Analysis of pseudoxanthoma elasticum-causing missense mutants of ABCC6 in vivo; pharmacological correction of the mislocalized proteins

J Invest Dermatol. 2014 Apr;134(4):946-953. doi: 10.1038/jid.2013.482. Epub 2013 Nov 11.

Abstract

Mutations in the ABCC6 gene cause soft-tissue calcification in pseudoxanthoma elasticum (PXE) and, in some patients, generalized arterial calcification of infancy (GACI). PXE is characterized by late onset and progressive mineralization of elastic fibers in dermal, ocular, and cardiovascular tissues. GACI patients present a more severe, often prenatal arterial calcification. We have tested 10 frequent disease-causing ABCC6 missense mutants for the transport activity by using Sf9 (Spodoptera frugiperda) cells, characterized the subcellular localization in MDCKII (Madin-Darby canine kidney (cell line)) cells and in mouse liver, and tested the phenotypic rescue in zebrafish. We aimed at identifying mutants with preserved transport activity but with improper plasma membrane localization for rescue by the chemical chaperone 4-phenylbutyrate (4-PBA). Seven of the mutants were transport-competent but mislocalized in mouse liver. The observed divergence in cellular localization of mutants in MDCKII cells versus mouse liver underlined the limitations of this 2D in vitro cell system. The functionality of ABCC6 mutants was tested in zebrafish, and minimal rescue of the morpholino-induced phenotype was found. However, 4-PBA, a drug approved for clinical use, restored the plasma membrane localization of four ABCC6 mutants (R1114P, S1121W, Q1347H, and R1314W), suggesting that allele-specific therapy may be useful for selected patients with PXE and GACI.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / genetics
  • Alleles
  • Animals
  • Cell Line
  • Cell Membrane / metabolism
  • Dogs
  • Humans
  • Insecta
  • Liver / metabolism
  • Madin Darby Canine Kidney Cells
  • Mice
  • Mice, Inbred C57BL
  • Multidrug Resistance-Associated Proteins / genetics*
  • Mutation
  • Mutation, Missense*
  • Phenotype
  • Phenylbutyrates / chemistry
  • Protein Conformation
  • Protein Folding
  • Pseudoxanthoma Elasticum / genetics*
  • Zebrafish

Substances

  • ABCC6 protein, human
  • ATP-Binding Cassette Transporters
  • Abcc6 protein, mouse
  • Multidrug Resistance-Associated Proteins
  • Phenylbutyrates
  • 4-phenylbutyric acid