Recombinant Expression and Functional Characterization of Human Hephaestin: A Multicopper Oxidase with Ferroxidase Activity

Abstract

Human hephaestin (Hp) is a transmembrane protein that has been implicated in duodenal iron export. Mutations in the murine hephaestin gene (sla) produce microcytic, hypochromic anemia that is refractory to oral iron therapy. Hp shares approximately 50% sequence identity with the plasma multicopper ferroxidase ceruloplasmin including conservation of residues involved in disulfide bond formation and metal coordination. On the basis of this similarity to ceruloplasmin, human hephaestin may also bind copper and possess ferroxidase activity. To test this hypothesis, human hephaestin cDNA has been cloned by reverse transcription of human duodenal mRNA. Following in vitro mutagenesis to make the encoded polypeptide suitable for expression and purification, the hephaestin cDNA was cloned into the expression vector pNUT and introduced into baby hamster kidney cells. After selection with methotrexate, the baby hamster kidney cells secreted the recombinant human hephaestin into the medium at a level of approximately 2 mg/L. Purification was achieved by a single immunoaffinity chromatography step. As judged by SDS-PAGE, N-terminal sequence analysis, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, the purified hephaestin was homogeneous with a mass of 129600 Da, suggesting a carbohydrate content of 7.7%. Inductively coupled plasma mass spectrometry revealed that recombinant hephaestin contained an average of 3.13 atoms of copper per protein molecule. A visible absorption maximum was observed at 607 nm, consistent with the presence of a Type 1 copper site. By using ferrous ammonium sulfate as a substrate, recombinant hephaestin was shown to have ferroxidase activity with a K(m) of 2.1 microM for Fe(II). Lastly, urea PAGE showed that hephaestin was able to catalyze formation of diferric transferrin from Fe(II) and apotransferrin.

Human hephaestin expression is not limited to enterocytes of the gastrointestinal tract but is also found in the antrum, the enteric nervous system, and pancreatic β-cells

“…Prior to immunization, blood was collected from each rabbit for later use as a preimmune serum. rHp was expressed and purified as described previously (18). The priming immunization consisted of an emulsion of equal parts complete Freund's adjuvant and purified human rHp [0.5 mg/ml in phosphate-buffered saline (PBS)].…”

“…The mucosa of the duodenum sample was scraped with a glass slide and the resulting tissue (30 mg) was lysed in RIPA buffer (25 mM Tris · HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) containing 1ϫ protease inhibitor cocktail (Complete, Roche, Mannheim, Germany). Pure Cp for the Western blot was obtained from Vital Products (Boynton Beach, FL), and pure rHp was produced as previously described (18). Proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes.…”

“…To study the function of Hp, we expressed recombinant human Hp by cloning Hp cDNA from human duodenal tissue and replaced the putative membrane-binding region at the COOH-terminus with a 1D4 affinity purification tag (18). The resulting recombinant soluble human Hp (rHp) was characterized by various physical-chemical techniques and enzymology and was found to be a multicopper ferroxidase of M r ϳ129,600 (18).…”

“…The resulting recombinant soluble human Hp (rHp) was characterized by various physical-chemical techniques and enzymology and was found to be a multicopper ferroxidase of M r ϳ129,600 (18).…”

Analysis of the High-Affinity Iron Uptake System at the Chlamydomonas reinhardtii Plasma Membrane

“…Incorporation of iron into Tf was monitored via a previously described method (16). Each reaction mixture contained 100 l of 6.25 M apo-Tf, 200 M ferrous ammonium sulfate, and 100 M ascorbate in 100 mM sodium acetate, pH 5.0.…”