Antibody Epitope of Human α‐Galactosidase A Revealed by Affinity Mass Spectrometry: A Basis for Reversing Immunoreactivity in Enzyme Replacement Therapy of Fabry Disease

Abstract

α-Galactosidase (αGal) is a lysosomal enzyme that hydrolyses the terminal α-galactosyl moiety from glycosphingolipids. Mutations in the encoding genes for αGal lead to defective or misfolded enzyme, which results in substrate accumulation and subsequent organ dysfunction. The metabolic disease caused by a deficiency of human α-galactosidase A is known as Fabry disease or Fabry-Anderson disease, and it belongs to a larger group known as lysosomal storage diseases. An effective treatment for Fabry disease has been developed by enzyme replacement therapy (ERT), which involves infusions of purified recombinant enzyme in order to increase enzyme levels and decrease the amounts of accumulated substrate. However, immunoreactivity and IgG antibody formation are major, therapy-limiting, and eventually life-threatening complications of ERT. The present study focused on the epitope determination of human α-galactosidase A against its antibody formed. Here we report the identification of the epitope of human αGal(309-332) recognized by a human monoclonal anti-αGal antibody, using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance biosensing mass spectrometry. The epitope peptide, αGal(309-332), was synthesized by solid-phase peptide synthesis. Determination of its affinity by surface plasmon resonance analysis revealed a high binding affinity for the antibody (K =39×10 m), which is nearly identical to that of the full-length enzyme (K =16×10 m). The proteolytic excision affinity mass spectrometry method is shown here to be an efficient tool for epitope identification of an immunogenic lysosomal enzyme. Because the full-length αGal and the antibody epitope showed similar binding affinities, this provides a basis for reversing immunogenicity upon ERT by: 1) treatment of patients with the epitope peptide to neutralize antibodies, or 2) removal of antibodies by apheresis, and thus significantly improving the response to ERT.

Molecular Epitope Determination of Aptamer Complexes of the Multidomain Protein C‐Met by Proteolytic Affinity‐Mass Spectrometry

“…The PROTEX-SPR-MS method has been successfully applied in a number of recent studies for epitope identifications of protein-antibody complexes, protein-peptide interactions, and carbohydrate-protein complexes. [26][27][28][29][30][31][32] SPR determinations of the C-Met-aptamer complexes revealed high affinities, consistent with the suitability of aptamers as specific inhibitors of C-Met. PROTEX-MS using immobilized aptamer affinitymatrices revealed specific epitopes on C-Met, with a linear epitope peptide identified for the CLN0004 aptamer, and a discontinuous epitope comprising two specific peptides for the CLN0003 aptamer.…”

“…In order to obtain molecular epitope identifications, proteolytic epitope extraction mass spectrometry (PROTEX‐MS) was performed as illustrated in Figure S1 . Prior to digestion, the protein was subjected to reduction of disulfide bonds and subsequent alkylation with iodoacetamide as described in the Experimental Section, followed by digestion with trypsin, and proteolytic fragment mixtures were characterized by mass spectrometric peptide mapping.…”

“…For the identification of aptamer epitopes of C‐Met and determination of binding affinities, selective proteolytic extraction of C‐Met‐aptamer complexes in combination with electrospray (ESI) and MALDI mass spectrometry, and SPR (surface plasmon resonance) biosensor analysis were used as principal tools (PROTEX‐SPR‐MS) (Supplementary Figure S1). The PROTEX‐SPR‐MS method has been successfully applied in a number of recent studies for epitope identifications of protein‐antibody complexes, protein‐peptide interactions, and carbohydrate‐protein complexes . SPR determinations of the C‐Met‐aptamer complexes revealed high affinities, consistent with the suitability of aptamers as specific inhibitors of C‐Met.…”

“…[31,32] All digestion mixtures were further characterized by gel electrophoresis; proteolytic peptide mixtures provided digestion yields > 95 % and were used for epitope analyses. [32]…”

“… Structure model of the recombinant human C‐Met/Fc Chimera protein according to I‐TASSER . Highlighted in yellow is the extracellular domain of human c‐Met precursor and in green the C‐terminal polyhistidine‐tagged Fc region of human IgG 1 used.…”