Elucidation of the substrate specificity of the C1s protease of the classical complement pathway.
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.
Kerr
Felicity K
FK
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
O'Brien
Grace
G
Quinsey
Noelene S
NS
Whisstock
James C
JC
Boyd
Sarah
S
de la Banda
Maria Garcia
MG
Kaiserman
Dion
D
Matthews
Antony Y
AY
Bird
Phillip I
PI
Pike
Robert N
RN
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D003173:Complement C1s / Q000737:chemistry / Q000378:metabolism*; D003171:Complement Pathway, Classical / Q000502:physiology*; D004247:DNA / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D013379:Substrate Specificity
D009842:Oligopeptides; D019151:Peptide Library; D004247:DNA; D003173:Complement C1s