Iron regulatory and bactericidal properties of human recombinant hepcidin expressed in Pichia pastoris

被引：34

作者：

Koliaraki, Vasiliki ^{[1
]}

Marinou, Martha ^{[1
]}

Satniotaki, Martina ^{[2
]}

Panayotou, George ^{[2
]}

Pantopoulos, Kostas ^{[3
]}

Marnalaki, Avgi ^{[1
]}

机构：

[1] Hellenic Pasteur Inst, Dept Biochem, Lab Mol Biol & Immunobiotechnol, Athens 11521, Greece

[2] BSRC A Fleming, Vari 16672, Greece

[3] McGill Univ, Jewish Gen Hosp, Lady Davis Inst Med Res, Montreal, PQ H3T 1E2, Canada

来源：

BIOCHIMIE | 2008年 / 90卷 / 05期

关键词：

hepcidin; P; pastoris; iron metabolism; antibacterial; ferroportin;

D O I：

10.1016/j.biochi.2008.01.012

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Hepcidin is a circulating cysteine-rich peptide with antimicrobial properties. It functions as a hormonal regulator of iron homeostasis by controlling iron efflux from target cells via ferroportin (FPN1), which is internalized and degraded upon hepcidin binding. Because of its profound biomedical significance, hepcidin has become the target of intense biochemical studies. The aim of this study was to produce functional recombinant hepcidin in sufficient quantities for advanced research or potential clinical use, as the native hepcidin can be isolated from urine in very low yield. We report the expression, purification and functional characterization of hepcidin variants in yeast P. pastoris. The yield of untagged hepcidin 20- and 25-mer peptides was too low for complete functional characterization. By contrast, Hep20 and Hep25 tagged with either single 6xHis or double Myc-6xHis epitopes were expressed at high quantities (5-7 mg/l of culture), yet mostly in oligomeric forms. Purification of monomeric tagged hepcidins was achieved by size exclusion chromatography, with a yield of 0.5-1 mg/l of culture. All recombinant hepcidins exhibited bacteriostatic activity and the ability to control cellular iron homeostasis, with Hep25-His being the most potent. Thus, Hep25-His promoted an increase in the levels of the labile iron pool (LIP) in macrophages and consistently bound to ferroportin (FPN1) causing its internalization and the subsequent downregulation of transferrin receptor 1 (TfR1) expression. Analysis by mass-spectrometry suggested that all eight cysteines participated in disulfide bond formation. Our results suggest that only the recombinant Hep25-His monomer was a fully active peptide. As Hep25-His faithfully recapitulates the functional properties of native Hep25, it represents a powerful tool for biochemical studies and potential diagnostic and therapeutic applications. (C) 2008 Elsevier Masson SAS. All rights reserved.

引用

页码：726 / 735

页数：10

共 29 条

[1]

[Anonymous], 1989, Molecular Cloning

[2] Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression [J].

Babitt, JL ;

Huang, FW ;

Wrighting, DM ;

Xia, Y ;

Sidis, Y ;

Samad, TA ;

Campagna, JA ;

Chung, RT ;

Schneyer, AL ;

Woolf, CJ ;

Andrews, NC ;

Lin, HY .

NATURE GENETICS, 2006, 38 (05) :531-539

[3] Disrupted hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body iron homoeostasis [J].

Bridle, KR ;

Frazer, DM ;

Wilkins, SJ ;

Dixon, JL ;

Purdie, DM ;

Crawford, DHG ;

Subramaniam, VN ;

Powell, LW ;

Anderson, GJ ;

Ramm, GA .

LANCET, 2003, 361 (9358) :669-673

[4] Presence of the iron exporter ferroportin at the plasma membrane of macrophages is enhanced by iron loading and down-regulated by hepcidin [J].

Delaby, C ;

Pilard, N ;

Gonçalves, AS ;

Beaumont, C ;

Canonne-Hergaux, F .

BLOOD, 2005, 106 (12) :3979-3984

[5] The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis [J].

Donovan, A ;

Lima, CA ;

Pinkus, JL ;

Pinkus, GS ;

Zon, LI ;

Robine, S ;

Andrews, NC .

CELL METABOLISM, 2005, 1 (03) :191-200

[6] Resistance to hepcidin is conferred by hemochromatosis-associated mutations of ferroportin [J].

Drakesmith, H ;

Schimanski, LM ;

Ormerod, E ;

Merryweather-Clarke, AT ;

Viprakasit, V ;

Edwards, JP ;

Sweetland, E ;

Bastin, JM ;

Cowley, D ;

Chinthammitr, Y ;

Robson, KJH ;

Townsend, ARM .

BLOOD, 2005, 106 (03) :1092-1097

[7] Hepcidin: A putative iron-regulatory hormone relevant to hereditary hemochromatosis and the anemia of chronic disease [J].

Fleming, RE ;

Sly, WS .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2001, 98 (15) :8160-8162

[8] Recombinant human hepcidin expressed in Escherichia coli isolates as an iron containing protein [J].

Gerardi, G ;

Biasiotto, G ;

Santambrogio, P ;

Zanella, I ;

Ingrassia, R ;

Corrado, M ;

Cavadini, P ;

Derosas, M ;

Levi, S ;

Arosio, P .

BLOOD CELLS MOLECULES AND DISEASES, 2005, 35 (02) :177-181

[9] Balancing acts: Molecular control of mammalian iron metabolism [J].

Hentze, MW ;

Muckenthaler, MU ;

Andrews, NC .

CELL, 2004, 117 (03) :285-297

[10] The solution structure of human hepcidin, a peptide hormone with antimicrobial activity that is involved in iron uptake and hereditary hemochromatosis [J].

Hunter, HN ;

Fulton, DB ;

Ganz, T ;

Vogel, HJ .

JOURNAL OF BIOLOGICAL CHEMISTRY, 2002, 277 (40) :37597-37603

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