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. 1991 Sep;49(3):646–655.

Gaucher disease: heterologous expression of two alleles associated with neuronopathic phenotypes.

M E Grace 1, A Berg 1, G S He 1, L Goldberg 1, M Horowitz 1, G A Grabowski 1
PMCID: PMC1683149  PMID: 1909090

Abstract

To investigate the molecular basis for the distinct neuronopathic phenotypes of Gaucher disease, acid beta-glucosidases expressed from mutant DNAs in Gaucher disease type 2 (acute) and type 3 (subacute) patients were characterized in fibroblasts and with the baculovirus expression system in insect cells. Expression of the mutant DNA encoding a proline-for-leucine substitution at amino acid 444 (L444P) resulted in a catalytically defective, unstable acid beta-glucosidase in either fibroblasts from L444P/L444P homozygotes or in insect cells. This mutation was found to be homoallelic in subacute neuronopathic (type 3) Gaucher disease. In comparison, expression of the mutant cDNA encoding an arginine-for-proline substitution at amino acid 415 (P415R) resulted in an inactive and unstable protein in insect cells. This allele was found only in a type 2 patient with the L444P/P415R genotype. The substantial variation in the type 3 phenotype (L444P homozygotes) suggests the complex nature of the molecular basis of phenotypic variation in Gaucher disease. Yet, the association of neuronopathic phenotypes with alleles producing severely compromised (L444P) or functionally null (P415R) enzymes indicates that the effective level of residual activity at the lysosome is likely to be a major determinant of the severity of Gaucher disease.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BRADY R. O., KANFER J., SHAPIRO D. THE METABOLISM OF GLUCOCEREBROSIDES. I. PURIFICATION AND PROPERTIES OF A GLUCOCEREBROSIDE-CLEAVING ENZYME FROM SPLEEN TISSUE. J Biol Chem. 1965 Jan;240:39–43. [PubMed] [Google Scholar]
  2. Barton N. W., Furbish F. S., Murray G. J., Garfield M., Brady R. O. Therapeutic response to intravenous infusions of glucocerebrosidase in a patient with Gaucher disease. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1913–1916. doi: 10.1073/pnas.87.5.1913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bergmann J. E., Grabowski G. A. Posttranslational processing of human lysosomal acid beta-glucosidase: a continuum of defects in Gaucher disease type 1 and type 2 fibroblasts. Am J Hum Genet. 1989 May;44(5):741–750. [PMC free article] [PubMed] [Google Scholar]
  4. Beutler E., Gelbart T. Gaucher disease associated with a unique KpnI restriction site: identification of the amino-acid substitution. Ann Hum Genet. 1990 May;54(Pt 2):149–153. doi: 10.1111/j.1469-1809.1990.tb00371.x. [DOI] [PubMed] [Google Scholar]
  5. Beutler E., Kuhl W. Glucocerebrosidase processing in normal fibroblasts and in fibroblasts from patients with type I, type II, and type III Gaucher disease. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7472–7474. doi: 10.1073/pnas.83.19.7472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  7. Choy F. Y., Woo M., Potier M. In situ radiation-inactivation size of fibroblast membrane-bound acid beta-glucosidase in Gaucher type 1, type 2 and type 3 disease. Biochim Biophys Acta. 1986 Mar 7;870(1):76–81. doi: 10.1016/0167-4838(86)90010-5. [DOI] [PubMed] [Google Scholar]
  8. Conzelmann E., Sandhoff K. Partial enzyme deficiencies: residual activities and the development of neurological disorders. Dev Neurosci. 1983;6(1):58–71. doi: 10.1159/000112332. [DOI] [PubMed] [Google Scholar]
  9. Dahl N., Lagerström M., Erikson A., Pettersson U. Gaucher disease type III (Norrbottnian type) is caused by a single mutation in exon 10 of the glucocerebrosidase gene. Am J Hum Genet. 1990 Aug;47(2):275–278. [PMC free article] [PubMed] [Google Scholar]
  10. Dinur T., Grabowski G. A., Desnick R. J., Gatt S. Synthesis of a fluorescent derivative of glucosyl ceramide for the sensitive determination of glucocerebrosidase activity. Anal Biochem. 1984 Jan;136(1):223–234. doi: 10.1016/0003-2697(84)90329-4. [DOI] [PubMed] [Google Scholar]
  11. Dinur T., Osiecki K. M., Legler G., Gatt S., Desnick R. J., Grabowski G. A. Human acid beta-glucosidase: isolation and amino acid sequence of a peptide containing the catalytic site. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1660–1664. doi: 10.1073/pnas.83.6.1660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dreborg S., Erikson A., Hagberg B. Gaucher disease--Norrbottnian type. I. General clinical description. Eur J Pediatr. 1980 Mar;133(2):107–118. doi: 10.1007/BF00441578. [DOI] [PubMed] [Google Scholar]
  13. Erickson A. H., Ginns E. I., Barranger J. A. Biosynthesis of the lysosomal enzyme glucocerebrosidase. J Biol Chem. 1985 Nov 15;260(26):14319–14324. [PubMed] [Google Scholar]
  14. Fabbro D., Desnick R. J., Grabowski G. A. Gaucher disease: genetic heterogeneity within and among the subtypes detected by immunoblotting. Am J Hum Genet. 1987 Jan;40(1):15–31. [PMC free article] [PubMed] [Google Scholar]
  15. Firon N., Eyal N., Kolodny E. H., Horowitz M. Genotype assignment in Gaucher disease by selective amplification of the active glucocerebrosidase gene. Am J Hum Genet. 1990 Mar;46(3):527–532. [PMC free article] [PubMed] [Google Scholar]
  16. Ginns E. I., Brady R. O., Pirruccello S., Moore C., Sorrell S., Furbish F. S., Murray G. J., Tager J., Barranger J. A. Mutations of glucocerebrosidase: discrimination of neurologic and non-neurologic phenotypes of Gaucher disease. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5607–5610. doi: 10.1073/pnas.79.18.5607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grabowski G. A., Gatt S., Horowitz M. Acid beta-glucosidase: enzymology and molecular biology of Gaucher disease. Crit Rev Biochem Mol Biol. 1990;25(6):385–414. doi: 10.3109/10409239009090616. [DOI] [PubMed] [Google Scholar]
  18. Grabowski G. A., Gatt S., Kruse J., Desnick R. J. Human lysosomal beta-glucosidase: kinetic characterization of the catalytic, aglycon, and hydrophobic binding sites. Arch Biochem Biophys. 1984 May 15;231(1):144–157. doi: 10.1016/0003-9861(84)90371-0. [DOI] [PubMed] [Google Scholar]
  19. Grabowski G. A., Goldblatt J., Dinur T., Kruse J., Svennerholm L., Gatt S., Desnick R. J. Genetic heterogeneity in Gaucher disease: physicokinetic and immunologic studies of the residual enzyme in cultured fibroblasts from non-neuronopathic and neuronopathic patients. Am J Med Genet. 1985 Jul;21(3):529–549. doi: 10.1002/ajmg.1320210316. [DOI] [PubMed] [Google Scholar]
  20. Grabowski G. A., White W. R., Grace M. E. Expression of functional human acid beta-glucosidase in COS-1 and Spodoptera frugiperda cells. Enzyme. 1989;41(3):131–142. doi: 10.1159/000469068. [DOI] [PubMed] [Google Scholar]
  21. Grace M. E., Graves P. N., Smith F. I., Grabowski G. A. Analyses of catalytic activity and inhibitor binding of human acid beta-glucosidase by site-directed mutagenesis. Identification of residues critical to catalysis and evidence for causality of two Ashkenazi Jewish Gaucher disease type 1 mutations. J Biol Chem. 1990 Apr 25;265(12):6827–6835. [PubMed] [Google Scholar]
  22. Graves P. N., Grabowski G. A., Eisner R., Palese P., Smith F. I. Gaucher disease type 1: cloning and characterization of a cDNA encoding acid beta-glucosidase from an Ashkenazi Jewish patient. DNA. 1988 Oct;7(8):521–528. doi: 10.1089/dna.1.1988.7.521. [DOI] [PubMed] [Google Scholar]
  23. Hong C. M., Ohashi T., Yu X. J., Weiler S., Barranger J. A. Sequence of two alleles responsible for Gaucher disease. DNA Cell Biol. 1990 May;9(4):233–241. doi: 10.1089/dna.1990.9.233. [DOI] [PubMed] [Google Scholar]
  24. Horowitz M., Wilder S., Horowitz Z., Reiner O., Gelbart T., Beutler E. The human glucocerebrosidase gene and pseudogene: structure and evolution. Genomics. 1989 Jan;4(1):87–96. doi: 10.1016/0888-7543(89)90319-4. [DOI] [PubMed] [Google Scholar]
  25. Kolodny E. H., Firon N., Eyal N., Horowitz M. Mutation analysis of an Ashkenazi Jewish family with Gaucher disease in three successive generations. Am J Med Genet. 1990 Aug;36(4):467–472. doi: 10.1002/ajmg.1320360419. [DOI] [PubMed] [Google Scholar]
  26. Latham T. E., Theophilus B. D., Grabowski G. A., Smith F. I. Heterogeneity of mutations in the acid beta-glucosidase gene of Gaucher disease patients. DNA Cell Biol. 1991 Jan-Feb;10(1):15–21. doi: 10.1089/dna.1991.10.15. [DOI] [PubMed] [Google Scholar]
  27. Latham T., Grabowski G. A., Theophilus B. D., Smith F. I. Complex alleles of the acid beta-glucosidase gene in Gaucher disease. Am J Hum Genet. 1990 Jul;47(1):79–86. [PMC free article] [PubMed] [Google Scholar]
  28. Leibovitz-BenGershon Z., Rosenthal J., Shinar E. A study of acid beta-glucosidase in a patient with Gaucher disease and leukemia. Prog Clin Biol Res. 1982;95:481–491. [PubMed] [Google Scholar]
  29. Maret A., Potier M., Salvayre R., Douste-Blazy L. Modification of subunit interaction in membrane-bound acid beta-glucosidase from Gaucher disease. FEBS Lett. 1983 Aug 22;160(1-2):93–97. doi: 10.1016/0014-5793(83)80943-0. [DOI] [PubMed] [Google Scholar]
  30. Maret A., Potier M., Salvayre R., Troly M., Beauregard G., Douste-Blazy L. In vitro detergent activation of lysosomal acid beta-glucosidase in the spleen of normal and type 1 Gaucher patients is not accompanied by change in aggregation state. Biochim Biophys Acta. 1989 Jul 6;996(3):254–256. doi: 10.1016/0167-4838(89)90255-0. [DOI] [PubMed] [Google Scholar]
  31. Masuno M., Tomatsu S., Sukegawa K., Orii T. Non-existence of a tight association between a 444leucine to proline mutation and phenotypes of Gaucher disease: high frequency of a NciI polymorphism in the non-neuronopathic form. Hum Genet. 1990 Jan;84(2):203–206. doi: 10.1007/BF00208943. [DOI] [PubMed] [Google Scholar]
  32. Mueller O. T., Rosenberg A. beta-Glucoside hydrolase activity of normal and glucosylceramidotic cultured human skin fibroblasts. J Biol Chem. 1977 Feb 10;252(3):825–829. [PubMed] [Google Scholar]
  33. Myerowitz R., Proia R. L. cDNA clone for the alpha-chain of human beta-hexosaminidase: deficiency of alpha-chain mRNA in Ashkenazi Tay-Sachs fibroblasts. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5394–5398. doi: 10.1073/pnas.81.17.5394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ohashi T., Hong C. M., Weiler S., Tomich J. M., Aerts J. M., Tager J. M., Barranger J. A. Characterization of human glucocerebrosidase from different mutant alleles. J Biol Chem. 1991 Feb 25;266(6):3661–3667. [PubMed] [Google Scholar]
  35. Osiecki-Newman K., Fabbro D., Legler G., Desnick R. J., Grabowski G. A. Human acid beta-glucosidase: use of inhibitors, alternative substrates and amphiphiles to investigate the properties of the normal and Gaucher disease active sites. Biochim Biophys Acta. 1987 Sep 2;915(1):87–100. doi: 10.1016/0167-4838(87)90128-2. [DOI] [PubMed] [Google Scholar]
  36. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schimmel P. Hazards and their exploitation in the applications of molecular biology to structure-function relationships. Biochemistry. 1990 Oct 16;29(41):9495–9502. doi: 10.1021/bi00493a001. [DOI] [PubMed] [Google Scholar]
  38. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Taylor J. W., Schmidt W., Cosstick R., Okruszek A., Eckstein F. The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8749–8764. doi: 10.1093/nar/13.24.8749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Theophilus B. D., Latham T., Grabowski G. A., Smith F. I. Comparison of RNase A, a chemical cleavage and GC-clamped denaturing gradient gel electrophoresis for the detection of mutations in exon 9 of the human acid beta-glucosidase gene. Nucleic Acids Res. 1989 Oct 11;17(19):7707–7722. doi: 10.1093/nar/17.19.7707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tsuji S., Choudary P. V., Martin B. M., Stubblefield B. K., Mayor J. A., Barranger J. A., Ginns E. I. A mutation in the human glucocerebrosidase gene in neuronopathic Gaucher's disease. N Engl J Med. 1987 Mar 5;316(10):570–575. doi: 10.1056/NEJM198703053161002. [DOI] [PubMed] [Google Scholar]
  42. Tsuji S., Martin B. M., Barranger J. A., Stubblefield B. K., LaMarca M. E., Ginns E. I. Genetic heterogeneity in type 1 Gaucher disease: multiple genotypes in Ashkenazic and non-Ashkenazic individuals. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2349–2352. doi: 10.1073/pnas.85.7.2349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wigderson M., Firon N., Horowitz Z., Wilder S., Frishberg Y., Reiner O., Horowitz M. Characterization of mutations in Gaucher patients by cDNA cloning. Am J Hum Genet. 1989 Mar;44(3):365–377. [PMC free article] [PubMed] [Google Scholar]
  44. Willemsen R., van Dongen J. M., Ginns E. I., Sips H. J., Schram A. W., Tager J. M., Barranger J. A., Reuser A. J. Ultrastructural localization of glucocerebrosidase in cultured Gaucher's disease fibroblasts by immunocytochemistry. J Neurol. 1987 Jan;234(1):44–51. doi: 10.1007/BF00314009. [DOI] [PubMed] [Google Scholar]
  45. Winkelman M. D., Banker B. Q., Victor M., Moser H. W. Non-infantile neuronopathic Gaucher's disease: a clinicopathologic study. Neurology. 1983 Aug;33(8):994–1008. doi: 10.1212/wnl.33.8.994. [DOI] [PubMed] [Google Scholar]
  46. Zimran A., Sorge J., Gross E., Kubitz M., West C., Beutler E. A glucocerebrosidase fusion gene in Gaucher disease. Implications for the molecular anatomy, pathogenesis, and diagnosis of this disorder. J Clin Invest. 1990 Jan;85(1):219–222. doi: 10.1172/JCI114415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zimran A., Sorge J., Gross E., Kubitz M., West C., Beutler E. Prediction of severity of Gaucher's disease by identification of mutations at DNA level. Lancet. 1989 Aug 12;2(8659):349–352. doi: 10.1016/s0140-6736(89)90536-9. [DOI] [PubMed] [Google Scholar]
  48. d'Azzo A., Proia R. L., Kolodny E. H., Kaback M. M., Neufeld E. F. Faulty association of alpha- and beta-subunits in some forms of beta-hexosaminidase A deficiency. J Biol Chem. 1984 Sep 10;259(17):11070–11074. [PubMed] [Google Scholar]

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