| 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 |
| Publication Type | Journal Article |
| Year of Publication | 2015 |
| Authors | Steward, M., Berezovskaya Y., Zhou H., Shediac R., Sun C., Miller N., and Rendle P.M. |
| Journal | Clinical Biochemistry |
| Volume | 48 |
| Issue | 12 |
| Pagination | 796 - 802 |
| Date Published | 2015 |
| ISSN | 00099120 (ISSN) |
| Keywords | adolescent, adult, article, Bacillus circulans, bacterial enzyme, bioinformatics, carboxy terminal sequence, Child, clinical article, Cloning, controlled study, disease marker, enzyme activity, enzyme purification, Escherichia coli, human, keratan sulfate, keratanase II, liquid chromatography, Mass spectrometry, Morquio A syndrome, Morquio syndrome, n acetylgalactosamine, nonhuman, priority journal, protein expression, quantitative analysis, unclassified drug, urinalysis |
| 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. © 2015. |
| URL | http://www.scopus.com/inward/record.url?eid=2-s2.0-84938285512&partnerID=40&md5=11919143264d24907d5a4598c5405802 |
| DOI | 10.1016/j.clinbiochem.2015.03.024 |
