Mutagenic Analysis of the Putative ABCC6 Substrate-Binding Cavity Using a New Homology Model.

Flora Szeri,Susan Cole,Fatemeh Niaziorimi,Sylvia Donnelly,Koen Van De Wetering,Valentina Corradi,Gwenaëlle Conseil,D Tieleman

doi:10.3390/ijms22136910

Abstract

Inactivating mutations in ABCC6 underlie the rare hereditary mineralization disorder pseudoxanthoma elasticum. ABCC6 is an ATP-binding cassette (ABC) integral membrane protein that mediates the release of ATP from hepatocytes into the bloodstream. The released ATP is extracellularly converted into pyrophosphate, a key mineralization inhibitor. Although ABCC6 is firmly linked to cellular ATP release, the molecular details of ABCC6-mediated ATP release remain elusive. Most of the currently available data support the hypothesis that ABCC6 is an ATP-dependent ATP efflux pump, an un-precedented function for an ABC transporter. This hypothesis implies the presence of an ATP-binding site in the substrate-binding cavity of ABCC6. We performed an extensive mutagenesis study using a new homology model based on recently published structures of its close homolog, bovine Abcc1, to characterize the substrate-binding cavity of ABCC6. Leukotriene C4 (LTC4), is a high-affinity substrate of ABCC1. We mutagenized fourteen amino acid residues in the rat ortholog of ABCC6, rAbcc6, that corresponded to the residues in ABCC1 found in the LTC4 binding cavity. Our functional characterization revealed that most of the amino acids in rAbcc6 corresponding to those found in the LTC4 binding pocket in bovine Abcc1 are not critical for ATP efflux. We conclude that the putative ATP binding site in the substrate-binding cavity of ABCC6/rAbcc6 is distinct from the bovine Abcc1 LTC4-binding site.

Highlights

Inactivating mutations in the gene encoding ATP-binding cassette (ABC) subfamily C member 6 (ABCC6) underlie the autosomal recessive disease pseudoxanthoma elasticum (PXE, OMIM #264800) [1,2,3], characterized by ectopic mineralization in the skin, eyes, and vascular system [4,5,6]
ABC transporters use the energy derived from ATP hydrolysis to mediate transport of indicated it could transport Leukotriene C4 (LTC4) [6], this was not the case for its ortholog rat Abcc6 (rAbcc6) a wide range of substrates across membranes
Low levels of LTC4 transport might have been has not been shown in vesicular uptake experiments, widely used to demonstrate that a given compound is actively transported by an ABC transporter [6,19]

Summary

Introduction

Inactivating mutations in the gene encoding ATP-binding cassette (ABC) subfamily C member 6 (ABCC6) underlie the autosomal recessive disease pseudoxanthoma elasticum (PXE, OMIM #264800) [1,2,3], characterized by ectopic mineralization in the skin, eyes, and vascular system [4,5,6]. There is currently no specific and effective therapy for PXE and the disease slowly progresses after initial diagnosis [8]. ABCC6 is predominantly expressed in the liver [9] where it mediates the release of ATP from hepatocytes into the bloodstream [10,11]. Outside the hepatocytes, yet still in the liver niche, the released ATP is converted into AMP and the mineralization inhibitor pyrophosphate (PPi), by ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) [12]. The absence of ABCC6-mediated ATP release in both PXE patients and Abcc null mice results in plasma PPi levels that are < 40% of those found in ABCC6-proficient individuals [11], providing a plausible biochemical explanation for their ectopic mineralization

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