Michael Steward1, Yana Berezovskaya2, Huiyu Zhou2, Renée Shediac2, Cynthia Sun3, Nicole Miller2, Phillip M Rendle4. 1. Christchurch Protein Science and Engineering, Callaghan Innovation, School of Biological Sciences, Canterbury University, Christchurch 8140, New Zealand. Electronic address: Michael.q.steward@gsk.com. 2. BioMarin Pharmaceutical Inc., Novato, CA, USA. 3. Integrated Bioactive Technologies, Callaghan Innovation, 69 Gracefield Road, Lower Hutt 5040, New Zealand. 4. Ferrier Research Institute, Victoria University of Wellington, 69 Gracefield Road, Lower Hutt 5040, New Zealand.
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. DESIGN AND 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.
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. DESIGN AND 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: Anabel Gonzalez-Gil; Ryan N Porell; Steve M Fernandes; Yadong Wei; Huifeng Yu; Daniela J Carroll; Ryan McBride; James C Paulson; Michael Tiemeyer; Kazuhiro Aoki; Bruce S Bochner; Ronald L Schnaar Journal: Glycobiology Date: 2018-10-01 Impact factor: 4.313
Authors: Anabel Gonzalez-Gil; T August Li; Ryan N Porell; Steve M Fernandes; Haley E Tarbox; Hyun Sil Lee; Kazuhiro Aoki; Michael Tiemeyer; Jean Kim; Ronald L Schnaar Journal: J Allergy Clin Immunol Date: 2020-08-11 Impact factor: 10.793