The Heparin-binding Domain of Extracellular Superoxide Dismutase Is Proteolytically Processed Intracellularly during Biosynthesis (original) (raw)
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Characterization of the heparin-binding domain of human extracellular superoxide dismutase
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1997
The C-terminal, heparin-binding domain of human extracellular superoxide dismutase hEC-SOD has been studied as a Ž. fusion to human carbonic anhydrase II HCAII. This technique allows the properties of the EC-SOD domain to be characterized. At the same time, it allows us to differentiate the contributions from the domain, from those properties originating from other parts of EC-SOD. The fusion of the 27 C-terminal amino acids of hEC-SOD to the C-terminal of Ž. HCAII FusCC resulted in the formation of a monomeric protein, which binds to heparin-Sepharose with approximately the same affinity as the tetrameric hEC-SOD. The structure of the fused C-terminal was characterized by CD and NMR spectroscopy and the data were compatible with the presence of a-helical structures as suggested by secondary structure predictions. The NMR data show that the C-terminal of FusCC moves independently from the rest of the protein and that its central part is involved in conformational exchange. The NOESY spectra demonstrate that the C-terminal in both FusCC and hEC-SOD binds to heparin, and that arginine side chains take part in the binding.
Journal of Biological Chemistry, 2004
Extracellular superoxide dismutase (EC-SOD) is a tetramer composed of either intact (Trp 1-Ala 222) or proteolytically cleaved (Trp 1-Glu 209) subunits. The latter form is processed intracellularly before secretion and lacks the C-terminal extracellular matrix (ECM)-binding region (210 RKKRRRESECKAA 222-COOH). We have previously suggested that the C-terminal processing of EC-SOD is either a one-step mechanism accomplished by a single intracellular endoproteolytic event cleaving the Glu 209-Arg 210 peptide bond or a two-step mechanism involving two proteinases (Enghild,
Biochemical Journal, 2005
The C-terminal region of EC-SOD (extracellular superoxide dismutase) mediates the binding to both heparin/heparan sulphate and type I collagen. A mutation (Arg 213 → Gly; R213G) within this extracellular matrix-binding region has recently been implicated in the development of heart disease. This relatively common mutation affects the heparin affinity, and the concentration of EC-SOD in the plasma of R213G homozygous individuals is increased 10-to 30-fold. In the present study we confirm, using R213G EC-SOD purified from a homozygous individual, that the heparin affinity is reduced. Significantly, the collagen affinity of the R213G EC-SOD variant was similarly affected and both the heparin and collagen affinities were reduced by 12-fold. Structural analysis of synthetic extracellular matrix-binding regions suggests that the mutation alters the secondary structure. We conclude that the increased concentration of EC-SOD in the plasma of R213G carriers is caused by a reduction in both heparin and collagen affinities.
Furin Proteolytically Processes the Heparin-binding Region of Extracellular Superoxide Dismutase
Journal of Biological Chemistry, 2002
Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that attenuates brain and lung injury from oxidative stress. A polybasic region in the carboxyl terminus distinguishes EC-SOD from other superoxide dismutases and determines EC-SOD's tissue half-life and affinity for heparin. There are two types of EC-SOD that differ based on the presence or absence of this heparin-binding region. It has recently been shown that proteolytic removal of the heparin-binding region is an intracellular event (Enghild,
Characterization of Heparin Binding of Human Extracellular Superoxide Dismutase
Biochemistry, 2000
The C-terminal domain of human extracellular superoxide dismutase (hEC-SOD) plays a crucial role in the protein's interaction with heparin. Here we investigated this interaction in more detail by comparing the heparin-binding characteristics of two variants of hEC-SOD: the two fusion proteins containing the hEC-SOD C-terminal domain and a synthetic peptide homologous to the C-terminal. The interaction studies were performed using a surface plasmon resonance based technique on a BIAcore system. It should be emphasized that this is a model system. However, the kinetic constants, as measured, are valid in a comparative sense. Comparison of affinities for size-fractionated heparins revealed that octa-or decasaccharides are the smallest heparin fragments that can efficiently interact with the C-terminal domain of hEC-SOD. At physiological salt concentration, and pH 7.4, the hEC-SOD/heparin interaction was found to be of a high-affinity type, with an equilibrium dissociation constant, K d , of 0.12 µM, which is 700 and 10-20 times lower than the K d values for the synthetic peptide and the fusion proteins, respectively. However, when an R-helical structure was induced in the synthetic peptide, by addition of 10% trifluoroethanol, the K d decreased to 0.64 µM. The differences in the K d values were mainly governed by differences in the association rate constants (k ass). The hEC-SOD/heparin interaction itself was found to have a fairly high dissociation rate constant (0.1 s-1), and a very high association rate constant (8 × 10 5 M-1 s-1), suggesting that the interaction is mainly controlled by the association. These results together with circular dichroism spectra of the synthetic peptide suggest that an R-helical structure in the C-terminal is essential for optimal binding to heparin and that other parts of hEC-SOD moderate the affinity. Our data also demonstrate that the tetramerization itself does not substantially increase the affinity.
Biochemical Journal, 1996
Studies examining the biochemical characteristics and pharmacological properties of extracellular superoxide dismutase (EC SOD) have been severely limited because of difficulties in purifying the enzyme. Recently EC SOD was found to exist in high concentrations in the arteries of most mammals examined and it is the predominant form of SOD activity in many arteries. We now describe a three-step, high-yield protocol for the purification of EC SOD from human aorta. In the first step, the high affinity of EC SOD for heparin is utilized to obtain a fraction in which EC SOD constitutes roughly 13% of the total protein compared with only 0.3% of that of the starting material. In addition, over 80% of the original EC SOD activity present in the aortic homogenate was retained after the first step of purification. EC SOD was further purified using a combination of cation- and anion-exchange chromatography. The overall yield of EC SOD from this purification procedure was 46%, with over 4 mg of...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2006
Objective-Extracellular superoxide dismutase (EC-SOD) is a secreted antioxidant enzyme that binds to the outer plasma membrane and extracellular matrix through its heparin-binding domain (HBD). Carriers of a common genetic variant of EC-SOD (EC-SOD R213G , within the HBD) have higher plasma concentration of EC-SOD and increased risk for vascular disease. In the present study, we used confocal fluorescence microscopy to examine mechanisms of endocytosis of EC-SOD to determine whether EC-SOD translocates to the nucleus of endothelial cells, and to test the hypothesis that EC-SOD, but not EC-SOD R213G , is endocytosed into endothelial cells. Methods and Results-Mouse endothelial cells (MS-1) were incubated with EC-SOD, EC-SOD R213G , or HBD-deleted EC-SOD (EC-SOD⌬HBD). Binding to MS-1 was observed only with EC-SOD, but not EC-SOD R213G or EC-SOD⌬HBD. Endocytosis of EC-SODs was monitored after coincubation of MS-1 cells with EC-SODs and BSA-Texas Red (BSA-TR), which marks endosomes and lysosomes. Only EC-SOD was endocytosed, colocalizing with BSA-TR. EC-SOD also colocalized with early endosome antigen 1 (EEA-1), a specific marker for endocytosis. Endocytosis of EC-SOD was inhibited by chlorpromazine, but not by methyl--cyclodextrin or nystatin, which suggests that endocytosis of EC-SOD is mediated by clathrin but not by caveolae. Minimal or no localization of EC-SOD in the nucleus of MS-1 cells was detected. Conclusions-Our findings indicate that EC-SOD, but not EC-SOD R213G , is endocytosed into endothelial cells through clathrin-mediated pathway, but does not translocate to the nucleus. We speculate that impairment of endocytosis may contribute to high plasma levels of EC-SOD R213G in R213G carriers. (Arterioscler Thromb Vasc Biol. 2006;26:1985
Biochemistry, 2002
An essential property of human extracellular superoxide dismutase (hEC-SOD) is its affinity for heparin and heparan sulfate proteoglycans located on cell surfaces and in the connective tissue matrix. The C-terminal domain of hEC-SOD plays the major role in this interaction. This domain has an unusually high content of charged amino acids: six arginine, three lysine, and five glutamic acid residues. In this study, we used alanine scanning mutagenesis of charged amino acids in the C-terminal domain to elucidate the requirements for the heparin/heparan sulfate interaction. As a tool in this study, we used a fusion protein comprising the C-terminal domain of hEC-SOD fused to human carbonic anhydrase II (HCAII). The interaction studies were performed using the surface plasmon resonance technique and heparin-Sepharose chromatography. Replacement of the glutamic acid residues by alanine resulted, in all cases, in tighter binding. All alanine substitutions of basic amino acid residues, except one (R205A), reduced heparin affinity. The arginine and lysine residues in the cluster of basic amino acid residues (residues 210-215), the RK-cluster, are of critical importance for the binding to heparin, and arginine residues promote stronger interactions than lysine residues.
The C-terminal proteolytic processing of extracellular superoxide dismutase is redox regulated
Free Radical Biology and Medicine, 2012
The antioxidant protein extracellular superoxide dismutase (EC-SOD) encompasses a C-terminal region that mediates interactions with a number of ligands in the extracellular matrix (ECM). This ECM-binding region can be removed by limited proteolysis before secretion, thus supporting the formation of EC-SOD tetramers with variable binding capacity. The ECM-binding region contains a cysteine residue (Cys219) that is known to be involved in an intersubunit disulfide bridge. We have determined the redox potential of this disulfide bridge and show that both EC-SOD dimers and EC-SOD monomers are present within the intracellular space. The proteolytic processing of the ECM-binding region in vitro was modulated by the redox status of Cys219, allowing cleavage under reducing conditions only. When wild-type EC-SOD or the monomeric variant Cys219Ser was expressed in mammalian cells proteolysis did not occur. However, when cells were exposed to oxidative stress conditions, proteolytic processing was observed for wild-type EC-SOD but not for the Cys219Ser variant. Although the cellular response to oxidative stress is complex, our data suggest that proteolytic removal of the ECM-binding region is regulated by the intracellular generation of an EC-SOD monomer and that Cys219 plays an important role as a redox switch allowing the cellular machinery to secrete cleaved EC-SOD.
2007
Background: Human extracellular superoxide dismutase (EC-SOD) is a tetrameric metalloenzyme responsible for the removal of superoxide anions from the extracellular space. We have previously shown that the EC-SOD subunit exists in two distinct folding variants based on differences in the disulfide bridge pattern (Petersen SV, Oury TD, Valnickova Z, Thøgersen IB, Højrup P, Crapo JD, Enghild JJ. Proc Natl Acad Sci USA. 2003;100(24):13875-80). One variant is enzymatically active (aEC-SOD) while the other is inactive (iEC-SOD). The EC-SOD subunits are associated into covalently linked dimers through an inter-subunit disulfide bridge creating the theoretical possibility of 3 dimers (aa, ai or ii) with different antioxidant potentials. We have analyzed the quaternary structure of the endogenous EC-SOD disulfide-linked dimer to investigate if these dimers in fact exist.