Processing of normal lysosomal and mutant N-acetylgalactosamine 4-sulphatase: BiP (immunoglobulin heavy-chain binding protein) may interact with critical protein contact sites

The lysosomal hydrolase N-acetylgalactosamine-4-sulphatase (4-sulphatase) i s essential for the sequential degradation of the glycosaminoglycans, derma tan and chondroitin sulphate and, when deficient, causes the lysosomal stor age disorder mucopolysaccharidosis type VI. The cysteine at codon 91 of hum an 4-sulphatase: was identified previously as a key residue in the active s ite 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 (C91 T). The C91T mutation caused a loss of 4-sulphatase activity, a detectable protein conformational change and a lower level of intracellular 4-sulphata se protein [Brooks, Robertson, Bindloss, Litjens, Anson, Peters, Morris and Hopwood(1995) Biochem. J. 307, 457-163], In the present study, we report t hat C91T is synthesized normally in the endoplasmic reticulum as a 66 kDa g lycosylated protein, which is very similar in size to wild-type 4-sulphatas e. However, C91T neither underwent normal Golgi processing, shown by lack o f 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 biosy nthetic compartment and was degraded. The molecular chaperone, immunoglobul in binding protein (BiP), was associated with newly-synthesized wild-type a nd mutant l-sulphatase proteins for extended periods, but no direct evidenc e was found for involvement of BiP in the retention or degradation of the C 91T protein. This suggested that prolonged association of mutant protein wi th BiP does not necessarily infer involvement of BiP in the quality control process, as previously implied in the literature. The predicted BIP bindin g sites on 4-sulphatase map to beta-strands and alpha-helices, which are co ordinated together in the folded molecule, indicating that BiP interacts wi th critical protein folding or contact sites on 4-sulphatase.