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The complement system is a central component of host defense but can also contribute to the inflammation seen in pathological conditions. The C1s protease of the first complement component, the C1 complex, initiates the pathway. In this study we have elucidated the full specificity of the enzyme for the first time using a randomized phage display library. It was found that, aside from the crucial P 1 position, the S 3 and S 2 subsites (in that order) played the greatest role in determining specificity. C1s prefers Leu or Val at P 3 and Gly or Ala residues at P 2 . Apart from the S 2 position, which showed specificity for Leu, prime subsites did not greatly affect specificity. It was evident, however, that together they significantly contributed to the efficiency of cleavage of a peptide. A peptide substrate based on the top sequence obtained in the phage display validated these results and produced the best kinetics of any C1s substrate to date. The results allow an understanding of the active site specificity of the C1s protease for the first time and provide a basis for the development of specific inhibitors aimed at controlling inflammation associated with complement activation in adverse pathological situations.Complement is an integral component of the adaptive and innate immune systems and represents one of the major effector systems for the immune response (1). However, inadvertent or unwanted complement activation can be harmful to the body (2); an example of this is the activation of the system during reperfusion injury after myocardial infarction (3). Thus, the control of complement may also have an important bearing on preventing or attenuating certain inflammatory diseases. The complement system consists of a highly regulated cascade of proteolytic enzymes and other proteins (1), and it is suggested that control of the system could be achieved through the use of highly targeted inhibitors of the serine proteases (2). In particular, the initiating proteases of the C1 complex of the classical pathway are of great relevance to such an approach since control of cascade systems is logically most efficiently achieved by shutting off the initiation steps.The C1 component is a complex of the binding protein C1q and two proenzymes, C1r and C1s (4). Upon binding of IgG to the heads of C1q, C1r undergoes autoactivation to cleave and activate C1s. C1s appears to be a highly specific protease, cleaving the C4 and C2 components to instigate a sequence of activation steps of other components of the complement system, culminating in the formation of the membrane attack complex, which induces cell lysis (5, 6). The only other protein known to interact with C1s physiologically is the serpin, C1 inhibitor, which inhibits and, thus, controls the enzyme. Several studies have revealed that C1s is able to cleave substrates outside complement (7-11), but the physiological relevance of these interactions remains to be understood.Understanding the substrate specificity of an enzyme is a prerequisite for the development of spe...

The complement system plays crucial roles in the immune system, but incorrect regulation causes inflammation and targeting of self-tissue, leading to diseases such as systemic lupus erythematosus, rheumatoid arthritis and age-related macular degeneration. In vivo, the initiating complexes of the classical complement and lectin pathways are controlled by SERPING1 [(C1 inhibitor) serpin peptidase inhibitor, clade G, member 1], which inactivates the components C1s and MASP-2 (mannan-binding lectin serine peptidase 2). GAGs (glycosaminoglycan) and DXS (dextran sulfate) are able to significantly accelerate SERPING1-mediated inactivation of C1s, the key effector enzyme of the classical C1 complex, although the mechanism is poorly understood. In the present study we have shown that C1s can bind to DXS and heparin and that these polyanions enhanced C1s proteolytic activity at low concentrations and inhibited it at higher concentrations. The recent determination of the crystal structure of SERPING1 has given rise to the hypothesis that both the serpin (serine protease inhibitor)-polyanion and protease-polyanion interactions might be required to accelerate the association rate of SERPING1 and C1s. To determine what proportion of the acceleration was due to protease-polyanion interactions, a chimaeric mutant of alpha1-antitrypsin containing the P4-P1 residues from the SERPING1 RCL (reactive-centre loop) was produced. Like SERPING1, this molecule is able to effectively inhibit C1s, but is unable to bind polyanions. DXS exerted a biphasic effect on the association rate of C1s which correlated strongly with the effect of DXS on C1s proteolytic activity. Thus, whereas polyanions are able to bind C1s and modulate its activity, polyanion interactions with SERPING1 must also play a vital role in the mechanism by which these cofactors accelerate the C1s-SERPING1 reaction.

Complement is a key component of the immune system, but can contribute to inflammatory diseases. The substrate specificity of C1s protease has been successfully investigated using a combinatorial approach, while a positional scanning method failed. The lack of success of the latter approach is possibly due to cooperativity in the active site, which could confound such analyses. With a panel of peptides devised using factorial design, we show pronounced cooperativity between the S4 and S1' subsites in the active site of the enzyme, and weaker cooperativity between the S1' and S3' subsites. The use of factorial design has promise as a methodology for determining cooperativity in protease active sites.

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