Elucidation of the Substrate Specificity of the C1s Protease of the Classical Complement Pathway

Abstract

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...

C1q Protein Binds to the Apoptotic Nucleolus and Causes C1 Protease Degradation of Nucleolar Proteins

“…Besides C4, C2, and the C1 inhibitor, only three other molecules, all expressed extracellularly, have been reported to be cleaved by C1s: insulin-like growth factor binding protein 5, MHC class I, and the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (42)(43)(44). However, on the basis of a new C1s substrate formula established using a library of peptides centered around the known C1s cleavage sites in C4, C2, and the C1 inhibitor, many intracellular proteins have been predicted to contain C1s cleavage sites (33). The prediction was largely made on the basis of sequence preference and local secondary structures, on the basis of which NCL and NPM1 were both predicted to contain multiple C1s cleavage sites (Fig.…”

Identification of a Catalytic Exosite for Complement Component C4 on the Serine Protease Domain of C1s

“…The CCP1-CCP2-SP and SP fragments were inhibited with EGRck, an irreversible inhibitor targeted to the active site. EGRck would be expected to occupy the P3-P1 subsites of C1s, which were shown to be vital for interaction with cleavage sites in peptide and most likely protein substrates (30). The active site-inhibited CCP1-CCP2-SP fragment showed a 3-fold increased K D value in comparison with the active form ( Table I), suggesting that the active site played a small role in the binding of the enzyme to C4.…”

Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpP

“…Following electroporation, a total of 1.1 ϫ 10 8 CFU were obtained. While the number of individual clones in the library represents only a small fraction of the amino acid sequences obtainable from randomization of an octamer peptide (2.6 ϫ 10 10 ), it should nonetheless be sufficient to capture the key properties of amino acids preferred by the protease (11). Sequencing of 12 randomly selected clones from this library revealed that each contained an insert with a unique octamer amino acid sequence.…”

“…In particular, the specificities of over 30 proteases, spanning all recognized classes, have been studied by techniques relying on phage display (5,11,19,20). Substrate phage display is based on the selective cleavage of specific peptide sequences sandwiched between the gene pIII minor coat protein of fd bacteriophage and an affinity tag.…”