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.

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: Michael Steward, Yana Berezovskaya, Huiyu Zhou, Renée Shediac, Cynthia Sun, Nicole Miller, Phillip M Rendle
• Journal: Clinical biochemistry (Clin Biochem) Vol. 48 Issue 12 Pg. 796-802 (Aug 2015) ISSN: 1873-2933 [Electronic] United States
• PMID: 25866399 (Publication Type: Journal Article)
• Copyright: Copyright © 2015. Published by Elsevier Inc.

Chemical References

• Biomarkers
• Keratan Sulfate
• keratanase II
• Acetylglucosaminidase

Topics

• Acetylglucosaminidase (biosynthesis, chemistry, genetics, metabolism)
• Adolescent
• Adult
• Animals
• Bacillus (enzymology, genetics)
• Bioengineering (methods)
• Biomarkers (urine)
• Case-Control Studies
• Catalysis
• Cattle
• Child
• Child, Preschool
• Chromatography, Liquid (methods)
• Cloning, Molecular
• Escherichia coli (enzymology, genetics)
• Humans
• Keratan Sulfate (urine)
• Mucopolysaccharidosis IV (urine)
• Protein Structure, Tertiary
• Tandem Mass Spectrometry (methods)
• Young Adult