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. 2002 Jul;106(3):336–342. doi: 10.1046/j.1365-2567.2002.01432.x

Cross-reactivity of the anti-La monoclonal antibody SW5 with early endosome antigen 2

Michael A Fouraux 1, Annemarie van der Heijden 1, Walther J van Venrooij 1, Ger J M Pruijn 1
PMCID: PMC1782732  PMID: 12100721

Abstract

Coimmunoprecipitation studies with SW5, a frequently used and specific mouse monoclonal antibody (mAb) directed against the human La autoantigen, led to the identification of a functionally unrelated 80000 MW protein, designated early endosome antigen 2 (EEA2). EEA2 appeared to be directly targeted by mAb SW5. Because an RNA-binding domain, a structural element of La containing the SW5-epitope, was not discernable in the primary structure of EEA2, the SW5-epitope on EEA2 was determined. Coiled-coil region 3 of EEA2 appeared to contain the epitope recognized by SW5. The SW5 epitope regions of La and EEA2 share a limited sequence homology and probably share a higher degree of structural similarity at the tertiary level. Most likely, the most critical determinants for recognition by SW5 reside in elements adopting alpha-helical conformations. These data indicate that the application of specific mAbs to purify and characterize (functionally) interacting proteins can be severely obscured by the cross-reactivity of mAbs with structurally, but not functionally, similar proteins.

Introduction

The human La (SS-B) protein is an RNA-binding phosphoprotein that is frequently targeted by autoantibodies occurring in sera from patients with diseases like systemic lupus erythematosus and Sjögren's syndrome.1,2 This abundant protein has been identified in many eukaryotic organisms including Saccharomyces cerevisiae, Drosophila melanogaster and Xenopus laevis.3–5 La has been implicated in RNA polymerase III transcription and internal initiation of translation.6–8 The best documented function of La is the binding to and stabilization of newly synthesized RNA polymerase III transcripts and therefore La has been proposed to act as an RNA chaperone.9 La binds to its target RNAs via three ribonucleoprotein (RNP) motifs (also known as RNA recognition motifs), designated RNP-1, RNP-2 and RNP-3.9 Structural data obtained with other RNP motif containing RNA-binding proteins, as for instance nucleolin, revealed that the RNP motif adopts a globular domain with a β-α-β-β-α-β topology.10 The four β strands form a β sheet, constituting a potential RNA interaction surface, while the two α helices are positioned on the opposing side of the β sheet.

The mouse anti-La monoclonal antibody (mAb) SW5 recognizes a conformational epitope in the RNP-2 motif of La.11 Recently, coimmunoprecipitation studies with SW5 led to the identification of a novel 80000 MW protein, designated early endosome antigen-2 (EEA2). EEA2 is a member of the FYVE-finger domain protein family (conserved cysteine-rich protein domain) and is localized to early endosomes, where it functions as a putative bifunctional effector of the small GTPases rab5 and rab4 (Fouraux et al. manuscript submitted for publication). The lack of a discernable RNP motif in EEA2 raised the question which region of EEA2 is responsible for the recognition by SW5. In this study we investigated the recognition of EEA2 by SW5 and show that the cross-reactive epitope is located in coiled-coil region 3 (CC3) of EEA2.

Materials and methods

Antibodies

The mAbs against the human La/SS-B autoantigen (SW5 and SW3) have been previously described.11,12 Polyclonal antisera against EEA2 were generated by immunizing rabbits with keyhole limpet haemocyanin (KLH)-conjugated peptides corresponding to amino acids (aa) 93–107, and 302–316, respectively. Polyclonal antisera against La have been described.13 Peroxidase-conjugated swine anti-rabbit and horseradish peroxidase-conjugated rabbit anti-mouse were from Dako Immunoglobulins (Glostrup, Denmark).

Cell extracts

HeLa S100 extracts for Western blot analyses and immunoprecipitation analyses were prepared using cells purchased from Computer Cell Culture Center (Mons, Belgium). Briefly, HeLa cells were washed twice in isotonic buffer (10 mm Tris–HCl, pH 7·9, 140 mm KCl, 1·5 mm MgCl2, 1 mm ethylenediaminetetraacetic acid (EDTA), 1 mm dithioerythritol (DTE), 20% glycerol), resuspended in 2 pellet volumes of buffer A (25 mm Tris–HCl, pH 7·4, 50 mm KCl, 1·5 mm MgCl2, 1 mm EDTA, 1 mm DTE, 20% glycerol) and disrupted by dounce homogenization. Nuclei were separated by centrifugation (3000 g, 5 min) and the supernatant was first centrifuged for 20 min at 20000 g, followed by centrifugation at 100000 g for 1 hr.

Constructs and recombinant protein expression

For in vitro transcription/translation an N-terminally vesicular stomatitis virus glycoprotein epitope (VSV-G)-tagged La protein construct was used.14 Recombinant human La was expressed and purified as described previously.15 An N-terminally VSV-G-tagged human EEA2 construct has been described (Fouraux et al., manuscript submitted for publication). Mutants of EEA2 were generated using a polymerase chain reaction (PCR)-based approach by amplifying the desired regions of EEA2 using gene-specific primers and the full-length EEA2 cDNA as template. The following mutants were made: N-RUN (aa 1–290), CC13-FYVE (aa 291–708), N-RUN-CC13 (aa 1–636), N-RUN-CC1 (aa 1–378), LZ1-CC13 (aa 205–636), LZ1-CC12 (aa 205–514) and LZ1-CC1 (aa 205–378). The integrity of all constructs was confirmed by DNA sequencing. A glutathione-S-transferase (GST)–EEA2 fusion-protein construct was generated by inserting the full-length EEA2 cDNA in-frame to a GST-encoding sequence from the pGEX-4 vector (Pharmacia, Roosendaal, the Netherlands). GST–EEA2 was expressed and purified according to protocols supplied by the manufacturer (Pharmacia).

Immunoprecipitation

Protein A-agarose beads (10 µl packed beads) were coated with anti-La mAbs SW3 or SW5 (500 µl of culture supernatant; mAb concentration approx. 20 µg/ml) by overnight incubation at 4° in IPP500 (10 mm Tris–HCl, pH 8·0, 500 mm NaCl, 0·05% NP-40). A HeLa S100 extract (from 5 × 106 cells) diluted in IPP100 (10 mm Tris–HCl, pH 8·0, 100 mm NaCl, 0·05% NP-40) was mixed with either the SW5-coated protein A-agarose beads, or SW3-coated beads, or beads alone, and rotated for 2 hr at 4°. After extensive washing, coprecipitated proteins were solubilized in sodium dodecyl sulphate (SDS)-sample buffer, separated by 12% SDS–polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose and immunoblotting.

Immunoblotting

Western blots containing either HeLa S100 extracts or recombinant EEA2 were prepared, blocked in blotting buffer (5% non-fat dried milk, 0·1% NP-40, PBS) for 1 hr at room temperature, and incubated with either mAbs SW5 or SW3 (culture supernatant; mAb concentration approx. 20 µg/ml) diluted 1:100 in wash buffer or anti-EEA2 rabbit serum SN569 diluted 1:500 in blotting buffer for 1 hr at room temperature. After washing with blotting buffer, bound antibodies were detected by incubation with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin G (IgG; for SW3 and SW5) or horseradish peroxidase-conjugated swine anti-rabbit IgG (for SN569), followed by chemiluminescence.

In vitro translation and immunoprecipitation

In vitro translated 35S-methionine labelled wild-type EEA2 or La protein were synthesized using the TNT T7 Quick Coupled Reticulocyte Lysate System (Promega, Leiden, the Netherlands), using the VSV-G-tagged versions of EEA2 and La in the pCI-neo vector as templates. For immunoprecipitation analysis, protein A-agarose beads were coated with SW5 or SW3 as described above. 35S-methionine labelled EEA2 or La proteins were incubated with the mAb-coated beads in IPP300 (10 mm Tris–HCl, pH 8·0, 300 mm NaCl, 0·05% NP-40) for 1 hr at 4°. After extensive washing, bound protein was eluted in SDS-sample buffer, separated by SDS–PAGE and visualized by autoradiography. For competition assays, the mAb-coated beads were preincubated with increasing amounts of recombinant La (0, 0·1, 1 and 10 µg recLa) prior to the incubation with 35S-methionine labelled EEA2 or La proteins.

Results

SW5 precipitates EEA2 from a HeLa S100 extract

To identify proteins interacting with La we performed preparative immunoprecipitations from HeLa S100 extracts with the mAb SW5. Co-precipitated proteins were eluted from SW5-coated protein A-agarose beads with 1 m NaCl and were fractionated by 12% SDS–PAGE. Several proteins were specifically isolated from the extract in addition to the expected La and Ro60 proteins and one of these was identified as EEA2 (Fouraux et al. manuscript submitted for publication). To investigate whether EEA2 also coimmunoprecipitated from HeLa S100 with a related anti-La mAb, SW3, we probed Western blots of SW5- and SW3-immunoprecipitates with the rabbit antiserum SN569, raised against EEA2. As shown in Fig. 1(a), EEA2 was clearly detected in the SW5 precipitate (lane 4), but not in the SW3 precipitate (lane 3). This indicates that EEA2 is specifically coimmunoprecipitated by SW5, and not by the related mAb SW3. Incubating the same blot with a rabbit antiserum raised against the human La protein showed that, as expected, the La protein was precipitated by both SW3 and SW5 (Fig. 1b, lanes 3 and 4).

Figure 1.

Figure 1

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The human anti La monoclonal antibody SW5 coprecipitates EEA2 from a HeLa S100 extract (a) SW5, but not SW3, precipitates EEA2 from a HeLa S100 extract. Protein A-agarose beads coated with the anti-La mAbs SW3 or SW5, or beads alone, were incubated with HeLa S100 extract. Bound proteins were eluted and EEA2 in the eluate was detected by immunoblotting using the anti-EEA2 rabbit serum SN569. Lane 1, HeLa S100 extract (2% of the amount used in the immunoprecipitations); lane 2, beads alone; lane 3, material precipitated by SW3; lane 4, material precipitated by SW5. (b) Both SW3 and SW5 precipitate La from a HeLa S100 extract. Reprobing of the Western blot from (a) with an anti-La rabbit serum. Lane 1, HeLa extract; lane 2, beads alone; lane 3, material precipitated by SW3; lane 4, material precipitated by SW5.

SW5 cross-reacts with recombinant GST-EEA2 protein on immunoblots

The efficient coimmunoprecipitation of EEA2 by SW5 can be caused by either an interaction between EEA2 and La or to direct recognition of EEA2 by SW5. To investigate this, we probed Western blots containing a bacterially expressed fusion protein of EEA2 and GST or a HeLa S100 extract with the anti-EEA2 rabbit serum SN569 (Fig. 2, lanes 1 and 2), the anti-La mAb SW5 (Fig. 2, lanes 3 and 4) or the anti-La mAb SW3 (Fig. 2, lanes 5 and 6). SW5 reacted with recombinant GST–EEA2 on immunoblots (Fig. 2, lane 4), in contrast to SW3, which did not react with GST–EEA2 (Fig. 2, lane 6). It should be noted that SW3 is suited for immunoblotting, as indicated by the efficient detection of La in the lane containing the HeLa S100 proteins (Fig. 2, lane 5). As expected, the anti-EEA2 serum, which was used as a positive control for this protein, was reactive with both the recombinant and the HeLa EEA2 protein (lanes 1 and 2). Moreover, overexposure of an immunoblot of a HeLa S100 cell extract probed with SW5 showed that SW5 also displays reactivity with a polypeptide comigrating with EEA2 (Fig. 2, lane 7). These results demonstrate that mAb SW5 directly interacts with EEA2 on Western blots. This strongly suggests that the immunoprecipitation of EEA2 from HeLa extracts was caused by direct targeting of EEA2 by SW5.

Figure 2.

Figure 2

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SW5 reacts with recombinant GST–EEA2 on immunoblots. A HeLa S100 extract (H) and recombinant GST–EEA2 protein (R) were separated using 10% SDS–PAGE, transferred to nitrocellulose membranes, and the blots were incubated with rabbit serum SN569 (anti-EEA2; lanes 1 and 2), mouse mAb SW5 (lanes 3, 4 and 7) and mouse mAb SW3 (lanes 5 and 6). Bound antibodies were visualized by peroxidase-conjugated secondary antibodies. Arrows indicate the positions of GST–EEA2, EEA2 and La, respectively.

SW5 recognizes a structurally related epitope in La and EEA2

The results of the immunoprecipitation and Western blotting experiments described above strongly suggest that SW5 is also reactive with the folded EEA2 protein in solution. To confirm this, we subjected in vitro translated, 35S-labelled EEA2 to immunoprecipitation with SW5. As shown in Fig. 3(a), lane 2, SW5 indeed immunoprecipitated EEA2 independent of the presence of the La protein. Similar immunoprecipitation experiments that were done in the presence of increasing amounts of bacterially expressed recombinant human La protein indicated that La competes for the binding of EEA2 to SW5 (Fig. 3a, lanes 3–5). This result indicates that EEA2 shares antigenic determinants with La, which are recognized by mAb SW5. As expected, under these conditions recombinant La also competed for the binding of in vitro translated La (Fig. 3b, lanes 3–5).

Figure 3.

Figure 3

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Recombinant La competes with in vitro translated EEA2 for binding to SW5. Protein A-agarose beads coated with the anti-La mAb SW5 were incubated with increasing concentrations (0, 0·1, 1 and 10 µg recLa) of recombinant La, followed by incubation with in vitro translated 35S-methionine-labelled EEA2 (a) or in vitro translated 35S-methionine-labelled La (b). Subsequently, the beads were extensively washed, followed by solubilization of the precipitated protein and analysis by 10% SDS–PAGE and autoradiography. Lane 1, in vitro translated protein (5% of the amount used in the precipitations); lane 2, SW5 immunoprecipitation; lanes 3–5, SW5 immunoprecipitation in the presence of 0·1, 1 and 10 µg recLa, respectively; lane 6, control precipitation with beads alone and 35S-methionine-labelled EEA2 (a) or 35S-methionine-labelled La (b).

SW5 recognizes an epitope in CC3 of EEA2

Since SW5 cross-reacted with recombinant EEA2 protein both on Western blots and in solution, we were interested to delineate the region of EEA2 responsible for the recognition by SW5. To investigate this, we constructed a series of deletion mutants of EEA2 (Fig. 4a). These mutants, as well as wild-type EEA2, were produced by in vitro transcription-translation (Fig. 4b, lanes 1–8) and subjected to immunoprecipitation with either SW5 (Fig. 4b, lanes 9–16) or SW3 (Fig. 4b, lanes 18–25). All in vitro translated EEA2 mutants were reactive with the anti-EEA2 rabbit serum in immunoprecipitation analyses, suggesting efficient (re)folding of these proteins (data not shown). The results demonstrated that all mutants of EEA2 lacking CC3, namely N-LZ1, N-LZ1-CC1, LZ1-CC12 and LZ1-CC1, were not or only very inefficiently precipitated by SW5 (Fig. 4b, lanes 10, 13, 15 and 16), indicating that CC3 plays an important role in the recognition of EEA2 by SW5. SW3 did not detectably precipitate 35S-labelled EEA2 nor its mutants (Fig. 4b, lanes 18–25), in agreement with the lack of recognition of EEA2 by SW3. The results of these deletion mutant analyses are summarized in Fig. 5(b).

Figure 4.

Figure 4

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SW5, but not SW3, binds to in vitro translated EEA2 via CC3 (a) Schematic structure of EEA2 and EEA2 mutants. The RUN domain, the coiled-coil domains (CC1, CC2 and CC3), the FYVE-finger domain and the leucine zipper regions (LZ1 and LZ2) are indicated. (b) Immunoprecipitations of in vitro translated EEA2 and deletion mutants of EEA2. Protein A-agarose beads coated with the anti-La mAbs SW3 or SW5 were incubated with in vitro translated 35S-methionine-labelled EEA2 and mutants thereof, and precipitated proteins were analysed by gel electrophoresis and autoradiography. Lanes 1–8, in vitro translated proteins (5% of the amount used in the precipitations); lanes 9–16, proteins precipitated by SW5; lanes 18–25, proteins precipitated by SW3; lanes 17 and 26, control precipitations with beads alone and wild-type 35S-methionine-labelled EEA2.

Figure 5.

Figure 5

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Comparison of the SW5 epitopes of La and EEA2 (a) Schematic structure of human La. The RNP motifs are indicated by RNP-1, RNP-2 and RNP-3, and the nuclear localization signal is indicated by NLS. The region recognized by SW5 is indicated. The predicted secondary structure of the region containing the SW5 epitope (RNP-2) is depicted in the lower scheme. The arrow marks the position of glutamate-132, which plays an important role in the recognition of La by SW5. Beta strands are indicated by β1, β2, β3 and β4, alpha helices by α1 and α2. (b) Schematic structure of human EEA2. The RUN domain, the coiled-coil domains (CC1, CC2 and CC3), the FYVE-finger domain and the leucine zipper regions (LZ1 and LZ2) are indicated. The region recognized by SW5 is indicated. (c) Sequence comparison of CC3 of EEA2 and RNP-2 motif of La. The glutamate at position 132 in the La protein is indicated by an asterisk.

CC3 of EEA2 and RNP-2 of La share a moderate sequence homology

To investigate the two SW5 epitopes in more detail, we compared the primary sequences of the RNP-2 motif of the La protein and CC3 of EEA2 using the CLUSTAL W algorithm (Fig. 5c16). The CLUSTAL W alignment was performed using amino acids 524–636 (CC3) of EEA2 and amino acids 112–183 (RNP-2) of human La. The sequences share an identity of 18% and a similarity of 28%.

Discussion

In this report we describe the recognition of EEA2 by the anti-La mAb SW5. The cross-reactivity of SW5 with both recombinant EEA2 in immunoblotting and 35S-labelled EEA2 in immunoprecipitation assays indicated that SW5 recognizes an epitope on EEA2. Our previous studies have shown that SW5 specifically interacts with a structural epitope located within the RNP-2 motif of La.11 Surprisingly, such a structural element does not seem to occur in EEA2. Nevertheless, the recombinant La protein was shown to compete for the binding of EEA2 by SW5. Analysis of a series of deletion mutants of EEA2 revealed that the epitope recognized by SW5 is located in the CC3 of EEA2. A sequence comparison of the CC3 region of EEA2 and the SW5 epitope region of La (RNP-2), showed that both regions share some amino acid sequence homology. A clue for the basis of the cross-reactivity of SW5 came from the observation that SW3 did not cross-react with EEA2. Both SW3 and SW5 recognize an epitope within the RNP-2 motif of La, but their epitopes are clearly distinct. The recognition by SW5 is critically dependent on a glutamate residue at position 132 (indicated by an arrow in Fig. 5a), whereas the recognition by SW3 is not influenced by the substitution of this residue. In addition, SW5 has been shown to coprecipitate most, if not all, La associated RNAs from a cell extract, whereas SW3 coprecipitates only a specific subset of La RNPs.11 These data indicate that SW5 and SW3 recognize structurally different epitopes in the RNP-2 motif of La. The SW3 epitope is probably at least in part formed by the β sheet (the putative RNA interaction site of RNP motifs17,18) of the RNP-2 motif, since the binding of many RNAs to La seems to interfere with the accessibility of the SW3 epitope.11 The SW5-epitope of La, on the other hand, is probably located on the opposite side of the β sheet (with respect to the RNA interaction surface), where the two α-helices of the RNP-2 motif are located, which is consistent with the coprecipitation of La associated RNAs.11 When the sequence of the RNP-2 motif of La is modelled on the existing structures for RNP motifs, the position of glutamate-132 is found at the C-terminal border of the α1-helix (Fig. 5a). The whole SW5-epitope on EEA2 (CC3 region, Fig. 5b) most likely adopts an α-helical structure.19 These data strongly suggest that the key residues of the SW5 epitope reside in α-helices, present in both La and EEA2. What do our findings tell us about the reactivity of SW5 with other cellular proteins containing coiled-coil regions? The fact that SW5 is only reactive with the CC3 region of EEA2, but not with CC1 or CC2, suggests that the reactivity of SW5 with coiled-coil structures is not a general phenomenon.

The human La protein is a well-known autoantigen and autoantibodies against this protein are commonly found in sera of patients suffering from systemic lupus erythematosus and Sjögren's syndrome.20 The observed cross-reactivity of the anti-La mAb SW5 prompted us to investigate whether human anti-La autoantibodies are also cross-reactive with EEA2. Fifty human sera including 25 sera with anti-La reactivity were tested for reactivity with recombinant EEA2. The results showed that none of the 50 sera tested reacted with recombinant EEA2 (data not shown). From these data we concluded that EEA2 is not autoantigenic, and that anti-La autoantibodies do not cross-react with EEA2.

Cross-reactivity of antibodies is not uncommon. For example, antibodies immunoadsorbed to laminin have been reported to cross-react with the human La protein.21,22 In this case the laminin/La epitope consisted of a linear epitope, present in both proteins.23 On the other hand, cross-reactivity due to epitopes that share higher order similarities is much less documented in the literature. Several reports have described the cross-reactivity of the anti-Sm mAb Y12, which recognizes the Sm proteins B, B′, N, D1, D3 and E proteins24–26 with the functionally unrelated ribosomal protein S10.27 Detailed analyses revealed that Y12 recognizes a conformational epitope within both the target Sm-proteins and the S10 protein.27 Similar to what we describe here for the SW5 epitope, the Y12 epitope spontaneously renatured on Western blots after SDS–PAGE.27

As illustrated in this report by the cross-reactivity of SW5, the application of specific mAbs to purify and characterize proteins interacting with the protein targeted by the antibody can be severely obscured by the cross-reaction of the antibody with structurally, but not functionally, related proteins. As a consequence data obtained by studies in which such antibodies are applied, e.g. for immunodepletion or immunopurification, should be interpreted with great care.

Acknowledgments

The authors thank Els van Genne for mental support. This research has been financially supported by the Council for Chemical Sciences of the Netherlands Organization for Scientific Research (CW-NWO).

Abbreviations

aa

amino acid

CC

coiled-coil

DTE

dithioerythritol

EEA2

early endosome antigen-2

mAb

monoclonal antibody

NLS

nuclear localization signal

VSV-G

vesicular stomatitis virus glycoprotein epitope

References

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