Tài liệu Báo cáo khoa học: Production of biologically active forms of recombinant hepcidin, the

Production of biologically active forms of recombinant

hepcidin, the iron-regulatory hormone

Bruno Gagliardo1

, Audrey Faye2,3, Maryse Jaouen1

, Jean-Christophe Deschemin2,3, Franc¸ois

Canonne-Hergaux4

, Sophie Vaulont2,3 and Marie-Agne` s Sari1

1 CNRS (UMR 8601), Universite´ Paris Descartes, France

2 Institut Cochin CNRS (UMR 8104), Universite´ Paris Descartes, France

3 Inserm (U567), Universite´ Paris Descartes, France

4 CNRS (ICSN), Gif-Sur-Yvette, France

Hepcidin is a 25 amino acid, cysteine-rich peptide, first

identified in human blood [1] and urine [2] as an anti￾microbial peptide of the defensin family. Human hep￾cidin is the product of the HAMP gene, which encodes

an 84 amino acid precursor protein [3,4]. In addition,

hepcidin genes have also been identified in several

species, including the mouse [5], rat [6], pig [7], dog [8]

and fish [9–11]. In mammals, the hepcidin gene is

expressed predominantly in hepatocytes and constitutes

the master regulator of iron homeostasis. Hepcidin

gene expression is positively regulated by iron and

inflammation and negatively regulated by anemia and

hypoxia [5,12]. There is compelling evidence that

dysregulation of hepcidin underlies many iron disorders.

Most of the iron overload syndromes (primary hemo￾chromatosis and secondary iron overloads) imply a

reduction of hepcidin secretion whereas, in contrast,

overexpression of hepcidin appears to play a deter￾mining role in anemia of inflammation or inflammation

of chronic disease [13] and iron-refractory iron

deficiency anemia [14].

HAMP and Hepc1 encode precursors of 84 and 83

amino acids, respectively, which contain typical 24 and

23 amino acid endoplasmic reticulum targeting signal

motifs at the N-terminus of the human and mouse

precursors, respectively. This signal is cleaved to

produce prohepcidin, harboring a 35 amino acid

proregion and the C-terminus 25 amino acid mature

Keywords

antimicrobial peptide; Escherichia coli

expression; ferroportin; hepcidin; iron

homeostasis

Correspondence

M.-A. Sari, UMR 8601, Universite´ Paris

Descartes, 45 rue des Saints-Pe`res, 75006

Paris, France

Fax: +33 1428 68387

Tel: +33 1428 62142

E-mail: [email protected]

(Received 1 April 2008, revised 22 May

2008, accepted 28 May 2008)

doi:10.1111/j.1742-4658.2008.06525.x

Hepcidin is a liver produced cysteine-rich peptide hormone that acts as the

central regulator of body iron metabolism. Hepcidin is synthesized under

the form of a precursor, prohepcidin, which is processed to produce the

biologically active mature 25 amino acid peptide. This peptide is secreted

and acts by controlling the concentration of the membrane iron exporter

ferroportin on intestinal enterocytes and macrophages. Hepcidin binds to

ferroportin, inducing its internalization and degradation, thus regulating

the export of iron from cells to plasma. The aim of the present study was

to develop a novel method to produce human and mouse recombinant hep￾cidins, and to compare their biological activity towards their natural recep￾tor ferroportin. Hepcidins were expressed in Escherichia coli as thioredoxin

fusion proteins. The corresponding peptides, purified after cleavage from

thioredoxin, were properly folded and contained the expected four-disulfide

bridges without the need of any renaturation or oxidation steps. Human

and mouse hepcidins were found to be biologically active, promoting ferro￾portin degradation in macrophages. Importantly, biologically inactive

aggregated forms of hepcidin were observed depending on purification

and storage conditions, but such forms were unrelated to disulfide bridge

formation.

Abbreviations

huhepc, human hepcidin encoded by HAMP gene with an extra N-terminal methionine; m1hepc, mouse hepcidin encoded by Hepc1 gene

with an extra N-terminal methionine; MES, 2(N-morpholino)ethanesulfonic acid; SELDI-TOF, surface enhanced laser desorption ionization

time of flight; TFA, trifluroacetic acid; TRX, thioredoxin.

FEBS Journal 275 (2008) 3793–3803 ª 2008 The Authors Journal compilation ª 2008 FEBS 3793