Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.

Evaluation of the C5a anaphylatoxin and its receptor (CD88) in allergic lung disease

Allergic asthma is characterized by airflow obstruction, airway hyperresponsiveness (AHR) and chronic airway inflammation. We and others have reported that complement component C3 and the anaphylatoxin C3a receptor promote while C5 protects against the development of the biological and physiological hallmarks of allergic lung disease in mice. In this study, we assessed if the protective responses could be mediated by C5a, an activation-induced C5 cleavage product. Mice with ablation of the C5a receptor (C5aR) either by genetic deletion or by pharmacological blockade exhibited significantly exacerbated AHR compared to allergen-challenged wild-type (WT) mice. However, there were no significant differences in many of the other hallmarks of asthma such as airway infiltration by eosinophils or lymphocytes, pulmonary IL-4-producing cell numbers, goblet cell metaplasia, mucus secretion or total serum IgE levels. In contrast to elevated AHR, numbers of IL-5 and IL-13 producing pulmonary cells, and IL-5 and IL-13 protein levels, were significantly reduced in allergen-challenged C5aR-/- mice compared to allergen-challenged WT mice. Administration of a specific cysteinyl leukotriene receptor 1 (cysLT1R) antagonist before each allergen-challenge abolished AHR in C5aR-/- as well as in WT mice. Pretreatment with a C3aR antagonist dose-dependently reduced AHR in allergen-challenged WT and C5aR-/- mice. Additionally, allergen-induced upregulation of pulmonary C3aR expression was exaggerated in C5aR-/- mice compared to WT mice. In summary, deficiency or antagonism of C5aR in a mouse model of pulmonary allergy increased AHR, which was reversed or reduced by blockade of the cysLT1R and C3aR, respectively. In conclusion, this study suggests that C5a and C5aR mediate protection against AHR by suppressing cysLT and C3aR signaling pathways, which are known to promote AHR. This also supports important and opposing roles of complement components C3a/C3aR and C5a/C5aR in AHR. ^

The function of the murine complement *C3a and *C5a anaphylatoxin receptors in endotoxemia, bacteremia and septic shock

The complement system functions as a major effector for both the innate and adaptive immune response. Activation of the complement cascade by either the classical, alternative, or lectin pathway promotes the proteolysis of C3 and C5 thereby generating C3a and C5a. Referred to as anaphylatoxins, the C3a and C5a peptides mediate biological effects upon binding to their respective receptors; C3a binds to the C3a receptor (C3aR) while C5a binds to the C5a receptor (C5aR, CD88). Both C3a and C5a are known for their broad proinflammatory effects. Elevated levels of both peptides have been isolated from patients with a variety of inflammatory diseases such as COPD, asthma, RA, SLE, and sepsis. Recent studies suggest that C5a is a critical component in the acquired neutrophil dysfunction, coagulopathy, and progressive multi-organ dysfunction characteristic of sepsis. The primary hypothesis of this dissertation was that preventing C3a-C3aR and C5a-C5aR mediated pro-inflammatory effects would improve survival in endotoxic, bacteremic and septic shock. To test this hypothesis, the murine C3aR and C5aR genes were disrupted. Following disruption of both the C3aR and C5aR genes, no abnormalities were identified other than the absence of their respective mRNA and protein. In models of both endotoxic and bacteremic shock, C3aR deficient mice suffered increased mortality when compared to their wild type littermates. C3aR deficient mice also had elevated circulating IL-1β levels. Using a model of sepsis, C3aR deficient mice had a higher circulating concentration of IL-6 and decreased peritoneal inflammatory infiltration. While these results were unexpected, they support an emerging role for C3a in immunomodulation. In contrast, following endotoxic or bacteremic shock, C5aR deficient mice experienced increased survival, less hemoconcentration and less thrombocytopenia. It was later determined that C5a mediated histamine release significantly contributes to host morbidity and mortality in bacteremic shock. These studies provide evidence that C5a functions primarily as a proinflammatory molecule in models of endotoxic and bacteremic shock. In the same models, C3a-C3aR interactions suppress the inflammatory response and protect the host. Collectively, these results present in vivo evidence that C3a and C5a have divergent biological functions. ^