title: Recombinant, truncated B. circulans keratanase-II: Description and characterisation of a novel enzyme for use in measuring urinary keratan sulphate levels via LC-MS/MS in Morquio A syndrome.
abstract: OBJECTIVE
  Morquio A syndrome (mucopolysaccharidosis IVA; MPS IVA) is an autosomal recessive lysosomal storage disorder caused by deficient N-acetylgalactosamine-6-sulphatase (GALNS) activity. Early and accurate diagnosis of this condition is critical for improved patient outcomes, particularly as enzyme replacement therapy has recently become available. An LC-MS/MS assay utilising keratan sulphate (KS) disaccharides derived from keratanase-II digestion provides a sensitive and specific means for quantitation of urinary KS, a screening biomarker for Morquio A (Oguma et al., 2007; Martell et al., 2011). To ensure a reliable supply of keratanase-II, we sought to produce a Bacillus circulans-derived enzyme via a recombinant approach in Escherichia coli.
  
  
  METHODS
  Bioinformatics analysis of the B. circulans keratanase-II enzyme identified likely dispensable C-terminal domains amenable to enhancement via protein engineering. A truncated form of the enzyme was designed to remove the domains predicted to be unnecessary for catalytic activity and detrimental to recombinant expression in E. coli.
  
  
  RESULTS
  C-terminally truncated, recombinant B. circulans keratanase-II was purified to >98% homogeneity and extensively characterised, demonstrating desired activity, specificity and utility in LC-MS-based quantitation of urinary KS from Morquio A and control samples, and is functionally indistinguishable from full-length, native B. circulans-derived keratanase-II.
  
  
  CONCLUSIONS
  This novel, recombinant keratanase-II meets all performance requirements and can be produced in a rapid and reproducible manner. We speculate that other related bacterial enzymes of biomedical or industrial interest may be amenable to similar engineered enhancements.
authors: Christchurch Protein Science and Engineering, Callaghan Innovation, School of Biological Sciences, Canterbury University, Christchurch 8140, New Zealand. Electronic address: Michael.q.steward@gsk.com.
authors: Michael
authors: M
authors: Steward
authors: BioMarin Pharmaceutical Inc., Novato, CA, USA.
authors: Yana
authors: Y
authors: Berezovskaya
authors: BioMarin Pharmaceutical Inc., Novato, CA, USA.
authors: Huiyu
authors: H
authors: Zhou
authors: BioMarin Pharmaceutical Inc., Novato, CA, USA.
authors: Renée
authors: R
authors: Shediac
authors: Integrated Bioactive Technologies, Callaghan Innovation, 69 Gracefield Road, Lower Hutt 5040, New Zealand.
authors: Cynthia
authors: C
authors: Sun
authors: BioMarin Pharmaceutical Inc., Novato, CA, USA.
authors: Nicole
authors: N
authors: Miller
authors: Ferrier Research Institute, Victoria University of Wellington, 69 Gracefield Road, Lower Hutt 5040, New Zealand.
authors: Phillip M
authors: PM
authors: Rendle
mesh_terms: D000118:Acetylglucosaminidase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D000818:Animals; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D057005:Bioengineering / Q000379:methods; D015415:Biomarkers / Q000652:urine; D016022:Case-Control Studies; D002384:Catalysis; D002417:Cattle; D002648:Child; D002675:Child, Preschool; D002853:Chromatography, Liquid / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007632:Keratan Sulfate / Q000652:urine*; D009085:Mucopolysaccharidosis IV / Q000652:urine*; D017434:Protein Structure, Tertiary; D053719:Tandem Mass Spectrometry / Q000379:methods; D055815:Young Adult
chemical_list: D015415:Biomarkers; D007632:Keratan Sulfate; C086950:keratanase II; D000118:Acetylglucosaminidase
keywords: Keratanase; LC/MS–MS; Morquio syndrome; Mucopolysaccharidosis IV; Recombinant enzyme