Anti-HSP90 autoantibodies in sera of infertile women identify a dominant, conserved epitope EP6 (380-389) of HSP90 beta protein

Abstract

BackgroundWe earlier reported a simple specific test for detection of anti-ovarian antibodies in infertile women and identified number of specific molecular and cellular targets of which human heat shock protein 90-beta (HSP90 beta) was found to be the most immunodominant. The present study focuses on prediction and validation of the immunodominant epitope/s of this protein using sera from infertile women having anti-HSP90 autoantibodies.MethodsDelineation of the immunodominant epitopes of HSP90 beta was done by using epitope prediction algorithms and 10 peptides (EP1-EP10) were custom synthesized. Their immunoreactivity was measured by ELISA using sera from patients and controls. To determine the most immunodominant epitope, the results were subjected to statistical analysis. The immunoreactivity of the immunodominant peptides were confirmed by dot blots using sera from patients. A rabbit polyclonal antibody against the immunodominant epitope was generated and its immunoreactivity to the parent protein in ovarian extracts as well in oocytes and embryos was investigated.ResultsExperimentally and statistically, peptide EP6 (380-389) seems to be the major antigenic epitope for the serum antibody binding followed by EP1 (1-12) and EP8 (488-498). Predicted 3D structures of these peptides demonstrated that they exist in the loop conformation which is the most mobile part of the protein. Also, analysis of the sequences of HSP90 beta across several species reveals that EP6 peptide forms a part of a well conserved motif. The polyclonal antibody generated to the immunodominant epitope- EP6 confirms similar biochemical and cellular immunoreactivity as seen with the patients' sera having anti-HSP90 autoantibodies.ConclusionsThe decapeptide EP6 is a major immunogenic epitope of HSP90 followed by EP1 and EP8. Knowledge of binding epitopes on the autoantigen is necessary to understand the subsequent pathologic events. The study might generate new tools for the detection of disease-inducing epitopes and a possible therapeutic intervention.

HSP90 Antibodies: A Detrimental Factor Responsible for Ovarian Dysfunction

“…The antibody titers of pre‐ and post‐immune sera ( n = 30) obtained from the control and experimental group of mice were checked by ELISA as previously described …”

“…Forty microgram of total ovarian protein or 250 ng of recombinant human HSP90 protein (Stressgen, Glanford Ave, Canada) was electrophoretically separated and transblotted onto Nitrocellulose membrane. Blots were processed to check the immunoreactivity of pre‐ and post‐immune sera ( n = 3) diluted to 1 in 10, as previously described . Uniform loading was checked by Western Blotting with anti‐GAPDH antibody (Millipore) diluted to 1:3000 in PBS as per the protocol previously described …”

“…Blots were processed to check the immunoreactivity of pre‐ and post‐immune sera ( n = 3) diluted to 1 in 10, as previously described . Uniform loading was checked by Western Blotting with anti‐GAPDH antibody (Millipore) diluted to 1:3000 in PBS as per the protocol previously described …”

Immune‐mediated destruction of ovarian follicles associated with the presence of HSP90 antibodies

“…In the quest to understand the pathology of this disease, our group developed a specific and sensitive test (Pires et al, ) that helped establish that autoantibodies to heat‐shock protein 90 (HSP90) are observed in 47% cases of AI‐POI and repeated IVF‐ET failures (Pires and Khole, ). Using the sera from these groups of patients, we demonstrated that the EP6 decapeptide was the most antigenic region of HSP90 (Pires et al, ). Immunizing C57BL/6 female mice with the EP6 peptide revealed that autoantibodies interfere with the process of normal ovarian folliculogenesis and affect oocyte and embryo development (Choudhury and Khole, ).…”

Selenium-Binding Protein 1 (SBP1) autoantibodies in ovarian disorders and ovarian cancer

“…Ovarian insufficiency is associated with ovarian autoantibodies (Luborsky 2002, Forges et al 2006, Sundblad et al 2006, Nelson 2009, Pires & Khole 2009). Specific autoantigens such as HSP90 (Pires & Khole 2009, Pires et al 2011, Choudhury & Khole 2015) and enolase (Sundblad et al 2006) were reported in POF and infertility, although autoantibodies to these antigens also occur as part of the background antibody repertoire in healthy individuals (Pashov et al 2002). Using immune-proteomics, we found several unique autoantigens including Selenium Binding Protein 1 (SBP1) associated with infertility and POF (Edassery et al 2010).…”