Tài liệu Báo cáo khoa học: A selenium-containing single-chain abzyme with potent antioxidant

A selenium-containing single-chain abzyme with potent

antioxidant activity

Delin You1

, Xiaojun Ren1,2, Yan Xue1

, Guimin Luo1

, Tongshu Yang1 and Jiacong Shen2

1

Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, P. R. China;

2

Key Laboratory for Supramolecular Structure and Materials of Ministry of Education, Jilin University, Changchun, P. R. China

Reactive oxygen species (ROS) are products of normal

metabolic activities and are thought to be the cause of many

diseases. A selenium-containing single-chain abzyme 2F3

(Se-2F3-scFv) that imitates glutathione peroxidase has been

produced which has the capacity to remove ROS. To

evaluate the antioxidant ability of Se-2F3-scFv, we con￾structed a ferrous sulfate/ascorbate (Vc/Fe2+)-induced mito￾chondrial damage model system and investigated the

capacity of Se-2F3-scFv to protect mitochondria from oxi￾dative damage. Se-2F3-scFv markedly decreased mito￾chondrial swelling, inhibited lipid peroxidation, and

maintained the activity of cytochrome c oxidase, in com￾parison with Ebselen, a well-studied glutathione peroxidase

mimic, indicating that Se-2F3-scFv has potential for treating

diseases mediated by ROS.

Keywords: antioxidant activity; glutathione peroxidase;

mitochondria; scFv; selenium.

Reactive oxygen species (ROS) include free radicals such as

superoxide anion (O2

–•

) and hydroxyl radical (•

OH), as well

as nonradical intermediates such as hydrogen peroxide

(H2O2), hydroperoxide (ROOH), nitric oxide (NO) and

singlet oxygen (1

O2) [1,2]. All these ROS are produced from

molecular oxygen by mitochondrial electron carriers and

from enzymes during normal metabolism of oxidative

phosphorylation of aerobic mammalian cells. In addition,

ROS are produced on irradiation, both ionizing and UV

irradiation.

To protect themselves from oxidative injury, aerobic

cells have evolved an enzymatic and nonenzymatic defense

system. The enzymatic antioxidant system is mainly

composed of glutathione peroxidase (GPX), catalase,

superoxide dismutase and thioredoxin peroxidase. The non￾enzymatic antioxidant system includes vitamin E, ascorbate,

glutathione (GSH) and uric acid. However, if the ROS

loading reaches a critical concentration, overwhelming the

antioxidative defense, oxidative damage to all cellular

components, such as DNA, proteins and lipids, eventually

occurs, resulting in ROS-mediated diseases [3–5]. Exam￾ples of such diseases are ischemia-reperfusion injury,

inflammation, age-related diseases, neuronal apoptosis,

cancer and cataract.

The individual antioxidant enzymes are located in

specific subcellular sites and reveal distinct substrate

specificity [6]. Superoxide dismutase is a metalloenzyme

that catalyzes the reduction of O2

–• to H2O2. H2O2

produced by the reduction of O2

–• is subsequently

detoxified by catalase present in peroxisomes or by the

selenoenzyme GPX located in the cytosol and mitochon￾dria. GPX, the most important selenium-containing

peroxidase, catalyzes the reduction of a variety of

hydroperoxides (ROOH and H2O2) by GSH, thereby

protecting mammalian cells against oxidative damage. At

least five GPX isoenzymes have been identified in

mammals. Although their expression is ubiquitous, the

levels of each isoform vary depending on the tissue type.

The classical cellular GPX (GPX1 or cGPX), found in the

cytosol and mitochondria, reduces fatty acid hydroper￾oxides and H2O2 [7–9]. Phospholipid hydroperoxide GPX

(GPX4 or PHGPX), found in most tissues and located in

both the cytosol and the membrane fraction, can directly

reduce the phospholipid hydroperoxides, fatty acid hydro￾peroxides, and cholesterol hydroperoxides that are

produced in peroxidized membranes and oxidized lipo￾proteins [10–12]. Cytosolic GPX2 (or giGPX) [13,14] and

extracellular GPX 3 (pGPX) [15,16] are weakly detected

in most tissues except gastrointestinal tract and kidney,

respectively. Recently, a new member, GPX5, expressed

specifically in mouse epididymis, is interestingly selenium￾independent [17]. The mechanism by which cGPX cata￾lyzes the reduction of hydroperoxide has been extensively

investigated.

Because production of selenium-containing peroxidase

is extremely difficult by traditional genetic engineering,

attempts have been made to generate compounds that

imitate the enzymatic action of GPX. The strategies used

to generate GPX-like catalysts include chemical synthesis

of a model system and mutation of naturally occurring

enzyme by chemical or protein engineering [18–20]. Three

different strategies have been tested for chemically

synthesizing a GPX mimic: one in which the selenium

atom binds directly to a heteroatom such as nitrogen

Correspondence to G. Luo, Key Laboratory of Molecular

Enzymology and Engineering of Ministry of Education,

Jilin University, Changchun, P. R. China.

Fax: + 86 431 8923907, Tel.: + 86 431 8498974,

E-mail: [email protected]

Abbreviations: ROS, reactive oxygen species; GSH, glutathione; GPX,

glutathione peroxidase; TBA, thiobarbituric acid; CCO, cytochrome c

oxidase; TBARS, thiobarbituric acid reactive substances.

(Received 20 April 2003, revised 6 July 2003,

accepted 22 August 2003)

Eur. J. Biochem. 270, 4326–4331 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03825.x