Tài liệu Báo cáo khoa học: Antioxidant protein 2 prevents methemoglobin formation in erythrocyte

Antioxidant protein 2 prevents methemoglobin formation

in erythrocyte hemolysates

Karl M. Stuhlmeier1

, Janet J. Kao2

, Pia Wallbrandt3

, Maria Lindberg3

, Barbro Hammarstro¨ m3

,

Hans Broell1 and Beverly Paigen4

1

Ludwig Boltzmann Institute for Rheumatology and Balneology, Vienna, Austria; 2

Maimonides Medical Center, Brooklyn, NY,

USA; 3

Department of Molecular Biology, AstraZeneca, Umea˚, Sweden; and 4

The Jackson Laboratory, Bar Harbor, ME, USA

Antioxidant protein 2 (AOP2) is a member of a family of

thiol-specific antioxidants, recently renamed peroxiredoxins,

that evolved as part of an elaborate system to counteract and

control detrimental effects of oxygen radicals. AOP2 is

found in endothelial cells, erythrocytes, monocytes, T and B

cells, but not in granulocytes. AOP2 was found solely in the

cytoplasm and was not associated with the nuclear or

membrane fractions; neither was it detectable in plasma.

Further experiments focused on the function of AOP2 in

erythrocytes where it is closely associated with the hemo￾globin complex, particularly with the heme. An investigation

of the mechanism of this interaction demonstrated that the

conserved cysteine-47 in AOP2 seems to play a role in

AOP2-heme interactions. Recombinant AOP2 prevented

induced as well as noninduced methemoglobin formation in

erythrocyte hemolysates, indicating its antioxidant proper￾ties.We conclude that AOP2 is part of a sophisticated system

developed to protect and support erythrocytes in their many

physiological functions.

Keywords: hemoglobin; erythrocytes; reactive oxygen species;

antioxidant protein 2.

Evolving antioxidant defence systems to protect against O2

toxicity has been a prerequisite for an organism’s use of O2

for efficient energy production. To benefit from O2 as an

energy source, multicellular organisms had to develop a

system to distribute O2. In mammals this function is carried

out by red blood cells (RBC), which utilize hemoglobin to

distribute O2 to cells. Not only are RBC highly specialized

O2 and CO2 carriers, they also serve an additional important

function, namely acting as a sink for reactive oxygen species

(ROS) [1]. Erythrocytes can take up O2-radicals as well as

H2O2 in plasma to protect the organism from damage by

such compounds [2–4]. These tasks make erythrocytes

especially vulnerable to damage by ROS. Furthermore,

carrying high concentrations of O2 and high levels of

potentially pro-oxidant heme protein inside a membrane

rich in polyunsaturated fatty acid side chains cause

additional problems. RBCs are therefore exposed to a

constant flow of hemoglobin auto-oxidation, as approxi￾mately 3% of the hemoglobin undergoes oxidation to

methemoglobin (metHb) every day. Moreover RBC are

also exposed to repeated physical stress through deforma￾tion. More importantly, RBC have low metabolic activities

with no ability to synthesize new proteins or lipids to replace

damaged molecules [1]. Due to these properties, RBC need

to be equipped with a series of enzymes that can protect cells

from damage by free radicals; such enzymes include Cu-Zn￾superoxide dismutase, catalase, glutathione peroxidase,

metHb reductase, and glucose 6-phosphate dehydrogenase.

Recently, a new type of antioxidant protein has been

reported to be present in RBC [5,6], the thiol-specific

antioxidant proteins, which are members of a large family of

more than 40 proteins found in prokaryotes as well as

eukaryotes [7–10]. The peroxiredoxin proteins show no

significant homology with previously identified antioxidant

proteins. The nomenclature of these proteins is still confu￾sing, as these molecules were originally described under

several names e.g. rehydrins, thioredoxin-dependent per￾oxide reductases, but this family has been renamed as

peroxiredoxins [11,12]. Peroxiredoxins are grouped into

1-Cys proteins with a conserved cysteine at amino acid

position 47 and 2-Cys proteins with a second conserved

amino acid at position 170 (relative to yeast peroxiredoxin).

They usually exist as homodimers. The substrates are alkyl

hydroperoxides [9], peroxynitrates [13] and hydrogen per￾oxides [14], and they detoxify these substrates by oxidation

of the Cys at amino acid 47 [9,15]. These proteins

enzymatically detoxify hydroxyradicals using reducing

equivalents from thiol-containing molecules such as thio￾redoxins and glutathione. As a major function of these

proteins is to regulate ROS levels, they not only protect

Correspondence to K. M. Stuhlmeier, Ludwig Boltzmann

Institute for Rheumatology and Balneology, Kurbadstrasse 10,

PO Box 78, A-1107 Vienna, Austria.

Fax: + 43 1 68009 9234, Tel.: + 43 1 68009 9237,

E-mail: [email protected]

Abbreviations: AOP2, antioxidant protein 2; metHb, methemoglobin;

MNCs, mononuclear cells; PMNs, polymorphonuclear cells;

RBC, red blood cells; ROS, reactive oxygen species.

Note: The nomenclature of antioxidant protein 2 is currently under￾going reconsideration. This protein is currently named antioxidant

protein 2 in humans and peroxiredoxin 5 in mice. However, peroxi￾redoxin 5 in humans refers to a different protein (named peroxiredoxin

6 in mouse). As the same protein is supposed to have the same name

in different species, we will use the old name of antioxidant protein 2

until this nomenclature issue is resolved by the human and mouse

nomenclature committees.

(Received 11 June 2002, revised 13 October 2002,

accepted 26 November 2002)

Eur. J. Biochem. 270, 334–341 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03393.x