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

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 80 000 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.

Figure 1

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.

Figure 2

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.

Figure 3

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).

Figure 4

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

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.