Abstract
The C1s protease of the classical complement pathway propagates the initial activation of this pathway of the system by cleaving and thereby activating the C4 and C2 complement components. This facilitates the formation of the classical pathway C3 convertase (C4bC2a). C1s has a Lys residue located at position 628 (192 in chymotrypsin numbering) of the SP domain that has the potential to partially occlude the S2–S2′ positions of the active site. The 192 residue of serine proteases generally plays an important role in interactions with substrates. We therefore investigated the role of Lys628 (192) in interactions with C4 by altering the Lys residue to either a Gln (found in many other serine proteases) or an Ala residue. The mutant enzymes had altered specificity profiles for a combinatorial peptide substrate library, suggesting that this residue does influence the active site specificity of the protease. Generally, the K628Q mutant had greater activity than wild type enzyme against peptide substrates, while the K628A residue had lowered activity, although this was not always the case. Against peptide substrates containing physiological substrate sequences, the K628Q mutant once again had generally higher activity, but the activity of the wild type and mutant enzymes against a C4 P4–P4′ substrate were similar. Interestingly, alteration of the K628 residue in C1s had a marked effect on the cleavage of C4, reducing cleavage efficiency for both mutants about fivefold. This indicates that this residue plays a different role in cleaving protein versus peptide substrates and that the Lys residue found in the wild type enzyme plays an important role in interacting with the C4 substrate. Understanding the basis of the interaction between C1s and its physiological substrates is likely to lead to insights that can be used to design efficient inhibitors of the enzyme for use in treating diseases caused by inflammation as result of over-activity of the classical complement pathway.
Highlights
The complement system is of major importance in innate and adaptive immunity [1], but has been shown to play a major role in several inflammatory diseases [2, 3]
The results indicate that the Lys residue at this position in the protease plays a role in facilitating efficient cleavage of C4 in particular
In general, the results obtained with the REPLi library reinforced previous specificity data for this enzyme [13], it was interesting to note that the I/L-S/T-K/R and I/L-A/V-K/R pools were not well cleaved by the wild type enzyme, somewhat contrary to previous data indicating a preference for Leu residues at the P3 position of substrates for C1s
Summary
The complement system is of major importance in innate and adaptive immunity [1], but has been shown to play a major role in several inflammatory diseases [2, 3]. It has recently been shown that the activation of the C1s protease is concomitant with the formation of an exosite on the serine protease domain [6, 7] that works in concert with a likely exosite on the CCP domains and the active site to efficiently catalyze the cleavage of C4 to yield the C4b and C4a fragments. In this regard, the interaction between C1s and C4 is likely to have similarity to the MASP-2-C4 interaction, which has been elucidated structurally [8] and biochemically [9,10,11]. Given that the exosite interactions for the classical and lectin pathway proteases are somewhat similar, the focus falls on the active sites of the proteases in order to provide selective inhibitors of the pathways for use in controlling diseases in which they are involved
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