Isoaspartylation appears to trigger small cell lung cancer-associated autoimmunity against neuronal protein ELAVL4

Abstract

Autoantibodies against SCLC-associated neuronal antigen ELAVL4 (HuD) have been linked to smaller tumors and improved survival, but the antigenic epitope and mechanism of autoimmunity have never been solved. We report that recombinant human ELAVL4 protein incubated under physiological conditions acquires isoaspartylation, a type of immunogenic protein damage. Specifically, the N-terminal region of ELAVL4, previously implicated in SCLC-associated autoimmunity, undergoes isoaspartylation in vitro, is recognized by sera from anti-ELAVL4 positive SCLC patients and is highly immunogenic in subcutaneously injected mice and in vitro stimulated human lymphocytes. Our data suggest that isoaspartylated ELAVL4 is the trigger for the SCLC-associated anti-ELAVL4 autoimmune response.

Data on isoaspartylation of neuronal ELAVL proteins

“…1B, bottom panel), the oligomers (which are resistant to denaturation) are present in modest amounts but appear to be unusually reactive. This figure is related to Figure 2 in Pulido et al [1].…”

“…The protein fragments contain the whole N-terminal RNA recognition motif (RRM) and amino acids N-terminal to it. Recombinant proteins were incubated under isoaspartyl-inducing conditions for 0, 1, 3 and 7 days (50 mM K-HEPES pH 7.4, 1 mM EGTA, 0.02% w/v sodium azide, 5% w/v glycerol at 37 °C as described [1]). 1 μg of each recombinant protein sample per lane was denatured for 10 min at 100 °C in reducing loading buffer (60 mM Tris–HCl pH 6.8, 2% SDS, 0.01% bromophenol blue, 5% 2-mercaptoethanol, 10% glycerol) and run on polyacrylamide gel electrophoresis-SDS gels (14%), transferred to Immun-Blot PVDF membranes (Bio-Rad, Hercules, CA, USA) and probed with an affinity-purified polyclonal rabbit antiserum [1] raised against CTSNTS-isoAsp-GPSSNNR-amide peptide carrying an isoaspartyl moiety at the central asparagine.…”

“…Recombinant proteins were incubated under isoaspartyl-inducing conditions for 0, 1, 3 and 7 days (50 mM K-HEPES pH 7.4, 1 mM EGTA, 0.02% w/v sodium azide, 5% w/v glycerol at 37 °C as described [1]). 1 μg of each recombinant protein sample per lane was denatured for 10 min at 100 °C in reducing loading buffer (60 mM Tris–HCl pH 6.8, 2% SDS, 0.01% bromophenol blue, 5% 2-mercaptoethanol, 10% glycerol) and run on polyacrylamide gel electrophoresis-SDS gels (14%), transferred to Immun-Blot PVDF membranes (Bio-Rad, Hercules, CA, USA) and probed with an affinity-purified polyclonal rabbit antiserum [1] raised against CTSNTS-isoAsp-GPSSNNR-amide peptide carrying an isoaspartyl moiety at the central asparagine. The affinity-purified anti-isoaspartyl-ELAVL4 antiserum was used at 1:250 dilution in TBST (20 mM Tris/HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) with 5% milk, washed in TBST and probed with a secondary goat anti-rabbit IgG HRP conjugate (at 1:20,000 in TBST with 3% milk) ( # sc-2004, Santa Cruz, CA, USA) followed by visualization with a chemiluminescent HRP substrate kit (Millipore, Danvers, MA, USA) on a Bio-Rad Fluor-STM MultiImager (BioRad, Hercules, CA, USA) according to the manufacturers’ instructions.…”

“…We have previously shown that the ELAVL4 aa 1–117 fragment, similar to the N-terminal fragment used to broadly characterize the ELAVL immune epitopes in numerous published studies (reviewed in [2]), becomes highly reactive with the affinity-purified anti-isoaspartyl-ELAVL4 rabbit antisera, and that both the monomer and oligomers that form during isoaspartyl conversion show reactivity to the sera [1]. Oligomerization is a known consequence of isoaspartylation [3], [4], [5], [6].…”

“…First, we provide the alignment of the N-terminal amino acid sequences of ELAVL proteins, indicating regions of homology between the proteins and a peptide used to generate a rabbit anti-isoaspartyl-ELAVL4 serum [1]. Secondly, we show Western blots documenting the reactivity of the affinity-purified rabbit antiserum with recombinant human ELAVL2, ELAVL3, ELAVL4, and ELAVL1 polypeptides (consisting of the N-terminal parts of the proteins, including the first RNA recognition motif).

Amino acid sequence alignments of neuronal ELAVL proteins (ELAVL2-4) and the ubiquitously-expressed ELAVL1 protein indicate any regions of homology with the peptide used to generate the rabbit anti-isoaspartyl-ELAVL4 antiserum.

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