The lysosomal hydrolase N-acetylgalactosamine-4-sulphatase (4-sulphatase) is essential for the sequential degradation of the glycosaminoglycans, dermatan and chondroitin sulphate and, when deficient, causes the lysosomal storage disorder mucopolysaccharidosis type VI. The cysteine at codon 91 of human 4-sulphatase was identified previously as a key residue in the active site of the enzyme and was mutated by site-directed mutagenesis to produce a 4-sulphatase in which cysteine-91 was replaced by a threonine residue (C91T). The C91T mutation caused a loss of 4-sulphatase activity, a detectable protein conformational change and a lower level of intracellular 4-sulphatase protein [Brooks, Robertson, Bindloss, Litjens, Anson, Peters, Morris and Hopwood (1995) Biochem. J. 307, 457-463]. In the present study, we report that C91T is synthesized normally in the endoplasmic reticulum as a 66 kDa glycosylated protein, which is very similar in size to wild-type 4-sulphatase. However, C91T neither underwent normal Golgi processing, shown by lack of modification to form mannose 6-phosphate residues on its oligosaccharide side chains, nor did it traffic to the lysosome to undergo normal endosomal-lysosomal proteolytic processing. Instead, C91T remained in an early biosynthetic compartment and was degraded. The molecular chaperone, immunoglobulin binding protein (BiP), was associated with newly-synthesized wild-type and mutant 4-sulphatase proteins for extended periods, but no direct evidence was found for involvement of BiP in the retention or degradation of the C91T protein. This suggested that prolonged association of mutant protein with BiP does not necessarily infer involvement of BiP in the quality control process, as previously implied in the literature. The predicted BiP binding sites on 4-sulphatase map to β-strands and α-helices, which are co-ordinated together in the folded molecule, indicating that BiP interacts with critical protein folding or contact sites on 4-sulphatase.
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Research Article|
June 24 1999
Processing of normal lysosomal and mutant N-acetylgalactosamine 4-sulphatase: BiP (immunoglobulin heavy-chain binding protein) may interact with critical protein contact sites
Tessa M. BRADFORD;
Tessa M. BRADFORD
*Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia 5006, Australia
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Mary-Jane GETHING;
Mary-Jane GETHING
†Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052, Australia
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Richard DAVEY;
Richard DAVEY
*Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia 5006, Australia
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John J. HOPWOOD;
John J. HOPWOOD
*Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia 5006, Australia
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Doug A. BROOKS
Doug A. BROOKS
1
*Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia 5006, Australia
1To whom correspondence should be addressed (e-mail dbrooks@medicine.adelaide.edu.au).
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Publisher: Portland Press Ltd
Received:
November 30 1998
Revision Received:
March 11 1999
Accepted:
April 23 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 341 (1): 193–201.
Article history
Received:
November 30 1998
Revision Received:
March 11 1999
Accepted:
April 23 1999
Citation
Tessa M. BRADFORD, Mary-Jane GETHING, Richard DAVEY, John J. HOPWOOD, Doug A. BROOKS; Processing of normal lysosomal and mutant N-acetylgalactosamine 4-sulphatase: BiP (immunoglobulin heavy-chain binding protein) may interact with critical protein contact sites. Biochem J 1 July 1999; 341 (1): 193–201. doi: https://doi.org/10.1042/bj3410193
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