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. 1999 Jul 1;341(Pt 1):193–201.

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

T M Bradford 1, M J Gething 1, R Davey 1, J J Hopwood 1, D A Brooks 1
PMCID: PMC1220347  PMID: 10377262

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.

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Selected References

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  1. Accili D., Kadowaki T., Kadowaki H., Mosthaf L., Ullrich A., Taylor S. I. Immunoglobulin heavy chain-binding protein binds to misfolded mutant insulin receptors with mutations in the extracellular domain. J Biol Chem. 1992 Jan 5;267(1):586–590. [PubMed] [Google Scholar]
  2. Arlt G., Brooks D. A., Isbrandt D., Hopwood J. J., Bielicki J., Bradford T. M., Bindloss-Petherbridge C. A., von Figura K., Peters C. Juvenile form of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). A C-terminal extension causes instability but increases catalytic efficiency of arylsulfatase B. J Biol Chem. 1994 Apr 1;269(13):9638–9643. [PubMed] [Google Scholar]
  3. Blond-Elguindi S., Cwirla S. E., Dower W. J., Lipshutz R. J., Sprang S. R., Sambrook J. F., Gething M. J. Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP. Cell. 1993 Nov 19;75(4):717–728. doi: 10.1016/0092-8674(93)90492-9. [DOI] [PubMed] [Google Scholar]
  4. Bond C. S., Clements P. R., Ashby S. J., Collyer C. A., Harrop S. J., Hopwood J. J., Guss J. M. Structure of a human lysosomal sulfatase. Structure. 1997 Feb 15;5(2):277–289. doi: 10.1016/s0969-2126(97)00185-8. [DOI] [PubMed] [Google Scholar]
  5. Brooks D. A., Harper G. S., Gibson G. J., Ashton L. J., Taylor J. A., McCourt P. A., Freeman C., Clements P. R., Hoffmann J. W., Hopwood J. J. Hurler syndrome: a patient with abnormally high levels of alpha-L-iduronidase protein. Biochem Med Metab Biol. 1992 Jun;47(3):211–220. doi: 10.1016/0885-4505(92)90028-w. [DOI] [PubMed] [Google Scholar]
  6. Brooks D. A., McCourt P. A., Gibson G. J., Ashton L. J., Shutter M., Hopwood J. J. Analysis of N-acetylgalactosamine-4-sulfatase protein and kinetics in mucopolysaccharidosis type VI patients. Am J Hum Genet. 1991 Apr;48(4):710–719. [PMC free article] [PubMed] [Google Scholar]
  7. Brooks D. A. Protein processing: a role in the pathophysiology of genetic disease. FEBS Lett. 1997 Jun 9;409(2):115–120. doi: 10.1016/s0014-5793(97)00423-7. [DOI] [PubMed] [Google Scholar]
  8. Brooks D. A. Review: the immunochemical analysis of enzyme from mucopolysaccharidoses patients. J Inherit Metab Dis. 1993;16(1):3–15. doi: 10.1007/BF00711309. [DOI] [PubMed] [Google Scholar]
  9. Brooks D. A., Robertson D. A., Bindloss C., Litjens T., Anson D. S., Peters C., Morris C. P., Hopwood J. J. Two site-directed mutations abrogate enzyme activity but have different effects on the conformation and cellular content of the N-acetylgalactosamine 4-sulphatase protein. Biochem J. 1995 Apr 15;307(Pt 2):457–463. doi: 10.1042/bj3070457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chessler S. D., Byers P. H. BiP binds type I procollagen pro alpha chains with mutations in the carboxyl-terminal propeptide synthesized by cells from patients with osteogenesis imperfecta. J Biol Chem. 1993 Aug 25;268(24):18226–18233. [PubMed] [Google Scholar]
  11. Dlott B., d'Azzo A., Quon D. V., Neufeld E. F. Two mutations produce intron insertion in mRNA and elongated beta-subunit of human beta-hexosaminidase. J Biol Chem. 1990 Oct 15;265(29):17921–17927. [PubMed] [Google Scholar]
  12. Fourie A. M., Hupp T. R., Lane D. P., Sang B. C., Barbosa M. S., Sambrook J. F., Gething M. J. HSP70 binding sites in the tumor suppressor protein p53. J Biol Chem. 1997 Aug 1;272(31):19471–19479. doi: 10.1074/jbc.272.31.19471. [DOI] [PubMed] [Google Scholar]
  13. Gething M. J., Blond-Elguindi S., Buchner J., Fourie A., Knarr G., Modrow S., Nanu L., Segal M., Sambrook J. Binding sites for Hsp70 molecular chaperones in natural proteins. Cold Spring Harb Symp Quant Biol. 1995;60:417–428. doi: 10.1101/sqb.1995.060.01.046. [DOI] [PubMed] [Google Scholar]
  14. Gibson G. J., Saccone G. T., Brooks D. A., Clements P. R., Hopwood J. J. Human N-acetylgalactosamine-4-sulphate sulphatase. Purification, monoclonal antibody production and native and subunit Mr values. Biochem J. 1987 Dec 15;248(3):755–764. doi: 10.1042/bj2480755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Groll M., Ditzel L., Löwe J., Stock D., Bochtler M., Bartunik H. D., Huber R. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature. 1997 Apr 3;386(6624):463–471. doi: 10.1038/386463a0. [DOI] [PubMed] [Google Scholar]
  16. Hermans M. M., Kroos M. A., de Graaff E., Oostra B. A., Reuser A. J. Two mutations affecting the transport and maturation of lysosomal alpha-glucosidase in an adult case of glycogen storage disease type II. Hum Mutat. 1993;2(4):268–273. doi: 10.1002/humu.1380020406. [DOI] [PubMed] [Google Scholar]
  17. Hopwood J. J., Morris C. P. The mucopolysaccharidoses. Diagnosis, molecular genetics and treatment. Mol Biol Med. 1990 Oct;7(5):381–404. [PubMed] [Google Scholar]
  18. Hu P., Reuser A. J., Janse H. C., Kleijer W. J., Schindler D., Sakuraba H., Tsuji A., Suzuki Y., van Diggelen O. P. Biosynthesis of human alpha-N-acetylgalactosaminidase: defective phosphorylation and maturation in infantile alpha-NAGA deficiency. Biochem Biophys Res Commun. 1991 Mar 29;175(3):1097–1103. doi: 10.1016/0006-291x(91)91678-6. [DOI] [PubMed] [Google Scholar]
  19. Katsumi A., Senda T., Yamashita Y., Yamazaki T., Hamaguchi M., Kojima T., Kobayashi S., Saito H. Protein C Nagoya, an elongated mutant of protein C, is retained within the endoplasmic reticulum and is associated with GRP78 and GRP94. Blood. 1996 May 15;87(10):4164–4175. [PubMed] [Google Scholar]
  20. Knarr G., Gething M. J., Modrow S., Buchner J. BiP binding sequences in antibodies. J Biol Chem. 1995 Nov 17;270(46):27589–27594. doi: 10.1074/jbc.270.46.27589. [DOI] [PubMed] [Google Scholar]
  21. Kobayashi T., Honke K., Jin T., Gasa S., Miyazaki T., Makita A. Components and proteolytic processing sites of arylsulfatase B from human placenta. Biochim Biophys Acta. 1992 Oct 20;1159(3):243–247. doi: 10.1016/0167-4838(92)90051-e. [DOI] [PubMed] [Google Scholar]
  22. Kopito R. R. ER quality control: the cytoplasmic connection. Cell. 1997 Feb 21;88(4):427–430. doi: 10.1016/s0092-8674(00)81881-4. [DOI] [PubMed] [Google Scholar]
  23. Kozutsumi Y., Normington K., Press E., Slaughter C., Sambrook J., Gething M. J. Identification of immunoglobulin heavy chain binding protein as glucose-regulated protein 78 on the basis of amino acid sequence, immunological cross-reactivity, and functional activity. J Cell Sci Suppl. 1989;11:115–137. doi: 10.1242/jcs.1989.supplement_11.10. [DOI] [PubMed] [Google Scholar]
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Litjens T., Brooks D. A., Peters C., Gibson G. J., Hopwood J. J. Identification, expression, and biochemical characterization of N-acetylgalactosamine-4-sulfatase mutations and relationship with clinical phenotype in MPS-VI patients. Am J Hum Genet. 1996 Jun;58(6):1127–1134. [PMC free article] [PubMed] [Google Scholar]
  26. Marquardt T., Ullrich K., Zimmer P., Hasilik A., Deufel T., Harms E. Carbohydrate-deficient glycoprotein syndrome (CDGS)--glycosylation, folding and intracellular transport of newly synthesized glycoproteins. Eur J Cell Biol. 1995 Mar;66(3):268–273. [PubMed] [Google Scholar]
  27. Pasyk E. A., Foskett J. K. Mutant (delta F508) cystic fibrosis transmembrane conductance regulator Cl- channel is functional when retained in endoplasmic reticulum of mammalian cells. J Biol Chem. 1995 May 26;270(21):12347–12350. doi: 10.1074/jbc.270.21.12347. [DOI] [PubMed] [Google Scholar]
  28. Paw B. H., Moskowitz S. M., Uhrhammer N., Wright N., Kaback M. M., Neufeld E. F. Juvenile GM2 gangliosidosis caused by substitution of histidine for arginine at position 499 or 504 of the alpha-subunit of beta-hexosaminidase. J Biol Chem. 1990 Jun 5;265(16):9452–9457. [PubMed] [Google Scholar]
  29. Pind S., Riordan J. R., Williams D. B. Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator. J Biol Chem. 1994 Apr 29;269(17):12784–12788. [PubMed] [Google Scholar]
  30. Sato S., Ward C. L., Krouse M. E., Wine J. J., Kopito R. R. Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation. J Biol Chem. 1996 Jan 12;271(2):635–638. doi: 10.1074/jbc.271.2.635. [DOI] [PubMed] [Google Scholar]
  31. Segal M. S., Bye J. M., Sambrook J. F., Gething M. J. Disulfide bond formation during the folding of influenza virus hemagglutinin. J Cell Biol. 1992 Jul;118(2):227–244. doi: 10.1083/jcb.118.2.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stuart D. I., Jones E. Y. Cutting complexity down to size. Nature. 1997 Apr 3;386(6624):437–438. doi: 10.1038/386437a0. [DOI] [PubMed] [Google Scholar]
  33. Taylor J. A., Gibson G. J., Brooks D. A., Hopwood J. J. Human N-acetylgalactosamine-4-sulphatase biosynthesis and maturation in normal, Maroteaux-Lamy and multiple-sulphatase-deficient fibroblasts. Biochem J. 1990 Jun 1;268(2):379–386. doi: 10.1042/bj2680379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Thomas P. J., Qu B. H., Pedersen P. L. Defective protein folding as a basis of human disease. Trends Biochem Sci. 1995 Nov;20(11):456–459. doi: 10.1016/s0968-0004(00)89100-8. [DOI] [PubMed] [Google Scholar]
  35. Weitz G., Proia R. L. Analysis of the glycosylation and phosphorylation of the alpha-subunit of the lysosomal enzyme, beta-hexosaminidase A, by site-directed mutagenesis. J Biol Chem. 1992 May 15;267(14):10039–10044. [PubMed] [Google Scholar]
  36. Zokaeem G., Bayleran J., Kaplan P., Hechtman P., Neufeld E. F. A shortened beta-hexosaminidase alpha-chain in an Italian patient with infantile Tay-Sachs disease. Am J Hum Genet. 1987 Jun;40(6):537–547. [PMC free article] [PubMed] [Google Scholar]

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