Sanfilippo Syndrome: Identification and Removal of a Cryptic Splice Site in Αlpha-N-Acetylglucosaminidase cDNA for Potential Improved Expression and Enzyme Replacement Therapy


Authors: Sarah N. Truelson, R. Rebecca Jantzen, and Francis Y. M. Choy
Page Range: 89-99
Published in: Journal of Biochemistry and Molecular Biology in the Post Genomic Era, 1#1 (2011)
ISSN: 2156-5732

Table of Contents


-N-Acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase that is deficient in patients with Mucopolysaccharidosis III type B (MPS IIIB). Naglu deficiency causes cytotoxic accumulation of heparan sulfate in lysosomes; this is especially detrimental to the central nervous system, resulting in neurodegeneration, behavioural problems, mental retardation, and shortened lifespan. Our laboratory is currently working to express Naglu fused to a synthetic protein transduction domain with the ultimate goal of developing an efficient system to produce human recombinant Naglu for enzyme replacement therapy. Conjugation of Naglu to a protein transduction domain may allow the enzyme to cross the blood-brain barrier to treat the neurological symptoms of MPS IIIB.

The Naglu fusion protein has been successfully produced in an active state in recombinant Spodoptera frugiperda 9 (Sf9) cultures; however, low resultant expression levels have prevented further characterization studies. Degradation of Naglu mRNA due to activation of a cryptic splice site in human Naglu cDNA was hypothesized, as we consistently detected the presence of a small, non-target DNA band in reverse transcription polymerase chain reaction (RT-PCR) experiments. Sequence analysis showed that the 426 bp band contained Naglu sequence corresponding to the 5’ and 3’ ends of the gene, with a large central portion missing, and was likely the result of cryptic splicing in the Sf9 expression system. Site-directed mutagenesis by fusion PCR was used to alter the splice donor and acceptor sites in Naglu cDNA; RT-PCR on RNA isolated from Sf9 cells transfected with the new construct indicated presence of the full length 2.2 Kb Naglu product and absence of the 426 bp band. Use of this construct, which successfully eliminated cryptic splicing, could potentially increase recombinant Naglu expression in Sf9 cells.

Keywords: Sanfilippo syndrome, -N-acetylglucosaminidase, cryptic splice site, site-directed mutagenesis, Sf9 cell expression system.

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