Thrombospondin is an inhibitor of angiogenesis that modulates endothelial cell adhesion, proliferation, and motility. Synthetic peptides from the second type I repeat of human thrombospondin containing the consensus sequence-Trp-Ser-Pro-Trp- and a recombinant heparin binding fragment from the amino-terminus of thrombospondin mimic several of the activities of the intact protein. The peptides and heparin-binding domain promote endothelial cell adhesion, inhibit endothelial cell chemotaxis to basic fibroblast growth factor (bFGF), and inhibit mitogenesis and proliferation of aortic and corneal endothelial cells. The peptides also inhibit heparin-dependent binding of bFGF to corneal endothelial cells. The antiproliferative activities of the peptides correlate with their ability to bind to heparin and to inhibit bFGF binding to heparin. Peptides containing amino acid substitutions that eliminate heparin-binding do not alter chemotaxis or proliferation of endothelial cells. Inhibition of proliferation by the peptide is time-dependent and reversible. Thus, the antiproliferative activities of the thrombospondin peptide and recombinant heparin-binding domain result at least in part from competition with heparin-dependent growth factors for binding to endothelial cell proteoglycans. These results suggest that both the Trp-Ser-Xaa-Trp sequences in the type I repeats and the amino-terminal domain play roles in the antiproliferative activity of thrombospondin.

Expression of human Cu/Zn superoxide dismutase (SOD) with activity comparable to the human erythrocyte enzyme was achieved in Escherichwa coli by using a vector containing a thermoinducible X PL promoter and a ,8-lactamase-derived ribosomosal binding site. The recombinant human SOD was found in the cytosol of disrupted bacteria and represented >10% of the total bacterial protein. The enzyme was purified to homogeneity by salt precipitation, gel filtration chromatography, and ion exchange chromatography. The active enzyme was obtained in high yield only when 1 mol of copper and 1 mol of zinc were incorporated into each mol of subunit during bacterial growth or by reconstitution of the apoenzyme. Human Cu/Zn SOD produced in bacteria has an apparent subunit molecular mass of 19 kDa on NaDodSO4/polyacrylamide gels. The native enzyme behaves as a dimer of 32 kDa as determined by gel filtration. Sequence analysis of the NH2 terminus revealed that the first 14 amino acids corresponded to authentic human SOD except that the NH2-terminal alanine was not acetylated. Thus, the bacterial processing system readily removes the NH2-terminal methionine residue from recombinant human SOD. and thus provide a defense against oxygen toxicity. There are three known forms of SOD that contain different metalsnamely, iron, manganese, or both copper and zinc. All of these catalyze the same reaction with high efficiency, and all operate by a similar mechanism in which the metal is the catalytic factor in the active site. These enzymes fall into several evolutionary groups. The Fe-containing SODs are found primarily in prokaryotic cells, while Cu/Zn SODs have been demonstrated in all higher eukaryotes. Mn SODs exist throughout the phylogenetic range, from microorganisms to humans (reviewed in ref. 4).Since every biological macromolecule can serve as a target for the damaging action of the abundant oxygen radical, interest has evolved in the therapeutic potential of SOD. A wide range of clinical applications has been suggested. These include prevention of oncogenesis and tumor promotion, reduction of the cytotoxic and cardiotoxic effects of anticancer drugs (5), anti-inflammatory action (6), and protection against reperfusion damage of ischemic tissues (7). In addition, there is much interest in studying the effects of SOD on the aging process (8).The exploration of the therapeutic potential of human SOD has been hindered by its limited availability. The enzyme is a dimeric metalloprotein composed of identical noncovalently linked subunits, each of 16 kDa and containing one atom of copper and one atom of zinc (9). Each subunit is composed of 153 amino acids of known sequence (10, 11). Recently, a cDNA clone containing the entire coding region of human SOD was isolated and sequenced (12, 13). The gene coding for human SOD was introduced by us into an efficient bacterial expression vector. We report here the production of gram quantities of enzymatically active human Cu/Zn SOD in Escherichia coli. MATERIALS AND METHODSBacterial Gro...

Human Mn superoxide dismutase (MnSOD) encoded by chromosome 6 is a mitochondrial matrix enzyme positioned to scavenge oxygen radicals produced by the extensive oxidation-reduction and electron transport reactions undergoing in that organelle. cDNA clones containing the entire coding region for human MnSOD were isolated from a T-lymphocyte cDNA library in A gt1O. The cDNA contains a 666 bp coding region followed by a 3' untranslated region which lacks the AATAAA polyadenylation signal. The predicted amino acid sequence is in accordance with the published amino acid sequence of human liver MnSOD (1) with the following exceptions: Glu instead of Gln at positions 42, 88 and 109 and an additional Gly-Trp after amino acids 123. The deduced protein sequence extends 24 amino acids upstream from the N-terminal Lys of human MnSOD, suggesting a pre-peptide. Hence human MnSOD is composed of 222 amino acids, 24 of which are removed during processing and maturation of the enzyme.

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In many pathological situations, tissue damage is caused by cellular generation of superoxide free radicals (O2-). These active species are generated during post-ischemic reperfusion of organs, in hyperoxic tissue, during acute and chronic inflammation and during exposure to ionizing radiation. Exogenous superoxide dismutase (SOD) was shown to significantly prevent such damage. The genes for human cytosolic Cu/ZnSOD and mitochondrial MnSOD were cloned and introduced into an E. coli expression system. The proteins were expressed in high yields and purified to homogeneity, yielding pharmaceutical-grade materials. These enzymes were used in a variety of in vivo animal models for the demonstration of their protective effects against oxidative damage. Comparative pharmacokinetic studies in rats have revealed that the half-life of Cu/ZnSOD was 6-10 min., while that of MnSOD was 5-6 hours, thus indicating that MnSOD may be superior to Cu/ZnSOD for the treatment of chronic diseases. Indeed, MnSOD was found to be effective as an anti-inflammatory agent in the rat carrageenan induced paw edema acute inflammation model. Both enzymes were also effective in ameliorating post-irradiation damage in mice exposed to whole-body or localized chest X-ray radiation.