Basic Research

Identification of the Immunodominant Epitope Region in Phospholipase A2 Receptor-Mediating Autoantibody Binding in Idiopathic Membranous Nephropathy

Liyo Kao, Vinson Lam, Meryl Waldman, Richard J. Glassock and Quansheng Zhu
JASN February 2015, 26 (2) 291-301; DOI: https://doi.org/10.1681/ASN.2013121315

Abstract

Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. Recent clinical studies established that >70% of patients with idiopathic (also called primary) MN (IMN) possess circulating autoantibodies targeting the M-type phospholipase A2 receptor-1 (PLA2R) on the surface of glomerular visceral epithelial cells (podocytes). In situ, these autoantibodies trigger the formation of immune complexes, which are hypothesized to cause enhanced glomerular permeability to plasma proteins. Indeed, the level of autoantibody in circulation correlates with the severity of proteinuria in patients. The autoantibody only recognizes the nonreduced form of PLA2R, suggesting that disulfide bonds determine the antigenic epitope conformation. Here, we identified the immunodominant epitope region in PLA2R by probing isolated truncated PLA2R extracellular domains with sera from patients with IMN that contain anti-PLA2R autoantibodies. Patient sera specifically recognized a protein complex consisting of the cysteine-rich (CysR), fibronectin-like type II (FnII), and C-type lectin-like domain 1 (CTLD1) domains of PLA2R only under nonreducing conditions. Moreover, absence of either the CysR or CTLD1 domain prevented autoantibody recognition of the remaining domains. Additional analysis suggested that this three-domain complex contains at least one disulfide bond required for conformational configuration and autoantibody binding. Notably, the three-domain complex completely blocked the reactivity of autoantibodies from patient sera with the full-length PLA2R, and the reactivity of patient sera with the three-domain complex on immunoblots equaled the reactivity with full-length PLA2R. These results indicate that the immunodominant epitope in PLA2R is exclusively located in the CysR-FnII-CTLD1 region.

Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. It can be a primary form (approximately 60%–80% of patients with MN depending on region) without identified causes (idiopathic MN [IMN]) or a secondary form associated with various autoimmune diseases, infections, and cancers.1–5 MN is characterized by the presence of immune deposits in the subepithelial space, resulting in thickened capillary walls and podocyte foot process effacement.6 In the rat MN model (Heymann nephritis), antibodies interact with the target antigen on the podocyte surface that triggers the formation of the membrane attack complex (complement C5b-9), resulting in podocyte injury.7 Intriguingly, in human IMN, the predominant IgG subclass in the immune deposits is IgG4,8 which is known to be ineffective to trigger the classic complement pathway activation, although the mannan-binding lectin pathway was recently predicted to be involved.9

The antigen in rat podocyte responsible for Heymann nephritis was identified to be megalin10,11; however, megalin is not found in the human podocyte.12,13 Two membrane proteins, neutral endopeptidase14 and M-type phospholipase A2 receptor (PLA2R),8 on the basal surface of podocytes have recently been identified as serving as the antigens for alloimmune or autoimmune antibody targeting in human IMN, respectively. Moreover, approximately 70%–80% of patients with IMN were found to possess anti-PLA2R autoantibodies in circulation, and the level of autoantibody correlates with the level of proteinuria.8,15 PLA2R is highly expressed in the kidney podocytes,8 alveolar type II epithelial cells,16 and neutrophils.17 Surprisingly, the anti-PLA2R autoantibody only induces proteinuria and nephrotic syndrome without apparent lung or other organ functional impairment.12 In addition, the autoantibody does not form immune complexes with the soluble form of PLA2R in the circulation.8 Taken together, these findings suggest that the autoantibody may only target a specific conformational region of PLA2R protein on the podocyte surface.

PLA2R is a type I transmembrane glycoprotein (molecular mass is approximately 180 kD) that belongs to the mannose receptor family.18,19 It is predicted to function as a scavenger receptor for binding and removal of the secreted PLA2 enzyme from circulation.20 PLA2R is also reported to serve as a signaling molecule on the cell surface that mediates cellular responses21,22 and cell senescence.23 PLA2R consists of a large glycosylated extracellular portion that interacts with ligands, a single transmembrane region, and a short cytoplasmic tail (Figure 1). The extracellular portion can be further divided into 10 domains: an N-terminal cysteine-rich domain (CysR), a fibronectin-like type II domain (FnII), and eight repeated C-type lectin-like domains (CTLDs). The PLA2R extracellular portion has been predicted to adopt either a bent or an extended conformation on the cell surface on the basis of mannose receptor24; however, whether each of the domains in PLA2R is arranged similarly to that of the mannose receptor in three dimensions is not clear.

Figure 1.
Figure 1.

Structure model of PLA2R. PLA2R protein consists of a larger extracellular portion, a single transmembrane region, and a short cytoplasmic tail. The large extracellular portion contains 10 domains: a CysR (dark gray hexagon), an FnII (gray square), and 8 repeated CTLD (light gray ovals) domains. The polymorphisms that may link to the occurrence of IMN are indicated as white circles. The positions of amino acids used for designing experimental constructs are indicated by arrows.

To understand the pathogenic mechanism of anti-PLA2R autoantibody binding-induced IMN, a clear picture of the antigenic epitope in PLA2R is a prerequisite. Genetic screens have identified multiple polymorphisms in the extracellular portion of PLA2R; however, only the polymorphisms M292V and H300D in C-type lectin-like domain 1 (CTLD1) and G1106S in the linker region between CTLD6 and CTLD7 may correlate to the occurrence of IMN in patients.25,26 The antigenic epitope in PLA2R is located in the extracellular portion and sensitive to reduction,8 suggesting that it is a conformational epitope that requires specific disulfide bonds. A recent peptide mapping study also suggested that the autoantibody may bind to various regions distributed throughout of the PLA2R extracellular portion.27

In this study, we examined in detail the antigenic epitope in PLA2R by using patient serum containing anti-PLA2R autoantibodies. Our results unambiguously showed that the immunodominant epitope region in PLA2R responsible for autoantibody interaction is formed by the CysR, FnII, and CTLD1 domains.

Results

Design and Construction of Truncated PLA2R Extracellular Domains

The autoantibody only recognizes the nonreduced PLA2R protein on immunoblot,8 suggesting that the antigenic epitope in PLA2R is a conformational epitope that requires precise formation of the involved disulfide bonds. PLA2R contains 56 endogenous cysteines that are distributed throughout the extracellular portion. How these cysteines are paired to form disulfide bonds is currently not known. To prevent potential structural alteration caused by protein truncation, we selectively truncated the PLA2R extracellular portion in the linker regions between each of the domains at amino acid positions of Ile-169 (1–1), Gly-228 (1–2), Glu-367 (1–3), Asp-510 (1–4), Glu-651 (1–5), Lys-805 (1–6), Leu-944 (1–7), Val-1107 (1–8), Pro-1236 (1–9), and Asn-1322 (1–10) (Figure 1). The 1–1 construct only contains the CysR domain; the 1–2 construct contains the CysR and FnII domains and proceeds to the 1–10 construct that contains the whole extracellular portion. A 9-amino acid peptide (TETSQVAPA) from rhodopsin (1D4 tag) was introduced at the C terminus of each construct (Figure 2A). The 1D4 tag was designed for detecting the truncated PLA2R extracellular domains by a mouse anti-1D4 mAb on immunoblots.

Figure 2.
Figure 2.

Design and expression of truncated PLA2R extracellular domains. (A) The cartoon shows the constructs containing the truncated PLA2R extracellular domains. Dark gray hexagon, CysR; gray line, 1D4 tag; gray square, FnII; light gray oval, CTLD. (B) The expressed truncated PLA2R extracellular domains were concentrated and resolved on a 4%–20% SDS-PAGE under nonreducing conditions. Protein samples were then transferred to a nitrocellulose membrane and detected with a mouse anti-1D4 mAb. The experiment was performed at least three times.

Expression of Truncated PLA2R Extracellular Domains

The truncated PLA2R extracellular domains were individually expressed in the human embryonic kidney (HEK) 293 cells by transient transfection. Figure 2B shows that all of the truncated PLA2R extracellular domains were well expressed, and interestingly, the amount of expressed proteins was inversely related to the size of truncated PLA2R extracellular domains, with the 1–10 construct having the lowest level of protein expression.

Immunodetection of Truncated PLA2R Extracellular Domains with Patient Serum Containing Anti-PLA2R Autoantibodies

To determine which of the PLA2R extracellular domains carries the antigenic epitope for autoantibody recognition, we tested immunoblotting of each of the constructs with a patient serum containing anti-PLA2R autoantibodies under the nonreducing condition. Before the test, we verified the presence of autoantibody in the serum by probing HEK 293 cell lysate containing full-length PLA2R. Figure 3 shows that the serum containing anti-PLA2R autoantibodies strongly recognized the PLA2R protein that was resolved under the nonreducing condition at 1:1000 dilutions but did not recognize the protein sample that was reduced by 2% β-mercaptoethanol (β-ME), indicating the anti-PLA2R autoantibody in the serum carries the same characteristics as those reported previously.8 The level of PLA2R protein in each of the samples was determined by a rabbit anti-human PLA2R antibody (Sigma-Aldrich), which showed that an equal amount of PLA2R protein was present (Figure 3).

Figure 3.
Figure 3.

Characterization of patient serum containing anti-PLA2R autoantibodies. HEK 293 cells expressing the full-length PLA2R protein were lysed in the lysis buffer containing protease inhibitors and resolved under the nonreducing and reduced conditions. Protein samples were transferred to a nitrocellulose membrane and probed with the patient serum at 1:1000 dilutions in the immunoblotting buffer. The membrane was then stripped and reprobed with a rabbit anti-PLA2R antibody (Sigma-Aldrich) to determine the level of protein in each sample. The assay was performed at least three times.

The truncated PLA2R extracellular domains were then resolved on a 4%–20% SDS-PAGE under the nonreducing condition, transferred to a nitrocellulose membrane, and probed with the characterized patient serum at 1:1000 dilutions. Figure 4A shows that the PLA2R 1–1 or 1–2 constructs were not recognized, whereas the constructs containing the first three domains were all strongly recognized by the patient serum. Anti-1D4 antibody probing of the stripped membrane showed that both of the 1–1 and 1–2 constructs were present at a high level (Figure 4B). This result indicates that the first three domains, CysR, FnII, and CTLD1, are involved in forming the conformational antigenic epitope in PLA2R.

Figure 4.
Figure 4.

Immunodetection of truncated PLA2R extracellular domains with the characterized patient serum containing anti-PLA2R autoantibodies. (A) The transferred nitrocellulose membrane was incubated with the characterized patient serum at 1:1000 dilutions in immunoblotting buffer for 2 hours at room temperature. The membrane was then washed with TBST and incubated with HRP-conjugated rabbit anti-human secondary antibody (1:10,000) in immunoblotting buffer. (B) The nitrocellulose membrane was stripped and then reprobed with a mouse anti-1D4 antibody. The experiment was performed three to five times.

To test if the 1–3 construct carries indispensable intramolecular disulfide bonds required for the conformational epitope formation, we resolved the 1–3 construct under the nonreducing and reduced conditions on SDS-PAGE. Figure 5 shows that the nonreduced 1–3 construct but not the reduced form was strongly recognized by the anti-PLA2R autoantibody, indicating that the secreted 1–3 construct contains the correctly formed disulfide bonds resembling those of the full-length PLA2R protein. Moreover, the reduced 1–3 construct appeared to migrate much slower on the SDS-PAGE, suggesting that breakage of the internal disulfide bonds changed the protein conformation. The negative control (1–2 construct) was not recognized by the anti-PLA2R autoantibody but also appeared to migrate slower under the reduced condition (Figure 5), indicating that the contained CysR and FnII domains are folded properly with intramolecular disulfide bonds formed.

Figure 5.
Figure 5.

Characterization of the isolated PLA2R 1–3 construct. The isolated 1–2 and 1–3 constructs were resolved under nonreducing and reduced conditions (2% β-ME) on a 4%–20% SDS-PAGE. The 1–2 construct was included to serve as a negative control. The membrane was then probed with the characterized patient serum and reprobed with the anti-1D4 antibody as described in Figure 4. The experiment was performed three to five times.

To further determine if the antigenic epitope is exclusively located in the CTLD1 domain, we cloned the isolated CTLD1 domain (Asp-203 to Glu-367; including the signal peptide) in a mammalian expression vector. However, the cloned protein was not well expressed in the HEK 293 cells and biochemically unstable. To circumvent this technical challenge, we engineered a proteolytic site (thrombin; LVPRGS) in the linker regions between CysR and FnII (1–1T; at Leu-166 to Gly-171) or FnII and CTLD1 (1–2T; at Thr-231 to Asp-236) domains, respectively (Figure 6A). Figure 6B shows that the autoantibody recognized the expressed 1–1T and 1–2T constructs well but had no recognition of these constructs after thrombin digestion, suggesting that thrombin cleavage dramatically altered the configuration of the antigenic epitope when resolved on the SDS-PAGE. Reprobing of the stripped membrane with anti-1D4 antibody revealed that a 25-kD protein fragment corresponding to the FnII-CTLD1 domain was released from the 1–1T construct but that no protein fragment was released from the digested 1–2T construct (Figure 6C). Additional treatment of the digested 1–2T construct with 2% β-ME showed that a 23-kD protein fragment corresponding to the CTLD1 domain was released (Figure 6D). These results showed that the FnII and CTLD1 domains are likely to be interlocked by intermolecular disulfide bond(s), and formation of the conformational antigenic epitope requires regions from the CysR, FnII, and CTLD1 domains.

Figure 6.
Figure 6.

Identification of the domains in isolated PLA2R 1–3 construct responsible for anti-PLA2R autoantibody recognition. (A) The cartoon indicates the locations of introduced thrombin digestion sites in the 1–3 construct. 1–1T is the thrombin digestion site that was introduced between CysR and FnII domains, and 1–2T is the thrombin digestion site that was introduced between FnII and CTLD1 domains. (B and C) The thrombin untreated and treated 1–3, 1–1T, and 1–2T constructs were resolved under nonreducing conditions on a 4%–20% SDS-PAGE. The 1–3 construct was included to serve as a positive control. The membrane was (B) first probed with the characterized patient serum and (C) then reprobed with the anti-1D4 antibody as described in Figure 4. Asterisks indicate thrombin-treated protein samples. (D) The thrombin untreated and treated 1–3, 1–1T, and 1–2T constructs were resolved under reducing conditions (2% β-ME) on a 4%–20% SDS-PAGE and probed with the anti-1D4 antibody. Each experiment was performed three to five times.

We next performed epitope competition assay to test if the natively folded 1–3 construct can sufficiently block the autoantibody binding to the full-length PLA2R protein in the liquid phase. Figure 7A shows that the 1–10 construct (control; whole extracellular portion) almost completely blocked the reactivity of the anti-PLA2R autoantibody with the full-length PLA2R, like the 1–3 construct at a stepwise increased concentration. We then individually preincubated 10 patient sera containing a high level of autoantibodies with 1–3 construct at the dilution of 1:9 (vol/vol) and applied the mixture on the PLA2R protein transferred to a nitrocellulose membrane. Figure 7, B and C shows that the 1–3 construct completely blocked the anti-PLA2R sera reactivity with the full-length PLA2R protein, indicating that the 1–3 construct sufficiently absorbed all of the autoantibodies in the sera.

Figure 7.
Figure 7.

Immunoblocking of the autoantibody reaction with the full-length PLA2R by the 1–3 construct. (A) The 1–3 construct was stepwise diluted in the TBS buffer and incubated with an anti-PLA2R serum for 2 hours at room temperature before application on a blot transferred with nonreduced full-length PLA2R protein. Control, 1–10 construct (undiluted) blocked anti-PLA2R serum; 1, TBS buffer only; 2–10, 1–3 construct at dilutions (vol/vol) of 1:600, 1:300, 1:150, 1:75, 1:37.5, 1:18.75, 1:9.38, 1:4.69, and 1:2.35, respectively. (B) Sera from 10 patients with high levels of anti-PLA2R autoantibodies were first incubated with the 1–3 construct at 1:9 dilutions (vol/vol) for 2 hours at room temperature and then applied on the full-length PLA2R protein on a blot (−, without the 1–3 construct; +, with the 1–3 construct). (C) Summary of the 1–3 construct blocked sera reactivity with the full-length PLA2R. Error bars represent the means±SEMs (n=5).

To further determine if 1–3 construct exclusively harbors the conformational epitope, we selectively deleted the 1–3 construct or CTLD1 domain from the constructs 1–8 and 1–10 (whole extracellular portion) and expressed it in the HEK 293 cells (Figure 8A). We observed that autoantibody strongly recognized the positive control (1–3 construct) but had no recognition on the negative control (1–2 construct). When the 1–3 construct (1–8* and 1–10*) or the CTLD1 domain (1–8** and 1–10**) was absent, the remaining PLA2R domains were well expressed; however, none could be recognized by the autoantibody (Figure 8B). We also engineered a construct with a thrombin digestion site introduced in the linker region between the CTLD1 and CTLD2 domains (Ile-365 to Ala-370) in the full-length PLA2R (PLA2R-T). PLA2R-T was expressed in the HEK 293 cells and digested with thrombin to produce a fragment containing 1–3 domains and a fragment containing the remaining PLA2R domains. Figure 8C shows that the autoantibody recognized the 1–3 domain fragment; however, it had no recognition of the remaining PLA2R domains. Figure 8D summarizes the reactivity of autoantibodies from 10 patient sera with the constructs of 1–8*, 1–10*, and thrombin-digested PLA2R-T. These findings show clearly that the 1–3 construct in PLA2R is solely responsible for the anti-PLA2R autoantibody binding in vitro.

Figure 8.
Figure 8.

Immunodetection of the 1–3 construct or CTLD1 domain-deleted PLA2R constructs with anti-PLA2R sera from patients with IMN. (A) The cartoon shows the constructs with the 1–3 construct or CTLD1 domain deletion and a full-length PLA2R with an introduced thrombin digestion site between CTLD1 and CTLD2 domains. Deleted domains are indicated by unfilled shapes. (B) Truncated PLA2R extracellular domains 1–2, 1–3, 1–8 with 1–3 deletion (1–8*), 1–8 with CTLD1 deletion (1–8**), 1–10 with 1–3 deletion (1–10*), and 1–10 with CTLD1 deletion (1–10**) were (left panel) resolved under nonreducing condition and probed with the characterized patient serum. The 1–2 and 1–3 constructs were used as a negative and a positive control, respectively. (Right panel) The level of protein in each sample was determined by the anti-1D4 antibody. (C) Full-length PLA2R, PLA2R-T, and thrombin-digested PLA2R-T (PLA2R-T*) were resolved under the nonreducing condition and processed as described above. (Left panel) Anti-PLA2R serum. (Right panel) Anti-PLA2R antibody (Sigma-Aldrich). (D) Summary of anti-PLA2R sera reactivity with PLA2R, PLA2R-T*, 1–8*, and 1–10* (+++, strongly recognized; −, background level). Each experiment was performed three to five times.

Effect of Polymorphism on the Affinity of PLA2R for Autoantibody Binding

Genetic screens have suggested that two polymorphisms, M292V and H300D, in the CTLD1 domain correlate strongly with the occurrence of IMN in patients.25,26 We predicted that, if the polymorphisms are responsible for generating the autoantibody, polymorphic PLA2R would have higher affinity for the antibody than the wild type. To test it, we individually substituted Met-292 with Val and His-300 with Asp in the wild-type PLA2R. Polymorphic proteins and the wild-type PLA2R were then expressed in the HEK 293 cells and probed with the characterized patient serum at three dilutions in immunoblotting buffer: 1:100, 1:1000, and 1:10,000. Figure 9 shows that no difference could be detected between the wild-type PLA2R and either of the polymorphic proteins, suggesting that the polymorphisms may not be solely responsible for developing the anti-PLA2R autoantibody.25

Figure 9.
Figure 9.

Immunodetection of PLA2R with polymorphism M292V and H300D. (A) Whole-cell lysates expressing wild-type PLA2R (wt-PLA2R), PLA2R-M292V, or PLA2R-H300D were resolved under nonreducing conditions and probed with the characterized patient serum at three dilutions in immunoblotting buffer. (B) The level of antibody binding to each sample was quantified by densitometry. In each experiment, the level of antibody binding was compared with that of wt-PLA2R, which had antibody binding set to 100%. Error bars represent the means±SEMs (n=3–5).

Comparison of the PLA2R 1–3 Construct and the Full-Length PLA2R for Patient Sera Immunoscreening

To determine if the 1–3 construct could serve as the antigenic epitope in a large patient pool, we analyzed 74 serum samples from patients with IMN before and after treatment (Table 1). We tested side by side using the 1–3 construct and the full-length PLA2R for serum probing on the immunoblots. Figure 10A and Table 1 show that the sera containing anti-PLA2R autoantibodies identified by the full-length PLA2R are also positive with the 1–3 construct. Moreover, the 1–3 construct was recognized equally as the full-length PLA2R at the three titers that we tested up to 1:10,000 dilutions (Figure 10B). This result clearly shows that the 1–3 construct is equally efficient as the full-length PLA2R for a large-scale patient sample screening and more importantly, that the immunodominant antigenic epitope in PLA2R is exclusively formed by the CysR-FnII-CTLD1 domain complex.

View this table:
Table 1.

Summary of patient sample anti-PLA2R autoantibody screen using full-length PLA2R and PLA2R 1–3 construct containing the CysR-FnII-CTLD1 domain

Figure 10.
Figure 10.

Representative results of immunoscreen of patient samples with the PLA2R 1–3 construct and the full-length PLA2R. (A) HEK 293 cell lysate containing heterologously expressed full-length PLA2R protein and the PLA2R 1–3 construct was resolved under nonreducing condition and transferred to nitrocellulose membranes. After 30 minutes of blocking with immunoblotting buffer, the membranes were assembled into a multiscreen apparatus and individually incubated with each of the patient samples at 1:100 dilutions for 2 hours at room temperature. (B) Representative results of full-length PLA2R and the 1–3 construct immunoblotted with the characterized patient serum at three dilutions: 1:100, 1:1000, and 1:10,000 (vol/vol). The assay was performed three times.

Discussion

In this study, we showed, for the first time, that the immunodominant antigenic epitope in PLA2R responsible for autoantibody binding is exclusively formed by a region encompassing the CysR, FnII, and CTLD1 domains. Our conclusion is supported by the following evidence. (1) Autoantibody did not recognize the CysR, CysR-FnII, or FnII-CTLD1 domain but strongly recognized the CysR-FnII-CTLD1 domain complex (1–3 construct). (2) Autoantibody only recognized the nonreduced 1–3 construct but not the reduced form. (3) When the 1–3 construct was absent, autoantibody did not recognize any of the remaining domains. (4) The 1–3 construct in its native conformation completely blocked the reactivity of 10 patient sera containing high levels of autoantibodies with the full-length PLA2R. (5) The 1–3 construct was recognized as effectively as the full-length PLA2R by autoantibodies from various serum samples from patients with IMN.

The anti-PLA2R autoantibody is known to recognize only the nonreduced form of PLA2R. Our results showed that the isolated 1–3 construct containing CysR, FnII, and CTLD1 domains indeed contains the critical intramolecular disulfide bonds required for the antigenic epitope formation and that autoantibody recognition of the 1–3 construct is sensitive to β-ME reduction. Because autoantibody does not recognize the isolated CysR, CysR-FnII, or FnII-CTLD1 domain, the CyR and CTLD1 domains are likely to be responsible for the 1–3 construct recognition, and potentially, a cryptic region in the FnII domain is also involved. This conclusion is strongly supported by the observation that the 1–3 construct completely blocked the reactivity of autoantibody with the full-length PLA2R in the liquid and solid phases, and the PLA2R domains without the 1–3 construct were not recognized by the autoantibody. Our results also showed that the CTLD1 domain is interlocked with the FnII domain through disulfide bond(s), suggesting that the FnII domain has an important structural role to bring the CysR and CTLD1 domains in a close proximity for the epitope formation.

To further determine if our finding is applicable to a large number of patients with IMN (but not only to a unique serum sample from a specific patient), we compared the efficiency of autoantibody recognition on the full-length PLA2R and 1–3 construct side by side with 74 serum samples from patients with IMN before and after treatment. Our results showed that patient sera recognizing the full-length PLA2R also strongly recognized the 1–3 construct, whereas patient sera negative on the full-length PLA2R were also negative on the 1–3 construct, showing that the CysR-FnII-CTLD1 domain complex can serve as the universal autologous antigen in all patients with IMN possessing the autoantibody.

Recent studies using small peptide mapping27 or physicochemical and imaging methods28 have indicated that the epitope for autoantibody binding may be located in either various regions distributed throughout the PLA2R extracellular portion or the CTLD3 domain exclusively. Because our experiments examined the location of the epitope using intact PLA2R extracellular domains and patient sera containing anti-PLA2R autoantibodies, the difference between our findings and these observations is potentially because of differences in the experimental approaches used. Our findings do not rule out the possibilities that other cryptic epitopes may appear in different experimental conditions.

Two polymorphisms, M292V and H300D, in CTLD1 domain have been linked to the occurrence of IMN in patients. Our finding that the CTLD1 domain harbors the autogenic antigen seems to support the prediction that polymorphism alters the conformation of the CTLD1 domain, leading to autoantibody generation. However, on 10,000 dilutions, the tested patient serum showed no differences in binding to the wild-type PLA2R, PLA2R-M292V, or PLA2R-H300D. Our data support the previous genetic findings that the potential conformational change on the surface of PLA2R may not be fully responsible for the generation of autoantibody in patients.25,29

Our findings have important clinical applications. We have shown that, compared with the full-length PLA2R, the 1–3 construct can be obtained at a high level in the HEK 293 cell culture medium and that it is equally efficient to the full-length PLA2R for large-scale patient sample screening for autoantibodies. Our finding offers a potentially valuable tool for developing sensitive and efficient ELISA assays for IMN diagnosis and prognosis.

Concise Methods

Patient Sera

Deidentified patient sera were collected at the Clinical Research Center, National Institute of Diabetes and Digestive and Kidney Diseases/Kidney Disease Section according to the Institutional Review Board.

Molecular Cloning

A wild-type human PLA2R cDNA was used as the template for constructing truncated PLA2R extracellular domains. The PCR fragments generated by Easy-A High-Fidelity PCR Cloning Enzyme (Agilent Technologies) were first cloned into the pCR2.1 TA cloning vector (Invitrogen) and then subcloned into the pcDNA3.1− vector using EcoRI site. The reverse primers for PCR reaction were all tagged with 27 nucleotides encoding 9 amino acids (TETSQVAPA, 1D4 tag) followed with a stop codon. In separate experiments, a thrombin digestion site was introduced into the 1–3 construct or wild-type PLA2R using the Strategene site-directed mutagenesis kit following the manufacturer’s protocol. The complete sequence of each construct was verified by DNA sequencing.

Protein Expression

The truncated PLA2R extracellular domains were transiently expressed in the HEK 293 cells (ATCC) maintained in Gibco FreeStyle 293 expression medium. Briefly, HEK 293 cells were plated onto 10-cm dishes in 10 ml serum-free medium 16 hours before transfection. HEK 293 cells were then transfected with various PLA2R constructs using TurboFect reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. Because each construct contains the endogenous signal peptide, the expressed proteins were secreted into the cell culture medium. To increase the amount of secreted proteins, we supplemented the transfected cells with fresh culture medium 48 hours post-transfection and further expressed the proteins for 24 hours.

Protein Sample Preparation

Seventy-two hours post-transfection, culture medium from each of the plates was collected into 50-ml Falcon tubes and centrifuged at 2000 rpm for 10 minutes at 4°C. The supernatant was then loaded onto Amicon Ultra-15 Centrifugal Filter Units (EMD Millipore) and centrifuged at 4000 rpm for 30 minutes at 4°C. The concentrated protein samples were collected, and the protein concentration of each of the samples was determined by the bicinchoninic acid assay method. The protein samples were then diluted with Tris-buffered saline (TBS; 20 mM Tris, 137 mM NaCl, pH 7.5) to equal volumes and stored at 4°C. In additional experiments, HEK 293 cells expressing full-length PLA2R (6-cm plate) were lysed in 200 µl lysis buffer (5 mM EDTA, 150 mM NaCl, 1% [vol/vol] Igepal, 0.5% [wt/vol] sodium deoxycholate, 10 mM Tris-HCl, pH 7.5) containing protease inhibitors (Roche), and PLA2R protein was then mixed with SDS-sample buffer and processed for SDS-PAGE.

Protease Digestion

Protein samples were mixed with human thrombin (EMD Millipore) at 1 unit/10 µl in TBS buffer and incubated on a rotating shaker for 6–12 hours at room temperature. Samples were then mixed with 2× SDS sample buffer and processed for SDS-PAGE. In separate experiments, plasma membranes from HEK 293 cells expressing full-length PLA2R with an introduced thrombin site were isolated and lysed in the lysis buffer, and PLA2R protein was then digested with thrombin at 1 unit/10 µl.

SDS-PAGE and Immunoblotting

Protein samples were mixed with 2× SDS sample buffer without or with 2% β-ME and heated at 100°C for 5 minutes. Truncated PLA2R extracellular domains were then resolved on 4%–20% SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad), and probed by the mouse anti-1D4 antibody (Flintbox) at 1:10,000 dilution in the immunoblotting buffer (Tris-buffered saline/Tween-20 [TBST]; 0.1% [vol/vol] Tween-20, 137 mM NaCl, and 20 mM Tris [pH 7.5] containing 5% [wt/vol] nonfat dry milk). The membrane was then washed with TBST buffer, incubated with horseradish peroxidase (HRP) -conjugated rabbit anti-mouse IgG (The Jackson Laboratory) antibodies at a dilution of 1:10,000, and processed with enhanced chemiluminescence (ECL) reagent (GE Healthcare). For patient serum probing, transferred nitrocellulose membranes were first blocked with immunoblotting buffer for 30 minutes, and then, patient serum was added at 1:1000 dilutions and incubated for 2 hours at room temperature. The membrane was then washed with TBST buffer, incubated with HRP-conjugated rabbit anti-human IgG (Santa Cruz Biotechnology) antibodies for 1 hour at a dilution of 1:10,000, and processed with ECL reagent.

Epitope Competition Assay

Patient sera were first diluted 10 times in TBS buffer; 6 µl diluted sera were then mixed with the 1–3 construct (in TBS buffer) at various dilutions (60 µl total volume) and incubated on a rotating shaker for 2 hours at room temperature. The samples were then mixed with 600 µl immunoblotting buffer and applied on full-length PLA2R protein using the Mini-PROTEAN II Multiscreen Apparatus (Bio-Rad) as described below.

Patient Sera Screening

The anti-PLA2R autoantibody level in patient serum was screened using the Mini-PROTEAN II Multiscreen Apparatus. Briefly, nitrocellulose membranes were first blocked with the immunoblotting buffer for 30 minutes and then assembled into the multiscreen apparatus. Each of the patient sera was applied at 1:100 dilutions (in immunoblotting buffer) onto the membrane and incubated at room temperature for 2 hours. The membrane was then washed three times with TBST buffer and incubated with HRP-conjugated rabbit anti-human IgG (Santa Cruz Biotechnology) antibodies at a dilution of 1:10,000. The membranes were incubated with ECL reagent for 1 minute and exposed to Amersham High Performance Chemiluminescence Film (GE Healthcare). The exposure times were 30–60 seconds for positive bands and up to 5 minutes for weak or negative bands.

Image and Data Analyses

Films from immunoblots were scanned with a Hewlett-Packard Scanjet 5590. Scanned images were quantified with UN-SCAN-IT Gel, version 6.1 software.

Statistical Analyses

Means±SEMs were calculated using SigmaPlot 10 software. Dunnett t test was used to assess statistical significance, with P<0.05 considered significant.

Disclosures

Q.Z. holds a patent to use the identified conformational epitope for developing ELISA essays for patient diagnosis and prognosis and developing therapeutic peptides for patient treatment.

Acknowledgments

This work was supported by the Norman S. Coplon Grant from Satellite Healthcare (to Q.Z.).

Footnotes

  • Published online ahead of print. Publication date available at www.jasn.org.

  • See related editorial, “The Dominant Humoral Epitope in Phospholipase A2 Receptor-1: Presentation Matters When Serving Up a Slice of π,” on pages 237–239.

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Identification of the Immunodominant Epitope Region in Phospholipase A2 Receptor-Mediating Autoantibody Binding in Idiopathic Membranous Nephropathy
Liyo Kao, Vinson Lam, Meryl Waldman, Richard J. Glassock, Quansheng Zhu
JASN Feb 2015, 26 (2) 291-301; DOI: 10.1681/ASN.2013121315
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