Expression, purification, and characterization of a human complement component C3 analog that lacks the C-terminal C345c domain.

The complement system consists of a series of soluble and cell-surface proteins that serve numerous roles in innate immunity, development, and homeostasis. Despite its many functions, the central event in the complement system is the proteolytic activation of the 185 kDa complement component 3 (C3) into its opsonin and anaphylatoxin fragments known as C3b (175 kDa) and C3a (10 kDa), respectively. The C3 protein is comprised of thirteen separate structural domains, several of which undergo extensive structural rearrangement upon activation to C3b. In addition to this, the C-terminal C345c domain found in C3, C3b, and the terminal degradation product, C3c (135 kDa), appears to adopt multiple conformations relative to the remainder of the molecule. To facilitate various structure/function studies, we designed two C3 analogs that could be activated to a C345c-less, C3c-like state following treatment with Tobacco Etch Virus (TEV) protease. We generated stably transfected Chinese Hamster Ovary (CHO) cell lines that secrete approximately 1.5 mg of the highest-expressing C3 analog per liter of conditioned culture medium. We purified this C3 analog by sequential immobilized metal ion affinity and size exclusion chromatographies, activated the protein by digestion with TEV protease, and purified the resulting C3c analog by a final size exclusion chromatography. The conformations and activities of our C3 and C3c analogs were assessed by measuring their binding profiles to known C3/b/c ligands by surface plasmon resonance. Together, this work demonstrates the feasibility of producing a C3 analog that can be site-specifically activated by an exogenous proteolytic enzyme.

Evidence for multiple modes of neutrophil serine protease recognition by the EAP family of Staphylococcal innate immune evasion proteins.

Neutrophils contain high levels of chymotrypsin-like serine proteases (NSPs) within their azurophilic granules that have a multitude of functions within the immune system. In response, the pathogen Staphylococcus aureus has evolved three potent inhibitors (Eap, EapH1, and EapH2) that protect the bacterium as well as several of its secreted virulence factors from the degradative action of NSPs. We previously showed that these so-called EAP domain proteins represent a novel class of NSP inhibitors characterized by a non-covalent inhibitory mechanism and a distinct target specificity profile. Based upon high levels of structural homology amongst the EAP proteins and the NSPs, as well as supporting biochemical data, we predicted that the inhibited complex would be similar for all EAP/NSP pairs. However, we present here evidence that EapH1 and EapH2 bind the canonical NSP, Neutrophil Elastase (NE), in distinct orientations. We discovered that alteration of EapH1 residues at the EapH1/NE interface caused a dramatic loss of affinity and inhibition of NE, while mutation of equivalent positions in EapH2 had no effect on NE binding or inhibition. Surprisingly, mutation of residues in an altogether different region of EapH2 severely impacted both the NE binding and inhibitory properties of EapH2. Even though EapH1 and EapH2 bind and inhibit NE and a second NSP, Cathepsin G, equally well, neither of these proteins interacts with the structurally related, but non-proteolytic granule protein, azurocidin. These studies expand our understanding of EAP/NSP interactions and suggest that members of this immune evasion protein family are capable of diverse target recognition modes.