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https://hdl.handle.net/2440/7021
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dc.contributor.author | Brooks, D. | - |
dc.contributor.author | Robertson, D. | - |
dc.contributor.author | Bindloss, C. | - |
dc.contributor.author | Litjens, T. | - |
dc.contributor.author | Anson, D. | - |
dc.contributor.author | Peters, C. | - |
dc.contributor.author | Morris, C. | - |
dc.contributor.author | Hopwood, J. | - |
dc.date.issued | 1995 | - |
dc.identifier.citation | Biochemical Journal, 1995; 307(Pt. 2):457-463 | - |
dc.identifier.issn | 0264-6021 | - |
dc.identifier.issn | 1470-8728 | - |
dc.identifier.uri | http://hdl.handle.net/2440/7021 | - |
dc.description.abstract | The sulphatase family of enzymes have regions of sequence similarity, but relatively little is known about either the structure-function relationships of sulphatases, or the role of highly conserved amino acids. The sequence of amino acids CTPSR at position 91-95 of 4-sulphatase has been shown to be highly conserved in all of the sequenced sulphatase enzymes. The cysteine at amino acid 91 of 4-sulphatase was selected for mutation analysis due to its potential role in either the active site, substrate-binding site or part of a key structural domain of 4-sulphatase and due to the absence of naturally occurring mutations in this residue in mucopolysaccharidosis type VI (MPS VI) patients. Two mutations, C91S and C91T, altering amino acid 91 of 4-sulphatase were generated and expressed in Chinese hamster ovary cells. Biochemical analysis of protein from a C91S cell line demonstrated no detectable 4-sulphatase enzyme activity but a relatively normal level of 4-sulphatase polypeptide (180% of the wild-type control protein level). Epitope detection, using a panel of ten monoclonal antibodies, demonstrated that the C91S polypeptide had a similar immunoreactivity to wild-type 4-sulphatase, suggesting that the C91S substitution does not induce a major structural change in the protein. Reduced catalytic activity associated with normal levels of 4-sulphatase protein have not been observed in any of the MPS VI patients tested and all show evidence of structural modification of 4-sulphatase protein with the same panel of antibodies [Brooks, McCourt, Gibson, Ashton, Shutter and Hopwood (1991) Am. J. Hum. Genet. 48, 710-719]. The loss of enzyme activity without a detectable protein conformation change suggests that Cys-91 may be a critical residue in the catalytic process. In contrast, analysis of protein from a C91T cell line revealed low levels of catalytically inactive 4-sulphatase polypeptide (0.37% of the wild-type control protein level) which had missing or masked epitopes, suggesting an altered protein structure or conformation. Subcellular fractionation studies of the C91T cell line demonstrated a high proportion of 4-sulphatase polypeptide content in organelles characteristic of microsomes. The aberrant intracellular localization and the reduced cellular content of 4-sulphatase polypeptide was consistent with the observed structural modification leading to retention and degradation of the protein within an early vacuolar compartment. | - |
dc.description.statementofresponsibility | D A Brooks, D A Robertson, C Bindloss, T Litjens, D S Anson, C Peters, C P Morris and J J Hopwood | - |
dc.language.iso | en | - |
dc.publisher | Biochemical Society | - |
dc.source.uri | http://dx.doi.org/10.1042/bj3070457 | - |
dc.subject | CHO Cells | - |
dc.subject | Subcellular Fractions | - |
dc.subject | Animals | - |
dc.subject | Sulfatases | - |
dc.subject | N-Acetylgalactosamine-4-Sulfatase | - |
dc.subject | DNA Primers | - |
dc.subject | Mutagenesis, Site-Directed | - |
dc.subject | Base Sequence | - |
dc.subject | Protein Conformation | - |
dc.subject | Molecular Sequence Data | - |
dc.subject | Cricetinae | - |
dc.title | Two site-directed mutations abrogate enzyme activity but have different effects on the conformation and cellular content of the N-acetylgalactosamine 4-sulphatase protein | - |
dc.type | Journal article | - |
dc.identifier.doi | 10.1042/bj3070457 | - |
pubs.publication-status | Published | - |
dc.identifier.orcid | Brooks, D. [0000-0001-9098-3626] | - |
Appears in Collections: | Aurora harvest 5 Paediatrics publications |
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