Mycobacterium bovis bacillus Calmette–Guérin (BCG)/Mycobacterium tuberculosis culture filtrate respectively, induce strong T-cell proliferation and IFN-γ production in most healthy individuals latently infected with M. tuberculosis and in BCG-vaccinated mice and humans but not in tuberculosis patients. Members of the Ag85 complex are highly conserved in other mycobacterial species. Mice and humans infected with Mycobacterium ulcerans or cattle infected with M. bovis or Mycobacterium avium subsp. paratuberculosis also show strong T-cell responses to this protein family. Using synthetic overlapping peptides, bio-informatic prediction programs and tetramer-binding studies, a number of immunodominant CD4+ and CD8+ T-cell epitopes have been identified in experimental animal models as well as in humans, using proliferation and Th1 cytokine secretion as main read-outs. The results from these studies are summarized in this review.The Ag85 complex is a 30–32 kDa family of three proteins , which all three possess enzymatic mycolyl-transferase activity involved in the coupling of mycolic acids to the arabinogalactan of the cell wall and in the biogenesis of cord factor. By virtue of their strong potential to induce Th1-type immune responses, important for the control of intracellular infections, members of the Ag85 family rank among the most promising TB vaccine candidate antigens. Ag85A and Ag85B, initially purified from  MtbAg85A: Rv3804c, P32, FbpA; Mtb85B: Rv1886c, 30 kDa antigen, α-antigen, FbpB; Mtb85C: Rv0129c, FbpC2; M. bovis85A: Mb3834c, MPB44; M. bovisAg85B:MPB59; M. bovis bacillus Calmette–Guérin (BCG) 85A: BCG_3866c; M. ulcerans85A: MUL4987; Map85A: MAP0216; Map85B: MAP 1609c.The Ag85 complex is actually a 30–32 kDa family of three proteins , which each possess enzymatic mycolyl-transferase activity involved in the coupling of mycolic acids to the arabinogalactan of the cell wall and in the biogenesis of cord factor . These pMtb virulence, and cell wall arabinogalactan-linked mycolic acids. A novel inhibitor of Ag85C, 2-amino-6-propyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile (I3-AG85) inhibits Mtb survival in infected primary macrophages and quantification of mycolic acid-linked lipids of the Mtb envelope showed a specific blockade of TDM synthesis  show cross-reactive Th1-type immune responses to Ag85 components purified from BCG CF or produced as recombinant E. coli derived proteins . The aa sequence of Ag85A of Mycobacterium bovis and M. bovis BCG (1173P2 strain) is identical to the aa sequence of the Ag85A component of Mtb (H37Rv) and is therefore not shown. The Ag85B sequence of M. bovis differs in one aa from the Ag85B sequence of Mtb: Phe100Leu. On the other hand, expression levels of these proteins may differ and whereas Ag85A is the major component in CF of surface-pellicle grown BCG, Ag85B is the major component in CF from Mtb and Map. The Ag85C component is found in lesser concentrations in the CF, as it is localized more internally in the cell wall. In 2000, M. Horwitz reported that recombinant BCG vaccines expressing the Mtb 30-kDa (Ag85B) major secretory protein induced greater protective immunity against tuberculosis than conventional BCG vaccines in a highly susceptible animal model, i.e., the guinea pig   reacted preferentially against the amino-terminal half of the protein, i.e., against peptide 5 (aa 41–60) and especially against peptide 11 (aa 101–120), which contains a predicted I-Ed binding motif. H-2b haplotype mice, on the other hand, reacted against peptides from both amino- and carboxy-terminal halves of the protein, peptide 25 (aa 241–260) and peptide 27 (aa 261–280) being the most potent stimulators of IL-2 and IFN-γ production. Finally, CBA/J (H-2k) and major histocompatibility complex class II mutant B6.C.bml2 mice, carrying a mutant I-Abml2 allele on an H-2b background, reacted only very weakly to the 85A peptides.  but responses were completely component specific. In C57BL/6 mice, a cross-reactive T-cell response was detected against two carboxy-terminal peptides spanning amino acids 241–260 and 261–280 of Ag85A and Ag85B. These regions were not recognized at all in C57BL/6 mice vaccinated with Ag85C DNA.Plasmid DNA vaccination is a powerful tool to identify protective antigens of tuberculosis and to identify immunodominant CD4epitopes . BALB/c epitopes . Furtherepitopes . T-cell epitopes . DespiteMtb infected mice  peptide motif” (http://www-bimas.cit.nih.gov/molbio/hla_bind/) showed that these three peptides spanned four predicted CD8+ T-cell epitopes   and the I-Ed restricted epitope TFLTSELPGWLQANRHVKPT (aa 99–119), respectively  and CD8+  T-cells  provided potent protection against airway  T-cells .b haplotype mice, no MHC class I restricted epitopes have been identified so far on Ag85A or Ag85B to our knowledge, neither in BCG or plasmid DNA vaccinated nor in TB infected mice.For H-2Mtb. Subsequently, we reported on T-cell epitope mapping of Ag85A from Mtb using peripheral blood mononuclear cell (PBMC) cultures from healthy tuberculin-positive volunteers and from patients with tuberculosis, using the same synthetic 20-mer peptides of the murine study. Peptide recognition was largely promiscuous, with a variety of HLA haplotypes reacting to the same peptides. PBMC from all tuberculin-positive subjects reacted to Ag85A, and the majority proliferated in response to peptide 6 (amino acids 51–70), peptides 13, 14, and 15 (amino acids 121–160), or peptides 20 and 21 (amino acids 191–220). PBMC from tuberculosis patients demonstrated a variable reactivity to Ag85 and its peptides, and the strongest proliferation was observed against peptide 7 (amino acids 61–80) , we reported that healthy Mantoux positive volunteers showed a much stronger lymphoproliferative and IFN-γ response to this antigen than tuberculosis patients . This was 61–80) . Nine oulunteers , 25. Ag8Mtb Ag85B using blastogenic responses of PBMC from 12 healthy purified protein derivative-positive subjects to a set of synthetic 15-mer peptides based on the full 325-amino-acid sequence (leader sequence included)  . Seven iMtb CF in Incomplete Freund’s adjuvant : LQVPSPSMGRDIKVQFSGG. This sequence is identical in Ag85A and Ag85B, but differs in two positions in Ag85C: Pro16Ala and Ser28Gly. Also in M. ulcerans, Map, and Maa, position 16 has the shift to Alanine. In M. leprae there is also one aa shift: Ser28Asn.Human leukocyte antigen-transgenic mice can be a powerful tool to identify human T-cell epitopes in an experimental mouse model. In 1998, A. Geluk reported on the identification of an HLA-class II restricted epitope of Ag85. HLA-DRA/B1*0302 (DR3) transgenic mice were vaccinated with Ag85 protein, purified from adjuvant . Using 2Mtb-specific CD4+ T-cells, using HLA-class II tetramers for a peptide shared between Ag85A and Ag85B, i.e., aa 90–104 AGCQTYKWETFLTSE in healthy PPD-positive donors, latently infected with Mtb  T-cells. Only a minor fraction appeared to be naïve or effector T-cells. Interesting to note that this sequence is shared with Ag85A of M. leprae that there is only Q93T shift in M. ulcerans, Map, and Maa but that the sequence of Mtb Ag85C differs in five positions.Whereas most of these studies on human T-cell epitope mapping were performed during the mid-nineties, one more recent paper of Lindestam Arlehamn et al. reported on the memory phenotype of with Mtb . Tetrame+ T-cell epitope of Ag85C  and also an IL-2/IFN-γ inducing region for C57BL/6 mice vaccinated with pAg85C (Mtb and the four non-tuberculous mycobacteria. WPTLIGLAM stimulated effector cells were able to kill Mtb or BCG infected macrophages and produced IFN-γ and TNF-α (d restricted epitope spanning the same aa 161–168 (PTLIGLAM) was identified in Ag85A DNA vaccinated BALB/c mice, which did not cross-react with the corresponding Ag85B peptide (because of the Thr162Ser shift).Less is known on human MHC class I restricted epitopes of Ag85. Klein et al. reported on a HLA-B*35 restricted CD8of Ag85C . Using rh pAg85C . Peptidend TNF-α . Interes1/2 off-rate) analysis for individual peptide species on HLA-A and HLA-B molecules revealed binding ranges between 10−3 and 10−7 M. After selection of the best matches, major histocompatibility complex class I/peptide tetramer complexes were constructed to measure the CD8+ T-cell responses directly ex vivo in PBMC derived from 57 patients with acute pulmonary tuberculosis. Three patterns of  specific CD8+ recognition were identified: (a) Focus on one dominant epitope, (b) Co-dominant recognition of two distinct groups of peptides, and (c) Diverse and broad recognition of peptides (presented by HLA-A*0201). Peptides that bound with slow off-rates to class I alleles, that is HLA-A*0201, were associated with low frequency of CD8+ T-cells in PBMCs from patients with tuberculosis. HLA-B alleles showed fast off-rates in peptide binding and restricted high numbers (up to 6%) of antigen-specific CD8+ T-cells in patients with pulmonary tuberculosis (in vitro IL-2 and IFN-γ production) revealed that tetramer-binding T-cells in PBMCs from these patients were little or not responsive to the nominal peptide epitope, confirming the notion of a deficient Ag85 specific T-cell response in TB patients. The study focused on TB patients and not on latently infected subjects, which could have been more relevant in the context of TB vaccine development.A comprehensive epitope mapping to HLA-A*0101, A*0201, A*1101, A*2402, B*0702, B*0801, and B*1501 of Ag85B was published in 2007 . Affinitrculosis . FunctioM. bovis (MPB 59) in BCG vaccinees and TB patients. The mature 85B protein of M. bovis (MPB59) has a high degree of amino-acid identity with the M. bovis 85A protein (76%) and the Mtb 85B (99%) and Mtb85A (76%) proteins. Proliferative assays with recombinant MPB59 demonstrated that PBMC from 95% of BCG vaccinees and 52% of tuberculosis patients responded to the whole mature protein. Using a set of synthetic 20-mer peptides, five peptides were found to be recognized in more than half of the MPB59 responders. The T-cell-reactive regions were essentially identical in the M. bovis and Mtb 85B proteins. Subjects with a variety of HLA-DR phenotypes responded to a number of these peptides and there was no difference in the pattern of responses between BCG vaccinees and TB patients. A promiscuous recognition pattern was observed in response to peptides spanning aa 51–70 (recognized by 87% of the responsive BCG vaccinees and 93% of the responding TB patients) and aa 11–30 (recognized by 73% of the responders). Peptides in the C-terminal region (aa 131–150 and aa 191–210) were more frequently recognized by patients than by BCG vaccinees , change that does not affect the binding affinity for HLA-A*0201 , peptide 5 (aa 41–60), and peptides 25 and 26 (aa 241–270). PBMC from paucibacillary patients reacted preferentially against peptide 1 (amino acids 1–20) and peptide 5. Multibacillary patients were not reactive to Ag85 or the Ag85A peptides  shares 84.1% amino-acid sequence identity and 91% conserved residues with the gene encoding Ag85A from Mtb  have been sequenced, and at the protein level, a 99% sequence identity with M. avium subsp. avium (Maa) was found, with a single amino-acid residue difference for each protein: Ag85A: Ser155Pro, Ag85B: Ser120Asn, and Ag85C: Ileu284Thr for Map vs. Maa. Compared to the mature protein sequences of M. bovis, Map85A (Map 0216) shares 82%, Map85B (Map 1609c) shares 86%, and the Map85C (Map 3531c) shares 87% identity  was strongly expressed in Map CF . Sp. Sp+ epimponents . Peptide+ epitopes of Ag85A, Ag85B, and Ag85C were also identified in C57BL/6 mice vaccinated with plasmid DNA encoding the Map antigens  on Ag85A, peptide 17 (aa 145–162) on Ag85B, and the corresponding region of Ag85C covered by peptides 15–16 (aa 141–170). The strongest Th1 epitope identified in this study spanned region 241–260 of the Ag85A and Ag85B sequence. These sequences are very similar to each other, but – as for M. tuberculosis – the Ag85C sequence is completely different.Cross-reactive CD4antigens . Plasmid8 CFU) of Map (ATCC 19698) cells per day for 10 consecutive days.  was identified in experimentally infected cattle.Ag85 specific T-cell epitopes were also mapped in experimentally infected cattle, the target species of Johne’s disease. Five 2- to 3-week-old calves were infected by the oral route with 10 mg , the same epitopes have been identified. More in particular, regions spanning aa 10–30, 60–80, 100–120, 140–160, and 199–207 of the mature Ag85A and Ag85B span these IFN-γ/IL-2 inducing Th1/CTL epitopes. The Ag85C component differs in its sequence from the two other components and overall T-cell responses against this third component are lower. The Ag85C molecules are buried more in the cell wall and hence may be less accessible for rapid antigenic processing and presentation to T-cells. It is also possible that because of this less exposed localization, there has been less evolutionary pressure on the gene of Ag85C to encode for such immunodominant, promiscuous T-cell epitopes. Members of the Ag85 complex are highly conserved in other mycobacterial species and cross-reactive T-cell responses against Mtb antigens can be found in M. ulcerans, Map, and M. leprae infection. Sequence comparisons indicate that the Ag85A sequences of these three non-tuberculous mycobacteria are more similar to the sequence of MtbAg85B than of MtbAg85A, particularly in aa stretches spanning the immunodominant epitope regions. Moreover, expression of the two components by different mycobacterial species seems to be differentially regulated, with a preference for the Ag85B orthologs in NTM.Members of the Ag85 complex are immunodominant mycobacterial antigens, which induce strong Th1-type immune responses in situations of controlled mycobacterial infection. By virtue of their role in cell wall integrity and synthesis of cord factor, these abundantly expressed proteins have long been considered as virulence factors. On the other hand, because of the strong T-cell responses they induce, these proteins can also be regarded as a means of both host and pathogen to reach a state of equilibrium, advantageous for both parties. An estimated one third of the world population is infected with quality of the memory response induced by the BCG vaccine may be insufficient  that the vaccine potential of live BCG can be augmented by coadministration with plasmid DNA encoding PPE44 and Ag85A respectively, as measured by Th1-type cytokine secretion, specific IgG antibodies, as well as specific IFN-γ producing CD8+ T-cells  and furthermore did not augment protective efficacy of BCG . The reafficient . More spesponses . More re T-cells  (Bruffaeproperly .The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The systematic organization of enzymes is a key feature for the efficient operation of cascade reactions in nature. Here, we demonstrate a facile method to create nanoscale enzyme cascades by using engineered bacterial outer membrane vesicles (OMVs) that are spheroid nanoparticles (roughly 50 nm in diameter) produced by Gram-negative bacteria during all phases of growth. By taking advantage of the fact that OMVs naturally contain proteins found in the outer cell membrane, we displayed a trivalent protein scaffold containing three divergent cohesin domains for the position-specific presentation of a three-enzyme cascade on OMVs through a truncated ice nucleation protein anchoring motif (INP). The positional assembly of three enzymes for cellulose hydrolysis was demonstrated. The enzyme-decorated OMVs provided synergistic cellulose hydrolysis resulting in 23-fold enhancement in glucose production than free enzymes. A trivalent scaffold, Scaf3 was amplified from pSctf GCGCGAGCTCCAACAGCAGCAATCCGGCGATTCTCTTAAAGTTACAGTAGGAACAGC) and scaf-R (TGCCGCTCGAGCTTAACAATGATAGCGCCATCAGTAAGAGTAACC) and inserted into pINP between SacI and XhoI, resulting in pINPScaf. The entire INP-scaf3 was amplified from pINPScaf with a forward primer IS-F (GCTCTAGAGAAGGAGATATACATATGAATATCGACAAAGCGTTG-GTACTGC) and a reverse primer IS-R (GCTCTAGATCAGTGGTGGTGGTGGTGGTGCTTAACAAT-GATAGCGCCATCAGTAAGAGTA) and the amplified fragment of INP-scaf3 was inserted into pVLT33 E. coli JC8031 E. coli JC8031 harboring pINPscaf3 were grown in 100 ml of TB medium containing 50 µg/ml kanamycin at 37 °C. Protein expression was induced by the addition of 20 µM IPTG and grown at 30 °C when the OD600 reached ∼0.5.Plasmid pINP was constructed by ligating the PCR-amplified the truncated ice nucleation protein gene from pPNC20 E. coli cells displaying Scaf3 were harvested by centrifugation, washed with phosphate buffered saline  and resuspended in PBS containing 0.1 % bovine serum albumin (BSA) and mouse anti-His immunoglobulin G . After 2 h incubation at room temperature with occasional mixing, cells were washed twice with the same buffer before resuspending in PBS with 0.1 % BSA and the goat anti-mouse IgG conjugate with Alexa488 (Invitrogen). Cells were washed twice after 1 h incubation, resuspended in PBS buffer, and examined by an Olympus BX51 fluorescent microscope.Vesicles were collected from the cell-free supernatant as described Samples in PBS buffer were applied to 400-mesh carbon-coated copper grids (Electron Microscopy Sciences), stained with 2% uranyl acetate, air dried, and visualized on a Zeiss CEM 902 transmission electron microscope operating at voltage of 120 kV. Dynamic light scattering (DLS) for characterization of size of engineered vesicles was performed on a ZetaPALS . Collected vesicles were suspended in PBS buffer, and 1 ml of the vesicle suspension was transferred to a square cuvette for DLS measurement. DLS evaluates the time-dependent fluctuations of spherical particles in a light scattering intensity caused by the Brownian motion and a hydrodynamic diameter is calculated via the Stokes-Einstein equation E. coli BL21 (DE3) cells harboring each plasmid were precultured overnight at 37 °C in LB medium with 100 µg/ml of ampicillin or 50 µg/ml kanamycin. Cells were inoculated again in the same medium with 1.5 % glycerol before adding 200 µM IPTG at an OD600 of 1. After 16 h, cells were harvested by centrifugation and lysed with a sonicator in Tris buffer containing 100 mM NaCl and 10 mM CaCl2. For binding, OMVs were incubated with different cell lysates for 2 h at room temperature in the same Tris buffer as above. After incubation, OMVs were washed and collected by centrifugation  and resuspended in the same buffer for protein analysis and activity assay.Construction of dockerin-fused cellulases were previously described Enzyme activity was determined as described Clostridium cellulolyticum (C), Clostridium thermocellum (T) and Ruminococcus flavefaciens (F) and an internal cellulose-binding module (CBM) from our previous work was adopted Saccharomyces cerevisiaeE. coli. To investigate this feasibility, the truncated ice nucleation protein anchor (INP), which has been used as an surface anchor to display proteins up to 119 kDa in E. coliE. coli strain BL21, which was used to express the different cellulases, produced virtually no OMV under the same growth condition. Successful production of INP-scaf3 was demonstrated by SDS-PAGE analysis  comprised of three orthogonal cohesin domains from analysis . A new panalysis . The suranalysis . Cells dAfter confirming the surface localization of Scaf3, we next investigated whether the displayed INP-scaf3 can be incorporated into the secreted OMVs. As OMVs are released into the culture medium during cell growth, they can be easily isolated from cell-free culture supernatants using sequential centrifugations with increasing speeds up to 141,000 g and analyzed on SDS-PAGE . SimilarTo probe the surface-accessibility and functionality of Scaf3, three different dockerin-tagged cellulases, an endoglucanase fused with DocT (AT), an exoglucanase fused with DocC (EC) and a β-glucosidase fused DocF (BF), were used. Each cellulase was individually assembled onto the OMVs. After removing unbound proteins, OMVs were washed and recovered by centrifugation. Enzyme docking onto Scaf3-decorated OMVs was verified by detecting enzyme activities for each cellulase . In contThe concept of sequential assembly of multiple enzymes onto OMVs was further demonstrated by assembling all three cellulases. Enzyme binding curves were first constructed with increasing amounts of cell lysates to determine the amount of cellulase required to saturate the corresponding cohesin domain on Scaf3  with multiple enzymatic activities. A trivalent protein scaffold was genetically tethered onto the OMVs to enable the positional specific recruitment of three different cellulases. The assembled enzyme complex not only retained full activity but also hydrolyzed cellulose 23-fold faster than non-complexed enzymes. Our synthetic biology approach excludes the use of any chemical conjugation and can provide a simple platform for generating nanobiocatalysts for highly efficient multi-enzyme reactions. The flexibility in designing scaffolds using a wide range of specific binding domains will permit the virtually unlimited number of functional proteins to be displayed on the OMVs.File S1Supporting Figures. Figure S1, Density-gradient fractionation of vesicles. Western blot analysis of different fractions using an anti-His6, anti-OmpA or anti-DsbA serum, respectively. Figure S2, Binding of CelA-DocCT onto wild-type and engineered OMVs. (A) SDS-PAGE analysis of OMVs before and after enzyme binding. (B) CelA activity of the resulting OMVs after binding. Figure S3, Hydrolysis of cellulose by single enzyme binding. Cellulose hydrolysis from the binding of increasing amount of CelA-DocT (CMC) (A), CelE-DocC (PASC) (B) and BglA-DocF (cellobiose) (C). Figure S4, SDS-PAGE analysis of OMVs loaded with all three enzymes. For better resolutions, binding of (A) AT or (B) EC and BF was confirmed using either 10% or 7% SDS-PAGE. Bands corresponding to either INP-scaf3 (107 kDa), AT (53 kDa), EC (90 kDa) or BF (70 kDa) are shown.(DOCX)Click here for additional data file.


Their structures were elucidated on the basis of 1D and 2D NMR spectra analysis. The structure of sinensine E was confirmed by X-ray crystallographic analysis of its acetyl product (4a). Four new alkaloids, sinensines B–E (Supplementary material is available for this article at 10.1007/s13659-011-0026-4 and is accessible for authorized users. Supplementary material, approximately 708 KB.


Retromer is a protein assembly that plays a central role in orchestrating export of transmembrane-spanning cargo proteins from endosomes into retrieval pathways destined for the Golgi apparatus and the plasma membrane . Recentl •VPS35(D620N) mutation leads to a defect in endosome-to-TGN transport•Mutant has a perturbed association with the actin-polymerizing WASH complex•Primary defect is a reduced affinity for binding WASH component FAM21•Provides new molecular insight into retromer deregulation in Parkinson disease Using quantitative proteomics and functional analysis in patient-derived fibroblasts, McGough et al. define the primary molecular defect associated with the Parkinson disease-linked retromer VPS35(D620N) mutation and, by revealing effects on retromer-mediated endosome-to-TGN transport, provide new insight into retromer deregulation in this disease. We sought to investigate the effect of the VPS35(D620N) mutation on trafficking of two known retromer cargos, the endosome-to-TGN transport of the cation-independent mannose 6-phosphate receptor (CI-MPR) , and theNext, we examined the endosome-to-TGN transport of the CI-MPR. After delivery to endosomes, CI-MPR dissociates from its ligand and is recognized by the retromer complex and retrieved to the TGN for further rounds of ligand binding and transport . In the To determine the molecular basis of the perturbed endosome-to-TGN trafficking observed with the VPS35(D620N) mutant, we first sought to determine whether the VPS35(D620N) mutation affected formation of the VPS26-VPS29-VPS35 heterotrimer or its endosomal localization. Like GFP-VPS35, GFP-VPS35(D620N) localized predominantly to a sorting nexin-1 (SNX1) positive early-to-late transitional endosomal compartment A and 2B.Retromer scaffolds the assembly of a number of accessory proteins that aid its role in endosomal sorting . To idenWhile there is comparative enrichment of VPS29, VPS26A, and VPS26B between wild-type and VPS35D620N) (left side of N left s and S2. To identify the primary defect in the VPS35(D620N) mutant, we performed a series of GFP-VPS35 immunoisolations from cells treated with RNAi’s targeting FAM21—the component of the WASH complex that directly binds to VPS35 , ANKRD50To correlate these data with the previously described endosome association of SDCCAG3 and RME-8 , we analThe VPS35(D620N) mutation has been suggested to inhibit the neuroprotective effect of VPS35 , so we sPrevious studies have suggested that the interaction between the retromer and FAM21 is mediated by the VPS35 and VPS29 (but not VPS26) subunits of the retromer heterotrimer and multiple LFa motifs from the C terminus of FAM21 . FAM21 cd = 4.6 ± 0.7 μM) was significantly lower (by 2.2 ± 0.5-fold) than that of the wild-type VPS35/VPS29 (Kd = 2.4 ± 0.6 μM)  A and 4B.In addition to R20-21, we also attempted to measure the affinity for a larger fragment of FAM21, R15-21. Although the ITC thermograms were qualitatively consistent with a reduced affinity for the VPS35(D620N) mutant, since the R15-21 fragment contains seven LFa motifs potentially interacting with VPS35/VPS29, it was not possible to quantitatively fit and interpret these data (data not shown).In summary, stemming from an unbiased quantitative proteomic analysis, we have identified that at the molecular level the primary effect of the Parkinson disease-linked VPS35(D620N) mutation is a decrease in the affinity for FAM21 and as a consequence a reduced association with the WASH complex and its associated proteins, including RME-8, and the newly identified retromer-associated proteins SDCCAG3 and ANKRD50. While not perturbing retromer-mediated endosome-to-plasma membrane recycling of GLUT1, this partial loss of FAM21 binding and the WASH complex-associated proteins does lead to a decrease in the efficiency of endosomes-to-TGN retrieval of the CI-MPR. While the primary defect of the VPS35(D620N) mutation is a decrease in affinity for the WASH complex, it is important to stress that the observed phenotypes maybe arise from a reduced activity of RME8, SDCCAG3, or ANKRD50 or a combination of these and FAM21. This is consistent with the current view that WASH-mediated actin polymerization is required for the organization and maturation of retromer sorting domains on early-to-late transition endosomes . PreciseDuring the final stages of reviewing our manuscript Zavodszky and colleagues, using a candidate approach, published work consistent with a defect in WASH binding to the VPS35(D620N) mutant . Our detI.J.M, F.S., D.D.B., M.K.R., and P.J.C. designed the project; I.J.M. and F.S. performed the cell biology experiments; K.J.H. performed the proteomics; D.J. performed the biochemical analysis of the VPS35 FAM21 interaction; P.A.B., K.J.M., A.L.W., and M.A.C. generated the VPS35(D620N) patient fibroblast cell line. I.J.M., F.S., D.J., D.D.B., M.K.R., and P.J.C. wrote the manuscript.


In this study, we hypothesized that anti-hnRNP A1 antibodies might cause stress granule formation and altered levels of RNAs and proteins that bind hnRNP A1.Multiple sclerosis (MS) is the most common demyelinating disorder of the central nervous system (CNS). Data suggest that antibodies to CNS targets contribute to the pathogenesis of MS. MS patients produce autoantibodies to heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). hnRNP A1 is an RNA binding protein (RBP) overexpressed in neurons that functions in pre-mRNA splicing, mRNA trafficking, and translation. Previously, we showed that anti-hnRNP A1 antibodies entered neuronal cells  4 and 7, which when mutated mimic progressive MS.Taken together, these data suggest potential mechanisms by which autoantibodies may contribute to neurodegeneration in MS. Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system (CNS) of unknown etiology. It is believed to occur in individuals with genetic susceptibility –5. DiseaMS patients have been shown to produce autoantibodies to various myelin and non-myelin antigens. For example, patients develop autoantibodies to myelin oligodendrocyte glycoprotein (MOG), myelin associated glycoprotein (MAG), myelin basic protein (MBP), and proteolipid protein (PLP) –9. TheseFurthermore, in contrast to healthy controls and patients with Alzheimer’s disease, MS patients specifically produce autoantibodies to the non-myelin antigen heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) –27. hnRNIn vitro, anti-hnRNP A1-M9 antibodies were found to enter neuronal cells through a mechanism involving clathrin-mediated endocytosis [Autoantibodies produced by MS patients are specific to the M9 region of hnRNP A1 ,22,24–27ocytosis . Once inocytosis . Furtherocytosis .Three main types of granules exist within neuronal cells: stress granules (SGs), processing bodies (P bodies), and neuronal transport RNPs  . SGs devIn summary, SGs harbor translationally arrested RBPs and their cargo during times of cellular stress until homeostasis is achieved, P bodies manage the waste of the cell, and neuronal transport granules are responsible for transport of RBPs and their translationally arrested cargoes, which upon arrival to designated sites in the neuron, undergo translation.In this paper we examined whether anti-hnRNP A1-M9 antibodies interacted with the three types of neuronal granules . Additionally, we tested whether the SPGs  are mRNA binding partners of hnRNP A1 protein. Furthermore, we determined the downstream effects of anti-hnRNP A1-M9 antibodies on translation of these clinically relevant mRNAs.5 SK-N-SH cells were seeded per well into 500 uL of complete DMEM/F12 media into 8 well chamber slides (Corning #354632) for RNA granule immunocytochemistry experiments.The SK-N-SH cell line , an immortalized human neuroblastoma cell line was maintained in Dulbecco’s modified Eagle’s medium/F12 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. 100 mm plates were seeded for the RNA immunoprecipitation, RT-PCR, and Western blot experiments. 10Anti-hnRNP A1 antibodies were obtained from Abcam  and Millipore . The anti-hnRNP A1 antibodies are specific for hnRNP A1-M9 and have been shown to overlap the M9 immunodominant epitope recognized by IgG isolated from MS patients . Isotype5 SK-N-SH cells were seeded per well into 500 uL of complete DMEM/F12 media into 8 well chamber slides (Corning #354632). Anti-hnRNP A1 and control isotype-matched IgG2b antibodies were labeled with Alexafluor 488 and added to the culture media of SK-N-SH cells at a concentration of 8 ug/ml for each assay as determined in previous studies [10SK-N-SH cells were cultured in 100 mm cell culture dishes. Cells were scraped and lysate extracted. 60 mcg of protein extracted from whole cell lysate was added to protein A/G beads labeled with either anti-hnRNP A1 antibody (ab4791) or anti-IgG (Millipore 12-370) as a control for non-specific binding. Samples were rotated overnight at 4°C. The following day, bound proteins were eluted from the protein A/G beads. Eluent from both samples as well as control SK-N-SH lysates were run on (sodium dodecyl sulfate) SDS gels and probed for hnRNP A1 to confirm specific binding of hnRNP A1 protein. RNA was then extracted from the eluents using the RNA-Stat 60 protocol . RNA conSK-N-SH cells seeded in 100 mm dishes were treated with either anti-hnRNP A1 antibodies, control anti-IgG antibodies (isotype matched), or were left untreated as previously described using protocols in our lab . AntibodSK-N-SH cells seeded in 100 mm dishes were treated with either anti-hnRNP A1 antibodies, anti-IgG antibodies, or were left untreated. Antibody treatments were at a concentration of 8 ug/ml in DMEM/F12 complete media for 48 hours. Following antibody treatment, cells were scraped and lysates were centrifuged 5 min at 2500 rpm. Supernatants were heated with 2X running buffer for 5 minutes at 95°C and then samples were loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and run at 100V for 1.5 hours. Gels were then transferred onto nitrocellulose membranes at 100V for 1.5 hours. Membranes were blocked with 6% milk-TBS for 40 minutes at RT. Primary antibodies were then added overnight at 4°C at manufacturer’s suggested concentration. Primary antibodies were as follows: SPG4 (Abcam ab38150), SPG7 (Abcam ab154989), SPG20 (Abcam ab94950), hnRNP A1 (Millipore 05-1521), GAPDH (Abcam ab9485), and Beta-actin (Novus NB600-503). The next day, membranes were washed 4 X 5 min with Tris buffered saline (TBS). Secondary antibodies were then added at a 1:10,000 dilution . Membranes were washed 4 X 5 minutes with TBS-Tween, developed and imaged. To compare relative changes in signal intensity for each protein tested, the following technique was utilized. Using Image J software, signal intensity was calculated for each Western blot. Next, percent signal intensity was calculated for each protein tested . For each protein, the relative signal intensity was calculated relative to the whole cell lysate signal. The adjusted signal intensity was normalized relative to the loading control (Beta-actin or GAPDH). Percent change of signal intensity of anti-hnRNP A1 antibodies was compared relative to control IgG for each protein tested .Neuronal granules play a crucial role in the trafficking of various RBPs (including hnRNP A1 ) and thehttp://rbpdb.ccbr.utoronto.ca/). This database contains the unique RNA binding sequences that are required for RNA to bind RNA binding domains of an RBP. The user enters an RNA sequence into the RBPDB. The RBPDB scans the RNA sequence and reports out all RBPs that might bind to it, and the exact sequence and its location within the RNA sequence input. We previously showed that anti-hnRNP A1 antibodies transfected into a neuronal cell line caused altered RNA levels of the SPGs  [After determining that both anti-hnRNP A1-M9 antibodies and control IgG colocalize within SGs, we examined whether anti-hnRNP A1-M9 antibodies might have a specific effect on the RNA metabolism of hnRNP A1 protein’s RNA cargo. In order to accomplish this goal we needed to define the RNA binding partners of hnRNP A1 protein. To evaluate which RNAs might theoretically bind hnRNP A1, we used the RBPDB  . Therefo and 20) . Thus, R and 20) .To test whether the target RNAs bound hnRNP A1, we performed RNA immunoprecipitation (IP), followed by RT-PCR of the resulting eluent. Specifically, protein A/G agarose beads were labeled with anti-hnRNP A1-M9 antibodies to elute hnRNP A1 protein with its bound RNA. In parallel, protein A/G agarose beads were labeled with isotype-matched IgG (control). SK-N-SH lysates were incubated with the antibody-bound beads, bound protein-RNA complexes were eluted and the eluents run on Western blot . FollowiAfter determining the specificity of the immunoprecipitation, RNA was extracted from the eluents using the RNA-Stat 60 protocol . FollowiAfter determining that SPG4 and SPG7 were RNA binding partners of hnRNP A1 protein, we tested whether the addition of anti-hnRNP A1-M9 antibodies altered RNA levels of these genes. We hypothesized that anti-hnRNP A1-M9 antibodies would alter RNA levels of hnRNP A1’s binding partners  compared to RNA that did not bind hnRNP A1 (SPG 20). We performed these studies by adding 8 ug/ml of anti-hnRNP A1-M9 antibodies or control IgG to SK-N-SH neuronal cells. Cells were incubated with antibodies for 48 hours, lysed and RNA extracted and amplified as described previously. Values obtained from the anti-hnRNP A1-M9 treated cells were compared to the control IgG treated cells to determine the effect on RNA expression due to the specificity of the anti-hnRNP A1-M9 antibodies . The resNext, we hypothesized that protein levels of hnRNP A1’s RNA binding partners  would be altered whereas RNAs that did not bind hnRNP A1 (SPG-20) would not. Additionally, since anti-hnRNP A1-M9 antibodies and isotype control IgG both initiated SG formation and colocalized within SGs, we wanted to determine if anti-hnRNP A1-M9 antibodies caused specific downstream effects  upon the bound RNAs compared to isotype control IgG. We performed these studies by adding 8 ug/ml of anti-hnRNP A1-M9 antibodies or control IgG to SK-N-SH neuronal cells. These data were compared to untreated SKN-SH neuronal cells run in parallel. Cells were incubated with antibodies for 48 hours, lysed and proteins extracted for Western blotting. We found dramatic differences in protein expression of SGP4 (100% in both experiments) and SPG 7  in the anti-hnRNP A1-M9 antibody compared to isotype control treated cells . DifferePrevious studies have shown that in contrast healthy controls as well as patients with other neurologic disease, MS patients develop antibodies to hnRNP A1 ,24–26,28Taken together, these studies suggest a potential mechanism by which autoantibodies might contribute to neurodegeneration. One of hnRNP A1’s many roles in RNA metabolism (‘ribostasis’) is to bind RNA, transport RNA from the nucleus to the cytoplasm, where it undergoes translation . hnRNP Ain vitro due to the addition of anti-hnRNP A1-M9 antibodies. In this article we show that anti-hnRNP A1-M9 antibodies alter protein levels of SPG7 in vitro. In concurrence with the studies in HSP patients with depleted mitochondrial respiratory chain complexes, our data suggests that altered SPG7 levels due to anti-hnRNP A1-M9 antibodies might act in a similar mechanism to the SPG7 mutations seen in patients with autosomal recessive HSP.It is important to note that mutations in the genes affected  result in various forms of hereditary spastic paraplegia (HSP) . HSP is in vivo studies are required to clarify the role of anti-hnRNP A1-M9 antibodies in the pathogenesis of neurodegeneration in MS, an important link is made because (1) the monoclonal antibodies used in this study overlap the human immunodominant epitope of hnRNP A1–M9 recognized by IgG isolated from MS patients, (2) the SPG data supports previous data shown in vitro and in neurons isolated from MS brains and (3) the SPGs are clinically relevant targets [In summary, we showed that anti-hnRNP A1-M9 antibodies cause cellular stress resulting in SGs to which the antibodies colocalize. Additionally, we determined that SPG4, and SPG7 are RNA binding partners of hnRNP A1, and that addition of anti-hnRNP A1 antibodies to a neuronal cell line resulted in decreased levels of both SPG4 and SPG7 RNA and protein. The exact mechanism by which this occurs requires further study. For example, although we showed decreased levels of target proteins by Western blotting, it is not yet know whether this is due to changes in protein translation, degradation or altered RNA levels. Alternatively, the changes we observed might also be due to changes in hnRNP A1 protein , which m targets ,24. TakeSupplementary file


Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive.T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC), namely sn-1 C18:1(delta 9)-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV) and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF.Here, we have purified and structurally characterized the putative T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the progression of Chagas disease and its biosynthesis could eventually be exploited as a potential target for new therapeutic interventions.Taken together, our data indicate that  Trypanosoma cruzi, was exclusively confined to Latin America but it has recently spread to other regions of the world. Chagas disease affects 8–10 million people and kills thousands of them every year. Lysophosphatidylcholine (LPC) is a major bioactive phospholipid of human plasma low-density lipoproteins (LDL). Platelet-activating factor (PAF) is a phospholipid similar to LPC and a potent intercellular mediator. Both PAF and LPC have been reported to act on mammalian cells through PAF receptor (PAFR). Previous data from our group suggested that T. cruzi produces a phospholipid with PAF activity. Here, we describe the structural and functional analysis of different species of LPC from T. cruzi, including a LPC with a fatty acid chain of 18 carbon atoms and one double bond (C18:1-LPC). We also show that C18:1-LPC is able to induce rabbit platelet aggregation, which is abrogated by a PAFR antagonist. In addition, a three-dimensional model of human PAFR was constructed. Contrary to other T. cruzi LPC molecules, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF. Further studies are needed to validate the biosynthesis of T. cruzi C18:1-LPC as a potential drug target in Chagas disease.Chagas disease, caused by the parasite  Trypanosoma cruzi is the etiological agent of Chagas disease, which is associated with myocarditis and vasculitis, accompanied by an increase in inflammatory mediators, such as cytokines, chemokines, phospholipids, and glycolipids T. cruzi-infected individuals from Latin American countries to other regions of the world. Several thousands of people in the United States, Canada, various European countries, Australia, and Japan are chronically infected with T. cruziT. cruzi has a complex life cycle, with two morphophysiological stages within a triatomine bug and two in a mammalian host. Infectious metacyclic trypomastigotes can be expelled with the insect's excreta during a bloodmeal, reaching the host bloodstream through the bite wound or exposed ocular or oral mucosa. In addition, the insect can acquire the parasite during blood feeding from an infected individual and continue the cycle Rhodnius prolixus, where it acts as an anti-hemostatic molecule and immunomodulator of T. cruzi infection in a mammalian model sn-glycero-3-phosphorylcholine) is a major plasma phospholipid of oxidized low-density lipoproteins (Ox-LDL), albumin and other carrier proteins, being a critical factor in the inflammatory processes and the atherogenic activity of Ox-LDL T. brucei, Leishmania spp., and T. cruzi) are known to synthesize phosphatidylcholine (PC) and LPC. Over 50% of the total lipids shed to the culture medium by T. cruzi were identified as PC and LPC LeishmaniaPlasmodium falciparumT. cruzi have not been defined to date.In general, lipid mediators O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is structurally very similar to LPC T. cruzi synthesizes a lipid with platelet-aggregating properties similar to PAF T. cruzi PAF-like lipid, metabolically labeled with 14C-acetate, was labile to mild-alkaline or hydrofluoric acid hydrolysis, suggesting a molecule containing a glycerolipid moiety with at least one acyl chain and a phosphate group T. cruzi PAF-like lipid remains elusive.Platelet-activating factor  to provide ample structural information. Moreover, we constructed a 3-D molecular model of PAFR and used molecular docking to predict the interactions of the T. cruzi PAF-like molecule and other lysophospholipids with the receptor.Here, we describe the identification and bioactivity of the as-yet elusive Rabbit platelets used in this study were obtained following the guidelines of the Committee for Evaluation of Animal Use for Research of the Federal University of Rio de Janeiro (CAUAP-UFRJ) and the NIH Guide for the Care and Use of Laboratory Animals. The vertebrate animal protocol was approved by CAUAP-UFRJ under registry number IBQM011.9)-, and C22:6-LPC, and C16:0-PAF were purchased from Avanti Polar Lipids . The competitive PAF antagonist WEB 2086 (4-Synthetic C16:0-, C18:0-, C18:1, maintained in RPMI-1640 medium supplemented with 2% FCS at 37°C in a 5% humidified CO2 atmosphere g for 5 min at 4°C). The supernatant was then harvested and passed through a DE-52 column and parasites were incubated for 2 h at 37°C in a humidified 5% CO2 atmosphere, after which the parasites were again passed through a DE-52 column, from which they were harvested and stored. For the viability testing of all parasite forms, cells were resuspended in a Trypan Blue solution and counted in a Neubauer chamber All 9 cells each), were suspended in 1.6-ml ice-cold HPLC-grade water and transferred to 13×100-mm Pyrex culture tubes with polytetrafluoroethylene (PTFE)-lined screw caps. HPLC-grade chloroform and methanol were added to each vial, giving a final ratio of chloroform/methanol/water (C/M/W) of 1∶2∶0.8 (v/v/v). The samples were mixed vigorously using a vortex for 2 min and then centrifuged for 15 min at 1,800× g at room temperature. After centrifugation, the supernatants were transferred to PTFE-lined Pyrex glass test tubes and the pellets were dried under a constant flow of N2 stream. The dry pellets were then extracted three times with C/M  and twice with C/M/W . After extraction, the supernatants were pooled together and dried before being subjected to Folch's partition g at room temperature. After centrifugation, the lower (organic) and upper (aqueous) phases were separated in PTFE-lined Pyrex glass test tubes. The Folch lower phase was then washed two times with a freshly prepared upper phase, dried under N2 steam, and stored at −70°C until use.Frozen pellets derived from Epis, Metas, ICAs, and TCTs , acetone (glycolipids), and methanol (phospholipids and free fatty acids). Each fraction was collected into a 7-ml amber glass vial with PTFE-lined screw top . All samples were immediately dried under a constant flow of N2 stream and stored at −70°C until use.Phospholipids derived from the lower phase of the Folch's partition, as described above, were purified from other classes of lipids using a three-step SPE protocol T. cruzi phospholipid mixture, a novel method was developed using perfusion chromatography lyso-PC (C16:0-LPC), C16:0-lyso-PAF (C16:0-LPAF), C16:0-PAF, and C18:0/C18:1-diacyl-PC) or T. cruzi phospholipids derived from the SPE procedure were suspended in HPLC-grade water, sonicated for 10 min in a bath sonicator, and added to the column. Both the standards and the phospholipid extracts were eluted using a 0%–50% n-propanol gradient in 5% n-propanol increments. Each fraction was stored in Axygen 2-ml microcentrifuge tube at −70°C until further use.With the purpose of enriching the putative PAF-like molecule from a complex n) by collision-induced dissociation (CID). Full-scan (MS) spectra were collected at the 400–1000 m/z range. Tandem mass fragmentation was carried out using normalized collision energies of 35, 40, and 45 for MS2, MS3, and MS4, respectively. The resulting spectra were compared to the aforementioned phospholipid standards, as well as to previously described results.All fractions derived from the POROS R1 mini-column purification, as well as the fractions obtained prior to this last procedure , were dissolved in MS-grade methanol containing 5 mM LiOH, as indicated. Samples were directly injected (at 300 nl/min) by chip-based infusion using a TriVersa NanoMate nanoelectrospray source , into an LTQXL ESI-linear ion trap-MS (ESI-LIT-MS) (Thermo Fisher Scientific), in positive-ion mode. The source voltage was set at 0.01 kV and current at 0.03 µA; capillary voltage and temperature were 36 V and 150°C, respectively; and tube lens voltage was set at 145 V. Select ions were subjected to sequential tandem fragmentation -PC  with either PLA2  or PLA1 . For PLA1 treatment, one mg of diacyl-PC standards were dried under N2 stream, redisolved in 200 µL of reaction buffer  and sonicated for 30 min. Afterwards, the samples were incubated at 37°C for 2 h in the presence of 12 units PLA1. The reaction was interrupted by the addition of 1.5 mL chloroform, followed by vortexing for 1 min. The resulting LPC species present in the organic phase of the mixtures were then purified by SPE, following the protocol described above. Finally, LPCs were recovered in the methanolic phase of the SPE column. PLA2 treatment was performed following the same procedure steps used for PLA1 treatment but using a different reaction buffer  and 5 units PLA2 sn-1 or sn-2) was performed by comparing the fragmentation pattern of standard sn-1 and sn-2 C18:1-LPCs with T. cruzi-derived 18:1-LPC samples.The location of the acyl chain on sn-1 C18:1(Δ6-cis)-LPC and sn-1 C18:1(Δ9-cis)-LPC standards were generated by treatment of both 18:1(Δ6-cis)-PC  and 18:1(Δ9-cis)-PC  with PLA2 , as described above. LPC species were recovered from incubation mixtures and analyzed by ESI-LIT-MS in 100% methanol containing 5 mM LiOH, as described above.The position of the double bond on fatty acid substituents was determined by MS4, essentially as described by Hsu et al. sn-glycero-3-phosphocholine, catalog # 855375, Avanti Polar Lipids) was used as an internal standard to quantify the most abundant LPC species found in T. cruzi samples. Briefly, 18 nmoles of standard C10:0-LPC were added to parasite pellets (7×108 cells) shortly before lipid extraction. Lipid extraction was conducted on freshly prepared parasite pellets following the protocol described above. Then, the Folch lower-phase fractions were analyzed by ESI-MS (at the 400–1000 m/z range) under the identical MS conditions used for the characterization of T. cruzi LPCs. The amount of each LPC species was calculated using the formula: [T. cruzi LPC peak intensity/C10:0-LPC peak intensity]×[concentration of C10:0-LPC (18 pmol/µl)/MRRF], where, MRRF stands for the molar relative response factor of each LPC species to the C10:0-LPC standard. The MRRF was calculated by dividing the intensity of the peak corresponding to a standard LPC  by the intensity of the peak corresponding to C10:0-LPC (m/z 418.5), when all molecules were in equimolar concentrations.Commercial C10:0-LPC  and EV-free supernatant or conditioned medium (VF) of Epis  and Metas , obtained from Epi and Meta pellets (9×109 parasites each), as previously described m/z 418) (18 nmoles per sample) was used as an internal standard. LPC species were extracted from Epi- and Meta-derived EVs, and respective total parasite pellets (ePellet and mPellet) as described above. The Folch lower phase fractions were analyzed by ESI-LIT-MS as above.2, at a final concentration of 3–4×105 cells/µl in Tyrode's buffer. Platelet aggregation experiments were performed with a Chronolog Aggregometer , with monitoring time of 5 min. Rabbit platelets used in this investigation were obtained following the guidelines for animal experimentation of the USA National Institutes of Health and the experimental protocol received official approval of the Institutional Animal Care and Use Committee, Universidade Federal do Rio de Janeiro.C16:0-PAF and various synthetic  or purified (C18:2-LPC) LPC species were tested in a platelet aggregation assay ab initio algorithms http://salilab.org/modeller/) was used to add a disulfide bridge between Cys90-Cys173 and, subsequently, to refine the best I-TASSER model. Thus, the final model was validated using three programs: PROCHECK http://nihserver.mbi.ucla.edu/SAVES_3/) and PROQM http://www.bioinfo.ifm.liu.se/services/ProQM/index.php?about=proqm). PROCHECK analyzes the stereochemical quality and ERRAT evaluates the non-bonded atomic interactions in the model structure, while PROQM uses a specific-scoring function for membrane protein, including GPCR, to assess local and global structural quality of the model.The amino acid sequence of the human PAF receptor  was obtained from ExPASy server http://www.ks.uiuc.edu/Research/vmd/vmd-1.9.1/).The ligand structures  were built in the Spartan'10 software . The docking of the ligands to the PAFR model binding site was performed using Molegro Virtual Docker (MVD) program , which uses a heuristic search algorithm that combines differential evolution with a cavity prediction algorithm. The MolDock scoring function used is based on a modified piecewise linear potential (PLP) with new hydrogen bonding and electrostatic terms included. Full description of the algorithm and its reliability compared to other common docking algorithm have been described T. cruzi synthesizes a phospholipid with platelet-aggregating activity similar to PAF T. cruzi PAF-like phospholipid from a complex phospholipid mixture, we developed a fractionation protocol, which included solvent extraction and Folch's partition, followed by SPE and perfusion chromatography (T. cruzi (data not shown). ESI-LIT-MS analysis of the Folch lower phase of the four parasite forms  showed major phospholipid species at the 700–900 m/z range, except for the ICA form (n) analysis of these lipid species revealed that, as expected, they were mostly diacyl-PC and sphingomyelin (SM) species . In contrast to other parasite forms, ICA is much richer in lipid species at the 500–600 m/z range, particularly m/z 526, 528, 530, and 574 (+) of synthetic LPAF, LPC, and PAF standards were found at this range of the spectrum , which would indicate an enrichment of potential LPC, LPAF, or PAF molecules. Nevertheless, most samples still contained high amounts of diacyl-PCs and, possibly, SMs (data not shown). Therefore, a novel protocol using POROS R1 beads was designed to enrich the putative PAF-like molecule, which as we predicted could have a structure similar to PAF, LPAF, or LPC. First, we tested the POROS R1 mini-column with a complex mixture of phospholipid standards containing LPAF, LPC, PAF, and diacyl-PCs. We were able to obtain highly enriched LPAF, LPC, and PAF species in the 20% and 25% n-propanol fractions  and flow rate (300 nl/min)  . The ionfraction  than in fraction . In contm/z 530) was compared to those of synthetic C18:0- and C18:1-LPC standards , because certain PAF and LPC species may be isobaric. The assignment of the lysophospholipid species found in the 25%-n-propanol POROS R1 fraction of all T. cruzi forms was based on the fragmentation pattern of these standards, as well as on previously reported results 2) analysis of singly-charged, lithiated C16:0-PAF, C18:0-LPC, and C18:1-LPC ion species gave rise to fragment ions at m/z 471, 471, and 469, respectively, corresponding to the neutral loss of 59 a.m.u. ( = trimethylamine group). The fragmentation of 16:0-PAF standard, however, also gave rise to a fragment ion at m/z 341, consistent with the neutral loss of the whole phosphocholine headgroup along with the lithium adduct (−189 a.m.u.). This fragment could not be detected on either LPC standards , gave rise to the major non-lithiated ion fragments at m/z 341, 341, and 339, respectively, corresponding to the loss of 130 a.m.u. ( = ethyl phosphate group+Li) , for C16:0-PAF, C18:0-LPC, and C18:1-LPC, respectively. Interestingly, C18:0-LPC and C18:1-LPC also gave rise to two fragment ions  that could not be detected in the C16:0-PAF standard. These fragment ions corresponded to the lithiated ([R1CO2H+Li]+) stearoyl (m/z 291) and oleyl chains (m/z 289), after the loss of choline (N+(CH3)3(CH2)2OH) from the precursor ions m/z 427 and 425, respectively  (sn-2 position of C16:0-PAF (m/z 281 and 263), and a fragment ion corresponding to the protonated C16:0-alkyl chain (R1+) (m/z 225). Moreover, a series of fragments resulting from the loss of methylene groups ( = 14 a.m.u.) from the PAF C16:0-alkyl chain could also be seen below m/z 225. Similar information could be obtained from the C18:0-LPC and C18:1-LPC, where fragment ions derived from the protonated stearoyl  and oleyl (m/z 265 and 247) acyl chains at the sn-1 position could be identified m/z 240 , non-lithiated  fragment ions of linoleyl , oleyl , and stearoyl  chains, respectively, corroborated our assignments of T. cruzi (Tc) m/z 526, 528, and 530 as C18:2-, C18:1, and C18:0-LPC (T. cruzi LPC species are represented in The fragmentation pattern of the synthetic C16:0-PAF standard (tandards . MS3 Fraroup+Li) . In addiectively  [54]. Fictively) . A compl m/z 240 . Based o18:0-LPC ,B. No den experiments carried out above, however, could not provide sufficient structural information to determine the position of the fatty acid (sn-1 or sn-2) and the location of the double bonds on three major LPCs of T. cruzi. To address this point, we first generated LPC standards with fatty acids on either the sn-1 or sn-2 position by treating diacyl-PCs with commercial PLA2 and PLA1, respectively. Following protocols by Hsu et al. T. cruzi LPCs was localized at the sn-1 position. In 2 and MS3) of sodiated and lithiated sn-1 C18:1-LPC, sn-2 C18:1-LPC, and T. cruzi C18:1-LPC (from ICA form) are shown. In agreement with Hsu et al. m/z 544) to the fragment ion (m/z 485), corresponding to the loss of trimethylamine (−59 a.m.u), could be used to differentiate between the two possible regioisomers (T. cruzi C18:1-LPC showed a fragmentation pattern consistent with an acyl chain located at sn-1. This result was corroborated by the fragmentation spectrum of the lithiated T. cruzi C18:1-LPC (m/z 425 [M – N+(CH3)3(CH2)2OH+Li+]+ to the ions at m/z 339 (M - 189) and m/z 345 (M - 183) was used to corroborate the sn-1 position of the acyl chain on T. cruzi C18:1-LPC. We carried out identical experiments with T. cruzi C18:0- and C18:2-LPC and found that both contained the acyl chain at the sn-1 position (data not shown).The MSoisomers . Clearly18:1-LPC . In thisT. cruzi C18:1- and C18:2-LPC, we followed the protocols described by Hsu and Turk 4 spectra of Δ9- and Δ6-C18:1-LPC standards, we observed noticeably different fragmentation patterns of the acyl chain, especially in the relative abundance of fragment ions C16H31 (m/z 223), C14H29 (m/z 197), C14H27 (m/z 195), C13H27 (m/z 183), C13H25 (m/z 181), C8H15 (m/z 111), and C8H13 (m/z 109) (T. cruzi C18:1-LPC (from ICA forms) was analyzed under the same MS conditions, the fragmentation pattern observed was consistent with a Δ9 double bond (T. cruzi C18:2-LPC (from ICA forms) and the resulting fragmentation was consistent with Δ9,12 double bonds (data not shown).To address the location of the double bonds in m/z 109) . When T.m/z 574), C22:4-LPC (m/z 578), C16:0-LPC (m/z 502), and C16:1-LPC (m/z 500) that were also characterized by MSn , we could not detect any trace amounts of PAF species in T. cruzi. This was true not only for the POROS R1 25% n-propanol fractions, but for all fractions described in this study. Therefore, we surmise that if there were any PAF species in this parasite, the concentration levels would likely be below the detection limit of the MS approaches used in the present study.The POROS R1 protocol we have described here also enriched other LPC species, which included C22:6-LPC  were much higher than in the insect-dwelling  forms (T. cruzi infection After characterizing the different a) forms . For insa) forms . InteresT. cruzi is also able to secrete LPC species to the extracellular milieu, we analyzed extracellular vesicles (EVs) and EV-free supernatant or conditioned medium (VF) of Epi and Meta forms, obtained as described 6 cells in mVF and an additional 0.5 pmol associated with mV2 fraction. Similar values were found for C18:2-LPC in mVF and mV2  was not feasible. Therefore, we carried out the platelet aggregation assays using synthetic  or purified (C18:2-LPC) LPC species, and synthetic C16:0-PAF . In a set of experiments platelets were pre-incubated for 30 min in the presence of 10 µM WEB 2086. As seen in Since we were unable to detect any PAF species in any of the four parasite stages analyzed, we hypothesized that the PAF-like molecule could be one or more of the lysophospholipids structurally characterized here. This assumption is also based on previous reports that certain LPC species seem to activate the PAFR To gain insights into the mechanism by which C18:1-LPC, but not C16:0, C18:0, C18:2, could induce platelet aggregation mediated by PAFR, we decided to build a 3D-structural model of the PAFR, since a high-quality model was not available in the literature. The structural features of our PAFR model are shown in The I-TASSER methodology is very accurate for the construction of protein models when the sequence identity between the target sequence and the template protein drops below 30%, where lack of a high-quality structure match may provide substantial alignment errors and, consequently, poor quality models. In addition, the I-TASSER methodology has already been used for modeling other GPCRs sn-1) to the carbonyl group of the acyl chain were chosen and all distances between the ligands are presented in To compare the binding mode of all ligands  to the PAFR model, we carried out ten docking simulations for each ligand  and, consequently, the poses with the lowest energy for each ligand was selected for analysis. The comparison between C16:0-PAF and each LPC species is represented in T. cruzi synthesizes a PAF-like phospholipid capable of aggregating rabbit platelets T. cruzi PAF-like phospholipid is in fact an LPC, namely sn-1 C18:1(Δ9)-LPC. However, we do not discard the possibility that very low amounts of a bona fide PAF species might still be synthesized by this parasite. The MSn assignments for the C18:1-LPC we provide here are in agreement with previous reports et al.m/z 265 was characterized as being derived from the neutral loss of acetyl and methyl groups from the fragment ion at m/z 339. In addition, a fragment ion at m/z 247 was proposed to be derived from the loss of water from the ion at m/z 265. Conversely, we show here that the non-lithiated [R1CO+]+ and [R1CO+ - H2O]+ fragment ions at m/z 265 and 247, respectively, are in fact derived from a C18:1-acyl chain at the sn-1 position. This is further confirmed by the presence of lithiated ([R1CO2H+Li]+) fragment ion from oleyl chain at m/z 289. Our assignments of C18:1-LPC are in complete agreement with those reported by Hsu et al. m/z 223 as being the protonated C16:1-alkenyl chain from the sn-1 position of that putative PAF species. We also observed the same fragment ion in both synthetic and T. cruzi-derived C18:1-LPC, but we assigned it as derived from the fragmentation of the acyl chain. Taking all these facts into consideration, it is likely that Smith et al. Previous results from our group showed that T. cruzi synthesizes at least five species of LPC and that only sn-1 C18:1(Δ9)-LPC was able to promote rabbit platelet aggregation. These LPCs could be generated by the action of host- and/or parasite-derived phospholipase(s) A1 and/or A2 on diacyl-PCs. A PLA1 has already been well characterized in T. cruzi trypomastigote and amastigote forms  T. bruceisn-1 C18:0-, C18:1-, and C18:2-LPC, we believe that the parasite PLA1 is not playing a major role in the generation of these LPCs, at least under the experimental conditions used in this study. Nevertheless, we could not discard the possibility that the T. cruzi PLA1 might be important for the generation of LPC species using different experimental conditions and/or other parasite strains, as previously described T. cruzi, despite the fact that at least four putative PLA2 genes have been annotated in the parasite genome  T. cruzi epimastigote supernatants. This raises the possibility that the T. cruzi LPCs identified here could have been generated by the action of a host-derived PLA2 and/or by a parasite-derived enzyme that is expressed in much higher levels in the mammal-dwelling stages. Further studies are needed to address this important point.Here we show that T. cruzi also synthesizes TXA2, which modulates several pathophysiological aspects of Chagasic cardiomyopathy T. cruzi produces at least two platelet activators, given the importance of platelet aggregation in the progression of Chagas disease. Our bioactivity data with C18:1-LPC are in agreement with other results in the present study, namely the platelet aggregation assays and molecular docking predictions. We had also previously demonstrated that the putative PAF-like phospholipid was able to trigger the differentiation of T. cruzi epimastigotes into metacyclic trypomastigotes in vitroT. cruzi epimastigotes with polyclonal antibody raised against mouse PAFR, in immunofluorescence assays, strongly indicated that T. cruzi might have putative PAFR both at the cell surface and intracellularly A direct correlation between increased platelet reactivity and the incidence of acute coronary diseases has been described T. cruzi-C18:1-LPC may be able to trigger platelet stimulation through a ligand-receptor system. Accordingly, LPC is known to induce intracellular signals R. prolixus-derived C16:0-LPC is able to prevent platelet aggregation triggered by PAF Schistosoma mansoni activates TLR-2-dependent signaling involved in eosinophil activation and recruitment, probably through cross-talk to a GPCR E. coli O111:B4 lipopolysaccharide (LPS), suggesting therefore a dual role of this lysophospholipid in immunoregulation.LPC has messenger functions, binding to a specific receptor and not acting through physicochemical effects on the plasma membrane of the target cell T. cruzi is able to generate different LPC species and suggest that the levels of individual LPC species are tightly regulated in the course of this parasite cell cycle. In fact, the LPC levels are notably high in the mammal-dwelling infectious trypomastigote and intracellular amastigote stages. Taken together with the fact that there was no exogenous source  of lipids in the TAU culture medium used for the differentiation of epimastigotes into metacyclic trypomastigotes, our data indicate that LPC species might be generated endogenously by this organism.Over time, LPC was found not to be exclusively of mammalian origin and was described in several other organisms R. prolixus is a source of LPC, which may act as an enhancing factor for Chagas disease T. cruzi is far more infective towards both mouse macrophages R. prolixusT. cruzi controls the proliferation of the parasite and resulting inflammatory response to infection in the mouse T. cruzi infection.The saliva of T. cruzi synthesizes at least five LPC species, C16:0-, C18:0, C18:1-, C18:2-, and C22:6-LPC. The most abundant species are C18:2- and C18:1-LPC, being the latter the only one able to aggregate rabbit platelets, probably through a PAFR. This result was supported by molecular docking study of the interactions between LPC species and a PAFR model. These analyses showed that C18:1-LPC was able to interact with the PAFR model in a fashion similar to PAF. More studies on LPC metabolism in T. cruzi and relationship of the parasite with the mammalian and invertebrate hosts could, therefore, lead to the discovery of putative targets for novel therapies and control for Chagas disease.In conclusion, here we demonstrate that Figure S1lyso-PAF (LPAF) (m/z 488.4), C16:0-lyso-LPC (LPC) (m/z 502.4), C16:0-PAF (PAF) (m/z 530.5), and purified diacyl-PCs (700–900 m/z range), was suspended in HPLC-grade water and applied onto the POROS R1 mini-column, which was eluted with a 0%–50% n-propanol gradient. All fractions were diluted in methanol containing 5 mM LiOH and analyzed by direct infusion in an LTQXL ESI-LIT-MS, in positive-ion mode.Fractionation of phospholipid standards using POROS R1 perfusion chromatography. A mixture of phospholipid standards, containing synthetic C16:0-(TIF)Click here for additional data file.Figure S2n analysis of major T. cruzi ion species enriched by POROS R1 fractionation. (A) ESI-LIT-MS2 spectra. Phospholipid standards  or purified T. cruzi phospholipids from the POROS R1 25% n-propanol fractions were diluted in methanol containing 5 mM LiOH and then infused directly into the LTQXL MS using an Advion Triversa NanoMate nanoelectrospray system. Major parent-ion species observed in the MS spectra (2 fragmentation. (B) ESI-LIT-MS3 spectra of selected ion species found in (A). Ion species corresponding to a neutral loss of 59 m/z (trimethylamine) from the parent ion were selected for MS3 fragmentation.ESI-LIT-MS spectra  were sub(TIF)Click here for additional data file.Figure S3T. cruzi C18:1-LPC. (A–B) Acyl chain position analysis. The sn-1 C18:1-LPC and sn-2 C18:1-LPC regioisomer standards were generated by treatment of 18:1(Δ9-cis)-diacyl-PC with PLA2 and PLA1, respectively, as described in A) MS2 analysis. All LPCs were diluted in methanol containing 2.5 mM NaCl and analyzed by direct infusion using an Advion Triversa NanoMate nanoelectrospray system coupled to an LTQXL MS. MS2 spectra were acquired in positive-ion mode. (B) MS3 analysis. All LPCs were diluted in methanol containing 2.5 mM LiOH and analyzed by MS3 (MS2 528.7→MS3 469.3), under the same MS experimental conditions. (C) Double-bond position analysis. The sn-1 C18:1(Δ6-cis)-LPC and sn-1 C18:1(Δ9-cis)-LPC standards were generated by treatment of 18:1(Δ6-cis)-diacyl-PC and 18:1(Δ9-Cis)-diacyl-PC with PLA2, respectively, as described in T. cruzi C18:1-LPC was determined by comparing the MS4 fragmentation pattern of this ion (MS2 528.7→MS3 469.3→MS4 339.3) to the fragmentation pattern of sn-1 C18:1(Δ6-cis)-LPC and sn-1 C18:1(Δ9-cis)-LPC under identical conditions to (B). Diagnostic ions (bold) are indicated.Determination of the acyl chain and double bond positions on (TIF)Click here for additional data file.Figure S4T. cruzi LPC-species enriched by POROS R1 fractionation. T. cruzi phospholipids from the 25% n-propanol fractions were diluted in methanol containing 5 mM LiOH and then infused directly into an LTQXL ESI-LIT-MS. Selected peaks were sequentially fragmented (MS2-MS4) by CID, as follows: C16:0-LPC (MS2 502→MS3 443→MS4 313); C16:1-LPC (MS2 500→MS3 441→MS4 311); C22:4-LPC (MS2 578→MS3 519→MS4 389); and C22:6-LPC (MS2 574→MS3 515→MS4 385).Tandem ESI-LIT-MS spectra of the low-abundance (TIF)Click here for additional data file.Figure S5T. cruzi. C10:0-LPC (m/z 418) was used as an internal standard for quantification of the most abundant T. cruzi LPC species. C10:0-LPC was added to fresh pellets or EV preparations from Epis and Metas, prior to lipid extraction with C∶M  and C∶M∶W , followed by Folch's partition. The Folch lower phase was analyzed by ESI-LIT-MS. C18:1- and C18:2-LPC are indicated at m/z 528 and 526, respectively. ePellet, Epi total pellet; eV2, Epi-derived ectosomes; eV16, Epi-derived exosomes; eVF, Epi-derived EV-free supernatant (or conditioned medium); mPellet, Meta total pellet; mV2, Meta-derived ectosomes; mV16, Meta-derived exosomes; mVF, Meta-derived EV-free supernatant.Quantification of LPC species in extracellular vesicles (EV) and EV-free supernatant of (TIF)Click here for additional data file.Figure S6A) Arrangement of the 7 transmembrane (TM) helices forming a cavity. (B) The extracellular loops (EL1-EL3). (C) The intracellular loops (CL1-CL3). Each TM  is indicated in a different color. TM1, blue; TM2, red; TM3, dark gray; TM4, orange; TM5, yellow; TM6, purple; TM7, green.Structural representation of the PAFR model. ((TIF)Click here for additional data file.Figure S7Main Ramachandran plot of the PAFR model. The main Ramachandran plot represents all amino acid residues by squares, whereas the glycine (Gly) residues are separately identified by black triangles, because these are not restricted to the regions in the generic Ramachandran plot. Red  and dark yellow  colors correspond to combinations of phi (Φ) and psi (Ψ) torsion angles of amino acid residues in the most favorable and additional allowed regions, respectively. In addition, pale yellow  and white colors represent less favorable and disallowed regions, respectively. The amino acid residues lying in those regions are highlighted in red.(TIF)Click here for additional data file.Figure S8Ramachandran plot for Gly residues of the PAFR model. The second Ramachandran plot considers only Φ and ψ angles for Gly residues, where the number in parenthesis indicates the total number of these residues in the primary sequence of PAFR. The favorable combinations of these angles are represented by green areas and the values of standard deviations greater than 2.5 Å are labeled in red in the graph, describing combinations of disallowed angles. All Gly residues are in allowed regions, with favorable combinations of angles Φ and Ψ.(TIF)Click here for additional data file.Figure S91-X2; side chain torsion angles) combination, X1 torsion for those residues that do not have a X2, combined X3 and X4 torsion angles, omega torsion angles, main chain bond lengths, and main chain bond angles.Analysis of the main-chain parameters of the PAFR model. The six graphs on the main chain parameters plot show how the PAFR model (represented by the black square) compares with a database of well-refined protein structures. The blue band in each graph represents the results from the well-refined protein structures; the central line is the least squares fitting to the mean trend as a function of resolution, whereas the width of the band on either side of it corresponds to a variation of one standard deviation from the mean. The six properties plotted are: (a) Ramachandran plot quality assessment; (b) peptide bond planarity; (c) number of bad contacts per 100 residues; (d) α-carbon tetrahedral distortion ; (e) main chain hydrogen bond energy; and (f) overall G-factor . The G-factor provides a measure of how “normal”, or alternatively how “unusual”, a given stereochemical property is and, in PROCHECK, it is computed for the following properties: Φ-Ψ combination, chi1-chi2 (X(TIF)Click here for additional data file.Figure S10gauche minus torsion angles; (b) standard deviation values of the Chi-1 trans; (c) standard deviation values of the Chi-1 gauche plus; (d) pooled standard deviation of Chi-1 torsion angles; and (e) standard deviation values of the Chi-2 trans torsion angles.Analysis of the side-chain parameters of the PAFR model. In a similar way to the main-chain analysis, the side-chain analysis shows how the PAFR model (black square) compares with well-refined protein structures. This analysis evaluates five properties: (a) standard deviation values of the Chi-1 (TIF)Click here for additional data file.Figure S11Analysis of geometrical distortions of the PAFR model. The parameters analyzed were: lengths and bond angles, including atoms of the main and side chains. This analysis shows the amino acid residues with distorted geometry, including their ideal values (in blue), those found in the model (in red), and the difference between these values (in green).(TIF)Click here for additional data file.Figure S12ERRAT analysis of the PAFR model. Errors in non-bonded atom-atom interactions of the PAFR model were verified by this analysis. The error values were plotted as a function of the position of a sliding residue in the window. An overall quality factor value of 87% was observed, thus validating the PAFR model.(TIF)Click here for additional data file.Figure S13ProQM analysis of the PAFR model. A score was given to each residue of the protein model, which lead to a global quality factor of 0.717, which corroborates the other modelling analyses.(TIF)Click here for additional data file.Figure S14Comparison of binding modes of PAF and each LPC species to PAFR. (A) C16:0-PAF (cyan) and C16:0-LPC (yellow); (B) C16:0-PAF (cyan) and C18:0-LPC (orange); (C) C16:0-PAF (cyan) and C18:1-LPC (green); (D) C16:0-PAF (cyan), and C18:2-LPC (gray). Transmembrane (TM) regions are represented as white rods.(TIF)Click here for additional data file.Figure S15Unfavorable steric interactions of PAF and LPCs with PAFR. Heteroatoms are represented by different colors in structures: nitrogen atoms are shown in blue, oxygen in red, and carbon atoms of amino acids in gray. In ligands, carbon chains are represented by different colors: (A) C16:0-PAF, cyan; (B) C16:0-LPC, yellow; (C) C18:0-LPC, orange; (D) C18:1-LPC, green; and (E) C18:2-LPC, pink.(TIF)Click here for additional data file.Table S1Quantification of LPC species in extracellular vesicles (EVs) and EV-free supernatant of epimastigotes and metacyclic trypomastigote forms.(DOCX)Click here for additional data file.


Sulfolobus tokodaii. GAOR1–3 belong to the xanthine oxidoreductase superfamily, and are composed of a molybdo-pyranopterin subunit (L), a flavin subunit (M), and an iron-sulfur subunit (S), forming an LMS hetero-trimer unit. We found that GAOR1 is a tetramer of the STK17810/STK17830/STK17820 hetero-trimer, GAOR2 is a dimer of the STK23390/STK05620/STK05610 hetero-trimer, and GAOR3 is the STK24840/STK05620/STK05610 hetero-trimer. GAOR1–3 exhibited diverse substrate specificities for their electron donors and acceptors, due to their different L-subunits, and probably participate in the non-phosphorylative Entner-Doudoroff glycolytic pathway. We determined the crystal structure of GAOR2, as the first three-dimensional structure of an archaeal molybdenum-containing hydroxylase, to obtain structural insights into their substrate specificities and subunit assemblies. The gene arrangement and the crystal structure suggested that the M/S-complex serves as a structural scaffold for the binding of the L-subunit, to construct the three enzymes with different specificities. Collectively, our findings illustrate a novel principle of a prokaryotic multicomponent isozyme system.Archaea use glycolytic pathways distinct from those found in bacteria and eukaryotes, where unique enzymes catalyze each reaction step. In this study, we isolated three isozymes of glyceraldehyde oxidoreductase  from the thermoacidophilic archaeon  The XDesulfovibrio gigas (DgAOR) and AOR from Desulfovibrio desulfuricans are homo-dimers composed of a 98-kDa polypeptide, comprising an N-terminal iron-sulfur domain and a C-terminal Mo-PCD domain.The XOR family enzymes are found throughout all three domains of life ,7,8 Fig. Most ofPicrophilus torridus and Thermoplasma acidophilum  I, [2Fe-2S] II and FAD are located almost linearly, with distances of 13.4, 11.5 and 7.0 Å, respectively . The Mo-. The two. The lig 33]. In. In(cis dithGly483L) . In the (Glu869)  (Glu869) . The ele2, 13,200 Å2 and 9,060 Å2 for L-, M- and S-subunits, respectively), the interface areas of L–S and L–L′ (dimer-related L-subunit) are relatively wide , whereas those of M–S and L–M are relatively narrow . There are no interactions between the other combinations of subunits. In GAOR2, the M–S and L–M interactions appear relatively weak, as in the OcCODH (LMS)2 dimer, in which its M-subunit can be removed with SDS and then reconstituted [The crystal structure and the sequence comparison provided mechanistic insights into the subunit assembly of the GAORs . The intstituted . There aL–Arg571L) in the region equivalent to the L–L′ interface (Phe546L–Asp553L) of GAOR2 (ST23390). In contrast to GAOR2 and GAOR3, GAOR1 forms a tetramer of hetero-trimers. GAOR1 (ST17810) has a unique 17-residue insertion (Ala70L–Arg96L) in the region equivalent to the solvent-exposed surface of GAOR2 (STK23390)  forms a homo-dimer. In contrast, STK24840 (L of GAOR3) is likely to be a monomer. Consistent with the differences in their subunit assemblies, the residues at the L–L′ interface of GAOR2 (STK23390)  are not TK23390) . These sS. tokodaii. The GAOR1 enzymes from S. acidocaldarius (SaGAOR) [S. tokodaii (previously referred to as indolepyruvate:MV oxidoreductase) [S. tokodaii, GAOR2 is more specific to GA, as compared with GAOR1, and indicated that GAOR2 is responsible for the GA oxidation in the npED glycolytic pathway in Sulfolobales.In this study, we purified three GAOR enzymes belonging to the XOR family, from the thermoacidophilic archaeon (SaGAOR)  and S. tductase)  were preductase) . HoweverPyrococcus furiosus (PfAFOR) has long been the sole three-dimensional structure of a metal-pyranopterin-containing enzyme from Archaea [In contrast to the mED pathway, a tungsto-bispyranopterin (W-bisPPT)-containing glyceraldehyde-3-phosphate:ferredoxin oxidoreductase is responsible for the oxidation of glyceraldehyde-3-phosphate into 3-phosphoglycerate, in the modified Embden-Meyerhof-Parnas glycolytic pathway of the anaerobic hyperthermophilic archaea . The cry Archaea . PfAFOR  Archaea . The preL with STK24840 Gln338L  with the homology models of GAOR1 (STK17810) and GAOR3 (STK24840) can partly explain their distinct substrate specificities (electron donor). In the homology model of STK24840, the substrate-binding pocket is narrower than that of STK23390, due to the replacement of STK23390 Ser330 Gln338L . This is Gln338L . In cont Val250L . This isstk23390 for GAOR2 and stk24840 for GAOR3) are located at distant loci from stk05610/stk05620, the two genes stk05610 (M subunit) and stk05620 (S subunit) are adjacent to each other. The stk05610 and stk05620 genes are preceded by a promotor-like sequence, suggesting that they constitute an operon. These observations imply that the M/S-subunits (STK05610/STK05620) may assemble into the M/S hetero-dimer, which then interacts with a different L-subunit, either STK23390 (GAOR2) or STK24840 (GAOR3). In contrast to GAOR2 and GAOR3, GAOR1 is encoded by three successive genes, stk17830 (M-subunit), stk17820 (S-subunit) and stk17810 (L-subunit). In GAOR1, the genes encoding the M/S-subunits also precede the genes encoding the L-subunit, thereby suggesting that in GAOR1, the M/S complex formation also occurs prior to the L-subunit association. These notions are supported by a recent study showing that bacterial gene organization into operons contributes to the effective assembly of protein complexes [The genes encoding the subunits of GAOR2 and GAOR3 are not clustered , suggestomplexes .2γ2 to α2β2 hemoglobin after the birth of the fetus is quite reasonable, given their different oxygen saturation modes [P. furiosus, form αβγδ hetero-tetramers, which share the same γ-subunit but show different specificities for 2-oxoacid substrates, pyruvate (POR) and 2-ketoisovalerate (VOR) [The exchange of subunit counterparts is observed in some eukaryotic hetero-oligomeric proteins, such as human hemoglobin and the G-protein heterotrimer complex. In the case of human hemoglobin, the α-subunit binds to the β- and γ-subunits in the periods before and after birth, respectively. The transition from αon modes . The humon modes . The α-son modes . In addite (VOR) . The thrte (VOR) . The subS. tokodaii, and found that GAOR2 and GAOR3 share the same M/S-subunits but contain different L-subunits, and exhibit distinct substrate specificities. We determined the crystal structure of GAOR2, as the first structure of an archaeal Mo-containing hydroxylase, thereby providing insights into the subunit assembly and the substrate specificity of the three GAORs. The structure of GAOR2 also enabled a comparative analysis of the Mo-hydroxylase enzymes from the three domains of life. Furthermore, our findings suggest that even primitive microbes are more complex than expected from their genomic information.In summary, we isolated and characterized the three GAORs from S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S1 File(PDB)Click here for additional data file.S2 File(PDB)Click here for additional data file.S1 References(DOCX)Click here for additional data file.S1 Supporting Information(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


One day prior to mass emergence of haematopoietic stem cells (HSCs) in the foetal liver at E12.5, the embryo contains only a few definitive HSCs. It is thought that the burst of HSC activity in the foetal liver is underpinned by rapid maturation of immature embryonic precursors of definitive HSCs, termed pre-HSCs. However, because pre-HSCs are not detectable by direct transplantations into adult irradiated recipients, the size and growth of this population, which represents the embryonic rudiment of the adult haematopoietic system, remains uncertain. Using a novel quantitative assay, we demonstrate that from E9.5 the pre-HSC pool undergoes dramatic growth in the aorta-gonad-mesonephros region and by E11.5 reaches the size that matches the number of definitive HSCs in the E12.5 foetal liver. Thus, this study provides for the first time a quantitative basis for our understanding of how the large population of definitive HSCs emerges in the foetal liver. Summary: A quantitative approach reveals how the pre-HSC pool undergoes dramatic growth in the AGM region and, by E11.5, reaches the size that matches the number of definitive HSCs in the E12.5 foetal liver. Although the first signs of haematopoietic differentiation in mouse development are observed in the embryonic day (E) 7.5 yolk sac of the mouse embryo, definitive HSCs (dHSCs), which generate the adult haematopoietic system and are detectable by direct transplantation into adult irradiated recipients, emerge only by E10.5-E11.5 in very small numbers . HoweverGiven that dHSCs that repopulate adult irradiated recipients appear only by the end of E10, it is broadly understood that dHSCs develop from immature HSC precursors (pre-HSCs), which are not detectable by direct transplantation . HoweverIn the E11.5 mouse embryo, the aorta-gonad-mesonephros (AGM) region, placenta, extra-embryonic arteries, yolk sac and head contain no more than one dHSC per tissue . Althougex vivo  cells, which sequentially upregulate CD41 (Itga2b), Runx1 (AML1), CD43 (Spn) and CD45 (Ptprc) haematopoietic markers, as in vivo embryonic precursors of dHSCs , but can be assayed by direct transplantation into adult irradiated recipients. All four types of pre-HSCs have been identified morphologically in the area of the dorsal aorta  population using extreme limiting dilution analysis  into calculations of pre-HSC numbers. Namely, cultured co-aggregates were transplanted individually into adult irradiated recipients (one co-aggregate per mouse). In these circumstances, any derivatives of a maturing pre-HSC or dHSCs remained contained within their original co-aggregates and injected into an individual recipient. Thus, if pre-HSC or dHSC expansion occurs in culture, it is constrained by each co-aggregate, and does not influence the number of positive co-aggregates. This allowed us to determine retrospectively which co-aggregates contained no pre-HSCs (i.e. gave no repopulation) and those which contained at least one pre-HSC (i.e. gave repopulation) prior to culture B. The pris ELDA; . For conFig. S2B,D). Notably, we observed a tendency of increasing pre-HSC numbers by the end of E11 in the placenta, in line with previous observations of placental dHSC dynamics . Practically no pre-HSCs were detected in somites and tail (S+T), body walls (BW), foetal liver (FL) or yolk sac (YS) carefully separated from EEVs . Saturated repopulation (lack of non-repopulated mice) with the AGM region, BA and EEVs in these experiments does not allow preHSC_LDA to be used for precise assessment of pre-HSC numbers in these tissues  and extra-embryonic vessels (EEVs), are enriched for pre-HSCs C,D. Indedynamics . In all rom EEVs D. Theref tissues D. To thi tissues C,D, furtin vivo, we assessed pre-HSC numbers at several developmental time points (stages) from E9 to E12.5 using preHSC_LDA (Table S2). Embryos were staged according to the number of somite pairs (sp). We found that the E9.5 caudal part contains no more than one or two pro-HSCs .To investigate developmental dynamics of pre-HSC expansion pro-HSCs A. By E10pro-HSCs . Pre-HSCpro-HSCs B. TherefRag2γc−/− mice from an earlier foetal liver that was cultured in the presence of thrombopoetin on OP9 cells  and those obtained by preHSC_LDA  coincide and therefore, by deduction, this strongly suggests that the E12.5 liver contains only dHSCs and no pre-HSCs.Because dHSCs are supported in these culture conditions  their numbers also contributed to preHSC_LDA estimates. Therefore, to obtain actual numbers of pre-HSCs, dHSC numbers detected + cells in the foetal liver, they are not related to dHSCs, in line with the view that the liver is colonised from outside.Remarkably, the number of pre-HSCs in E11.5 AGM region assessed by preHSC_LDA coincides with dHSC numbers in the E12.5 foetal liver (identified by direct transplantations). Thus, despite the considerable presence of VE-cadex vivo HSC maturation system does not artificially stimulate dHSC generation from cells outside the pre-HSC lineage. However, this quantitative matching also suggests that practically all immature pre-HSCs are captured, strongly indicating that the ex vivo assay employed here adequately identifies pre-HSC populations in vivo.The quantitative matching of pre-HSC numbers in the AGM region and dHSC numbers in the foetal liver allows us to conclude with high confidence that the − (pro-HSC and Type I pre-HSC) and more advanced CD45+ (Type II pre-HSC) embryonic precursors was assessed. To this end, we sorted E10.5 VE-cad+CD45− and VE-cad+CD45+ populations and co-aggregated them with OP9 stromal cells . After culture, co-aggregates were pooled and transplanted into irradiated recipients, confirming that 30-38 sp (E10) AGM regions contain only CD45− HSC precursors. We found that Type II pre-HSCs appear only after the end of E10  (n=6 experiments) (Table S1). As the average number of pre-HSCs in the AGM region at this stage is 54±5.8 (Table S2), the quantitative composition of the HSC hierarchy (pre-HSC Type I: pre-HSC Type II: dHSC) is 11:42:1, given the presence of approximately one dHSC as detected by direct transplantation .We next investigated the composition dynamics of the developing HSC hierarchy throughout E9.5-E12.5 stages. ig. S1B) . At E11.ig. S1B) . To evalig. S1B) B, we estantation A  robust homing of mature dHSCs into adult recipients, compared with engraftment of immature pre-HSCs in newborn animals. As single adult bone marrow HSCs can engraft at a very high frequency of 70-96% upon intravenous injections  although the first dHSCs appear in the AGM region, sequential maturation of the following ones occur followed by immediate relocation into the foetal liver ; (2) a short synchronous pulse of dHSC maturation inside the AGM region occurs sometime between E11 and E12, e.g. during night time, but this would be practically impossible to capture experimentally; (3) pre-HSCs can gradually migrate through EEVs to the placenta where they mature and subsequently relocate to the foetal liver. As an indication for the third possibility, we see a tendency for pre-HSC numbers in EEVs to increase towards the end of E11 C. This aIn summary, our data show that between E9.5 and E11.5 the pool of HSC precursors gradually matures and expands dramatically predominantly in the AGM region and at lower levels in extra-embryonic arteries. By E11.5, the size of the pre-HSC pool remarkably matches the number of mature HSCs appearing within less than 24 h in the foetal liver. Although some other embryonic tissues also contain pre-HSCs, their numbers are negligible compared with the AGM region. This finding offers a simple explanation for the abrupt appearance of the large pool of definitive HSCs in the foetal liver from pre-HSCs through a short maturation step, which in principle does not require any significant proliferation. The transition from the AGM region to the liver stage is reflected by a rapid decrease of pre-HSCs in the AGM region from 40-65 to ten, concurrent with a dramatic rise in the mature dHSC pool in the foetal liver between E11.5 and E12.5 A,C. ThusStaged embryos were obtained by crossing C57BL/6 (CD45.2/2). The morning of discovery of the vaginal plug was designated as E0.5. Accurate embryo staging was performed by counting somite pairs (sp) and grouping as follows: E9.0 (15-23 sp), E9.5 (24-30 sp), E10.0 (31-34 sp), E10.5 (35-39 sp), E11.0 (40-45 sp) E11.5 (46-49 sp), E12.0 (51-55 sp), E12.5 (<56 sp). All experiments using animals were performed under a Project Licence granted by the Home Office (UK), approved by the University of Edinburgh Ethical Review Committee, and conducted in accordance with local guidelines.Wild-type C57BL/6 CD45.2/2 cells were injected intravenously into adult γ-irradiated CD45.1/2 heterozygous recipients [split dose (471+471 Rad) with 3 h interval] along with 100,000 CD45.1/1 nucleated bone marrow carrier cells per recipient.Number of cells injected was expressed in embryo equivalents (ee). For sorted cells, ee values were adjusted for percentage of dead cells. For quantification of dHSC numbers, embryonic tissues isolated from different developmental stages were dissected, dissociated and injected directly in limiting dilutions into cohorts of recipients A. Donor-Embryonic tissues were dissected as previously described for the AGM region , for extThe following antibodies were used: anti-CD45 FITC ; anti-mouse VE-cadherin biotin-conjugated antibody  following streptavidin APC staining . Cells were sorted using a FACSAria-II sorter (BD Biosciences) followed by purity checks as described before . Gating At the core of this method is the co-aggregation culture system, which allows maturation of HSC precursors. In these culture conditions, all types of HSC precursors isolated from E9.5-E11.5 embryos can mature into dHSCs . Howeverin vivo  contained pre-HSCs, independently of their proliferative activity inside the co-aggregate. This is because all the progeny of the pre-HSC remains in the same co-aggregate and goes only into one recipient mouse. Using Poisson's statistics , 20% of preselected, heat-inactivated foetal calf serum (PAA/HyClone) and L-glutamine, penicillin/streptomycin]. Cells were centrifuged at 450 g for 12 min in 200 μl pipette tips sealed with parafilm as described previously . Mice with donor-derived chimerism >5% following 13.5-18 weeks post-transplantation were considered to be repopulated (Table S2).Throughout this study, pre-HSC_LDA was mainly used without distinction (no cell purification) between types of pre-HSCs D; Fig. 22 is 0.84-0.98) (P>0.01) were found  (Table S2).The number of pro-HSCs and HSCs and the 95% confidential interval were calculated by the single-hit Poisson model using ELDA software . Coeffic