Molecular Medicine

, Volume 19, Issue 1, pp 18–25 | Cite as

Molecular Expression and Characterization of Erythroid-Specific 5-Aminolevulinate Synthase Gain-of-Function Mutations Causing X-Linked Protoporphyria

  • David F. Bishop
  • Vassili Tchaikovskii
  • Irina Nazarenko
  • Robert J. Desnick
Research Article


X-linked protoporphyria (XLP) (MIM 300752) is a recently recognized erythropoietic porphyria due to gain-of-function mutations in the erythroid-specific aminolevulinate synthase gene (ALAS2). Previously, two exon 11 small deletions, c.1699_1670ΔAT (ΔAT) and c.1706_1709ΔAGTG (ΔAGTG), that prematurely truncated or elongated the ALAS2 polypeptide, were reported to increase enzymatic activity 20- to 40-fold, causing the erythroid accumulation of protoporphyrins, cutaneous photosensitivity and liver disease. The mutant ΔAT and ΔAGTG ALAS2 enzymes, two novel mutations, c.1734ΔG (ΔG) and c.1642C>T (p.Q548X), and an engineered deletion c.1670–1671TC>GA p.F557X were expressed, and their purified enzymes were characterized. Wild-type and ΔAGTG enzymes exhibited similar amounts of 54- and 52-kDa polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas the ΔAT and p.F557X had only 52-kDa polypeptides. Compared to the purified wild-type enzyme, ΔAT, ΔAGTG and Q548X enzymes had increased specific activities that were only 1.8-, 3.1- and 1.6-fold, respectively. Interestingly, binding studies demonstrated that the increased activity Q548X enzyme did not bind to succinyl-CoA synthetase. The elongated ΔG enzyme had wild-type specific activity, kinetics and thermostability; twice the wild-type purification yield (56 versus 25%); and was primarily a 54-kDa form, suggesting greater stability in vivo. On the basis of studies of mutant enzymes, the maximal gain-of function region spanned 57 amino acids between 533 and 580. Thus, these ALAS2 gain-of-function mutations increased the specific activity (ΔAT, ΔAGTG and p.Q548X) or stability (ΔG) of the enzyme, thereby leading to the increased erythroid protoporphyrin accumulation causing XLP.



This research was supported in part by grants from the National Institutes of Health (NIH), including a research grant (5 R01 DK026824) and a grant (1 U54 DK083909) for the Porphyria Consortium of the NIH Rare Diseases Clinical Research Network as well as a research grant (C024404) from the New York State Department of Health. Funding and/or programmatic support for this project was provided by the NIH Office of Rare Disease Clinical Research Network. The views expressed in written materials or publications do not necessarily reflect the official policies of the Department of Health and Human Services.

Supplementary material

10020_2013_1901018_MOESM1_ESM.pdf (333 kb)
Supplementary material, approximately 333 KB.


  1. 1.
    Whatley SD, et al. (2008) C-terminal deletions in the ALAS2 gene lead to gain of function and cause X-linked dominant protoporphyria without anemia or iron overload. Am. J. Hum. Genet. 83:408–14.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Anderson KE, Sassa S, Bishop DF, Desnick RJ. (2001) Disorders of heme biosynthesis: X-linked sideroblastic anemia and the porphyrias. In: The Metabolic and Molecular Bases of Inherited Disease. Scriver CR, et al. (eds.) McGraw-Hill, New York, pp. 2991–3062.Google Scholar
  3. 3.
    Magnus IA, Jarrett A, Prankerd TA, Rimington C. (1961) Erythropoietic protoporphyria: a new porphyria syndrome with solar urticaria due to protoporphyrinaemia. Lancet 2:448–51.CrossRefPubMedGoogle Scholar
  4. 4.
    Bonkowsky HL, Bloomer JR, Ebert PS, Mahoney MJ. (1975) Heme synthetase deficiency in human protoporphyria: demonstration of the defect in liver and cultured skin fibroblasts. J. Clin. Invest. 56:1139–48.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Anderson KE. (2008) The porphyrias. In: Cecil Medicine. Goldman L, Ausiello D (eds.) Saunders, Philadelphia, pp. 1585–93.Google Scholar
  6. 6.
    Gross U, Frank M, Doss MO. (1998) Hepatic complications of erythropoietic protoporphyria. Photodermatol. Photoimmunol. Photomed. 14:52–7.CrossRefPubMedGoogle Scholar
  7. 7.
    Harms J, Lautenschlager S, Minder CE, Minder EI. (2009) An alpha-melanocyte-stimulating hormone analogue in erythropoietic protoporphyria. N. Engl. J. Med. 360:306–7.CrossRefPubMedGoogle Scholar
  8. 8.
    Wahlin S, et al. (2010) Combined liver and kidney transplantation in acute intermittent porphyria. Transpl. Int. 23:e18–21.CrossRefPubMedGoogle Scholar
  9. 9.
    Balwani M, et al. (2013) Loss-of-function ferrochelatase and gain-of-function erythroid-specific 5-aminolevulinate synthase mutations causing erythropoietic protoporphyria and X-linked protoporphyria in North American patients reveal novel mutations and a high prevalence of X-linked protoporphyria. Mol. Med. 19:26–35.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Cotter PD, Rucknagel DL, Bishop DF. (1994) X-linked sideroblastic anemia: identification of the mutation in the erythroid-specific delta-aminolevulinate synthase gene (ALAS2) in the original family described by Cooley. Blood. 84:3915–24.PubMedGoogle Scholar
  11. 11.
    Bishop DF, Tchaikovskii V, Hoffbrand AV, Fraser ME, Margolis S. (2012) X-linked sideroblastic anemia due to carboxy-terminal ALAS2 mutations that cause loss of binding to the beta-subunit of succinyl-CoA synthetase (SUCLA2). J. Biol. Chem. 287:28943–55.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Furuyama K, et al. (1997) Pyridoxine refractory X-linked sideroblastic anemia caused by a point mutation in the erythroid 5-aminolevulinate synthase gene. Blood. 90:822–30.PubMedGoogle Scholar
  13. 13.
    Laemmli UK, Favre M. (1973) Maturation of the head of bacteriophage T4. I. DNA packaging events. J. Mol. Biol. 80:575–99.CrossRefPubMedGoogle Scholar
  14. 14.
    Urata G, Granick S. (1963) Biosynthesis of alphaaminoketones and the metabolism of aminoacetone. J. Biol. Chem. 238:811–20.PubMedGoogle Scholar
  15. 15.
    Bishop DF, et al. (1978) Pilot scale purification of alpha-galactosidase A from Cohn fraction IV-1 of human plasma. Biochim. Biophys. Acta. 524:109–20.CrossRefPubMedGoogle Scholar
  16. 16.
    Kadirvel S, et al. (2012) The carboxyl-terminal region of erythroid-specific 5-aminolevulinate synthase acts as an intrinsic modifier for its catalytic activity and protein stability. Exp. Hematol. 40:477–86.e1.CrossRefPubMedGoogle Scholar
  17. 17.
    Shah DI, et al. (2012) Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts. Nature. 491:608–12.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Surinya KH, Cox TC, Masy B. (1997) Transcriptional regulation of the human erythroid 5-aminolevulinase synthase gene. J. Biol. Chem. 272:26585–94.CrossRefPubMedGoogle Scholar
  19. 19.
    Lendrihas T, Hunter GA, Ferreira GC. (2010) Targeting the active site gate to yield hyperactive variants of 5-aminolevulinate synthase. J. Biol. Chem. 285:13704–11.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Chen W, Dailey HA, Paw BH. (2010) Ferrochelatase forms an oligomeric complex with mitoferrin-1 and Abcb10 for erythroid heme biosynthesis. Blood. 116:628–30.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Ducamp S, et al. (2013) Molecular and functional analysis of the C-terminal region of human erythroid-specific 5-aminolevulinic synthase associated with X-linked dominant protoporphyria (XLDPP). Hum. Mol. Genet. 2013, Jan 3 [Epub ahead of print].Google Scholar

Copyright information

© The Author(s) 2013

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this license to share adapted material derived from this article or parts of it.

The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this license, visit (

Authors and Affiliations

  • David F. Bishop
    • 1
  • Vassili Tchaikovskii
    • 1
  • Irina Nazarenko
    • 1
  • Robert J. Desnick
    • 1
  1. 1.Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount SinaiMount Sinai Medical CenterNew YorkUSA

Personalised recommendations