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

Felicity K Kerr,Grace O'Brien,Noelene S Quinsey,James C Whisstock,Sarah Boyd,Maria Garcia De La Banda,Dion Kaiserman,Antony Y Matthews,Phillip I Bird,Robert N Pike

doi:10.1074/jbc.m506131200

Felicity K Kerr, Grace O'Brien + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m506131200

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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.

Highlights

The C1 component is a complex of the binding protein C1q and two proenzymes, C1r and C1s [4]
Understanding the substrate specificity of an enzyme is a prerequisite for the development of specific inhibitors and allows insights into overall function
C1s is interesting in that it apparently only interacts with its major substrates (C2 and C4) and C1-inhibitor, which suggests that it is highly restricted in terms of its substrate specificity

Summary

Introduction

The C1 component is a complex of the binding protein C1q and two proenzymes, C1r and C1s [4]. The use of substrates based on the cleavage sequences of C4 and C2, the physiological substrates of human C1s, to investigate the kinetic parameters of the interaction of these substrates with the active site of the enzyme has previously been reported [14].

Results

Conclusion