Microfold (M) cells are a kind of intestinal epithelial cell in the follicle-associated epithelium (FAE) of Peyer’s patches. They can transport antigens and microorganisms to lymphoid tissues. Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder in cattle. It is linked to variant Creutzfeldt-Jakob disease in humans. Although it is thought that M cells transport the BSE agent, the exact mechanism by which it crosses the intestinal barrier is not clear. We have bovine intestinal epithelial cell line (BIE cells), which can differentiate into the M cell type <i>in vitro</i> after stimulation, and which is able to transport the BSE agent. We show here that M cells are able to incorporate large numbers of PrP coated magnetic particles into intracellular vesicles, which we collected. The results of 2-DE show a specific protein associated with the PrP-coated particles, compared with non-coated particles. This protein was identified as aldolase A, a glycolytic pathway enzyme, using LC-MS/MS analysis. Aldolase A was synthesized and secreted by BIE cells, and increased during M cell differentiation. In the villi of the bovine intestine, aldolase A was detected on the surface of the epithelium and in the mucus droplet of goblet cells. In the FAE of bovine jejunal and ileal Peyer’s patches, aldolase A was localized on the surface and the apical part of the M cells. The binding of rbPrP to aldolase A was clearly detected and inhibited by pre-treatment of anti-aldolase A antibody. Aldolase A was co-stained with incorporated PrP<sup>Sc</sup> in M-BIE cells. These results suggest that bovine M cells and goblet cells synthesize aldolase A, and that aldolase A may have the ability to bind PrP and associate with PrP in cellular vesicles. Therefore, aldolase A-positive M cells may play a key role in the invasion of BSE into the body.
The mucosal surface of the gastrointestinal tract is continuously exposed to large numbers of commensal microorganisms and sporadically to pathogens. The gastrointestinal epithelium is covered by a protective mucus gel composed predominantly of mucin glycoproteins that are synthesized and secreted by goblet cells [
Microfold (M) cells are a kind of intestinal epithelial cell in the follicle-associated epithelium (FAE) of Peyer’s patches. In general, M cells have a characteristic morphology different from the absorptive epithelium. Typically, M cells are characterized by a lack of almost all microvilli. In addition, the basement membrane of M cells forms a pocket structure that is deeply recessed, and they have a close connection with dendritic cells and lymphocytes [
Transmissible spongiform encephalopathies (TSE) or prion diseases, including human Creutzfeldt-Jakob disease (CJD) and endemic sheep scrapie, are fatal neurodegenerative disorders. Bovine spongiform encephalopathy (BSE) is a TSE of cattle. The first case of BSE in the world was found in the United Kingdom in 1986. By the end of 2000, more than 180,000 cases had been reported in the UK. BSE spread to continental Europe, North America, and Japan [
Studies have shown that PrPSc first have to cross M cells [
The duodenum, jejunum, ileum and colon were obtained from 6, 8 and 10-wk-old male Holstein calves (Miyagi Agriculture Public Corporation, Sendai, Japan). All animals were clinically healthy and free of infectious diseases. All experiments were permitted by the Tohoku University Environmental & Safety Committee and conducted in accordance with the Guidelines for Animals Experimentation of Tohoku University, which have been sanctioned by the relevant committee of the Government of Japan based on the Declaration of Helsinki.
Goat polyclonal anti-aldolase A antibody (LSB-1461, LSBio, Seattle, WA, diluted to 1:1000) was used for the immunohistochemistry, Western blot analysis, far-Western blot analysis and ELISA. Rabbit polyclonal anti-ly- sosome-associated membrane protein-1 (LAMP1), as a endosomal marker, antibody (ab24170, Abcam, Cambridge, UK; diluted to 1:200), rabbit polyclonal anti-mucin core protein 2 (MUC2), as a mucous marker, antibody (ab76774, Abcam, diluted to 1:1000) and mouse monoclonal cytokeratin (CK) 18 antibody, as a marker of bovine intestinal M cell (clone CY90, Sigma, St. Louis, MO, diluted to 1:1000) were used for the immunohistochemistry. The mouse monoclonal anti-His-probe antibody (clone H-3, Santa Cruz, Dallas, Texas, diluted in 1: 300) was used for far-Western blot analysis. The mouse monoclonal anti-PrP antibody (clone 132) [
A DNA fragment encoding amino acid residues 25 to 242 of bovine PrP was amplified by PCR from genomic DNA purified from bovine liver using the forward primer containing a NdeI site (5’-TCTAGCTGTCATATGAAGAAGCGACCAAAACCTGG-3’) and reverse primer containing a BamHI site (5’-AGCTGTGGATCCTCATCAACTTGCCCCTCGTTGGTAATAAG-3’). The PCR reaction was performed using a high fidelity heat resistant DNA polymerase (KOD, Toyobo, Osaka, Japan) and PCR conditions were 1 cycle of 2 min at 95˚C 10 sec at 58˚C, and 20 sec at 68˚C, and finishing with 2 min incubation at 68˚C. The PCR fragment was cloned into pGEM-T Easy vector (Promega, Madison, WI) and the sequences of the inserts were confirmed by DNA sequencing. The inserts were excised with NdeI and BamHI from the plasmids, and were re-cloned into pET15b (Novagen, Madison, WI) encoding an N-terminal His-probe sequence, at NdeI andBamHI sites. The vectors carrying bovine PrP were transformed into E. coli BL21 (DE3) (Novagen).
The expression was carried out according to the manufacturer’s instructions. The collected cells were solubilized in Bug buster (Novagen) containing 200 µg/ml lysozyme and 200 µg/ml DNase and then were centrifuged at 6000 g at 4˚C for 30 min. The pellet was separated into soluble and insoluble fractions by centrifugation at 6000 g at 4˚C for 30 min. The pellet was then solubilized by protein denaturation buffer (pH 8.0; 6 M GuHCl/10 mM Tris-HCl/100 mM sodium phosphate/20 mM imidazole) and then subjected to SDS-PAGE for Coomassie brilliant Blue staining, and Western blot analysis using an anti-PrP monoclonal antibody (clone 6H4, Prionics, Schlieren-Zurich, Switzerland) and anti-His probe monoclonal antibody. Soluble proteins were applied to a Ni-NTA affinity column (Ni-NTA Superflow Cartridges, Qiagen, Hilden, Germany), which was equilibrated by denaturation buffer for 10 min. After the column was washed with protein denaturation buffer for 10 min, soluble proteins were refolded for 22 h by a linear gradient from denaturation buffer to binding buffer (pH 8.0; 10 mM Tris-HCl/100 mM sodium phosphate/20 mM imidazole). Soluble proteins were eluted by elution buffer (pH 5.8; 10 mM Tris-HCl/100 mM sodium phosphate/500 mM Imidazole) and dialyzed to 10 mM sodium phosphate using a dialysis membrane (Eidia, Tokyo, JP). The purity of rbPrP was separated by SDS-PAGE, stained by Coomassie brilliant blue and determined using CS Analyzer software.
The bovine intestinal epithelial cell line (BIE cells) [
To induce M cell differentiation, BIE cells were seeded onto each well of 12-Transwell inserts (9 × 105 cells/ well, 3.0-µm-pore-size Transwell filter polycarbonate membrane; Corning, NY, US) and cultured for 3 days. The cells were treated with or without the IL-2-stimulated PBMC culture supernatant for another 3 days [
BIE cells were seeded on Transwell-inserts at 9.0 × 105 cells/1.12 cm2/well and cultured for 3 days. The cells were treated with or without the IL-2-stimulated PBMC culture supernatant for another 3 days. These cells were washed once with 0.1 M phosphate buffer (pH 7.4) and then fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB) for 1 h. Following three times washing with 0.1 M PB, the cells were dehydrated by passage through graded dilutions of ethanol and substituted with t-butylalcohol. The cells were freeze-dried, coated with platinum-palladium and observed by SEM (SU8000, Hitachi, Tokyo, Japan).
The caudate nucleus was removed from a cow (8 months old) after slaughter, and transferred to serum-free DMEM supplemented with penicillin (GIBCO, 10 U/ml) and streptomycin (10 mg/ml). The caudate nucleus was cut finely, washed three times with serum-free DMEM, and centrifuged at 200 g for 7 min. The cells were re-suspended in 10% FBS-DMEM and seeded into a flask (Nunc, Rothskilde, Denmark). All cultures were maintained at 37˚C and 5% CO2 in a humidified incubator. The cells were treated with a EDTA buffer (pH 7.5; 0.36% EDTA, 0.1% BSA in PBS) for a few minutes, detached by 0.04% trypsin/PBS, and then collected and passaged when they reached 70% confluence. The cells in primary culture were transformed by the SV40 large T antigen gene and cloned by limiting dilution, according to the method used for the BIE cell line [
Murine neuroblastoma N2aC24 cells overexpressing mouse PrPc were exposed to the murine-adapted 22 L scrapie prion, and the 22 L prion-infected N2aC24 clone, designed N2aC24L1-3, were obtained by limiting dilution [
After M cell differentiation, non-coated or PrP-coated magnetic particles were added to the surface of M-BIE cells. After 9 h of incubation, these cells were homogenized in Tris-Triton X buffer (10 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride). The vesicles containing magnetic particles were collected from the lysate of M-BIE cells using the magnetic stand and the membrane proteins were extracted using a Transmembrane protein Extraction Kit (Novagen). The electrophoresis protein samples were diluted 1:9 in sample buffer (40 mM Tris-HCl pH 6.8, 0.25 mM DTT, 7.5 M urea, 2.3% (w/v) SDS), centrifuged at 22,000 g at 4˚C for 30 min, and then the supernatants were collected. The supernatant was diluted 9:1 in 1 M acrylamide (Sigma). The protein samples were separated on agar gels (pH 3 - 10, 7.5 cm long, ATTO) using isoelectric point electrophoresis apparatus, (Disc-Run ATTO) at 300 V for 210 min. Each well was loaded with 1.5 mg of total proteins. The agar gels were fixed with 2.5% trichloroacetic acid for 3 min and then washed three times in Milli Q water. Second-dimension electrophoresis was performed on 12.5% polyacrylamide gradient gels in PAGE Run (ATTO). The agar gels were run at 20 mA for 90 min. Proteins were visualized using a Silver Stain Kit (Wako, Osaka, JP). The specific spots in PrP-coated magnetic particles were identified, compared with those in non-coated particles. The protein spots indicated were excised and digested with trypsin overnight at 35˚C and the resulting peptides separated by LC-MS/MS analysis (Japan Proteomics Co, Ltd.; Sendai, JP) (
BIE cells and M-BIE cells were homogenized in Tris-Triton X buffer. The total protein concentration was mea- sured by the Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). The lysates were re-suspended in SDS loading buffer (50 mM Tris-HCl [pH 6.8] containing 5% glycerol, 1.6% SDS, and 100 mM dithiothreitol) and loaded onto a 12% polyacrylamide gel after boiling for 5 min. The proteins were transferred onto an Immobilon-P membrane (Millipore, Billerica, MA) in transfer buffer containing 20% methanol at 1.2 mA/cm2 for 60 min, and then the membrane was blocked in 3% skim milk in TBST (100 mM Tris-HCl [pH 7.8], 100 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature (RT). After blocking, the membrane was incubated with goat polyclonal anti-aldolase A antibody or rabbit polyclonal anti-GAPDH antibody for 14 h at 4˚C. Following three TBST washing cycles, the blot was incubated with alkaline phosphatase (AP)-conjugated rabbit anti-goat IgG (81-1622, Zymed Labs, CA, USA, diluted in 1:10,000) or AP-conjugated rabbit anti-rabbit IgG (A3687, Sigma, diluted in 1:10,000) as the secondary antibody for 1 h at RT. After three TBST washing cycles, the immunoreactive bands were visualized by an enhanced chemiluminescence system (ECL plus; Amersham Pharmacia Biotech, Piscataway, NJ).The apical and basal media from the Transwell culture of BIE and M-BIE cells were diluted 1:5 in SDS loading buffer. After boiling for 5 min, 15 µl of samples were loaded onto a 12% polyacrylamide gel and transferred onto an Immobilon-P membrane (Millipore) in transfer buffer containing 20% methanol at 1.2 mA/cm2 for 60 min. The membrane was blocked in3% skim milk in TBST for 1 h at RT.
kDa/PI | Peptide matches (n)e | Scoref | |||
---|---|---|---|---|---|
Protein namea | Accnob | Theoreticalc | Observedd | ||
Fructose-bisphoaphate aldolase A [Bostauras] | NP_001095385 | 39/8.4 | 40/8.4 | 4 | 213 |
Aldolase [Mustelaputoriusfuro] | ABN58928 | 34/7.9 | 40/8.4 | 2 | 99 |
Fructose-bisphoaphate aldolase C [Salmosalar] | NP_001135182 | 40/6.6 | 40/8.4 | 1 | 62 |
Fructose-bisphoaphate aldolase C [Callorhinchuscallorynchus] | BAD17946 | 37/5.1 | 40/8.4 | 1 | 57 |
Anionic tripsin precursor [Rattusnorvegicus] | NP_036767 | 26/4.7 | 40/8.4 | 2 | 52 |
Cobalt-containing nitrile hydratase subunit alpha | P21219 | 23/4.8 | 40/8.4 | 1 | 47 |
Protease, serine, 1 [Musmusculus] | AAH94921 | 26/4.8 | 40/8.4 | 1 | 44 |
Hypothetical transcriptional regulator, TetR family protein | ZP_01221408.1 | 23/5.2 | 40/8.4 | 2 | 38 |
Orf3 [Pseudomonas putida] | BAB62048 | 27/9.9 | 40/8.4 | 1 | 31 |
All these proteins were obtained from the single circled spot in Figures 3(A). a,bProtein names and accession numbers are given according to National Center of Biotechnology Information, NCBI; cTheoretical isoelectric point (PI) and molecular mass (kDa); dObserved isoelectric point (PI) and molecular mass (kDa); eNumber of identified peptide matches; fProbability-based MOWSE scores in MASCOT program greater than 61 were considered significant (p < 0.05).
After blocking, the membrane was incubated with goat anti-aldolase A polyclonal antibody for 14 h at 4˚C. Following three TBST washing cycles, the blot was incubated with HRP-conjugated bovine anti-goat IgG as the secondary antibody (805-005-180, Jackson Immuno Research Europe Ltd., Suffolk, UK) for 1 h at RT. After three TBST washing cycles, immunoreactive bands were visualized by an enhanced chemiluminescence system (EZ-Capture MG, ATTO).
The lysate of BNP cells was separated by SDS-PAGE and transferred onto an Immobilon-P membrane in transfer buffer containing 20% methanol for 60 min. The lysate was separated by SDS-PAGE and subjected to the Western blot analysis described above. The membrane was blocked in 3% skim milk in TBST for 1 h at RT and the membrane was incubated with or without goat polyclonal anti-aldolase A antibody for 14 h at 4˚C. After the washing three times in TBST, the membrane was incubated with or without 100 µg/mL rbPrP in blocking buffer for 14 h at 4˚C. The membrane was washed three times for 5 min with TBST. The rbPrP-bounding on the membrane was incubated with the mouse monoclonal anti-His-probe antibody in blocking buffer for 1 h at RT. Following three TBST washing cycles, the membrane was incubated with AP-conjugated goat anti-mouse IgG as the secondary antibody (A3562, Sigma, diluted in 1:50,000) for 1 h at RT. After three TBST washing cycles, the immunoreactive bands were visualized by an enhanced chemiluminescence system.
Protein (40 µl) G-Sepharose 50% solution (GE Healthcare Life Sciences, Uppsala, Sweden) was added to a 40 µl mixture of rbPrP (1 µg) and aldolase from rabbit muscles (1 µg; Sigma), and incubated under rotational agitation at 4˚C for 30 min in Tris-Triton X buffer. The mixture was centrifuged at 9200 g at 4˚C for 1 min. The supernatant was collected. Anti-aldolase A antibody (5 µg) was added to it and incubated under rotational agitation at 4˚C for 30 min by Tris-Triton X buffer. Protein G-Sepharose 50% solution (40 µl) was added to it, then suspended and incubated under rotation at 4˚C for 30 min. Beads were centrifuged at 9200 g at 4˚C for 1 min. The pellet was washed with PBS, incubated in 20 µl of SDS loading buffer at 95˚C for 5 min, and then subjected to the SDS-PAGE analysis.
rbPrP in carbonate-bicarbonate buffer (Sigma) was incubated at 2 µg/ml in 96 well ELISA plates (C96 Maxisorp cert. Nunc-Immuno Plate, Thermo Fisher Scientific, Waltham, MA) for 14 h at 4˚C. After three TBST washing cycles, the wells were incubated with apical and basal media from the Transwell culture of BIE and M-BIE cells for 1 h at RT. After three TBST washing cycles, and the wells were then blocked with 1% BSA for 1 h to prevent nonspecific reactions and then the wells were treated with goat anti-aldolase A antibody for 2 h at RT. Following three TBST washing cycles, the wells were incubated with HRP-conjugated bovine anti-goat IgG as the secondary antibody (805-005-180, Jackson ImmunoResearch Europe Ltd.) for 1 h at RT. The freshly prepared substrate was added and the OD was taken at 450 nm using the TMB microwell peroxidase substrate system (KPL, Gaithersburg, Maryland). Negative control assays were performed without the apical and basal media.
Tissue samples from 6, 8 or 10-wk-old calves were fixed with 4% paraformaldehyde solution in 0.1 M phosphate buffer (PB) for 24 h at 4˚C. Tissues were dehydrated through a series of graded ethanol solutions and embedded in paraffin. Three serial sections were cut at 4 µm and mounted on glass slides (MAS-coated Superfrost; Matsunami, Osaka, JP). The sections were incubated with 0.05% proteinase (Type XXIV: Bacterial, P8038; Sigma) in Tris-HCl buffer (pH 7.5) for 5 min at 37˚C, and treated with 3% normal chicken serum for 30 min to prevent nonspecific reactions of antibodies. Subsequently, the sections were incubated with goat anti-aldolase A polyclonal antibody at 4˚C overnight. After the section was washed three times with PBS, Alexa Fluor 488 or 594-conjugated chicken anti-goat IgG (Molecular Probes, Leiden, The Netherlands, 1:400) was used as the secondary antibody for 1 h at RT, and then counterstained with 4, 6-diamidino-2-phenylindole (DAPI) and then examined using a confocal laser microscope (LSM7000, Carl Zeiss, Oberkochen, Germany). In order to determine the specificity of the immunostaining, negative controls were run in which the primary antibody was omitted (data not shown). After M cell differentiation, FITC-conjugated particles or PrPSc of N2aC24 cells were added to the surface of the BIE or M-BIE cells on Transwell inserts. After 9 h incubation, the cells were washed with PBS, fixed in 4% PFA for 30 min at RT, and treated with 3% normal chicken serum for 30 min to prevent nonspecific reactions. Subsequently, the samples were incubated with anti-aldolase A antibody at 4˚C overnight. After three PBS washing cycles, the samples were treated with Alexa Fluor 594-conjugated chicken anti-goat IgG as the secondary antibody for 1 h, counterstained with DAPI for 5 min, and then examined by a confocal laser microscope. For the detection of PrPSc, the sections were treated with 5 M guanidine hydrochloride (Wako, Osaka, JP) for 10 min at RT and treated with 3% normal chicken serum for 30 min to prevent nonspecific reactions. Subsequently, the samples were incubated with anti-PrP antibody clone 132 (diluted 1:1000) at 4˚C overnight. After three PBS washing cycles, the samples were treated with Alexa Fluor 488-conjugated chicken anti- goat IgG as the secondary antibody for 1 h, and then counterstained with DAPI for 5 min, and examined by a confocal laser microscope.
Bovine ileal tissues were fixed in 4% paraformaldehyde in 0.1 M PB (pH 7.4) for 14 h at 4˚C. The fixed samples were washed three times with PB, dehydrated in a graded ethanol series, infiltrated with LR White (Plano, München, Germany), and sectioned into 60 - 80 nm thicknesses by ultramicrotome. The sections were mounted on formvar-coated nickel grids, and subsequently blocked with 1% bovine serum albumin, 3% skim milk and goat serum for 30 min at RT. The grids were then incubated for 14 h with goat anti-aldolase A antibody at a 1:1000 dilution. After washing with PB, the preparations were incubated for 1 h with rabbit anti-goat IgG conjugated to 15 nm gold particles (BBI solution, Cardiff, UK) diluted in 1:200. Sections were washed five times with PB and were fixed with 2.5% glutaraldehyde in 0.1 M PB (pH 7.4) for 10 min and then were washed in distilled water. The sections were examined by TEM (H8100, Hitachi).
The data for the semi-quantitative analysis of protein expression and volume of transported particles are presented as mean ± SD. Student’s t-tests were performed to determine if the relative change was statistically significant. A significant difference was determined at p < 0.05.
Recombinant bovine PrP (rbPrP) (aa 23 - 242) was expressed in E.coli from the modified pET-15b expression vector. Following purification and refolding in vitro, the recombinant protein was analyzed by Coomassie staining (
affinity column, the soluble protein was detected as a single band at 26 kDa with Coomassie staining. The purity of rbPrP stained by Coomassie staining and Western blotting was estimated to be approximately 57% before the refolding, 92% after the refolding, and 97% after the dialysis using a CS Analyzer.
Initially, we confirmed the TER of BIE cell and M cell differentiated cells (M-BIE cells) cultured on Transwell-inserts. TER is widely used to describe the permeable properties of tight junctions between epithelial cells [
We next performed a proteomic analysis of PrP-binding proteins in vesicles of M-BIE cells. After M cell differentiation of BIE cells, non-coated or PrP-coated magnetic particles were added to the surface of M-BIE cells. After 9 h of incubation, the vesicles containing magnetic particles were collected from M-BIE cells using the magnetic stand. The results of the 2-DE show that there is a specific spot identified in PrP-coated particles, indicated by a circle, compared with non-coated particles (
In order to confirm the binding between aldolase A and PrP, we performed a far-Western blot analysis. Aldolase A of BNP cells were detected in 39 kDa using goat anti-aldolase A antibody (ALD-Ab) (
We examined whether M cells were able to secrete aldolase A or not. After M cell differentiation, BIE and M- BIE cells on Transwell inserts were incubated for 9 hours, and the apical and basal media were collected. Western blot analysis shows that the production of aldolase A by BIE and M-BIE cells is greater to the apical medium than to the basal medium. As the concentration of aldolase A from M-BIE cells was fourfold greater than that from BIE cells, the secretory ability of aldolase A in BIE cells increased following M cell differentiation (
We next investigated whether aldolase A was concerned with the transcytosis of PrP in M cells. After the addition of non- or PrP-coated particles, M-BIE cells incorporated a large number of each particle into their cytosol during a 9 h incubation (
number of incorporated particles in M-BIE cells was larger when non-coated particles were used than when PrP- coated particles were used. It is possibly for this reason why M-BIE cells incorporated PrP-coated particles and transported them to the basal medium to a greater extent than BIE cells. PrPSc of N2aC24L1-3 cells were added onto BIE and M-BIE cells, and incubated for 9 h. The incorporated PrPSc was co-stained with aldolase A in M- BIE cells, but not stained in BIE cells (
We investigated the effect of aldolase A on the transcytosis of M cells (
In the villous epithelium, aldolase A was detected on the surface of the epithelium facing the lumen and in the mucus droplet of goblet cells in duodenum, jejunum, ileum and colon (Figures 8(A)-(D)). Lamp-1, a late endos- omal maker [
the localization of aldolase A in the M-BIE cells in vitro (
In M cells, aldolase A was visualized with gold particles in microfold and small granule vesicles by immunoelectron microscopic analysis (Figures 10(A)-(C)). In addition, there were a lot of large vesicles containing aldolase A in M cells, including transport vesicles for transcytosis (
For oral transmission, PrPSc first must cross the intestinal epithelium to successfully infect the body. We previously reported that orally delivered PrPSc was incorporated by M cells in FAE of Peyer’s patches [
Aldolase A in the FAE of the Peyer’s patch was specifically stained in both the microfold and apical part of M cells, but not stained in CK-18-negative cells. The amount of aldolase A was greater in M cells following differentiation than in the undifferentiated BIE cells. We previously reported that a specific marker for bovine M cells was an intermediate filament protein CK18 [
We show that a large amount of aldolase A is detected in the micro villi of the small intestine, and this was shown to be secreted from goblet cells. A recent study reported that goblet cells delivered luminal antigen to CD103+ DCs in the small intestine, and that the preferential delivery of antigens to DCs with tolerogenic properties implies a key role for this goblet cells function in intestinal immune homeostasis [
We have demonstrated using far-western blot analysis, ELISA and immunoprecipitation, that aldolase A has the ability to bind to PrP. Many studies have identified PrP-interacting and -binding partners, such as Cu2+, Ni2+, glycosaminoglycans, DNA, RNA, a number of signaling proteins, histone H3, and thiamine etc. [
In conclusion, we have identified aldolase A as a PrP-binding protein in the vesicles of M cells. Far-Western blot, immunoprecipitation and ELISA analysis revealed the specific binding ability of aldolase A to PrP. Aldolase A was localized at the epithelia of the mucus droplet of goblet cells and M cells of FAE of in bovine Peyer’s patches. Aldolase A may therefore be an intestinal M cell marker in the FAE. Further, aldolase A was abundantly synthesized and secreted in M cells. These results suggest that aldolase A-positive M cells in Peyer’s patches may actively transcytosePrPSc into the basolateral side of the epithelium and can play a key role in promoting intestinal immune homeostasis by delivering a luminal antigen. This study contributes to the understanding of the function of M cells and the invasion mechanisms of BSE agents by bovine intestinal epithelial cells. After the text edit has been completed, the paper is ready for the template. Duplicate the template file by using the Save As command, and use the naming convention prescribed by your journal for the name of your paper. In this newly created file, highlight all of the contents and import your prepared text file. You are now ready to style your paper.
This research was supported by a Grant-in-Aid for Scientific Research (24380150) from the Ministry of Education, Culture, Sports, Science and Technology, BSE Control Project from the Ministry of Agriculture, Forestry and Fisheries and Cooperative Research Grant of the Institute for Enzyme Research and Cooperative Research Grant of the Institute for Enzyme Research, the University of Tokushima. Yuya Nagasawa was also supported by a Grant-in-Aid for Scientific Research (25-2872) from the Ministry of Education, Culture, Sports, Science and Technology, Japan Society for the Promotion of Science (JSPS), Research Fellowship for Young Scientists Program.