O53
Understanding the structure of PLA2R: insights into formation of the immunodominant
epitope recognised by autoantibodies in patients with idiopathic membranous nephropathy
Maryline Fresquet1, Thomas Jowitt1, Jennet Gummadova2, Edward McKenzie2, Rachel Lennon3,
Paul Brenchley4
1
University of Manchester; 2Manchester Institute of Biotechnology; 3Department of Paediatric
Nephrology, Royal Manchester Children's Hospital; 4Manchester Royal Infirmary.
BACKGROUND: 75% of patients with idiopathic membranous nephropathy (IMN) have
autoantibodies against the phospholipase A2 receptor (PLA2R). The epitope(s) within PLA2R and
its 3D structure are unknown.
OBJECTIVES: To understand how autoantibodies to PLA2R interact with the target receptor.
METHODS: To identify the dominant epitope, PLA2R was trypsin digested, separated on
unreduced gels and peptides reacting to patient sera were identified by western blotting. Bands of
16, 20 and 37kDa were excised, reduced and analysed by MS. The structure of both PLA2R and an
immunoreactive PLA2R fragment, NCTLD3, were investigated using sedimentation, SAXS and
3D electron tomography. A Surface Plasmon Resonance (SPR) method was developed to assess
binding kinetics of patient antibodies to PLA2R and NCTLD3.
RESULTS: MS analyses suggest 3 peptides constitute part of the PLA2R epitope. The distribution
of these peptides is discontinuous on the linear sequence but could be in proximity within the
folded molecule.
Hydrodynamic experiments showed PLA2R exists in two distinct pH-dependent conformations
similar to other mannose receptor family members. Low resolution ab initio structures from SAXS
and EM have enabled us to construct a model of both full-length and NCTLD3 PLA2R.
SPR analysis of patient sera revealed equivalent autoantibody binding kinetics to both full-length
PLA2R and NCTLD3. Antibody affinity was also similar in all five patient sera tested.
CONCLUSIONS: We identified a specific region on PLA2R as the potential immunodominant
epitope involved in the interaction with its autoantibody. This epitope appears to be solely within
the N-terminal CTLD3 region as shown by SPR, and there appears to be no difference in affinity
between patients. PLA2R fluctuates between an extended conformation at neutral pH and a bent
conformation at lower pH possibly involved in the regulation of ligand recognition or receptor
oligomerization. This has implications for formation of neo-epitopes that may become
immunogenic and induce autoimmunity.