Oxidative post-translational modifications of calprotectin during inflammation

Abstract

Calprotectin, a heterodimer of S100A8 and S100A9 subunits, is an abundant cytosolic neutrophil protein. It is released from neutrophils upon their activation or as a result of cell disruption or death. Extracellular levels of calprotectin are increased during infection and inflammation and thus calprotectin can be used as a non-invasive biomarker of neutrophil activation in many inflammatory diseases. Calprotectin plays an important role in the innate immune system by inhibiting bacterial growth through chelation of nutrient metal ions such as zinc. Activated neutrophils also generate large amounts of reactive oxygen species such as hypochlorous acid that can react with a wide range of biological molecules. One of the major protein targets for reactive oxygen species is likely to be calprotectin, due to its high abundance in neutrophils and increased extracellular levels at sites of infection and inflammation. The purpose of this thesis was to characterise oxidative post-translational modifications of calprotectin generated after exposure of calprotectin to neutrophil-derived oxidants, and to determine if these modifications can be detected in physiological samples. In addition, I investigated whether oxidation of calprotectin had an effect on the biological function of calprotectin.

Calprotectin was successfully purified from human neutrophil lysate using a two-step ion exchange protocol. It was purified as a non-covalent heterodimer of A8 and A9 (~24 kDa), and in accordance with other studies was able to form heterotetramers (~48 kDa) in the presence of excess calcium. Hypochlorous acid rapidly oxidised calprotectin to produce covalent dimers of A8 and A9, with low doses of reagent hypochlorous acid promoting reversible cross-linking of calprotectin monomers in vitro. Western blot and mass spectrometry analysis of these dimers revealed that they were either A9-A9 homodimers, or A8-A9 heterodimers coupled via disulfide bonds between cysteine residues.

Reversible cross-linking of calprotectin monomers also occurred when human neutrophils were stimulated with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) or when they phagocytosed Staphylococcus aureus. Cross-linking was generated by hypochlorous acid produced by the neutrophils and occurred predominantly extracellularly. In addition, reversible cross-linking of calprotectin was detected in saline washings from the lungs of children with cystic fibrosis, from adults with respiratory infections or lung cancer, and in saliva from healthy adults, highlighting the importance of understanding the functional implications of calprotectin cross-linking.

The quaternary structure of calprotectin was not dramatically affected by low levels of oxidation, with calprotectin still able to tetramerise in the presence of excess calcium. In contrast, more extensively oxidised calprotectin was less effective at forming the tetramer in the presence of excess calcium, suggesting that tetramer-dependent functions may be lost. Oxidation with low doses of hypochlorous acid did not alter the number of zinc ions bound by calprotectin, or have an effect on LPS-induced TNF-α release from human peripheral blood mononuclear cells. However, hypochlorous acid-mediated oxidation of calprotectin rendered the protein more susceptible to proteolysis. This is likely to occur in vivo, due to the high levels of calprotectin and neutrophil-resident proteolytic enzymes released at sites of infection and inflammation. As a result, detection of calprotectin specific peptides may be effective biomarkers of neutrophil activation and protease activity in various infective and inflammatory conditions. In addition, proteolysis of calprotectin may result in a loss of protein function or a gain of function through generation of calprotectin peptides.