NDT Advance Access originally published online on January 23, 2006
Nephrology Dialysis Transplantation 2006 21(4):1073-1081; doi:10.1093/ndt/gfk101
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Characteristics of polyclonal anti-human nephrin antibodies induced by genetic immunization using nephrin cDNA
1 Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, Sagamihara, Kanagawa and 2 Department of Rehabilitation Engineering, Research Institute, National Rehabilitation Center for the Disabled, Tokorozawa, Saitama, Japan
Correspondence and offprint requests to: Togo Aoyama, Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, 1-15-1 Kitasato, Sagamihara, Kanagawa 288-8555, Japan. Email: dm02001x{at}st.kitasato-u.ac.jp
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Abstract |
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Background. Nephrin is an essential protein for maintaining the normal structure of the podocyte foot process and the glomerular filtration barrier. To analyse the mechanism of proteinuria and treatment of nephrotic syndrome, we produced and characterized polyclonal anti-human nephrin antibodies using a newly developed genetic immunization technique.
Methods. An expression vector with full-length or fragmented cDNA of human nephrin protein was administered to female Lewis rats once a week for 12 weeks using a gene-gun method. Antibody production against different fragments of nephrin protein was then analysed by ELISA, Western blot analysis, immunoprecipitation and FACS analysis. We also analysed recognition of native nephrin protein using immunohistochemistry and electron microscopy, and conducted a functional analysis of in vitro clustering of nephrin protein.
Results. Serum anti-nephrin antibody titers reached a maximum level at 8 or 12 weeks. Four of five antibodies induced with cDNA of five different Ig-like motif fragments showed antigen-specific binding to Escherichia coli and produced recombinant human nephrin protein. Only two of these four antibodies, plus one antibody induced by full-length human nephrin protein cDNA, bound to solubilized native nephrin protein. These three IgG antibodies, subclasses IgG1, IgG2a and IgG2b, showed fine granular staining along the glomerular basement membrane of normal human glomeruli and clustering of human nephrin protein on plasma membranes of HEK293 cells.
Conclusions. We successfully produced polyclonal anti-human nephrin antibodies by genetic immunization using nephrin cDNA. These new antigen-specific polyclonal antibodies will be useful for functional analysis and tissue staining of native nephrin protein.
Keywords: anti-nephrin antibody; clustering; DNA immunization; gene-gun; nephrin; proteinuria
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Introduction |
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The gene responsible for Finnish type congenital nephrotic syndrome (NPHS1) was cloned in 1998 [1]. Its product, nephrin, is a 185 kDa transmembrane protein of the immunoglobulin superfamily that acts as an adhesion receptor and signalling protein. Nephrin has a large extracellular domain consisting of a 1060-amino acid residue with a single-pass transmembrane region and an intracellular domain consisting of a 154-amino acid residue. In situ hybridization and immunoelectron microscopy studies show that human nephrin is located on the cell surface of the slit diaphragm in glomerular visceral epithelial cells [2]. A single injection of monoclonal antibody 5-1-6 was previously shown to react with the extracellular domain of rat nephrin, causing severe proteinuria within a few days in Brown–Norway rats, but no reaction with human nephrin protein was observed [3,4]. These observations indicate that the slit diaphragm might act as a critical barrier for plasma proteins in glomerular ultrafiltration in humans.
Recent advances in molecular biology have made it possible to induce polyclonal antibodies against a single putative protein by administration of an expression vector into which DNA of the putative protein has been inserted. The benefits of using plasmid DNA and the gene-gun method instead of the protein injection method are: (1) plasmid DNA remains stable for a long time; (2) pleural DNA can be simultaneously administered in vivo; (3) a small amount of plasmid DNA can be used to induce a polyclonal antibody against product produced by cDNA fragment; (4) the gene-gun method as a physical delivery system is safe with respect to retrovirus infection or carcinogenesis [5–9] and (5) it is applicable in cases where it is hard to obtain a sufficient amount of purified native protein for immunization. Using this method, we previously developed a molecular-based experimental model of renal disease using an expression vector, pKA1, into which monkey DNA of alpha-2 macroglobulin receptor-associated protein was inserted (alpha-2MRAP) [10]. In the present study, we produced and characterized polyclonal anti-human nephrin antibodies using a newly developed genetic immunization technique.
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Experimental animals
Eight-week-old female Lewis rats weighing 180–200 g were obtained from the Charles River Breeding Laboratory (Atsugi, Japan). The rats were kept on a constant dark and light cycle of 12 h and fed standard laboratory chow (SLC Japan; Shizuoka, Japan) with free access to water. All animal experiments were performed under an experimental protocol approved by the Ethical Review Committee for Animal Experiments of Kitasato University School of Medicine.
Immunization plasmids
Full-length cDNA encoding human nephrin protein was synthesized and purified by TaKaRa Custom Services (Ohtsu, Japan). Nephrin protein has a COOH-terminal site, eight immunoglobulin (Ig)-like motifs and a fibronectin-like motif near the transmembrane region of the extracellular domain. Five fragments of the extracellular domain nephrin cDNA encoding Ig-like motif 1–2 (amino acids 1–257), 3–4 (amino acids 258–458), 5–6 (amino acids 459–755), 7–8 (amino acids 756–940) and 1–8 (amino acids 1–940), respectively, were generated by PCR using full-length human nephrin cDNA as a template. The 3' primers were designed to contain a stop codon in-frame with the site for NotI digestion, and the 5' primers were chosen upstream of the EcoRI restriction site. PCR amplified-cDNA fragments were cleaved using restriction enzymes of EcoRI and NotI, and then purified with gel filtration. Full-length and fragmented nephrin cDNAs were subsequently constructed and inserted into pTARGETTM mammalian expression vectors using a CMV promoter (Promega, Madison, WI). Expression vectors including the cDNA fragments were inserted into Escherichia coli JM 109 Competent Cells (TaKaRa) according to the manufacturer's protocol. After overnight incubation, each plasmid was collected using a QIAGEN Plasmid Maxi Kit (QIAGEN, Tokyo). The authenticity of the cDNA construct was confirmed by sequencing with a DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences AB, Uppsala, Sweden).
Immunization schedules and sampling
Seven groups of five or six female Lewis rats were used. The plasmid vectors were bound to gold particles 1 µm in diameter (Bio-Rad, Hercules, CA) according to the manufacturer's protocol. The coated gold particles were then transferred into the subcutaneous tissue of both inner thighs using the gene-gun method (Bio-Rad). 100 µg of plasmid DNA was used for the initial administration, then 10 µg was administered 11 times, once a week from week 2. As a control, expression vectors without nephrin DNA were administered to rats in the same manner. Blood samples were taken every 2 weeks from the start of the experiment and stored at –80°C until use.
Glutathione-S-transferase (GST)-tagged protein expression and purification
A full-length nephrin cDNA and seven nephrin cDNA fragments encoding amino acids 1–257 (Ig-like motif 1–2), 258–459 (Ig-like motif 3–4), 460–755 (Ig-like motif 5–6), 756–940 (Ig-like motif 7–8), 941–1060 (fibronectin-like motif), 1061–1086 (transmembrane domain) and 1087–1241 (intracellular domain), respectively, were inserted into a modified pGEX-5X-1 vector (Amersham Biosciences AB). GST fusion proteins were expressed in E. coli BL21 strain cultured in LB medium with 1mM isopropyl-ß-D-thiogalactopyranoside at 37°C. Cells were pelleted at 3000 g for 10 min at 4°C then resuspended in lysis buffer with phosphate buffered saline (PBS) containing 2% Triton X-100 and protease inhibitors. After one freeze–thaw cycle, the samples were sonicated on ice then centrifuged at 13 000 g for 30 min at 4°C. The majority of the GST fusion proteins were included in the cell pellets. The proteins were purified under denaturing conditions with phosphate buffer containing 8 M urea then allowed to refold by removing the urea with dialysis. Following this, they were purified with a prepacked Glutathione Sepharose 4B column (Amersham Biosciences AB) according to the manufacturer's instructions. Molecular weights of the GST-tagged Ig-like motifs (1–2, 3–4, 5–6 and 7–8) and the fibronectin-like motif, and the transmembrane and intracellular domains were 54, 48, 59, 47, 40, 35 and 64 kDa, respectively. The molecular weight of GST without fragmented nephrin protein was 27 kDa; however, a GST-tagged full-length nephrin protein could not be produced.
V5-tagged protein expression and purification
cDNA encoding full-length human nephrin protein was cloned into the mammalian expression vector pcDNA3.1/V5 (Invitrogen Corp., Carlsbad, CA) then inserted into HEK293 cells, a human embryonic kidney cell line, using an electroporation method. The HEK293 cells were cultured with RPMI-1640 medium (Sigma Chemical, St Louis, MO) supplemented with 10% (V/V) heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate and 2 mM extra L-glutamine in 5% CO2 and 95% air at 37°C. Stable clones were obtained by incubation with selection buffer containing 1 mg/ml G-418 sulfate (Calbiochem, Darmstadt). HEK293 cells with V5-tagged nephrin protein on their plasma membranes were cloned and cultured under almost confluent conditions. They were then washed three times with ice-cold PBS and lysed with 1 ml of lysis buffer containing 140 mM NaCl, 3 mM MgCl2, 0.5% (v/v) Nonidet-P 40 in 10 mM Tris-HCl (pH 8.4) and protease inhibitors. Cell lysates were removed by centrifugation at 14 000 g for 5 min and then used for immnoprecipitation studies.
Normal human kidney samples and isolation of glomeruli
Human kidney cortex was obtained from the histologically normal regions of kidneys removed from renal or ureter cancer patients at the Department of Urology, Kitasato University Hospital. Informed consent was obtained from all patients. Human glomeruli were isolated by differential sieving through 400 and 200 µm brass sieves then washed extensively with ice-cold PBS containing protease inhibitors. Isolated glomeruli were collected by centrifugation at 10 000 g. Proteins were extracted from isolated glomeruli with lysis buffer consisting of 140 mM NaCl, 3 mM MgCl2, 0.5% (v/v) Nonidet-P 40 and protease inhibitors in 10 mM Tris-HCl (pH 8.4). Extraction was performed for 30 min on ice. The supernatant obtained after centrifugation at 14 000 g for 5 min was used for immunoprecipitation studies.
Enzyme-linked immunosorbent assay (ELISA)
Sera were taken at 0, 4, 8 and 12 weeks from rats immunized with different fragments of nephrin cDNA. Antibody titers in the sera were then measured using ELISA coated with E. coli-produced GST fusion proteins containing four different extracellular domain fragments (Ig-like motifs 1–2, 3–4, 5–6 and 7–8, respectively). Microtiter plates were coated with 50 µl of GST fusion protein in PBS at a concentration of 1µg/ml (pH 7.4). After blocking the wells with PBS containing 20% (v/v) Block Ace (Dainippon Pharmacy, Osaka, Japan) for 2 h, the plates were washed with PBS five times. Sera were then diluted to 1/200 with assay buffer consisting of 1% Block Ace and 0.5% Tween-20 in PBS. Samples (50 µl) were then applied to each well and incubated for 1 hr at 37°C. After incubation, the plates were washed three times with PBS assay buffer, and then 50 µl of 1/500 diluted peroxidase-conjugated rabbit anti-rat IgG (Zymed, South San Francisco, CA) was added. The plates were incubated for 1 hr and then washed. Peroxidase activity was visualized at an absorbance of 492 nm with O-phenylenediamine dihydrochloride buffer.
SDS-PAGE and western blot analyses
The E. coli-produced GST fusion proteins containing nephrin protein fragments were separated using 5–20% sodium dodecyl sulfate (SDS)-gradient gel under reducing conditions. After electrophoresis, proteins were stained with Coomassie blue or transferred to polyvinylidene difluoride (PVDF) membranes. Blot membranes were incubated with blocking solution of PBS containing 20% (v/v) Block Ace overnight at 4°C, and then reacted for 2 h with 1/200 diluted sera derived at 12 weeks from rats immunized with different fragments of nephrin cDNA. Thereafter, membranes were incubated with horseradish peroxidase-conjugated rabbit anti-rat IgG (1/4000) and visualized with chemiluminescent reagent (ECL, Amersham Bioscience, Buckinghamshire, UK).
Immunoprecipitation studies
HEK293 cell lysates containing V5-tagged full-length nephrin protein were incubated with an Immunoprecipitation Starter Pack (Amersham Biosciences AB) containing 50 µl Protein A Fast Flow and 50 µl Protein G Fast Flow with 50% slurry for 1 hr at 4°C to avoid nonspecific binding of lysates to protein A and G beads. Immunoprecipitation was performed for 1 h at 4°C by incubating 500 µl of cell lysate and 50 µl of sera derived at 12 weeks from rats immunized with different fragments of nephrin cDNA. After adding 80 µl of the Immunoprecipitation Starter Pack containing 40 µl Protein A Fast Flow and 40 µl Protein G Fast Flow with 50% slurry, incubation was continued for 1 h at 4°C. The immune complexes on Sepharose beads were collected by centrifugation then washed three times with ice-cold lysis buffer and once with washing buffer containing 140 mM NaCl, 0.5% (v/v) Nonidet-P 40 in 10 mM Tris-HCl (pH 8.4) and protease inhibitors. The immunoprecipitated samples were suspended in Laemmli sample buffer and boiled for 5 min. Proteins were separated using 5–20% SDS-gradient gel under reducing conditions, transferred to PVDF membranes, immunostained with guinea pig antiserum raised against the intracellular domain of human nephrin protein (Progen Biotechnik, Heidelberg, Germany) then visualized with HRP-labelled rabbit anti-guinea pig IgG antibody (Cappel ICN, Aurora, OH).
Flow cytometry analyses
Full-length human nephrin protein expressed in HEK293 cells was used as the antigen for detecting the reactivity of polyclonal anti-human nephrin antibodies. Rat sera derived at 12 weeks from rats immunized with five different fragments of human nephrin cDNA were used for analysis. HEK293 cells (2 x 105) in 100 µl Hanks’ balanced salt solution (HBSS) with 0.5% BSA and 10 µl rat serum containing anti-human nephrin antibody were mixed and incubated at 4°C for 30 min. After centrifugation, the cell pellet was resuspended with 100 µl fluorescent isothionate (FITC)-labelled rabbit anti-rat IgG (1/100) (Zymed) and incubated at 4°C for 30 min. Following this, cells were washed twice with HBSS and assayed using flow cytometry (Becton Dickinson, Mountain View, CA). Data obtained were analysed with Cell Quest Software (Becton Dickinson).
Indirect immunofluorescence
Reactivities of rat anti-human nephrin antibodies to normal human kidney tissue were tested with indirect immunofluorescence. Normal human kidney tissue was obtained from kidneys removed because of renal malignancy. Small blocks of normal human kidney cortex were then taken from the opposite pole to the renal malignancy, embedded in OCT compound Tissue-Tek, Sukura Finetek, Torrance, CA, and snap-frozen in liquid nitrogen. The frozen samples were sliced into 5 µm sections then fixed with ice-cold acetone for 5 min. After blocking with 1% BSA/PBS for 1 h, the cryostat sections were overlaid with rat sera containing anti-nephrin antibodies, and incubated at 4°C overnight. After removing the rat sera, the sections were reacted with FITC-labelled rabbit anti-rat IgG (Zymed) for 1 h. Then, after washing three times with PBS, they were evaluated using a fluorescence microscope equipped with appropriate filters (Olympus, BX 51, Japan).
IgG subclasses of anti-nephrin antibody
The IgG subclasses of anti-nephrin antibody were analysed as follows. Sera obtained from rats at 12 weeks after cDNA immunization were used. Cryostat sections of normal human kidney cortex were fixed with ice-cold acetone for 5 min then, after blocking with 1% BSA in PBS for 1 h, overlaid with 80-fold diluted serum for 2 h at room temperature. After removing the rat serum, the cryostat sections were overlaid with rabbit monospecific antibodies against rat IgG1, IgG2a, IgG2b or IgG2c heavy chains (Bethyl Laboratories, Montgomery, TX) for 1 h at room temperature. After washing five times, the sections were incubated with 1/1000 diluted FITC-labelled goat anti-rabbit IgG (Zymed) for 1 h at room temperature then, after washing a further five times, the cryostat sections were evaluated by fluorescence microscopy (Olympus, BX 51, Japan).
Immunogold electron microscopic analysis of glomeruli
The pre-embedding technique for immunoelectron microscopy newly developed by Katsumata et al. [11] was used for this experiment. Briefly, cryosections on glass slides were incubated with a blocking solution then incubated overnight with 100-fold diluted rat serum containing anti-nephrin antibody followed by anti-rat IgG antibody conjugated with 0.8 nm gold collides. The sections were rinsed with three changes of PBS at each step. After incubation, they were post-fixed with 2.5% glutaraldehyde, 0.2% tannic acid and 1% OsO4 in 0.1 M phosphate buffer for 30 min at room temperature then rinsed with triple-distilled water. Following this, cryosections were soaked in a solution for silver enhancement then with three changes of distilled water, embedded in a chitosan solution, dehydrated and processed routinely. Finally, thin sections were stained with uranyl acetate and lead citrate and examined by electron microscopy (model JEX-1200EX; JEOL Ltd, Tokyo, Japan).
Clustering
HEK293 cells stably expressing V5-tagged full-length human nephrin protein on their plasma membranes were cultured on coverslips in 25 mm culture dishes with 1 ml of RPMI-1640 medium supplemented with 10% (V/V) heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate and 2 mM extra L-glutamine in 5% CO2 and 95% air at 37°C to nearly confluent culture conditions. After culture, the cells were washed three times with ice-cold PBS, and then incubated with 1ml of RPMI-1640 medium containing 25 mmol/l Hepes, pH 7.4 and 5 µl of rat serum containing polyclonal anti-nephrin antibody for 30 min at 4°C. The medium was then removed and cells were incubated with 500 µl of RPMI-1640 medium containing 25 mmol/l Hepes, pH 7.4 and 5 µl of 0.75 mg/ml FITC-conjugated rabbit anti-rat IgG for 10 min at 37°C. After removing the second antibody, plates were washed three times with ice-cold PBS and fixed by adding 2 ml of 4% paraformaldehyde in PBS for 20 min at room temperature. A fluorescence microscope equipped with appropriate filters was then used for analysis.
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Serum anti-nephrin antibody titers against E. coli-produced nephrin proteins detected with ELISA
The time courses of six different anti-nephrin antibody titers are shown in Figure 1. Serum anti-nephrin antibody titers increased at 4 weeks, reaching a maximum at 8 or 12 weeks. Rats immunized with cDNA of Ig-like motifs 1–2, 3–4 or 5–6 produced antigen-site specific polyclonal anti-nephrin antibodies, while those immunized with cDNA of Ig-like motif 1–8 produced a polyclonal antibody that reacted with Ig-like motifs 1–2, 3–4, 5–6 and 7–8. Rats immunized with cDNA of Ig-like motif 7–8 produced no anti-nephrin antibody, while, similarly, those immunized with full-length nephrin cDNA did not produce significantly high levels of antibody against Ig-like motifs 1–2, 3–4, 5–6 and 7–8 of E. coli-produced nephrin protein.
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), 3–4 (258–458 amino acids) (
), 5–6 (459–755 amino acids) (
), 7–8 (756–940 amino acids) (
), and 1–8 (1–940 amino acids) (
), full-length cDNA (1–1263 amino acids) (
) and an empty vector (•), respectively, are shown in each panel.E. coli-produced GST fusion proteins recognized by the six different polyclonal anti-nephrin antibodies
GST fusion proteins recognized are shown in Figure 2. Preimmune sera (lane 1) and sera of rats immunized with empty vector (lane 2) did not react with any proteins of the Ig-like motifs 1–2, 3–4, 5–6 and 7–8, or the fibronectin-like motif. Anti-nephrin antibody induced by immunization with cDNA of Ig-like motif 1–2 recognized the protein of Ig-like motif 1–2 only (lane 3). Antibodies induced by immunization with cDNA of Ig-like motifs 3–4 and 5–6 also showed antigen-specific recognition (lanes 4 and 5). The antibody induced by administration of cDNA of Ig-like motif 7–8 reacted with no fragments of the E. coli-produced nephrin protein (lane 6), while that induced by immunization with cDNA of Ig-like motif 1–8 recognized the proteins of Ig-like motifs 7–8 and 5–6, and minimally, Ig-like motifs 1–2 and 3–4 (lane 7). The antibody induced by administration of full-length nephrin cDNA only recognized the fibronection-like motif of nephrin protein (lane 8). GST fusion proteins of the transmembrane and intracellular domains, on the other hand, could not be recognized by any of these six anti-nephrin antibodies (data not shown). GST without fragmented nephrin protein could not be recognized by any of the pre-immune or the seven different post immune sera used in lanes 1–8 (data not shown).
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The recognition abilities of polyclonal anti-nephrin antibodies for native nephrin protein
Solubilized native nephrin protein from isolated normal human glomeruli and HEK293 cells expressing full-length human nephrin protein were used as antigens for immunoprecipitation (Figure 3). Only three polyclonal anti-nephrin antibodies from rats immunized with cDNA of Ig-like motifs 1–2 and 1–8 and full-length nephrin, respectively, recognized both the solubilized native nephrin proteins from normal human kidney and HEK293 cells expressing full-length human nephrin protein. Immunoprecipitated nephrin protein of normal human kidney migrated to 185 kDa, while immunoprecipitated protein from HEK293 cells showed double bands at 180 and 170 kDa, consistent with previous reports [12]. This size difference is thought to be the result of differential glycosylation. Flow cytometry analysis (Figure 4) also revealed that only the same three anti-nephrin antibodies recognized nephrin protein on the plasma membranes of HEK293 cells.
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Immunohistochemistry of native nephrin protein recognized by the polyclonal anti-nephrin antibodies in normal human kidney
Only three samples derived at 12 weeks from rats immunized with cDNA of Ig-like motifs 1–2 (Figure 5, panel B) and 1–8 (panel F) and full-length nephrin (panel G), respectively, showed fine granular staining along the glomerular basement membrane. This staining pattern was consistent with the typical staining pattern of nephrin protein in normal human kidney. No appreciable staining was seen in the kidney, except for the glomeruli.
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The ultrastructure localization of nephrin protein recognized by antibodies from rats immunized with full-length nephrin cDNA was shown by pre-embedding immunoelectron microscopy (Figure 6). Immunogold particles localized at the filtration slits between the podocyte foot processes in normal human kidney glomeruli.
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IgG subclasses of polyclonal rat anti-human nephrin antibodies
Anti-IgG subclass staining of anti-nephrin antibodies are shown in Figure 7. The IgG subclass of anti-nephrin antibody induced by cDNA of Ig-like motif 1–2 was predominantly IgG1 with a modicum of IgG2a (lane A). Those induced by cDNA of Ig-like motif 1–8 were IgG1 and IgG2a (lane B), while IgG2b was predominantly induced by the full-length nephrin cDNA, with a modicum of IgG2a and an insignificant degree of IgG1 (lane C).
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Clustering of nephrin protein on plasma membranes of HEK293 cells
Normally expressed nephrin protein and its clustering after treatment with anti-nephrin antibodies are visualized in Figure 8. Normally expressed nephrin protein localized mainly at the cell–cell adhesion sites, while clustering removed the nephrin from the cell–cell adhesion sites to large aggregates on apical sites of plasma membrane of HEK293 cells. The clustering of nephrin protein was induced only by antibodies produced by cDNA administration of Ig-like motif 1–2, Ig-like motif 1–8 and full-length nephrin, but not by antibodies produced by the cDNA of Ig-like motif 3–4 and Ig-like motif 5–6.
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Discussion |
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We successfully produced highly specific polyclonal antibodies against human nephrin protein by administration of nephrin cDNA using a gene-gun method. Four of the five antibodies induced by administration of cDNA of five different Ig-like motif fragments showed antigen-specific binding to fragments of E. coli-produced recombinant human nephrin protein. The remaining antibody, induced by administration of cDNA of Ig-like motif 7–8, was not able to bind any fragments of the E. coli-produced recombinant human nephrin protein. In ELISA, antibody titers induced by administration of cDNA of Ig-like motif 1–8 initially raised against the Ig-like motif 7–8 fragment at 4 weeks, reaching a maximum level against Ig-like motifs 5–6 and 7–8 at 8 weeks. This antibody also showed reactivity to Ig-like motifs 1–2 and 3–4 from 8 weeks, reaching moderate to high levels at 12 weeks. In Western blot analysis, a representative of this antibody bound mostly to Ig-like motif 7–8, moderately to Ig-like motif 5–6, and minimally to Ig-like motifs 1–2 and 3–4.
Although the antibody induced by full-length nephrin cDNA showed no reactivity to fusion protein fragments of Ig-like motifs 1–2, 3–4, 5–6 and 7–8 in ELISA, it reacted with the fibronectin-like motif of E. coli-produced recombinant human nephrin protein. It was previously reported that mouse monoclonal anti-nephrin antibodies were raised against recombinant human protein composed of Ig-like motif 1–8 and fibronectin-like motif [13]. In this previous study, of 11 antibodies, seven were raised against fibronectin-motif, three against Ig-like motif 8 and one against Ig-like motif 2. In the present study, the antibody induced by full-length cDNA reacted with the fibronectin motif only. Consequently, the fibronectin motif in the extracellular domain might be a distinguishable epitope for induction of anti-nephrin antibody. On the other hand, the cDNA of Ig-like motif 1-8, without the fibronectin motif, produced polyclonal antibody that strongly reacted with Ig-like motif 7–8. Ig-like motif 8 in the extracellular domain is therefore another possible antigenic epitope for the induction of anti-nephrin antibody.
Only three antibodies induced by administration of cDNA of Ig-like motif 1–2, Ig-like motif 1–8 and full-length nephrin, respectively, were able to bind to the native nephrin protein of normal glomeruli and plasma membranes of the HEK293 cells. Two of these three antibodies seemed to bind to the Ig-like motif without N-glycosylation, while the remaining antibody bound to the fibronectin motif after N-glycosylation. Ruotsalainen et al. [13] produced three mouse monoclonal antibodies against Ig-like motif 1–2 of human nephrin protein; however, while these three monoclonal antibodies were suitable for application where denatured nephrin was detected. A polyclonal antibody from rats immunized with cDNA of Ig-like motif 1–2 of human nephrin protein was suitable for application where both native and denatured nephrin were detected. The antibodies induced by administration of cDNA of Ig-like motifs 3–4 and 5–6 were able to bind to Ig-like motifs 3–4 and 5–6 of the fusion protein, respectively, but not the native nephrin protein. Though the products generated in rats from cDNA of Ig-like motifs 3–4 or 5–6 retained potential epitopes, the cDNA products might be either heterogeneously glycosylated or heterogeneously folded and so the antibody might not be produced against natively glycosilation sites of nephrin protein. Because the amino acid sequence of nephrin protein has six potential N-glycosylation sites in Ig-like motifs 3–6 [1]. Another problem is that the cDNA of Ig-like motifs 3–4, 5–6 and 7–8 did not have a signal array in the extracellular domain of nephrin protein, possibly influencing the structure and function of anti-nephrin antibodies. These valid antibodies reacted with what was presumed to be nephrin protein at the slit diaphragm between the foot processes of podocytes on electron microscopy.
Nephrin is an essential protein for maintaining the normal structure and filter integrity of the podocyte foot process [2]. In this study, nephrin protein appeared to form large aggregates on the apical side of the plasma membranes of HEK 293 cells after treatment with anti-nephrin antibodies. During this cluster formation, tyrosine in the intracellular domain of nephrin is phosphorylated by the Src family protein kinase p59fyn (fyn) [14]. Nephrin-dependent signalling might therefore mediate foot process effacement of glomerular podocytes. The three antibodies that reacted with Ig-like motif 1–2, Ig-like motif 1–8 and the fibronectin-like motif of the extracellular domain of native nephrin protein could therefore be used in functional and histological analyses of the glomerular foot process structure and the filtration barrier of plasma proteins in the slit diaphragm.
Genetic immunization of human thyrotropin receptor cDNA was previously shown to induce thyroiditis in rats [15]. Here, rats immunized with cDNA of full-length or fragmented human nephrin did not induce massive proteinuria in rats. Moreover, cross reactivity of the gene-gun mediated anti-human nephrin antibodies to rat nephrin protein on cryostat sections was not obvious. Further investigation is, however, needed to reveal cross reactivity to rat nephrin protein. IgG subclasses of antibodies induced by genetic immunization have been reported in mice [16,17], but not rats. In the present study, IgG1 and IgG2a antibodies were produced by fragments of extracellular domain cDNA. IgG2b antibody was predominantly induced by full-length nephrin cDNA, which contains a single transmembrane region. These three antibodies could be used in a complement-dependent assay because of the high complement activating capacity of IgG2a and IgG2b, and low activating capacity of IgG1 [18].
In a preliminary experiment, these three valid antibodies were shown to be effective in tissue staining, showing the decrease in and redistribution of human nephrin protein in diseased kidney.
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Acknowledgments |
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We thank Mr Osamu Katsumata and Mrs Naoko Ishigaki for technical assistance and Dr. Koichi Ito for helpful suggestions.
Conflict of interest statement. None declared.
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Accepted in revised form: 5. 1.06
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