Crystal structure of the TLDc domain of oxidation resistance protein 2 from zebrafish.

The oxidation resistance proteins (OXR) help to protect eukaryotes from reactive oxygen species. The sole C-terminal domain of the OXR, named TLDc is sufficient to perform this function. However, the mechanism by which oxidation resistance occurs is poorly understood. We present here the crystal structure of the TLDc domain of the oxidation resistance protein 2 from zebrafish. The structure was determined by X-ray crystallography to atomic resolution (0.97Å) and adopts an overall globular shape. Two antiparallel ß-sheets form a central ß-sandwich, surrounded by two helices and two one-turn helices. The fold shares low structural similarity to known structures.

Osteopontin is cleaved at multiple sites close to its integrin-binding motifs in milk and is a novel substrate for plasmin and cathepsin D.

Osteopontin (OPN) is a highly modified integrin-binding protein present in most tissues and body fluids where it has been implicated in numerous biological processes. A significant regulation of OPN function is mediated through phosphorylation and proteolytic processing. Proteolytic cleavage by thrombin and matrix metalloproteinases close to the integrin-binding Arg-Gly-Asp sequence modulates the function of OPN and its integrin binding properties. In this study, seven N-terminal OPN fragments originating from proteolytic cleavage have been characterized from human milk. Identification of the cleavage sites revealed that all fragments contained the Arg-Gly-Asp(145) sequence and were generated by cleavage of the Leu(151)-Arg(152), Arg(152)-Ser(153), Ser(153)-Lys(154), Lys(154)-Ser(155), Ser(155)-Lys(156), Lys(156)-Lys(157), or Phe(158)-Arg(159) peptide bonds. Six cleavages cannot be ascribed to thrombin or matrix metalloproteinase activity, whereas the cleavage at Arg(152)-Ser(153) matches thrombin specificity for OPN. The principal protease in milk, plasmin, hydrolyzed the same peptide bond as thrombin, but its main cleavage site was identified to be Lys(154)-Ser(155). Another endogenous milk protease, cathepsin D, cleaved the Leu(151)-Arg(152) bond. OPN fragments corresponding to plasmin activity were also identified in urine showing that plasmin cleavage of OPN is not restricted to milk. Plasmin, but not cathepsin D, cleavage of OPN increased cell adhesion mediated by the alpha(V)beta(3)- or alpha(5)beta(1)-integrins. Similar cellular adhesion was mediated by plasmin and thrombin-cleaved OPN showing that plasmin can be a potent regulator of OPN activity. These data show that OPN is highly susceptible to cleavage near its integrin-binding motifs, and the protein is a novel substrate for plasmin and cathepsin D.

Osteopontin enhances phagocytosis through a novel osteopontin receptor, the alphaXbeta2 integrin.

Osteopontin (OPN) is a cytokine with multiple functions, including immune defense mechanisms against invading microorganisms. OPN-deficient mice are impaired in clearing intracellular pathogens, suggesting an important role of OPN during phagocytosis, but it remains to be defined how OPN may enhance this innate immune process. Here, we demonstrate that OPN binds to monocytes, but not resting T cells, NK cells, or B cells, and mediates chemoattraction of IL-1-activated human monocytes. Moreover, OPN binds in a specific manner to all known serotypes of the two bacterial species Streptococcus agalactiae and Staphylococcus aureus and opsonizes these bacteria for phagocytosis. We identify the integrin alpha(X)beta(2) (CD11c/CD18), which is highly expressed on the cell surface of monocytes, as a novel OPN receptor. To eliminate the contribution from other molecular interactions between the bacteria and the phagocyte, we show that OPN-coated synthetic beads are phagocytosed in an alpha(X)beta(2) integrin-dependent manner. The ligand recognition does not involve the RGD motif previously reported to support binding of OPN to integrins. Taken together, these data identify the alpha(X)beta(2) integrin as a novel OPN receptor that is required for OPN-mediated phagocytosis, thereby elucidating an important mechanism of an innate immune function of OPN.

Plasminogen activator inhibitor-1 polymers, induced by inactivating amphipathic organochemical ligands.

Negatively charged organochemical inactivators of the anti-proteolytic activity of plasminogen activator inhibitor-1 (PAI-1) convert it to inactive polymers. As investigated by native gel electrophoresis, the size of the PAI-1 polymers ranged from dimers to multimers of more than 20 units. As compared with native PAI-1, the polymers exhibited an increased resistance to temperature-induced unfolding. Polymerization was associated with specific changes in patterns of digestion with non-target proteases. During incubation with urokinase-type plasminogen activator, the polymers were slowly converted to reactive centre-cleaved monomers, indicating substrate behaviour of the terminal PAI-1 molecules in the polymers. A quadruple mutant of PAI-1 with a retarded rate of latency transition also had a retarded rate of polymerization. Studying a number of serpins by native gel electrophoresis, ligand-induced polymerization was observed only with PAI-1 and heparin cofactor II, which were also able to copolymerize. On the basis of these results, we suggest that the binding of ligands in a specific region of PAI-1 leads to so-called loop-sheet polymerization, in which the reactive centre loop of one molecule binds to beta-sheet A in another molecule. Induction of serpin polymerization by small organochemical ligands is a novel finding and is of protein chemical interest in relation to pathological protein polymerization in general.