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

Abstract

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 beta-strands and alpha-helices, which are co-ordinated together in the folded molecule, indicating that BiP interacts with critical protein folding or contact sites on 4-sulphatase.

A Phaseolin Domain Involved Directly in Trimer Assembly Is a Determinant for Binding by the Chaperone BiP

“…The main parameter for the evaluation of affinity for BiP was the ability of a given peptide to stimulate in vitro the ATPase activity of the chaperone. In immunoglobulin heavy and light chains (Knarr et al, 1995;Augustine et al, 2001), in gp160 (Knarr et al, 1999), and in the lysosomal hydrolase N -acetylglucosamine-4-sulfatase (Bradford et al, 1999), most of the predicted potential sites of BiP binding involve interdomain or subunit contact sites or hydrophobic residues that are buried within the folded protein, as revealed by crystallography. However, at present, there is scarce evidence regarding whether these predictions apply to in vivo situations: direct information on sequences recognized in vivo on real proteins, in any eukaryotic system, is very limited.…”

Heparan N‐sulfatase: cysteine 70 plays a role in the enzyme catalysis and processing

“…Studies performed in MSD fibroblasts showed that arylsulfatase A, arylsulfatase B and steroid sulfatase, although inactive, were normally processed [23]; results obtained from expression studies on the cysteine 84 mutant of iduronate sulfatase [20] showed that a C84A mutation had a drastic effect on processing, while C84T replacement produced a small amount of IDS inactive mature forms. Experiments performed on the cysteine 91 of N ‐acetylgalactosamine 4‐sulfatase showed that the C91T replacement caused a lower level of intracellular protein [19]; however, a subsequent study revealed that the C91T was synthesized normally in the RER where it was apparently retained and degraded by the quality control system, but was not processed [24]. Also in this study, since RT‐PCR analysis showed that the C70M mutant was correctly translated in vitro, the C70M protein seems to be synthesized but not proteolytically processed.…”

“…Previous studies have demonstrated that point mutations may directly affect the protein function or alter the correct folding and cause subsequent premature degradation of the mutant proteins within the RER; the resulting protein misfolding has been implicated in the pathogenesis of some genetic diseases [25,26]. The mechanism involved in the degradation of the mutant protein in lysosomal storage disorders has yet to be fully characterized, but recent evidence indicates the involvement of the degradation system in the RER quality control processes [24]; furthermore, protein conformation changes have been observed in the mutant 4‐sulfatase protein in patient fibroblasts [19].…”

Chloroplast Hsp70s are not involved in the import of ferredoxin‐NADP+ reductase precursor

“…We performed BiP‐binding predictions on the FNR TP using a program based on a mathematical algorithm previously reported (Blond‐Elguindi et al 1993). The algorithm has been successfully used to predict the presence of BiP‐binding sequences in several polypeptides such as the light and heavy chains of antibodies (Knarr et al 1995), the precursor of the HIV type I envelope glycoprotein (gp 160) (Knarr et al 1999) and along the primary sequence of the lysosomal 4‐sulfatase (Bradford et al 1999). In a previous work we analyzed in depth the interaction of the E. coli Hsp70 DnaK with the TP of preFNR.…”