Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1996 Oct 15;98(8):1809–1817. doi: 10.1172/JCI118981

A carboxy-terminal truncation of human alpha-galactosidase A in a heterozygous female with Fabry disease and modification of the enzymatic activity by the carboxy-terminal domain. Increased, reduced, or absent enzyme activity depending on number of amino acid residues deleted.

N Miyamura 1, E Araki 1, K Matsuda 1, R Yoshimura 1, N Furukawa 1, K Tsuruzoe 1, T Shirotani 1, H Kishikawa 1, K Yamaguchi 1, M Shichiri 1
PMCID: PMC507620  PMID: 8878432

Abstract

Fabry disease is an X-linked disorder of glycosphingolipid metabolism caused by a deficiency of alpha-galactosidase A (alpha-Gal A). We identified a novel mutation of alpha-Gal A gene in a family with Fabry disease, which converted a tyrosine at codon 365 to a stop and resulted in a truncation of the carboxy (C) terminus by 65 amino acid (AA) residues. In a heterozygote of this family, although the mutant and normal alleles were equally transcribed in cultured fibroblasts, lymphocyte alpha-Gal A activity was approximately 30% of the normal control and severe clinical symptoms were apparent. COS-1 cells transfected with this mutant cDNA showed a complete loss of its enzymatic activity. Furthermore, those cotransfected with mutant and wildtype cDNAs showed a lower alpha-Gal A activity than those with wild type alone (approximately 30% of wild type alone), which suggested the dominant negative effect of this mutation and implied the importance of the C terminus for its activity. Thus, we generated mutant cDNAs with various deletion of the C terminus, and analyzed. Unexpectedly, alpha-Gal A activity was enhanced by up to sixfold compared with wild-type when from 2 to 10 AA residues were deleted. In contrast, deletion of 12 or more AA acid residues resulted in a complete loss of enzyme activity. Our data suggest that the C-terminal region of alpha-Gal A plays an important role in the regulation of its enzyme activity.

Full Text

The Full Text of this article is available as a PDF (384.1 KB).

Selected References

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

  1. Avila J. L., Convit J., Velazquez-Avila G. Fabry's disease: normal alpha-galactosidase activity and urinary-sediment glycosphingolipid levels in two obligate heterozygotes. Br J Dermatol. 1973 Aug;89(2):149–157. doi: 10.1111/j.1365-2133.1973.tb02951.x. [DOI] [PubMed] [Google Scholar]
  2. Barton N. W., Brady R. O., Dambrosia J. M., Di Bisceglie A. M., Doppelt S. H., Hill S. C., Mankin H. J., Murray G. J., Parker R. I., Argoff C. E. Replacement therapy for inherited enzyme deficiency--macrophage-targeted glucocerebrosidase for Gaucher's disease. N Engl J Med. 1991 May 23;324(21):1464–1470. doi: 10.1056/NEJM199105233242104. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bernstein H. S., Bishop D. F., Astrin K. H., Kornreich R., Eng C. M., Sakuraba H., Desnick R. J. Fabry disease: six gene rearrangements and an exonic point mutation in the alpha-galactosidase gene. J Clin Invest. 1989 Apr;83(4):1390–1399. doi: 10.1172/JCI114027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beutler E., Kuhl W. Biochemical and electrophoretic studies of -galactosidase in normal man, in patients with Fabry's disease, and in Equidae. Am J Hum Genet. 1972 May;24(3):237–249. [PMC free article] [PubMed] [Google Scholar]
  6. Bishop D. F., Calhoun D. H., Bernstein H. S., Hantzopoulos P., Quinn M., Desnick R. J. Human alpha-galactosidase A: nucleotide sequence of a cDNA clone encoding the mature enzyme. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4859–4863. doi: 10.1073/pnas.83.13.4859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bishop D. F., Kornreich R., Desnick R. J. Structural organization of the human alpha-galactosidase A gene: further evidence for the absence of a 3' untranslated region. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3903–3907. doi: 10.1073/pnas.85.11.3903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  9. Calhoun D. H., Bishop D. F., Bernstein H. S., Quinn M., Hantzopoulos P., Desnick R. J. Fabry disease: isolation of a cDNA clone encoding human alpha-galactosidase A. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7364–7368. doi: 10.1073/pnas.82.21.7364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Davies J. P., Winchester B. G., Malcolm S. Mutation analysis in patients with the typical form of Anderson-Fabry disease. Hum Mol Genet. 1993 Jul;2(7):1051–1053. doi: 10.1093/hmg/2.7.1051. [DOI] [PubMed] [Google Scholar]
  11. Desnick R. J., Simmons R. L., Allen K. Y., Woods J. E., Anderson C. F., Najarian J. S., Krivit W. Correction of enzymatic deficiencies by renal transplantation: Fabry's disease. Surgery. 1972 Aug;72(2):203–211. [PubMed] [Google Scholar]
  12. Eng C. M., Desnick R. J. Molecular basis of Fabry disease: mutations and polymorphisms in the human alpha-galactosidase A gene. Hum Mutat. 1994;3(2):103–111. doi: 10.1002/humu.1380030204. [DOI] [PubMed] [Google Scholar]
  13. Eng C. M., Niehaus D. J., Enriquez A. L., Burgert T. S., Ludman M. D., Desnick R. J. Fabry disease: twenty-three mutations including sense and antisense CpG alterations and identification of a deletional hot-spot in the alpha-galactosidase A gene. Hum Mol Genet. 1994 Oct;3(10):1795–1799. doi: 10.1093/hmg/3.10.1795. [DOI] [PubMed] [Google Scholar]
  14. Eng C. M., Resnick-Silverman L. A., Niehaus D. J., Astrin K. H., Desnick R. J. Nature and frequency of mutations in the alpha-galactosidase A gene that cause Fabry disease. Am J Hum Genet. 1993 Dec;53(6):1186–1197. [PMC free article] [PubMed] [Google Scholar]
  15. Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gould S. J., Keller G. A., Hosken N., Wilkinson J., Subramani S. A conserved tripeptide sorts proteins to peroxisomes. J Cell Biol. 1989 May;108(5):1657–1664. doi: 10.1083/jcb.108.5.1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gross-Bellard M., Oudet P., Chambon P. Isolation of high-molecular-weight DNA from mammalian cells. Eur J Biochem. 1973 Jul 2;36(1):32–38. doi: 10.1111/j.1432-1033.1973.tb02881.x. [DOI] [PubMed] [Google Scholar]
  18. Howey D. C., Bowsher R. R., Brunelle R. L., Woodworth J. R. [Lys(B28), Pro(B29)]-human insulin. A rapidly absorbed analogue of human insulin. Diabetes. 1994 Mar;43(3):396–402. doi: 10.2337/diab.43.3.396. [DOI] [PubMed] [Google Scholar]
  19. Ishii S., Sakuraba H., Shimmoto M., Minamikawa-Tachino R., Suzuki T., Suzuki Y. Fabry disease: detection of 13-bp deletion in alpha-galactosidase A gene and its application to gene diagnosis of heterozygotes. Ann Neurol. 1991 May;29(5):560–564. doi: 10.1002/ana.410290517. [DOI] [PubMed] [Google Scholar]
  20. Jackson M. R., Nilsson T., Peterson P. A. Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum. EMBO J. 1990 Oct;9(10):3153–3162. doi: 10.1002/j.1460-2075.1990.tb07513.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Koide T., Ishiura M., Iwai K., Inoue M., Kaneda Y., Okada Y., Uchida T. A case of Fabry's disease in a patient with no alpha-galactosidase A activity caused by a single amino acid substitution of Pro-40 by Ser. FEBS Lett. 1990 Jan 1;259(2):353–356. doi: 10.1016/0014-5793(90)80046-l. [DOI] [PubMed] [Google Scholar]
  22. Kornreich R., Bishop D. F., Desnick R. J. Alpha-galactosidase A gene rearrangements causing Fabry disease. Identification of short direct repeats at breakpoints in an Alu-rich gene. J Biol Chem. 1990 Jun 5;265(16):9319–9326. [PubMed] [Google Scholar]
  23. Kornreich R., Desnick R. J., Bishop D. F. Nucleotide sequence of the human alpha-galactosidase A gene. Nucleic Acids Res. 1989 Apr 25;17(8):3301–3302. doi: 10.1093/nar/17.8.3301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kusukawa N., Uemori T., Asada K., Kato I. Rapid and reliable protocol for direct sequencing of material amplified by the polymerase chain reaction. Biotechniques. 1990 Jul;9(1):66-8, 70, 72. [PubMed] [Google Scholar]
  25. LYON M. F. Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature. 1961 Apr 22;190:372–373. doi: 10.1038/190372a0. [DOI] [PubMed] [Google Scholar]
  26. Lei K. J., Pan C. J., Shelly L. L., Liu J. L., Chou J. Y. Identification of mutations in the gene for glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type 1a. J Clin Invest. 1994 May;93(5):1994–1999. doi: 10.1172/JCI117192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lemansky P., Bishop D. F., Desnick R. J., Hasilik A., von Figura K. Synthesis and processing of alpha-galactosidase A in human fibroblasts. Evidence for different mutations in Fabry disease. J Biol Chem. 1987 Feb 15;262(5):2062–2065. [PubMed] [Google Scholar]
  28. Mayes J. S., Scheerer J. B., Sifers R. N., Donaldson M. L. Differential assay for lysosomal alpha-galactosidases in human tissues and its application to Fabry's disease. Clin Chim Acta. 1981 May 5;112(2):247–251. doi: 10.1016/0009-8981(81)90384-3. [DOI] [PubMed] [Google Scholar]
  29. Meaney C., Blanch L. C., Morris C. P. A nonsense mutation (R220X) in the alpha-galactosidase A gene detected in a female carrier of Fabry disease. Hum Mol Genet. 1994 Jun;3(6):1019–1020. doi: 10.1093/hmg/3.6.1019. [DOI] [PubMed] [Google Scholar]
  30. Miyazawa S., Osumi T., Hashimoto T., Ohno K., Miura S., Fujiki Y. Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus. Mol Cell Biol. 1989 Jan;9(1):83–91. doi: 10.1128/mcb.9.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Munro S., Pelham H. R. A C-terminal signal prevents secretion of luminal ER proteins. Cell. 1987 Mar 13;48(5):899–907. doi: 10.1016/0092-8674(87)90086-9. [DOI] [PubMed] [Google Scholar]
  32. Nagao Y., Nakashima H., Fukuhara Y., Shimmoto M., Oshima A., Ikari Y., Mori Y., Sakuraba H., Suzuki Y. Hypertrophic cardiomyopathy in late-onset variant of Fabry disease with high residual activity of alpha-galactosidase A. Clin Genet. 1991 Mar;39(3):233–237. doi: 10.1111/j.1399-0004.1991.tb03018.x. [DOI] [PubMed] [Google Scholar]
  33. Nakao S., Takenaka T., Maeda M., Kodama C., Tanaka A., Tahara M., Yoshida A., Kuriyama M., Hayashibe H., Sakuraba H. An atypical variant of Fabry's disease in men with left ventricular hypertrophy. N Engl J Med. 1995 Aug 3;333(5):288–293. doi: 10.1056/NEJM199508033330504. [DOI] [PubMed] [Google Scholar]
  34. Nilsson T., Jackson M., Peterson P. A. Short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum. Cell. 1989 Aug 25;58(4):707–718. doi: 10.1016/0092-8674(89)90105-0. [DOI] [PubMed] [Google Scholar]
  35. Quinn M., Hantzopoulos P., Fidanza V., Calhoun D. H. A genomic clone containing the promoter for the gene encoding the human lysosomal enzyme, alpha-galactosidase A. Gene. 1987;58(2-3):177–188. doi: 10.1016/0378-1119(87)90374-x. [DOI] [PubMed] [Google Scholar]
  36. Romeo G., Migeon B. R. Genetic inactivation of the alpha-galactosidase locus in carriers of Fabry's disease. Science. 1970 Oct 9;170(3954):180–181. doi: 10.1126/science.170.3954.180. [DOI] [PubMed] [Google Scholar]
  37. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  38. Sakuraba H., Eng C. M., Desnick R. J., Bishop D. F. Invariant exon skipping in the human alpha-galactosidase A pre-mRNA: Ag+1 to t substitution in a 5'-splice site causing Fabry disease. Genomics. 1992 Apr;12(4):643–650. doi: 10.1016/0888-7543(92)90288-4. [DOI] [PubMed] [Google Scholar]
  39. Sakuraba H., Oshima A., Fukuhara Y., Shimmoto M., Nagao Y., Bishop D. F., Desnick R. J., Suzuki Y. Identification of point mutations in the alpha-galactosidase A gene in classical and atypical hemizygotes with Fabry disease. Am J Hum Genet. 1990 Nov;47(5):784–789. [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Tsuji A., Oda R., Sakiyama K., Nagamune H., Itoh K., Kase R., Sakuraba H., Suzuki Y., Matsuda Y. Lysosomal enzyme replacement using alpha 2-macroglobulin as a transport vehicle. J Biochem. 1994 May;115(5):937–944. doi: 10.1093/oxfordjournals.jbchem.a124442. [DOI] [PubMed] [Google Scholar]
  42. Tsuji S., Martin B. M., Kaslow D. C., Migeon B. R., Choudary P. V., Stubbleflied B. K., Mayor J. A., Murray G. J., Barranger J. A., Ginns E. I. Signal sequence and DNA-mediated expression of human lysosomal alpha-galactosidase A. Eur J Biochem. 1987 Jun 1;165(2):275–280. doi: 10.1111/j.1432-1033.1987.tb11438.x. [DOI] [PubMed] [Google Scholar]
  43. Wang A. M., Bishop D. F., Desnick R. J. Human alpha-N-acetylgalactosaminidase-molecular cloning, nucleotide sequence, and expression of a full-length cDNA. Homology with human alpha-galactosidase A suggests evolution from a common ancestral gene. J Biol Chem. 1990 Dec 15;265(35):21859–21866. [PubMed] [Google Scholar]
  44. von Scheidt W., Eng C. M., Fitzmaurice T. F., Erdmann E., Hübner G., Olsen E. G., Christomanou H., Kandolf R., Bishop D. F., Desnick R. J. An atypical variant of Fabry's disease with manifestations confined to the myocardium. N Engl J Med. 1991 Feb 7;324(6):395–399. doi: 10.1056/NEJM199102073240607. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES