Recombinant staphylokinase variants with altered immunoreactivity. I: Construction and characterization

Area of Science:

• Biochemistry
• Protein Engineering
• Immunology

Background:

• Recombinant staphylokinase is a promising thrombolytic agent for acute myocardial infarction.
• Its clinical use is limited by immunogenicity.
• Protein engineering strategies to reduce heterologous protein immunogenicity are underexplored.

Purpose of the Study:

• To reduce the immunogenicity of recombinant staphylokinase (SakSTAR) through site-specific mutagenesis.
• To identify and eliminate key immunodominant epitopes.
• To assess the impact of mutations on SakSTAR's activity and antigenicity.

Main Methods:

• Monoclonal antibody epitope mapping identified three nonoverlapping immunodominant epitopes in SakSTAR.
• Site-directed mutagenesis was used to substitute charged amino acids with alanine, targeting two epitopes.
• Combination mutants (SakSTAR.M38 and SakSTAR.M89) were constructed, expressed, and purified.
• Specific activity and antibody absorption assays were performed.

Main Results:

• Two major immunodominant epitopes were successfully eliminated through mutagenesis.
• Mutant variants (SakSTAR.M38, SakSTAR.M89) showed reduced reactivity with monoclonal antibodies.
• These mutants retained approximately 50% of SakSTAR's specific activity.
• Antibody absorption studies indicated incomplete reduction (<90%) of patient-elicited antibodies by the mutants.

Conclusions:

• SakSTAR possesses three immunodominant epitopes; two were eliminated via mutagenesis.
• The resulting combination mutants exhibit maintained specific activity and reduced recognition by patient antibodies.
• These engineered staphylokinase variants warrant further investigation for their thrombolytic and antigenic properties.