Human α-N-acetylglucosaminidase: cDNA cryptic site removal and native secretion signal addition significantly enhance enzyme expression and secretion |
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Authors: | Roni Rebecca Jantzen Sarah N Truelson Francis YM Choy |
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Institution: | Department of Biology, Biomedical Research Centre, University of Victoria, Victoria, British Columbia, Canada |
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Abstract: | Human α-N-acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase implicated in Mucopolysaccharidosis type IIIB (MPS IIIB). We utilize a Spodoptera frugiperda (Sf9) system to express Naglu fused to a synthetic protein transduction domain in hopes to facilitate delivery of Naglu across the blood–brain barrier, thus allowing enzyme replacement therapy to treat neurological symptoms.Although human recombinant Naglu was previously produced in Sf9, low expression levels suggested degradation via cryptic mRNA splicing. Two cryptic splice sites discovered within Naglu cDNA were altered by site-directed mutagenesis, reducing Naglu mRNA degradation. A native Naglu secretion-signaling peptide was efficiently recognized by the Sf9 system. Significantly higher enzyme activity was seen from multiple adherent Sf9 cultures stably expressing mutagenized Naglu over those expressing wildtype Naglu (P = 0.000; 3.4-fold average increased specific activity). Suspension cultures demonstrated a 4.0-fold increase in overall enzyme activity secreted post-mutagenesis. Thus elimination of cryptic splicing directly resulted in higher Naglu expression. |
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Keywords: | Mucopolysaccharidosis IIIB or Sanfilippo syndrome Human α-N-acetylglucosaminidase Cryptic splice site Site-directed mutagenesis Sf9 cell expression system Native secretion signal peptide |
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