Abstract
Hephaestin is a large membrane-anchored multicopper ferroxidase involved in mammalian iron metabolism. Newly absorbed dietary iron is exported across the enterocyte basolateral membrane by the ferrous iron transporter ferroportin, but hephaestin increases the efficiency of this process by oxidizing the transported iron to its ferric form and promoting its release from ferroportin. Deletion or mutation of the hephaestin gene leads to systemic anemia with iron accumulation in the intestinal epithelium. The crystal structure of human ceruloplasmin, another multicopper ferroxidase with 50% sequence identity to hephaestin, has provided a framework for comparative analysis and modelling. However, detailed structural information for hephaestin is still absent, leaving questions relating to metal coordination and binding sites unanswered. To obtain structural information for hephaestin, a reliable protocol for large-scale purification is required. Here, we present an expression and purification protocol of soluble mouse hephaestin, yielding milligram amounts of enzymatically active, purified protein using the baculovirus/insect cell system.
Highlights
Iron is a critical element for many cellular processes
Iron is toxic in high concentrations due to its ability to catalyze reactions leading to the generation of reactive oxygen species, requiring iron homeostasis to be tightly regulated
Dietary iron is absorbed through the apical membrane of enterocytes via the membrane transporter divalent metal-ion transporter 1 (DMT1)
Summary
Iron is a critical element for many cellular processes. iron is toxic in high concentrations due to its ability to catalyze reactions leading to the generation of reactive oxygen species, requiring iron homeostasis to be tightly regulated. Dietary iron is absorbed through the apical membrane of enterocytes via the membrane transporter divalent metal-ion transporter 1 (DMT1). It is subsequently either stored in intracellular ferritin, or transported across the basolateral membrane of enterocytes (and into the circulation) via the ferrous iron exporter ferroportin (FPN). Iron transported through FPN is not released efficiently into the plasma in the absence of a multicopper ferroxidase [1,2], which oxidizes Fe2+ to Fe3+. Hephaestin (HEPH), a membrane anchored ferroxidase with a large ~120kDa ectodomain, is the main ferroxidase associated with the transport activity of FPN in the small
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