Recombinant C1 inhibitor P5/P3 variants display resistance to catalytic inactivation by stimulated neutrophils.
Proteolytic inactivation of serine protease inhibitors (serpins) by neutrophil elastase (HNE) is presumed to contribute to the deregulation of plasma cascade systems in septic shock. Here, we report a supplementary approach to construct serpins, in our case C1 inhibitor, that are resistant to catalytic inactivation by HNE. Instead of shifting the specificity of alpha 1-antitrypsin towards the proteases of the contact activation and complement systems, we attempted to obtain a C1 inhibitor species which resists proteolytic inactivation by HNE. 12 recombinant C1 inhibitor variants were produced with mainly conservative substitutions at the cleavage sites for HNE, 440-Ile and/or 442-Val. Three variants significantly resisted proteolytic inactivation, both by purified HNE, as well as by activated neutrophils. The increase in functional half-life in the presence of FMLP-stimulated cells was found to be 18-fold for the 440-Leu/442-Ala variant. Inhibitory function of these variants was relatively unimpaired, as examined by the formation of stable complexes with C1s, beta-Factor XIIa, kallikrein, and plasmin, and as determined by kinetic analysis. The calculated association rate constants (k(on)) were reduced twofold at most for C1s, and appeared unaffected for beta-Factor XIIa. The effect on the k(on) with kallikrein was more pronounced, ranging from a significant ninefold reduction to an unmodified rate. The results show that the reactive centre loop of C1 inhibitor can be modified towards decreased sensitivity for nontarget proteases without loss of specificity for target proteases. We conclude that this approach extends the possibilities of applying recombinant serpin variants for therapeutic use in inflammatory diseases.
원문보기 무료다운로드 유료다운로드
- 원문이 없습니다.
유료 다운로드의 경우 해당 사이트의 정책에 따라 신규 회원가입, 로그인, 유료 구매 등이 필요할 수 있습니다. 해당 사이트에서 발생하는 귀하의 모든 정보활동은 NDSL의 서비스 정책과 무관합니다.
NDSL에서는 해당 원문을 복사서비스하고 있습니다. 아래의 원문복사신청 또는 장바구니 담기를 통하여 원문복사서비스 이용이 가능합니다.