科研之友
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摘要
-Galactosidase (Gal) is a lysosomal enzyme that hydrolyses the terminal -galactosyl moiety from glycosphingolipids. Mutations in the encoding genes for Gal lead to defective or misfolded enzyme, which results in substrate accumulation and subsequent organ dysfunction. The metabolic disease caused by a deficiency of human -galactosidaseA 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 -galactosidaseA against its antibody formed. Here we report the identification of the epitope of human Gal(309-332) recognized by a human monoclonal anti-Gal antibody, using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance biosensing mass spectrometry. The epitope peptide, Gal(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-D=39x10(-9)m), which is nearly identical to that of the full-length enzyme (K-D=16x10(-9)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 Gal 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.
- 出版日期2018-5-8
