations are missense mutations, with all but three located in the C-terminal glycine-rich domain  and the M337V mutation, a pathogenic mutation associated with familial ALS , in the NM_007375.3 for human TARDBP. The WT construct was cloned into the pCMV6-AN-myc vector for expression of N-terminally tagged WT or mutant TDP-43 protein. Custom mutagenesis to generate the A90V (point mutation c.269C>T) and M337V (point mutation c.1009A>G) TDP-43 amino acid substitutions were carried out by Origene. The presence of each mutation was confirmed by DNA sequencing performed by GENEWIZ .All plasmids were purchased from and customized by Origene. cDNA sequences were based on the accession number 2 in a humidified atmosphere in minimum essential medium  supplemented with L-glutamine, 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were subcultured every 3–4 days. For immunofluorescence analysis, cells were grown on glass coverslips coated with 1 mg/mL poly-L-lysine (Sigma) in 24-well plates (Cellstar). For Western blot analysis, cells were grown in 6-well plates (Costar). Cells were transiently transfected using Fugene HD (Promega) following the manufacturer’s instructions 24 h after seeding (Fugene HD:DNA ratio 3:1). Total transfection time was 48 h.SH-SY5Y cells were grown at 37°C with 5% COThe following antibodies were used for immunofluorescence staining: rabbit N-terminal TDP-43 , mouse anti-myc 9E10 , mouse anti-scaffold attachment factor B (SAFB) 6F7 , mouse anti-protein-associated splicing factor (PSF) , mouse anti-ubiquitin P4D1 . Anti-mouse and anti-rabbit secondary antibodies coupled to Alexa-488, Alexa-555, and Alexa-568 were used for detection . For Western blot analysis, the following antibodies were used: rabbit C- and N-terminal TDP-43 , mouse S409/410 phospho-TDP-43 , mouse α-tubulin , rabbit HDAC6 , mouse G3BP , mouse acetylated α-tubulin , mouse GAPDH 6C5 . Anti-mouse and anti-rabbit horseradish peroxidase-coupled secondary antibodies were purchased from Jackson Immunoresearch .Cells were washed in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (in PBS) for 15 min followed by permeabilization in 0.25% Triton-X (in PBS) for 10 min. Ten percent normal goat serum  was used as a blocking reagent. Cells were blocked for 1 h at room temperature and incubated overnight at 4°C in primary antibody solution. The next day, cells were washed with PBS and incubated for 1 h in secondary antibody solution. Coverslips were mounted with Prolong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Image acquisition was performed using a Nikon A1 confocal/Eclipse Ti inverted microscope system and NIS Elements software (Nikon).g and 4°C (Beckman Optima TLX ultracentrifuge with TLA100.3 rotor and Delrin adaptors). The supernatant was collected as the RIPA-soluble fraction. The pellet was washed in RIPA buffer and centrifuged for an additional 30 min at 100,000 × g and 4°C. The supernatant was discarded and the pellet was re-extracted in 100 μL urea buffer -1-propanesulfonate ]. The samples were sonicated 2x 15 s and centrifuged at room temperature for 30 min at 100,000 × g. The supernatant was collected as the urea-soluble fraction.The extraction protocol was adapted from Cells were lysed in RIPA buffer supplemented with 2 mM EDTA, 1 mM EGTA, protease inhibitors  and phosphatase inhibitors , and the protein concentration was determined by bicinchoninic acid (BCA) assay. RIPA and urea fractions obtained from sequential extractions were diluted 1:1 with RIPA buffer. Proteins were separated using standard Tris-glycine SDS-PAGE gels  and transferred onto nitrocellulose (BioRad) or polyvinylidene difluoride  membranes. All blocking and antibody incubation steps were performed either in 5% milk in Tris-buffered saline (TBS) supplemented with 0.1% Tween-20 (TBS-T) or in 3% bovine serum albumin (BSA) in TBS-T. Western blots were developed with enhanced chemiluminescent substrates (ECL). Digital images were acquired with a ChemiDoc MP imaging system (BioRad) and quantified with Image Lab 5 (BioRad). Where necessary, blots were stripped with stripping buffer for 15 min  and reprobed with loading control antibodies.The composite schematic of the full structure of human TDP-43 was produced from the nuclear magnetic resonance (NMR) structures 2N4P , 2016, 2p ≤ 0.05. Experiments were replicated a minimum of three times.All data were expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 6 software . Normality was established using the Shapiro–Wilk test. Mean differences between groups were analyzed by one-way ANOVA and Tukey’s multiple comparisons test. The threshold for statistical significance was in vitro efforts in elucidating its effects. Thus, we attempted to collate all structural information obtained from NMR and X-ray crystallography available in the public domain and obtain a complete 3D model of TDP-43 . However, structures including all domains which might enable a comprehensive examination of the effects of specific mutations on overall structure and stability are challenging and not yet realized. The human TDP-43 sequence  and NMR structures  in Figure 1 highlight the NLS, NES, RRM1, RRM2, glycine-rich domain, as well as the A90V and M337V mutation sites and the S409/S410 phosphorylation site  and thus the impact of the A90V and M337V amino acid substitutions. Without a priori assumptions of the impact of the A90V variant based on a complete model, we then proceeded to investigate the consequences of expressing the A90V variant on common pathological phenotypes associated with disease: cytoplasmic relocalization, and the formation of insoluble TDP-43 and TDP-43 protein fragments. We contrasted the A90V variant with the well-characterized mutant M337V, which causes autosomal-dominant familial ALS . Steady state levels of total TDP-43 protein were significantly increased in cells expressing M337V mutant TDP-43 compared to WT expressing cells , whereas there was no significant difference in cells expressing the A90V TDP-43 variant compared to WT protein . We confirmed that this increase in M337V TDP-43 protein levels was not caused by increased levels of mRNA, which were comparable between genotypes . In agreement with the previous report (p = 0.041). Interestingly, the proportion of transfected cells with cytoplasmic TDP-43 localization with A90V was very similar compared to the highly pathogenic mutant M337V .We then performed immunofluorescence staining of transiently transfected cells in order to confirm the relocalization phenotype observed by s report , we obseFigure 2E). These TDP-43-positive puncta were only rarely observed in SH-SY5Y cells transfected with WT TDP-43 (0.28 ± 0.10% of transfected cells), but were found in significantly higher numbers in cells expressing the pathogenic mutant M337V . However, we saw no significant difference in puncta formation between WT and A90V TDP-43 expressing cells . In order to more closely investigate the composition of these TDP-43-containing puncta, we performed co-staining experiments with two nuclear markers: the nuclear stress body marker SAFB and the paraspeckle marker PSF . We qualitatively distinguished between transfected cells with small, numerous nuclear TDP-43-positive puncta  and cells with few, but large, puncta . We observed no co-localization between TDP-43 protein and either SAFB or PSF in small puncta . In cells harboring large TDP-43-positive puncta, both SAFB and PSF were found to be excluded from these structures. We hypothesized that these puncta represent intranuclear inclusions of insoluble, aggregated TDP-43 protein. As aggregated TDP-43 is found pathologically ubiquitinated . As expected, we detected diffuse ubiquitin staining in both the nucleus and cytoplasm. Both small and large TDP-43 nuclear puncta were shown to co-localize with ubiquitin, indicating that they are bona fide aggregates of ubiquitinated TDP-43 protein.In a small number of transfected cells analyzed by immuno fluorescence staining we detected a punctate staining pattern for TDP-43 in the nucleus . In line with previous reports . While an enrichment of TDP-43 in the urea fraction was also observed in A90V-expressing cells (1.59 ± 0.38-fold increase compared to WT), the increase was not statistically significant .In order to further investigate how the A90V and M337V mutations affect the cell biology of TDP-43, we measured a range of cellular phenotypes which have been implicated in disease pathogenesis. Initially, we looked at the formation of insoluble TDP-43 aggregates. We fractionated lysates of transfected SH-SY5Y cells into RIPA-soluble and RIPA-insoluble, urea-soluble fractions by high speed centrifugation and separated proteins by SDS-PAGE. We used a TDP-43 antibody recognizing the N-terminus of TDP-43 to assess total TDP-43 protein levels in the RIPA and urea fractions . We normalized the phospho-signal in the urea-soluble fractions to the total TDP-43 detected in these fractions to control for the relative increase in insoluble TDP-43 protein in mutant expressing cells . Immunoblotting with this antibody revealed significantly increased phosphorylation levels at the S409/S410 residues in the urea-soluble fraction of M337V TDP-43 expressing cells compared to cells expressing WT TDP-43 . We detected a non-statistically significant increase in S409/S410 phosphorylation in cells transfected with the A90V variant . Taken together, these results indicate that while the A90V and M337V mutations induce similar levels of cytoplasmic relocalization of TDP-43, a pathology commonly associated with disease, the effects of the M337V variant on protein solubility and phosphorylation are far greater than those of the A90V mutation. In fact there looks to be a spectrum of pathology in these transfected cell models with WT and M337V TDP-43 at the opposite extremes, with the A90V TDP-43 somewhere in between, in the absence of any additional stressors.We used a phospho-specific TDP-43 antibody raised against the disease-relevant phosphorylation sites at S409/S410  to assesFigure 4 we extracted soluble and insoluble TDP-43 from transfected SH-SY5Y cells and used a C-terminal antibody . We identified two smaller protein species present only in TDP-43-transfected cells, but not in the vector control cells . The larger fragment ran close to the 34 kDa marker, while the smaller fragment ran close to the 26 kDa marker. C-terminal TDP-43 fragments with molecular weights of 35 kDa (CTF35) and 25 kDa (CTF25) have been described in a variety of cellular and animal models . However, we did not detect a significant increase of the smaller CTF25 in mutant-transfected cells . In the urea-soluble fractions the effect of the M337V mutant on the formation of these C-terminal fragments was even more striking: we detected a 20.0 ± 6.6-fold increase in the 25 kDa fragment in the M337V mutant compared to WT  and a 4.3 ± 1.7-fold increase compared to the A90V variant . We could not detect any 35 kDa fragment in the urea fraction of WT-TDP-43 transfected cells and found a 5.4 ± 1.1-fold increase in CTF35 levels in M337V- compared to A90V-transfected cells .In addition to abnormal phosphorylation as well as decreased protein solubility and aberrant cellular localization, another pathological feature of TDP-43 is the formation of C-terminal truncated protein fragments . In ordel models , and we p = 0.01; 23.0 ± 7.8% compared to mock, p = 0.03; Tukey’s multiple comparisons analysis; one-way ANOVA F3,20 = 4.861; P = 0.0106; Figures 6A,C). Furthermore, levels of HDAC6, a known target of TDP-43 that is likewise downregulated upon TDP-43 silencing . We also probed for levels of acetylated α-tubulin, a substrate for HDAC6 (p = 0.99) or A90V (p = 0.99) TDP-43 , suggesting that the decrease in HDAC6 protein levels in M337V-expressing cells is not sufficient to impair acetylation of α-tubulin. In comparison to mock-transfected controls, both WT and A90V-transfected cells showed a small, but significant upregulation of acetylated α-tubulin  and M337V-transfected cells showed a similar trend . An increase in α-tubulin acetylation has been described in conditions of cellular stress . We then resorted to more traditional in vitro studies to measure TDP-43 mislocalization, changes in protein solubility and phosphorylation, and the effects on their expression on known targets of TDP-43. We compared the impact of A90V with that of the M337V mutation, a disease-associated mutation frequently used for in vitro and in vivo studies of ALS pathogenesis (To date, genetic studies have revealed 52 missense mutations in the TDP-43 gene mutation . While m and FTD . Further (TARDBP . Genetic (TARDBP . It is togenesis .When we initially looked to reproduce the nuclear localization phenotype observed by in vitro have been described in the literature: overexpression of certain deletion mutants or the M337V mutant has been shown to cause the formation of nuclear inclusions of aggregated TDP-43 (In a small subset of transfected cells, we detected dot-like structures in the nucleus that stained positive for TDP-43. These TDP-43-positive nuclear structures were observed more frequently in cells transfected with the M337V, but not the A90V, mutant compared to WT. Instances of defined, patterned nuclear staining of TDP-43 d TDP-43 , while od TDP-43 . We sougin vitro, in vivo, and in culture systems (We investigated the aggregation propensity of the A90V and M337V mutants further by sequential extraction of total cell lysates. We found a stark increase in M337V in the insoluble fraction, in line with published literature showing increased aggregate formation of M337V-TDP-43  systems . Compare systems . While tC-terminally cleaved TDP-43 fragments are another pathological hallmark of ALS and FTD, and expression of TDP-43 in cell culture models can give rise to cleavage products of 35 and 25 kDa in the absence or presence of various stressors . We founIn addition to the effects of the A90V variant on TDP-43 aggregation, post-translational modifications and truncation, we investigated changes in known targets of TDP-43. We found that protein levels of G3BP are reduced in SH-SY5Y cells expressing M337V, but not A90V TDP-43. G3BP is a component of cytoplasmic stress granules, transient structures made from proteins and untranslated mRNAs, which form after insults such as heat shock and oxidative stress . Similarin vitro effects observed with the A90V variant, carriers might not develop a disease phenotype in the absence of a “second hit.” However, it is possible that the A90V mutant could lower the threshold required for an additional stressor or other genetic risk factor to cause the catastrophic effects on TDP-43 and its functions observed in ALS and FTD. Thus, we conclude that the A90V variant of TDP-43 could be a rare genetic risk factor for TDP-43 proteinopathies. However, further studies will be required to gain an insight into the interplay between A90V as a genetic risk factor and other genetic and environmental risk factors associated with ALS and FTD.In summary, the results of this study show that the A90V variant of TDP-43, which has been described as a rare variant found both in ALS and FTD patients and healthy controls, shows modest effects on protein solubility, phosphorylation, and truncation when compared to the disease-causing M337V mutation. In addition, in contrast to the M337V mutant, the A90V variant does not cause impairment of HDAC6 or G3BP expression. These findings are especially striking since we observed comparable levels of cytoplasmic TDP-43 relocalization in both A90V- and M337V-transfected cells, suggesting that cytoplasmic relocalization of TDP-43 protein is not by itself sufficient to cause the other molecular phenotypes associated with disease. Furthermore, we speculate that due to these modest HW and NB designed experiments; HW, JC, and LD carried out experiments; HW and JC analyzed the data; SW designed the composite NMR structure; JM and TD provided technical assistance; HW, NB, SW, and SM wrote the manuscript. All authors reviewed and approved the final version of the manuscript.SM serves as a consultant for AstraZeneca and SAGE Therapeutics in relationships that are regulated by Tufts University and do not impact on this study. At the time this work was conducted SW, SJ, JD, and NB were full-time employees and shareholders in AstraZeneca.The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


S-nitrosylation and S-acylation of cysteine residues regulate many functions of cytosolic proteins. We therefore used a resin-assisted capture approach to identify these modifications in Htt. In contrast to many proteins that have only a single S-nitrosylation or S-acylation site, we identified sites along much of the length of Htt. Moreover, analysis of cells expressing full-length Htt or a large N-terminal fragment of Htt shows that polyQ expansion strongly increases Htt S-nitrosylation. This effect appears to be general since it is also observed in Ataxin-1, which causes spinocerebellar ataxia type 1 (SCA1) when its polyQ tract is expanded. Overexpression of nitric oxide synthase increases the S-nitrosylation of normal Htt and the frequency of conspicuous juxtanuclear inclusions of Htt N-terminal fragments in transfected cells. Taken together with the evidence that S-nitrosylation of Htt is widespread and parallels polyQ expansion, these subcellular changes show that S-nitrosylation affects the biology of this protein  Huntington’s disease (HD) is an inherited movement disorder characterized by progressive degeneration of medium spiny neurons of the striatum, as well as other neuronal cell types. The pathogenic mutations of the HTT gene are (CAG) trinucleotide repeats that exceed 40. The (CAG) repeats encode a polyglutamine tract (polyQ) near the N-terminus of the Htt protein  1–6].–6.1–6].Full length Htt is conserved among vertebrates and chordates and is widely expressed in nucleated cells –9. ExpreMany proteins interact with both normal and polyQ-expanded Htt. These include proteins implicated in 14-3-3 signaling, microtubule-based transport, proteostasis, and fission of mitochondria . SeveralIn addition to proteolytic cleavage, Htt undergoes multiple posttranslational modifications –35. For Cysteine modifications, such as S-nitrosylation and S-acylation regulate the functions, physical properties and localization of many proteins –44, inclAlterations of protein S-nitrosylation and S-acylation have been reported in several neurodegenerative diseases, including HD . HoweverCollagen (C5533), HEPES (H4034), EDTA (E5134), Diethylene triamine pentaacetic acid , Neocuproine hydrochloride (72090), SDS (L6026), S-Methyl methanethiosulfonate , N-Ethylmaleimide , N-Methylmaleimide , Iodoacetamide (I1149), Sodium L-ascorbate (11140), Hydroxylamine hydrochloride (55459), Anti-FLAG antibody , Anti-actin antibody , poly-L-lysine solution (P4707), and histology mounting medium  were from Sigma. DMEM medium (11965092), F12K medium (21127022), Horse serum (26050088), FBS (26140079), and HBSS buffer (14025076) were from Gibco. Protease inhibitor cocktail (05892791001) and Protein G-agarose (11719416001) were from Roche. Anti-NOS1 , anti-NOS2 , and anti-NOS3  were from Santa Cruz Biotechnology. Anti-Htt  and Anti-Htt  were from EMD Millipore. SDS-Out SDS Precipitation kit (20308) and Imperial protein stain (24615) were from Thermo. BCA assay (23225) was from Pierce. Anti-Htt  was from AbD Serotec. Immun-Star AP chemiluminescence (1705010) was from Bio-rad. Polyclonal anti-GFP  was from Abcam. Monoclonal anti-GFP  was from Clontech. Thiopropyl Sepharose 6B (17042001) was from GE. MS grade trypsin (V5111) was from Promega. Lipofectamine 3000 (L3000015) was from Invitrogen. Ponasterone A (P-1083) was from A.G. Scientific. NP40 (19628) was from USB.2. HEK293T  and COS7  cells were grown in DMEM with 5% FBS. Recombinant proteins were expressed in these cell lines by using Lipofectamine 3000, following the instructions of the manufacturer. Cells in a well of 6 well plate was transfected with 2.5 μg. Cells were harvested 1–2 days after transfection. No obvious growth reduction or toxicity was observed in our cell models expressing Htt N548 +/- polyQ expansion over a week.Cells were grown at 37°C, with 5% COPC12 cells were grown in F12K medium with 2.5% FBS and 15% horse serum. Tissue culture plates were coated with collagen before use. For this purpose, a stock of 1 mg/mL collagen in HBSS buffer/0.25% acetic acid was diluted to 0.2 mg/mL in HBSS buffer. Plates were coated for 20 min, the liquid was aspirated and the plates were then air-dried overnight. PC12 cells expressing inducible full-length Htt constructs (HttQ23 and HttQ73) were the kind gift from Dr. X. Qi (CWRU). Recombinant HttQ23 and HttQ73 were induced by adding ponasterone A (5 μM) for two days.After harvesting by scraping cells in the presence of PBS including the chelator, DTPA (100 μM), cell pellets were washed twice with PBS/DTPA and snap frozen at -80°C.Tissue samples were from a B6 mice (3 months), originating from the Jackson Laboratory. Tissue homogenates were prepared in the buffer containing protease inhibitor cocktail that is also used for SNO-RAC/acyl-RAC (see below). All experimental procedures conducted upon live animals were first approved by the Institutional Animal Care and Use Committee of CWRU (Case Western Reserve University) School of Medicine and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. CWRU is a PHS-assured institution (Assurance # A-3145-01) and the institution is fully accredited by AAALAC .Htt constructs are shown in To create the C204S mutant, the Htt N-terminal 548 a.a. constructs were used as template. Site-directed mutagenesis was done using a kit . The C204S forward primer was:5’-cggcctcagaaaagcaggccttacctggtgaac-3’.The C204S reverse primer was:5’- gttcaccaggtaaggcctgcttttctgaggccg-3’.Resin-assisted capture of S-nitrosylated proteins (SNO-RAC) or S-acylated proteins (acyl-RAC) was described in previous studies , 53. CelProtein pellets were washed with 70% acetone (4°C) three times and then resuspended in the binding buffer . Protein was quantitated using a BCA assay.For selective capture, the binding mixture contained Thiopropyl Sepharose 6B beads and the appropriate specific reducing reagent. For SNO-RAC, S-nitrosylated Cys residues were reduced by adding ascorbic acid (50 mM) so that beads covalently bound to them. For acyl-RAC, hydroxylamine (200 mM) was used to reduce S-acylated residues. A “buffer control” (no reducing reagent) was performed in parallel to detect any non-specific binding. Binding was performed on a shaker in the dark at room temperature for 3 hrs. The beads were then washed four times with HENS buffer and two times with 10% HEN/1% SDS buffer.After elimination of excess liquid, protein was eluted from the beads using SDS sample buffer with β-mercaptoethanol. After 20 min incubation at room temperature and 4 min at 90°C, the eluate was collected and the samples were run in 7.5% gel for SDS-PAGE and analyzed by Western blotting. Quantification of images was with ImageJ.Free thiol-groups of proteins in cell extracts were first blocked with NEM, followed by acetone precipitation and resuspension in HENS buffer containing a protease inhibitor cocktail. Samples were then divided into two equal parts. One part was incubated with ascorbate (50 mM) and IAA (100 mM) for 1 hr to reduce S-nitrosylated residues and label them. The other part was incubated with hydroxylamine (200 mM) and IAA (100 mM) for 1 hr to reduce S-acylated residues and label them.When completing the labeling reaction, protein was again acetone-precipitated and then resuspended in IP buffer  plus SDS 1%. After resuspension, SDS was eliminated using a SDS precipitation kit . Samples were pre-cleared by incubating with a protein G-agarose slurry  in the cold for 1 hr. The supernatant was collected and incubated with antibody (~30 μg) in the cold for 1 hr. EGFP-tagged Htt N548 fragments were immunoprecipitated by anti-GFP  and full-length Htt proteins by anti-Htt . The bead slurry  was then added for an additional 3 hr. After washing three times with PBS, protein was eluted by incubating with SDS sample buffer containing β-mercaptoethanol. After 20 min incubation at the room temperature and 4 min at 90°C, the eluate was recovered and proteins in samples were resolved by SDS-PAGE on gradient gels. For the high molecular weight (HMW) species, a group of samples were incubated at 37°C to determine these HMW species were not induced by heat.After staining with Imperial Protein Stain (Thermo), protein bands were excised, destained in a mixture of 50% 100 mM ammonium biocarbonate and 50% acetonitrile and reduced with DTT, followed by excess NMM to reduce disulfides and to block the resulting free cysteine residues. After washing with 100 mM ammonium carbonate and 100% acetonitrile—alternately—three times, gel bands were spin-dried. A sequencing grade trypsin (Promega) solution (500 ng in 50 μL 100 mM ammonium bicarbonate) was incubated with the dried bands overnight at 37°C.Htt tryptic peptides were subjected to LC-MS/MS analysis using an Orbitrap Elite hybrid mass spectrometer (Thermo Scientific). Nano-reverse phase liquid chromatography separations were performed on a UPLC  directly connected to a nanospray emitter . LC separation was conducted by using mobile phases A (0.1% formic acid in water) and B (100% acetonitrile) with a 90 min linear gradient starting with 1% B and increasing to 40% phase B. All data were acquired in positive ion modes. Full MS scans (m/z 300–2000) were followed by MS2 scans of the ten most abundant peptide ions at normalized collision energy of 35%. High mass accuracy FT/MS was performed to detect precursor ions .Bioinformatics software MassMatrix   was usedPrecursor ions were searched with 10 ppm mass accuracy and product ions were searched with 1 Da mass accuracy. Peptide identifications with or without modifications were determined with PP scores greater than 5.0 and PPtage scores greater than 2.0. All of the detected modified peptides mass spectra were manually checked. LC-MS/MS and MS analysis were performed in the Center for Proteomics and Bioinformatics, Case Western Reserve University.HEK293T cells were grown in 12 well tissue culture plate with poly-lysine-coated cover slips. Htt N548-EGFP construct mixed with empty vector or NOS construct in 1:1 ratio (ng) was used to transfect HEK293T cells by Lipofectamine 3000 (L3000015). After two days expression, cells were fixed by 4% formaldehyde at room temperature in dark for 10 minutes. Then rinsed the samples with PBS for three times. The mounting medium (with DAPI) was then applied to the sample incubation at the room temperature in dark for 5 minutes, followed by sealing with nail polish. The EGFP-positive cells were counted to calculate the percentage of cells with N548-EGFP inclusion(s). Fluorescent signals from DAPI and EGFP were acquired by LEICA DMI4000 B microscopy/LEICA DFC345 FX camera. To obtain high resolution image, DeltaVision RT wide-field fluorescence imaging system  and CCD digital camera (Photometrics CoolSnap HQ) were used. To remove out-of-focus light, deconvolution software  was applied.http://manaslu.aecom.yu.edu/MMM) [et al., 2008) [et al., 1999) [The MMM server (edu/MMM)  was used., 2008)  using th., 1999) . The cho., 1999) , which sin vivo cleavage products derived from full-length Htt [To learn whether cysteine residues of Htt are S-nitrosylated or S-acylated, we expressed both full-length Htt and various Htt fragment constructs in cultured cell lines . The N-tngth Htt , 58.These experiments employed a “resin-assisted capture” (RAC) protocol that is based on selective retrieval of proteins from cell lysates using Thiopropyl Sepharose beads that covalently bind to the sulfhydryl groups which were exposed upon reduction of S-nitrosylated cysteine residues (by ascorbic acid) or S-acylated residues (by hydroxylamine). The bound proteins were then eluted with sample buffer containing β-mercaptoethanol and analyzed by Western blotting , 53.After one day transfection of COS7 cells (green monkey) with plasmids encoding N548 Q15 or N548 Q128, the proteins were readily detectable in the respective cell lysates . A more When similar experiments were performed with HEK293T (human) cells, we again detected both modifications and noted that polyQ expansion stimulates S-nitrosylation of Htt much more strongly than S-acylation . When noA striking increase of S-nitrosylation and a lesser increase of S-acylation was also observed when full-length Htt +/- polyglutamine expansion was expressed in HEK293T cells . Thus, iTo learn whether the relation between polyQ expansion and increased S-nitrosylation is specific to Htt, we conducted similar studies of Ataxin-1, which causes spinocerebellar ataxia type 1 (SCA1) when its N-terminal polyQ tract is expanded. For this purpose, we expressed a wild-type control, FLAG-tagged Ataxin-1Q30, or pathogenic Ataxin-1Q85  in HEK29The monomeric size of FLAG-Ataxin-1Q30 and FLAG-Ataxin-1Q85, based on protein sequence, are ~88 and ~95 kDa, respectively . NeverthSurprisingly, a high molecular weight (HMW) species ~300 kDa for Ataxin-1Q85 was also seen in the samples that were RAC-enriched either for SNO or for S-acylation. These species were barely observed in the input . ImportaPolyQ-dependent HMW species were also observed in extracts of cells expressing Htt N548Q128 when processed by SNO-RAC or acyl-RAC . The calIn all experiments, samples were prepared for SDS-PAGE by addition of β-mercaptoethanol (β-ME) and boiling; however, the HMW material was also detected when reduction by β-ME was at 37°C . Thus, tIn vivo cleavage at any of several sites, as indicated in The primary structure of huntingtin includes several distinct domains . At the To locate the cysteine residues that are modified, COS7 and HEK293T cells were transfected to express the N548 N-terminal fragment Q15 or Q128) of Htt and processed. COS7 cells were of particular interest since they had been used to study S-palmitoylation of Htt [ or Q128 To identify the modified cysteine residues, it is necessary to avoid changes of cysteine sulfhydryl status upon cell lysis. For this purpose, the cell lysis buffer included N-ethylmaleimide (NEM). After elimination of excess NEM and reduction with ascorbate to selectively expose the cysteine sulfhydryl groups that had participated in S-nitrosylation, the samples were blocked with iodoacetamide and then immunoprecipitated. In parallel, to detect sites that were S-acylated, samples were reduced with hydroxylamine before iodoacetamide-labeling and immunoprecipitation. Samples were then separated by SDS-PAGE. The bands of purified Htt proteins were recovered for in-gel digestion. Tryptic peptides were then separated and analyzed by liquid chromatography coupled with mass spectrometry.As summarized in Residues C204 and C214 were both in the same tryptic peptide, but SNO and S-acylation on these residues could be individually identified on the basis of analysis of corresponding Cys→Ser point mutants (C204S and C214S) that we expressed in the same host cells. Two other sites  were also in a single tryptic peptide. MS detected both single and double modifications of this peptide .By expressing full-length Htt in HEK293T cells, we identified S-nitrosylation and S-acylation of additional sites beyond the N-terminal 548 amino acids. Owing to the large size of Htt, the sequence coverage of the protein was incomplete (44%). Six additional SNO sites and eight additional S-acylation sites beyond N548 were identified by mass spectrometry, as summarized in Given that there are multiple SNO and S-acylation sites, some residues could be more frequently modified than others. Since S-palmitoylation of Htt on C214 has been reported in COS7 cells , we begaIn the context of normal polyQ (Htt N548Q15), Htt S-nitrosylation and S-acylation were reduced but not eliminated by this mutation . SNO wasThe Htt N548 fragment forms cytoplasmic inclusions in cultured cells in a polyQ-dependent manner . It has p-values for these differences upon nNOS coexpression may be due to the small sample size (n = 3). Statistic “effect size” has been found to be a useful indicator of the treatment effect on the observed outcome [d; d value), d> 0.80 indicates a large effect that cannot be neglected. The effect size calculated in this study was d = 1.02 for N548Q15 (nNOS coexpression versus vector coexpression control) and d = 1.85 for N548Q128 (nNOS coexpression versus vector coexpression control). Therefore the effect of the nNOS expression cannot be neglected.In three experiments, cells co-expressing nNOS, eNOS, or iNOS had increased levels of inclusions of N548Q15 and N548Q128 , as compared to empty vector controls . The mod outcome . It is np<0.05) upon expression of eNOS or iNOS, respectively  and the N548 fragment did not show remarkable co-immunoprecipitation of N548 and NOS due to polyQ expansion .Huntingtin is highly conserved among vertebrates and chordates  and mostA fundamental question that emerges from these studies is the mechanism by which polyQ expansion increases cysteine modifications. One might envisage a change in levels of nitric oxide (NO) or of transferases and in fact there has been a report of elevated NO in cells expressing polyQ-expanded Htt . We haveConsidering that cysteine residues are broadly distributed along the length of the protein , the obsWe have in fact noticed one indication of higher-level reorganization of Ataxin-1 and Htt upon polyQ expansion. This is the presence of high molecular weight species that are S-nitrosylated and S-acylated. None of these species is eliminated by reduction with β-ME, suggesting novel chemistry and perhaps linkage to other proteins. A recent report has found that disulfide formation, which may be promoted by S-nitrosylation  can medi2-D/E) that is predicted to be nitrosylated by the iNOS/S100A8/S100A9 complex [Our mass spectrometry data identified multiple cysteine residues that are S-nitrosylated. When we align the amino acid sequence of 21-mers centered on these S-nitrosylated residues, we find that seven of nine are flanked by both negatively charged (D/E) and positively charged (K/R/H) residues , fitting complex . We do n complex .a priori reason to expect selected residues to be preferentially modified. However, we observe that removal of a single site (C214) has a significant effect on the level of S-nitrosylation and S-acylation of wild-type Htt, but only a modest effect on polyQ expanded Htt. C214 is also a site of Htt S-palmitoylation by HIP14 [There is no by HIP14 , a palmiby HIP14 , 71.in cis, between separate copies of Htt, or between Htt and other proteins. In several other proteins, S-nitrosylation and S-acylation also occur on the same cysteine residues or are regulated in reciprocal fashion. Well-established examples concern GAP-43, SNAP-25 and PSD-95. PSD-95 is of central importance for regulation of NMDA receptors and neuronal survival [Since the chemical properties of NO and acyl moieties are very different in size and hydrophobicity, it is likely that a shift from one to the other will cause changes of spatial relations with nearby residues. These potential interactions could be either survival , 72, 73.Many proteins undergo S-nitrosylation or S-acylation but the extent of modification is often unknown. Some of these modifications could regulate specific functions and reactivities, while others could have more global effects on protein structure. In this view, considering the length of huntingtin and the broad distribution of its cysteine residues, its entire structure might undergo repeated fluctuations due to these (and other) modifications. It is also possible that these modifications reflect the age of Htt molecule.Htt, Elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) [et al. [et al. classified Htt HEAT motifs into the group represented by PR65/A [Htt is a defining member of the HEAT motif group of proteins (se TOR1) . Individse TOR1) . HEAT rese TOR1) . For exase TOR1) –78. S-nise TOR1) , 77. We  [et al.  S7 Fig)Htt, Elony PR65/A . Severaly PR65/A , 79  fragment on which we have concentrated includes the first cluster  and the In summary, we document a general effect of polyglutamine expansion on protein S-nitrosylation and S-acylation. These changes could modulate protein conformation and the progression of disease. We also observe that Htt inclusions increase in response to NOS overexpression and that the inclusions show an intimate relation to nuclei. Additionally, we found that endogenous Htt is S-nitrosylated in multiple mouse tissues . PolyQ-dS1 Fig(A) PolyQ expansion increases full-length Htt S-nitrosylation. Full-length HttQ15 and HttQ128 were expressed in HEK293T cells for one day. Cell extracts were processed for SNO-RAC and acyl-RAC as in (TIF)Click here for additional data file.S2 FigProg Neurobiol. 2012). The vertical lines indicates all 6 Cys residues. Cysteine residue numbers are indicated. Polyglutamine is interrupted by His residues. Ataxin-1 with uninterrupted polyQ≥ 39 is pathogenic. (B) FLAG-tagged full-length Ataxin-1 proteins. Q30 and Q85 were used in this study. (C) SNO-RAC and acyl-RAC were performed (3 hr bead binding) to purify S-nitrosylated and S-acylated Ataxin-1Q85. The supernatant of RAC was reserved to run the Western. In parallel, input control was incubated with or without reducer (ascorbic acid or NH2OH) for 3 hr. FLAG-tagged Ataxin-1Q85 was transiently expressed in HET293T cells for 1 day. ASC: ascorbic acid. SNO: S-nitrosylated proteins. S-acyl: S-acylated proteins. Negative control: no reagents to reduce S-nitrosylated/S-acylated proteins for pull-down.(A) Diagram of full-length Ataxin-1. The horizontal open boxes indicate the AXH domain, Ataxin-1/HBP1 (HMG box-containing protein 1 transcription factor). AXH of Ataxin-1 is a protein-protein interacting domain Click here for additional data file.S3 FigAlthough their size and shape are variable, the inclusions in different preparation are similar to each other. EGFP-tagged Htt N548 fragments were expressed in HEK293T cells for two days. Empty vector co-expression serves as the control for co-expression of nNOS, eNOS, or iNOS. DAPI signal is shown in blue and EGFP-tagged N548 signal in green. All images were acquired under the same parameters. Two fields are illustrated for each condition.(TIF)Click here for additional data file.S4 Fig(A), (B), and (C) NOS overexpression increases Htt N548Q15 S-nitrosylation. Recombinant proteins were expressed in HEK293T cells for one day. Htt N548Q15 was co-expressed with empty vector control (v), nNOS (n), eNOS (e), or iNOS (i). SNO-RAC and acyl-RAC were used to recover S-nitrosylated and S-acylated proteins, respectively. Western blotting was used to detect Htt, nNOS, eNOS, and iNOS. (A) Input loading controls for Htt N548Q15 co-expressed with empty vector (v), nNOS (n), eNOS (e), or iNOS (i). SNO: S-nitrosylation. (B) S-nitrosylation of N548Q15 co-expressed with empty vector control (v), nNOS (n), eNOS (e), or iNOS (i). (C) The quantification of S-nitrosylated N548Q15. SNO N548Q15 content is normalized to input N548Q15. Empty vector co-expression is set to one fold. ImageJ was used to determine band intensity. (D) and (E) PolyQ expansion in Htt N548 fragment does not significantly affect physical association of nitric oxide synthase (NOS) and N548 fragments. Htt N548 fragments were co-expressed with nNOS, eNOS, or iNOS in HEK239T cells for one day. Immunoprecipitation (IP) and Western blotting (WB) were used to detect NOS-Htt interaction. (D) N548 fragments recovered by IP with anti-EGFP. (E) Co-precipitated NOS with N548 fragments. Co-precipitation of nNOS or eNOS was detected whereas coprecipitated iNOS was not detectable. PolyQ expansion in N548 did not significantly change the N548-nNOS or N548-eNOS interaction.(TIF)Click here for additional data file.S5 Fig(A) Htt expression does not significantly change global S-nitrosylation and S-acylation. Full-length Htt proteins or N548 fragments was expressed in HEK293T cells. Extracted proteins were used for SNO-RAC and acyl-RAC. Purified S-nitrosylated and S-acylated proteomes were detected by Coomassie blue staining. N15: cell expressing N548Q15; N128:N548Q128; NT: no transfection; F15: full-length HttQ15; F128: full-length HttQ128. The band indicated by the star has an expected gel mobility of N548Q128. (B) to (G) PolyQ-expanded Htt does not significantly increase S-nitrosylation and S-acylation of wild-type Htt. Wild-type  Htt protein (full-length or N548) was co-expressed with the polyQ-expanded Htt fragment (N548 or exon1-coding region). For the control samples, wild-type Htt was co-expressed with an empty vector control (v), wild-type N548 or wild-type exon1. SNO-RAC and acyl-RAC followed by Western blotting were used to detect S-nitrosylation and S-acylation of wild-type Htt co-expressed with other constructs. In this experiments, MAB2166 recognizes full-length Htt and N548 but not exon1. MAB2168 recognizes the C-terminal region of full-length Htt but not N548/exon1. N548 and exon1 are EGFP-tagged. (B), (C), and (D) Co-expression of Htt exon1Q97 or N548Q128 does not increase S-nitrosylation and S-acylation of N548Q15 (bands indicated by stars). (B) Input loading control. (C) S-nitrosylation of N548Q15 co-expressed with other constructs. (D) S-acylation of N548Q15 co-expressed with other constructs. (E), (F), and (G) Co-expression of Htt exon1Q97 or N548Q128 does not increase S-nitrosylation and S-acylation of full-length HttQ15 (bands indicated by stars). (E) Input loading control. (F) S-nitrosylation of HttQ15 co-expressed with other constructs. (G) S-acylation of HttQ15 co-expressed with other constructs. SNO: S-nitrosylation. S-acyl: S-acylation.(TIF)Click here for additional data file.S6 FigThe 21-mers centered on cysteine residues are presented. The iNOS complex contains iNOS, S100A8, and S100A9 proteins. CRM1 C517 is the SNO and leptomycin alkylation site.(TIF)Click here for additional data file.S7 FigPPPPPPPPPPPQLPQPPPQAQPLLPQPQPPPPPPPPPP). (B) Enlarged side view of 79–165 a.a. region from (A). Residues beyond Leu165 are masked.(A) Computer-simulated Htt HEAT repeat cluster 1 (79–397 a.a.). MS-identified S-nitrosylation and S-acylation sites are indicated in red. In three independent experiments, we found no evidence of modification of C137. The sequence between polyQ and Gly79 is the 38-residue long polyproline tract ((TIF)Click here for additional data file.


Artemisia rupestris L. (AEAR) could be employed as an efficient adjuvant for influenza virus vaccine (V) to enhance immune responses and reduce the antigen doses required for initiating immunity, without compromising the immune response. ICR mice were subcutaneously co-administrated with V combined with different concentrations of AEAR demonstrated that 300 μg AEAR could significantly improve hemagglutination inhibition (HI) and increase IgG antibody titers in serum (P<0.05) and the population of CD4+CD44+ and CD8+CD44+ (P<0.05). Next, 300 μg AEAR combined with different doses of V in vivo markedly increased HI and specific IgG antibody level(P<0.05). It also significantly increased the amount of CD4+ and CD8+ T cells, CD4+CD44+ and CD8+CD44+ T cells (P<0.05), improved lymphocyte proliferation, the secretion of CD4+IL-4, CD4+IFN-γ and CD8+IFN-γ (P<0.05), and the killing efficacy of cytotoxic T lymphocyte (CTL) (P<0.05). Furthermore, the combination increased the expression of major histocompatibility complex-II (MHC-II) and co-stimulatory molecules including CD40, CD80, and CD86 on dendritic cells (DCs), and downregulated the expression of CD25+Foxp3+Treg cells (P<0.05). No significant difference was observed between high-dose V and low-dose AEAR-V (10-fold lower) vaccination group (P>0.05), indicating a 10-fold reduction of antigen required for V vaccine administration. In conclusion, this study demonstrated that AEAR, as an adjuvant for influenza vaccine, could stimulate potent humoral and cellular immune responses and reduce the antigen dose required for effective vaccination, which were mediated by promoting DCs activation and repressing Treg expression.Potent adjuvant can improve the effectiveness of vaccines and reduce the antigen doses required for initiating the protective immunity. In this study, we identified that aqueous extract of  Influenza, mainly caused by influenza virus, is an acute respiratory disease featuring strong and fast infectivity. It would cause death when complicated with pneumonia in elderly or patients with cardiopulmonary diseases , 2. CurrTraditional Chinese herbal medicine plays a major role in treating diseases . StudiesArtemisia rupestris L. is the perennial herb of rupestris belonging to Artemisia (Compositae), which is wildly distributed in Xinjiang (China), middle Asia, and Europe .A detailed description can be found in references . In brieP<0.01 indicates that difference is extremely significant.All data were presented as mean ± SD. Prism 5.0 was used for data analysis. To compare between groups, one-way ANOVA and Tukey's Multiple-Comparison Test, or Student’s t-test were used. * P<0.05 indicates a significant difference; ** P<0.05), and the difference between AEAR medium-dose group and Alum group was not significant (P>0.05). Indirect ELISA was performed to measure the IgG antibody level in serum on day 14 and 21 after first vaccination (P<0.05), and the difference between AEAR medium-dose group and Alum group was not significant (P>0.05). These findings suggested that AEAR could enhance serum HI titer and antibody level.In order to identify the optimal AEAR doses, 100 μg (low-dose group), 300 μg (medium-dose group), and 500 μg (high-dose group) of AEAR combined with 0.5 μg V were used respectively to immunize ICR mice. Alum was used as the positive control. On day 21 after first vaccination, the titer of HI antibody in serum was measured . As showcination . We foun+CD44+ and CD8+CD44+ T cells in spleen was measured by FACS (+CD44+T cells was higher compared with V-only group (P<0.05). There was no significant difference (P>0.05) when compared with Alum group. The amount of CD8+CD44+T cells in both AEAR low-dose and medium-dose group was higher than in V-only group (P<0.05), there was no significant difference when compared with Alum group (P>0.05). These results indicated that AEAR could effectively improve antigen-specific T cell immune responses.On day 7 after second vaccination, the amount of CD4 by FACS . The perP<0.05). No significant difference can be observed between AEAR-V-low-dose group and V-only-high-dose group (P>0.05), which indicated that the antigen dose required for immunization could be reduced by 10 folds.In order to further identify if ARAR can reduce V antigen dose required for immunization, we vaccinated the ICR mice with the optimized 300 μg AEAR together with 0.05 μg (low-dose), 0.1 μg (medium-dose), or 0.5 μg (high-dose) V respectively. HI test was next performed to measure the HI antibody titer on day 21 after first vaccination . The HI 1, and IgG2a in all  groups are dramatically higher than in V-only group (P<0.01). There was no significant difference between AEAR-V-low-dose group and V-only-high-dose group (P>0.05). It indicated that antigen dose required for immunization can be reduced by 10 folds . No significant difference was detected between AEAR-V-low-dose group and V-only-high-dose group (P>0.05). It indicated that antigen dose required for immunization could be reduced by 10 folds.To further determine the effect of AEAR on cellular immune response, Lymphocyte proliferation assay was performed. The proliferation of lymphocytes from mice was measured by MTT on day 21 after first vaccination. First, the suspension of spleen cells in mice was prepared. Then, SI was measured after adding ConA or LPS . Fig 4 s+, CD8+, CD4+CD44+, CD8+CD44+ T cells in spleen on day 21 after first vaccination (+ and CD8+ T cells in AEAR-V-low-dose and medium-dose groups compared with the V-only group (P<0.05) . No significant different can be observed between AEAR-V-low-dose group and V-only-high-dose group (P>0.05). Antigen dose required for immunizations is reduced by 10 folds (P<0.05).T cell response plays a major role in immune response against influenza virus. Using FACS, we measured the percentages of CD4cination . There w(P<0.05) . The per+IL-4 in AEAR-V-low-dose, medium-dose, and high-dose groups were significantly higher than in V-only group (P<0.05)  (P>0.05). These results indicated that AEAR could stimulate both humoral and cellular immune response, especially T-cell mediated immune response.Cytokines play an important role in immune response. We measured the expression of cytokines in spleen cells by FACS on day 7 after second vaccination . The sec(P<0.05) . The exp(P<0.05) . No signP<0.05). There was no significant difference between AEAR-V-low-dose group and V-only-high-dose group (P>0.05). The expression of CD25+Foxp3+ Treg in AEAR-V-low-dose, medium-dose and high-dose group were remarkably lower than in V-only group (P<0.05), which indicates that AEAR could stimulate the maturation of DCs and downregulate the expression of Tregs.It is well known that the activation of DCs and the expression of Tregs are important in initiating specific immune response and immunoregulation. To investigate the mechanisms of AEAR enhancing the immune response to V and antigen sparing, we determined the expression of surface molecules CD40, CD80, CD86 and MHC-II on DCs from splenocytes in mice on day 3 after first vaccination , and theP<0.05), indicating that AEAR could stimulate CTL response, which promoted the elimination of influenza viruses.CTL plays an important role in immune response against influenza virus. To investigate the CTL response in mice after vaccination, the CTL specific killing rate was measured by FACS on day 21 after first vaccination . As showThe effective components of Chinese herbal medicine have various pharmacological functions including antivirus and anti-inflammatory, which can be used as a treatment for autoimmune diseases ,31. EspeIt is very important to influenza vaccines that elicit robust humoral and cellular immune responses. One of these strategies of improving vaccine-mediated protection against influenza is to add adjuvants into the vaccine. Adjuvants, by promoting antigen presentation, activation and maturation of dendritic cells and production of inflammatory cytokines, can drive the desired humoral and T-cell-mediated responses to influenza vaccines.+ T cell responses were significantly improved by AEAR, and the optimal doses for vaccination were 300 μg in our experimental settings. We further investigated the role of AEAR in the reduction of antigen dose required for immunization by using the optimal dose of AEAR (300 μg) combined with low, medium, or high doses of V for vaccination respectively. HI and IgG antibody level results demonstrated that 300 μg AEAR remarkably boosted humoral immune responses even with 10-fold lower dose of V. IgG antibody levels in mice on day 7 after immunization confirmed that AEAR could be used as a V adjuvant to rapidly stimulate high antibody levels in the serum, which would provide early prevention from influenza infection.In this study, we investigated the role of AEAR as a V adjuvant in improving immune responses and reducing antigen dose needed for effective V vaccination. Our results demonstrated that ideal immune effect was achieved with combinations of appropriate concentration of AEAR and V antigen. We first investigated the effect of different concentrations of AEAR on immune responses including HI and IgG antibody levels in serum . Our res+ T cells can promote the maturation of B cells or facilitate the antigen presentation of APCs, while CD8+ T cells are capable of directly killing the target cells [+, CD8+, CD4+CD44+, and CD8+CD44+ T cells, improved CTL efficacy, and achieved a 10-fold reduction of the antigen dose required for immunization without compromising the immune response. These results suggested that AEAR could increase V-induced T-cell immunity via affecting T lymphocytes to control influenza virus infection.The efficacy of influenza vaccine is associated with stimulation of cell-mediated and cytotoxic T-lymphocyte responses. Cell-mediated immunity, an immune response mediated mainly by T cells, plays an important role in the prevention and control of influenza virus . T cellset cells . In thisDuring the antigen recognition and activation of immune responses, cytokines stimulate the proliferation of immune cells, IFN-γ induces CTL activation to initiate the elimination of influenza virus –41, IL-4The maturation and activation of DCs is a key step to initiate the antigen-specific immune responses. Treg cells are a subgroup of T lymphocytes with immunosuppression and regulation function . To undeIn conclusion, AEAR, as an influenza virus vaccine adjuvant, can induce specific humoral and cellular immunity, especially the T-cell mediated immune responses, and reduce the antigen dose required to initiate protective immunity, by promoting the maturation of DCs and downregulating Treg expression. Therefore, AEAR can be developed as an efficient commercial adjuvant for influenza virus vaccine. Furthermore, this study provides a novel and efficient strategy to address the potential shortage of vaccine supply during flu pandemic.


A single intramuscular vaccination of African green monkeys with M51R or M51R-F induced VSV-specific, dose-dependent humoral immune responses. Flagellin induced a significant increase in antibody production  at the low vaccination dose. A VSV-specific cellular response was detected at 6 weeks post-vaccination, but was neither dose-dependent nor enhanced by flagellin; similar numbers of VSV-specific, IFNγ-producing cells were detected in lymph node and spleen of all animals. These results indicate that virus-directed, intracellular flagellin production may improve VSV-based vaccines encoding heterologous antigens by lowering the dose required to achieve humoral immunity.Recombinant vesicular stomatitis virus (VSV) is a promising platform for vaccine development. M51R VSV, an attenuated, M protein mutant strain, is an effective inducer of Type I interferon and dendritic cell (DC) maturation, which are desirable properties to exploit for vaccine design. We have previously evaluated M51R VSV (M51R) and M51R VSV that produces flagellin (M51R-F) as vaccine vectors using murine models, and found that flagellin enhanced DC activation and VSV-specific antibody production after low-dose vaccination. In this report, the immunogenicity of M51R vectors and the adjuvant effect of virus-produced flagellin were evaluated in nonhuman primates following high-dose (10 Vaccines based on a live, attenuated vesicular stomatitis virus (VSV) platform are being developed for a wide range of infectious diseases and cancer ,2. For eSalmonella enterica fliC gene (M51R-F) [Bacterial flagellin has been widely investigated as an adjuvant for non-living vaccines, including killed and protein nanoparticle influenza virus vaccines ,21, and (M51R-F) ,27. In t(M51R-F) ,27,28. I(M51R-F) . Based ofliC gene encoding flagellin from Salmonella enterica, serovar Enteritidis, between the viral M and G genes, were described previously [fliC gene was inserted as an independent transcription unit between the M and G genes of the VSV genome. The production of flagellin in the intracellular compartment following infection of permissive cells with M51R-F VSV was confirmed by western blot [Recombinant M protein-mutant VSV (M51R VSV) and M51R VSV that constitutively produces flagellin (M51R-F VSV) as a result of introduction of the eviously ,27. Brieern blot . All virChlorocebus aethiops sabaeus) were used for the study. AGM were bred and housed at the Wake Forest School of Medicine Primate Center. Animals ranged in age from 8.5 to 15.5 years, and in weight from 4.2 to 7.89 kg, and were cared for and handled according to guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Wake Forest Institutional Animal Care and Use Committee (protocol A14-184). Animals were chosen for the study based on low pre-existing immunity to VSV or flagellin as measured by the presence of antibodies in pre-immune serum. All procedures were performed on animals that were anesthetized using ketamine . After collecting pre-immune blood samples by femoral venipuncture using standard heparin vacutainers, animals were split into 4 cohorts  and vaccinated in the right bicep muscle with 0.5 mL of purified live, recombinant M51R VSV (M51R) or M51R VSV expressing the flagellin gene (M51R-F) at 2 different doses . At weeks 1, 2 and 4 post-vaccination, blood was collected, processed to obtain plasma, and frozen at −80 °C. At 6 weeks post-vaccination animals were euthanized , and blood and tissue samples were harvested. For the current study, spleens and axillary lymph nodes  were processed to obtain single cell suspensions devoid of red blood cells, frozen and stored in liquid N2.Sixteen female, adult African green monkeys ). The cells were then used as antigen presenting cells or as responder cells in IFNγ Elispot assays, as specified below.VSV-permissive mouse EL4 cells  were culNunc MaxiSorp ELISA plates were coated with 0.2 µg/well purified VSV (Orsay strain) in sodium carbonate coating buffer (pH 9.6). Wells without virus served as a negative control. Plates were blocked with phosphate buffered saline containing 0.05% Tween 20 (PBST) + 5% skim milk. Plasma samples were serially diluted in PBST (wash buffer). Antibodies specific for monkey IgG  and IgM  directly conjugated to horseradish peroxidase (HRP) were used for detection. Plates were developed with 1-step Turbo TMB-ELISA substrate , and the absorbance at 450 nm was measured on a BMG Labtech POLARstar Omega microplate reader . The absorbance for each sample was calculated by subtracting the absorbance obtained from corresponding wells that were not coated with virus. The concentration of IgG or IgM was determined from standard curves prepared with monkey IgG (#017-0102-0001) and IgM (#017-0107) Rockland Immunochemicals .450 value relative to the monkey IgG standard curve. After a 2 h incubation of duplicate plasma samples on VSV-coated and blocked wells, the wells were washed 5 times with PBST and 100 uL of buffer containing sodium thiocyanate  was added for 15 min. The wells were again washed 5 times with PBST before proceeding with the secondary antibody incubation (30 min) and substrate steps (15 min). The % IgG remaining bound to the wells after NaSCN treatment relative to controls not treated with NaSCN, was calculated as follows: (OD450 + NaSCN)/(OD450 − NaSCN) × 100. The data were expressed as the concentration of NaSCN that removed 50% of bound IgG (EC50), and reported as the mean EC50 ± SD for each cohort.IgG affinity was measured by thiocyanate elution according to a published protocol  by modif5 pfu/well) was added to the wells, and samples were incubated for 1 h at 37 °C, 5% CO2 to allow for antibody binding to the virus. In the next step, 100 uL of pre-incubated sample was added to an equal volume of EL4 cells (3 × 106/mL in DMEM supplemented with 7% heat-inactivated FCS and 2 mM glutamine) in a 96-well round bottom dish. After a 5 h, 37 °C, 5% CO2 incubation to allow for virus infection, EL4 cells were harvested from the wells, washed and fixed with 4% paraformaldehyde. Samples were acquired on a BD FACSCalibur Flow Cytometer and analyzed with FlowJo software. The neutralizing antibody titer was defined as the dilution of plasma that inhibited the infection of EL4 cells by 50%, which was determined using GraphPad Prism software. Controls consisted of EL4 cells with no virus added, and EL4 cells infected with M51R-eGFP in the absence of plasma (for maximum infection level). The infection protocol was calibrated such that approximately 30% of the cells were infected with M51R-eGFP in the absence of neutralizing antibodies.A flow cytometry-based virus neutralization assay was developed according to a published protocol , with mo6 cells/mL with M51R VSV at MOI = 50, or were mock-treated, for 18 h at 37 °C, 5% CO2. Mock- or virus-infected APC were irradiated using an MDS Nordion Gammacell 1000 Elite  irradiation source (2000R). APC (2.5 × 105/well) were mixed with an equal volume (100 μL) of autologous responder cells (2 × 105/well). Cells were cultured for 48 h in ELISPOT plates  coated with anti-IFN-γ capture antibody. Spot-forming cells were detected using biotinylated secondary antibody and True Blue peroxidase substrate  according to manufacturer instructions. Spots were photographed and quantitated using an ImmunoSpot system .To measure the cellular immune response against VSV vectors, an IFNγ ELISPOT assay kit was used according to manufacturer’s instructions . Cells from the left axillary lymph node (LLN) were used as antigen presenting cells (APC), and autologous cells from the right (draining) axillary lymph node (RLN) or spleen were used as responding cells. LLN cells were infected at a concentration of 1.0 × 105 PFU of M51R or M51R-F. VSV-specific T cell responses were measured in spleen on days 6, 8, 10 and 12. Splenocytes were re-stimulated in vitro with peptides corresponding to an immunodominant N protein epitope for CD8+ T cells [+ T cells [Female 8 weeks old C57BL/6 mice  were immunized intranasally with 5 × 10 T cells  or G pro T cells . Intracep < 0.05 after correction for multiple comparisons. Cellular immune response data were analyzed by one-way ANOVA for comparison of multiple cohorts.Antibody data were analyzed as either the antibody titer  or plasma antibody concentration (virus-specific IgM and IgG). Data were analyzed by two-factor analysis of variance (ANOVA), with time and cohort as the two factors, using SigmaStat for Windows version 3.5 software . Post-hoc multiple comparisons were made by the Holm-Sidak and Bonferroni methods with an alpha level of 5 PFU); C2: high dose M51R ; C4: high dose M51R-F (1 × 108 PFU). For each animal and time point, plasma samples were tested to quantify VSV-specific neutralizing antibodies (NA), IgM and IgG from low-dose-  induced minimal or undetectable IgG responses (<2-fold above pre-immune) : Low dose M51R: 1.00 ± 0.70 M; Low dose M51R-F: 0.84 ± 0.33 M; High dose M51R: 1.20 ± 0.05 M; High dose M51R-F: 1.17 ± 0.14 M . Therefore by this criteria, expression of flagelin by the M51R vector did not modulate affinity maturation of the anti-VSV IgG response.As an additional measure of flagellin adjuvant potential, IgG affinity was measured. The rationale for this experiment was based on the report of a vaccine comprised of  and AGM ,36. We t and AGM . The dat6 responder cells (6 cells regardless of vaccine dose or the presence of flagellin. Monkey 1286 from the M51R high-dose group (cohort 2), was a low-responder as measured by IFNγ-producing cells, consistent with its low responder status with regard to antibody production  were prepared by infection of left axillary lymph node cells with M51R VSV for 24 h followed by X-irradiation prior to addition of responder cells from the draining (right) lymph node. Results are expressed as the number of spot forming cells (SFC) per 10er cells . Lymph noduction . Using soduction . Therefo8 PFU of M51R VSV induced substantial VSV-specific humoral responses as measured by VSV-specific IgM, IgG and neutralizing antibodies. Second, a VSV-specific cellular response was detected in lymph node and spleen at 6 weeks post-vaccination, as measured by IFNγ production after in vitro restimulation with VSV. Third, the VSV-specific humoral but not cellular immune response was dose-dependent. Fourth, M51R-directed intracellular flagellin production enhanced anti-VSV antibody production in the context of low dose vaccination only (105 PFU), and did not affect the generation of a cellular response at either vaccination dose.This study presents data using nonhuman primates to evaluate dosage and adjuvant function of flagellin when produced by M51R VSV, which has been previously evaluated in mice as a live virus vaccine vector . The res5 PFU), which is consistent with our previously published data using the same vectors to vaccinate mice via an intranasal route [Pseudomonas aeruginosa [Many studies to date have tested flagellin mixed with antigen, flagellin-antigen fusion proteins, or flagellin associated with noninfectious particles, all of which stimulate cells through TLR5 ,22,25,37al route . This fial route ,39. Withal route ,41 and Pruginosa . With reruginosa  and N prruginosa  peptides+ and CD3− cells, suggesting that in addition to T cells, IFNγ-producing long-lived NK cells [A VSV-specific cellular response, as measured by the number of IFNγ-producing cells in lymph node and spleen after in vitro re-stimulation, was detected in all animals at 6 weeks post-vaccination. Interestingly, the generation or maintenance of these cells did not depend on vaccine dose; similar numbers of IFNγ producing cells were detected in all animals in both spleen and lymph node, regardless of vaccine dose. Further, the presence of flagellin in the system  had no effect at either vaccination dose. The cells produced IFNγ in response to VSV-infected antigen presenting cells, with minimal cytokine produced in cultures that were stimulated with mock-infected APC , indicatNK cells  or B celNK cells  could coM protein mutant VSV is one of a number of attenuated VSV strains that have potential as vaccine vectors ,47. An afliC gene as a separate transcription unit between the M and G genes did not affect viral titers [Further development of M51R and M51R-F as vaccine platforms raises several considerations. First, an assessment of efficacy in inducing an immune response against a cloned heterologous antigen  is needed. Second, to more rigorously evaluate vector-directed flagellin production will require engineering the virus to efficiently produce both antigen and adjuvant. Third, a safety evaluation will be necessary. There is precedent for addressing these issues successfully ,51. We’vl titers . One posl titers . The aval titers  could be8 PFU of M51R VSV for inducing substantial adaptive immune responses in the absence of a boost. This may be attributed in part to the robust response of DC to infection with M protein mutant VSV strains [5 PFU of vector, raising the possibility that virus-directed intracellular flagellin production could improve the performance of M51R VSV as a vaccine vector by lowering the dose necessary to achieve humoral immunity against target antigens. These results form the basis for further development of M51R VSV as an alternative vaccine platform.This study demonstrates efficacy of 10 strains ,19,27,53


Pichia pastoris. We found that recombinant I62-CFH (protective against age-related macular degeneration) and V62-CFH functioned equivalently, matching or outperforming plasma-derived CFH, whereas R53H-CFH, linked to atypical hemolytic uremic syndrome (aHUS), was defective in C3bBb decay-accelerating activity (DAA) and factor I cofactor activity (CA). The aHUS-linked CCP 19 mutant D1119G-CFH had virtually no CA on (self-like) sheep erythrocytes (ES) but retained DAA. The aHUS-linked CCP 20 mutant S1191L/V1197A-CFH (LA-CFH) had dramatically reduced CA on ES but was less compromised in DAA. D1119G-CFH and LA-CFH both performed poorly at preventing complement-mediated hemolysis of ES. PspCN, a CFH-binding Streptococcus pneumoniae protein domain, binds CFH tightly and increases accessibility of CCPs 19 and 20. PspCN did not improve the DAA of any CFH variant on ES. Conversely, PspCN boosted the CA, on ES, of I62-CFH, R53H-CFH, and LA-CFH and also enhanced hemolysis protection by I62-CFH and LA-CFH. We conclude that CCPs 19 and 20 are critical for efficient CA on self-surfaces but less important for DAA. Exposing CCPs 19 and 20 with PspCN and thus enhancing CA on self-surfaces may reverse deficiencies of some CFH variants.Spontaneous activation enables the complement system to respond very rapidly to diverse threats. This activation is efficiently suppressed by complement factor H (CFH) on self-surfaces but not on foreign surfaces. The surface selectivity of CFH, a soluble protein containing 20 complement-control protein modules (CCPs 1–20), may be compromised by disease-linked mutations. However, which of the several functions of CFH drives this self-surface selectivity remains unknown. To address this, we expressed human CFH mutants in  Transiently, nascent C3b can utilize its freshly activated thioester to covalently attach to surfaces  destabilizes it; this is called decay-accelerating activity (DAA). CFH also has cofactor activity (CA) for cleavage of C3b by complement factor I (CFI). The product, iC3b, cannot bind CFB but is an opsonin in its own right , CFB, C3, CFI, CFHRs, and, in particular, CFH are linked to diseases , 16. Num17– and AMD –26 but pand AMD –. Most muand AMD –. Other pand AMD ––31.Penetrance is low for many of the CFH mutations linked to diseases. Variation in the genes for other complement proteins and environmental factors may also contribute to disease risk . The com34–B). For example, a fragment consisting of CFH CCPs 1–4 (CFH 1–4) binds C3b and has CA (ES)  , whereas CA (ES) .versus non-self discriminatory properties and to establish the phenotypic consequences of disease-related mutations.Crucially, the use of small CFH truncations cannot capture the collaboration between multiple binding sites that characterizes CFH function in its biological setting. CFH probably undergoes a conformational adjustment –50 in a –B). We investigated the activities of these CFH mutants on the man-made surfaces of surface plasmon resonance (SPR) chips and the foreign surface of rabbit erythrocytes (ER) as well as on the more hostlike surface of ES. We additionally investigated the functions of our mutants when in complex with PspCN. PspCN is a domain from a bacterial complement evasion protein that binds very tightly to CFH , D1119G-CFH , and S1191L/V1197A-CFH (henceforth LA-CFH) (on Ile-62 background). The P. pastoris-secreted proteins were harvested prior to endoglycosidase treatment to cleave mannose-rich and hence non-native N-glycans back to the first GlcNAc residues. Following purification, homogeneity was assessed using SDS-PAGE (C). The absence of extra bands in the gel, under both reducing and non-reducing conditions, indicates minimal proteolytic cleavage. Note that cleavage within a CCP might have been evident only in reduced samples due to the interlaced disulfide bonds within each CCP (i.e. Cys(i) to Cys(iii) and Cys(ii) to Cys(iv))  in recombinant SDS-PAGE C. The abCys(iv)) . All proC). Aliquots were stored at −20 °C. An aliquot was freshly thawed for each experiment. The C3b sample  had been stored at −80 °C and was also thawed immediately before use. The C3b was immobilized on a Biacore C1-sensor chip, using amine coupling. By flowing CFH solutions over amine-coupled C3b (A and B), we obtained similar DK values (in the range of 0.21–0.33 μm) for all three proteins . Both recombinant protein samples were freshly produced, purified, and assayed in parallel. The plasma CFH sample was purified from pooled human plasma and contained no CFH-related proteins detectable by Coomassie Blue-stained SDS-PAGE C. Aliquopled C3b , A and Bproteins .A and C). Both I62-CFH and V62-CFH bound to immobilized C3d with DK values of ∼1.9 μm (A). The lack of binding between plasma CFH and C3d was previously attributed to lack of access to the C3d-binding site in CCPs 19 and 20. Indeed, premixing 0.6 μm plasma CFH with 1.2 μm PspCN transformed it into a C3d binder  with a 2-fold molar excess of PspCN. Thus, both plasma CFH and recombinant CFH bind PspCN, via their central CCPs  amine-coupled to a different flow cell on the same C1-sensor chip as used for the C3b-binding experiments , A and C ∼1.9 μm . Convers ∼1.9 μm , 49, 50, ∼1.9 μm A. The lam, had identical DAA in this assay, whereas 20 nm plasma CFH was less active (A). The presence of PspCN (at a 2:1 ratio to CFH) enhanced the DAA of I62-CFH and V62-CFH to identical extents (B). As expected, PspCN also enhanced the DAA of plasma CFH in this assay.In SPR-based assays on a CM5-sensor chip (Biacore), we compared the abilities of recombinant and plasma CFH to accelerate decay of the preassembled C3bBb, the alternative pathway C3 convertase. Both I62-CFH and V62-CFH, at 20 ns active A. The pr extents B. As expES are susceptible to lysis by complement unless protected by added CFH. ER, in contrast, lack human-like SAs and are not effectively protected by human CFH. Both I62-CFH and V62-CFH proved equally effective (e.g. at 250 nm) in preventing ES hemolysis (A) in this assay, whereas both were, as expected, equally ineffective, at 1 μm, in protecting ER (B). Both I62-CFH and V62-CFH showed more ability to prevent ER hemolysis when in complex with PspCN.Functional similarities between I62-CFH and V62-CFH extended to hemolysis protection assays. Upon exposure to increasing percentages of CFH-depleted normal human serum (NHS), emolysis A in thiscting ER B. Both IEs-surface assay  and V62-CFH (IC50 = 9.5 nm) had similar potencies . Conversely, and interestingly, PspCN improved the CA of I62-CFH on ES-bound C3b by a factor of 2  exhibited poorer DAA on a CM5-sensor chip (B) in comparison with 40 nm I62-CFH. Similarly, in assays of DAA performed on C3bBb-decorated ES, R53H-CFH (IC50 = 6.6 nm) performed less well than I62-CFH (IC50 = 1.4 nm)  was likewise less effective in CA than I62-CFH (IC50 = 18 nm) . PspCN (2:1 molar ratio to CFH) did not noticeably improve the DAA of R53H-CFH measured on ES . A 2-fold ratio of PspCN to R53-CFH did improve its CA on ES , it was able to protect the more “self-like” ES (C). Protection of ES in this case may reflect the inability of R53H-CFH to prevent turnover and hence “futile” depletion of C3 in the fluid phase, yet a 1:1 mixture of R53H-CFH with I62-CFH, included in an effort to prevent fluid-phase C3 depletion, was likewise protective in this assay (E). By competing with I62-CFH for binding to soluble C3b, R53H-CFH might antagonize the fluid-phase C3-conserving regulatory action of I62-CFH. In a further investigation of this phenomenon, R53H-CFH was mixed 1:1 with the S1191L/V1197A-CFH (LA-CFH) mutant. As described below, LA-CFH prevents C3 depletion in fluid phase, although it exhibits impaired binding to SA-decorated surfaces. The R53H-CFH/LA-CFH mixture was less able to prevent ES hemolysis than R53H-CFH alone (E). This is consistent with non-depletion of C3, an expected consequence of the fluid-phase regulatory action of LA-CFH. Importantly, it confirms the lack of ability of either LA-CFH or R53-CFH, for different reasons, to fully control C3b amplification on the ES surface.These functional deficiencies of R53H-CFH were not immediately evident in hemolysis protection assays. Although R53H-CFH, with or without PspCN, did not protect foreign ER D, it waslike” ES C. Protecis assay E. By comFH alone E. This iES is consistent with its poor ES hemolysis protection properties under circumstances where fluid-phase C3 levels are not depleted.To summarize, R53H-CFH is functionally deficient despite retaining good affinity for C3b. The inability of R53H-CFH to regulate C3b amplification on ES . The negative effect of D1119G on the CA of CFH for cleavage of C3b bound to ES is greater still (IC50 = 12 nm for I62-CFH versus IC50 > 500 nm for D1119G-CFH) . PspCN (at a 2:1 molar ratio to D1119G-CFH) had negligible effects on the C3bBb-DAA  and C3b-CA of D1119G-CFH measured on ES .Asp-1119 of CCP 19 is a key interface residue in the CFH 19-20–C3d complex . In recoES , 39. In n assays . To explES (DK = 0.8 μm for LA-CFH versus 1.2 μm for I62-CFH in one experiment (A) and 0.9 μm for LA-CFH versus 1.0 μm for I62-CFH in a second experiment . Taken together, these results suggest that the N-terminal region of CFH dominates its properties on the foreign surface of a SPR chip such that C-terminal mutations have undetectable effects.Ser-1191 and Val-1197 in CCP 20 are critical for recognition of SA according to a crystal structure of a C3d–CFH 19-20–SA complex . In prevES , 53 but ES . Here, wperiment A and 0.9periment . In thesES cells, S1191L/V1197A had a smaller effect on DAA than on CA (C and D). The effect on DAA was measurable but minor (IC50 = 1.6 nm for I62-CFH versus IC50 = 2.9 nm for LA-CFH)  . As is apparent from the results of further experiments on ES, PspCN did not measurably increase the DAA of LA-CFH, but it clearly enhanced the CA of LA-CFH (E and F), although the wild-type IC50 was not restored , just as it had previously been found to partially rescue another C-terminal mutant, D1119G-CFH (ER (D).When assayed on hostlike on assay C. As is restored . In ES he LA-CFH C, just a-CFH . The E936D SNP is not unique to at-risk or protective haplotypes . Our pooled plasma-derived CFH contains a mixture of common haplotypic variants, but these are located in CCPs 1, 7, and 15, so are unlikely to impact directly on C3d binding that involves only CCPs 19 and 20. Contamination with CFH-related proteins in the plasma CFH sample is unlikely because these were removed during purification. Whereas chemical modifications may be present in aging plasma proteins  . Comparisons of I62- and V62-CFH are important because Ile-62 is encoded by an SNP unique to a protective haplotype for AMD and C3G  much less tightly than full-length CFH.I62-CFH bound marginally better than V62-CFH to C3b in our SPR-based comparison using identically prepared samples , but morsma CFH)   negative impacts of R53H on the DAA of CFH on an SPR surface. Encouragingly, these benchmark results closely parallel those obtained for R53H-CFH 1–4.We mutated another CCP 1 residue (R53H) in CFH and compared the consequences with those reported for R53H in the autonomously functional CFH 1–4 fragment , 60. We m   . The cry/V1197A) . Convers97A) (ES . Subsequ97A)  and CA (larger), of the C-terminal mutations S1191L/V1197A and, to a less pronounced extent, D1119G -binding and self-recognition site in CCPs 19 and 20 of CFH becomes exposed upon binding PspCN , perhaps context . The disf LA-CFH . This di, D1119G .Both DAA and CA reside in CFH CCPs 1–4 and can act in a concerted manner, but each involves a distinct sequence of events . To acceES surfaces support this line of reasoning because they show that retention of CFH on a surface via its CCPs 19 and 20 is more important for CA than for DAA. Our results with PspCN likewise support this reasoning because they confirm that the PspCN-induced increase in access to the surface-engaging C-terminal modules has little effect on DAA but a positive effect on CA.The results obtained here for disease-linked C-terminal mutants on C)) or the availability or unavailability (tested by the inclusion or not of PspCN (E)) of the self-recognition site in CCPs 19 and 20. On the other hand, CA (for CFI on C3b bound to ES) is sensitive to the presence or absence (mutagenesis) of the self-recognition site (D) and its availability as enhanced by PspCN (F).To summarize, our results show that acceleration of C3bBb decay on a hostlike surface is not very responsive to the presence or the absence (as tested by mutagenesis C) or theof PspCN E) of theion site D and itsby PspCN F.ER in hemolysis assays  and less straightforward to interpret. Nonetheless, we infer that PspCN can work as a protector of self-surfaces mainly by enhancing CFH CA on these surfaces, and this suggests new therapeutic strategies based on CFH activators.Soluble PspCN has a protective effect on s assays . This waHuman CFH was either purchased from Complement Technologies or purified from pooled plasma (TCS Biosciences Ltd.). Other plasma-derived human complement proteins  and CFH-depleted normal human serum (Complement Technologies) were stored as recommended by the supplier and used without further purification.f to trim N-glycans to GlcNAc residues, as described (Pichia pastoris expression vector pPICZaB (Invitrogen). This was amplified in XL1 Blu Escherichia coli cells (Agilent) and then used to transform the KM71H strain of P. pastoris (Invitrogen). Single colonies grown on yeast-peptone-dextran (YPD) agar plates with 300 μg/ml Zeocin were picked. The colony with the highest level of CFH production in small-scale trials was used to inoculate a BioFlow 4500 (New Brunswick Scientific) 10-liter fermenter. After a glycerol-fed batch phase, protein production was induced by a 4-day methanol/glycerol continuous feed. Secreted proteins were chromatographically purified , was produced and purified, following treatment with EndoHescribed . SimilarStreptococcus pneumoniae was expression-optimized for E. coli (GeneArt-Life Technologies). It was cloned into E. coli vector pE-SumoPro-Kan  and then expressed in BL21 (DE3) E. coli cells (Lucigen). The resultant N-terminally His6-SUMO-tagged protein was captured on nickel-affinity resin, eluted with imidazole, and then further purified by cation-exchange chromatography. The His6-SUMO tag was cleaved from the fusion protein using ULP1, a SUMO-specific protease, and removed by re-passing over the nickel-affinity resin. Cleaved material was purified by size-exclusion chromatography. Homogeneity was confirmed by SDS-PAGE and MS.The protein PspCN was produced as described . In briem HEPES buffer, 150 mm NaCl, 0.05% (v/v) surfactant P20 , preincubated with or without a 2-fold molar excess of PspCN, was injected for 150 s at 50 μl/min and a dissociation time of 600 s. The sensor chip was regenerated by three injections of 1 m NaCl. Data were analyzed using the Biacore Evaluation software and a 1:1 steady-state binding model.Experiments were performed on a Biacore T200 at 25 °C as described previously . Brieflym CFB and 50 nm CFD. Formation of C3bBb (∼150 RU) was monitored, and then an initial dissociation phase (of 240-s duration) allowed observation of intrinsic convertase decay (i.e. a decline in RUs attributable to loss of Bb). Subsequently, CFH or CFH–PspCN was injected (10 μl/min for 180 s), allowing observation of a further dissociation phase. The chip was regenerated between cycles by a 30-s injection of 0.1 μm CFH and three 30-s injections of 1 m NaCl.Decay-accelerating activity was measured in an SPR-based assay as described . In brieet al. (ES in Alsever's solution (TCS Biosciences) were pelleted and resuspended three times in complement-fixation diluent (C-FxD) . The washed cells, at 2% (w/v) in C-FxD, were sensitized by incubation with anti-ES stroma antibody from rabbit (Sigma) (diluted 1:4000 (v/v) in the C-FxD) for 30 min at 37 °C. Sensitized sheep erythrocytes (EAS) were washed three times in C-FxD and then resuspended in C-FxD to a final cell density of 2% (w/v). C3b was then deposited on the EAS surface by adding one volume of 4% (v/v) CFB-deficient and CFH-deficient normal human serum (ΔBΔH-NHS)  in C-FxD, followed by a 20-min incubation at 37 °C. The C3b-coated EAS (EAS-C3b) were washed three times in AP buffer . Finally, EAS-C3b cells were resuspended at 2% (v/v) in AP buffer.According to Tortajada et al. , ES in ABΔH-NHS)  containiEAS-C3b in AP buffer containing 2.5 μg/ml CFI. After 15 min (25 °C) of C3b proteolysis, cells were washed three times in 200 μl of AP buffer and pelleted. Washed cells were resuspended in 50 μl of AP buffer in preparation for C3bBb formation as an indirect measure of surviving C3b  to the suspended cells and incubating for 20 min at 37 °C. To quantify lysis, cells were pelleted, and the absorbance (A420 nm) of the supernatant was measured on a Multiskan Ascent Plate Reader (Agilent). The no-lysis controls, for blank subtraction, contained no CFB or CFD. Maximal lysis was determined by an A420 measurement following the addition of PBS, instead of CFH solution, to the EAS-C3b. Mean readings from experiments performed in triplicate were calculated. Total protection from lysis for each concentration of CFH was determined by subtracting, from 1, the following: ((mean A420 for a particular CFH sample) − (mean A420 for a no-lysis control sample))/(A420 for a sample where PBS was used rather than CFH). This was then multiplied by 100. A sigmoidal curve was fitted using SigmaPlot and employed to determine the concentration at which CFH gave 50% protection from lysis (IC50).Cofactor activity was determined by adding (in triplicate) 50 μl from a dilution series of wild-type or mutant CFH solutions, with or without PspCN at a 2:1 PspCN/CFH molar ratio, to 50 μl of a 2% (w/v) suspension of ving C3b . To thisEAS-C3b by adding 50 μl of AP buffer containing 40 μg/ml CFB and 0.2 μg/ml CFD to 50 μl of a cell suspension  CFH solutions (with or without PspCN at a 2:1 ratio) in PBS, C3bBb was allowed to decay for 15 min (25 °C). The extent of lysis then served as a measure of remaining C3bBb, as described above. As above, mean readings of experiments in triplicate were calculated. Total protection from lysis for each concentration of CFH was determined as above, and a sigmoidal curve was fitted using SigmaPlot to determine the concentration at which CFH gave 50% protection from lysis (IC50).For these assays, C3bBb was formed on spension . After 1ES and ER (TCS Biosciences) were prepared by washing three times with buffer E1  gelatin , 10 mm EDTA, pH 7.3) and then buffer E2  gelatin, 10 μm EDTA, pH 7.3). A stock of E was suspended in E2 at a density such that an A412 nm = 1.0–1.2 resulted following complete lysis in water. CFH-depleted serum (ΔH-NHS) was reconstituted with a stated, final concentration (250 nm to 1.0 μm) CFH, with or without PspCN (at a 2:1 PspCN/CFH ratio), and then aliquots of varying size were combined, to give various percentages of ΔH-NHS/CFH, with 5 μl of E stock and buffer E2 to a final volume of 45 μl. Lysis was initiated by adding 5 μl of 50 mm MgEGTA, pH 7.3. The reaction was incubated (37 °C) for 30 min and then quenched with 200 μl of buffer E1. The plate was spun  to pellet non-lysed cells, and then 100 μl of supernatant from each well was transferred to another 96-well plate for A412 measurements.For this 96-well plate assay , ES and H. K. produced the CFH mutants and carried out some of the assays; E. W. performed the erythrocyte-based assays of CA and DAA; E. M. contributed to the production, and carried out the SPR-based comparisons, of V62-CFH and I62-CFH; Y. Y. carried out the hemolysis protection assays; K. M. designed experiments and contributed to writing the manuscript; D. K. and A. R. helped to conceive the study and contributed to the experimental design; A. P. H. contributed to the experimental design and to the production and purification of proteins; P. N. B. helped to conceive the study, contributed to the experimental design, and wrote the manuscript.