Design and synthesis of heterotrimeric collagen peptides with a built‐in cystine‐knot Models for collagen catabolism by matrix‐metalloproteases

FEBS Letters

Tschesche, Harald¹  Kühn, Klaus²  Ottl, Johannes²  Pieper, Michael¹  Bode, Wolfram²  Battistuta, Roberto²  Moroder, Luis² 

[1]Universität Bielefeld, Fakultät Chemie und Biochemie I, Bielefeld, Germany

[2]Max-Planck-Institut für Biochemie, AG Bioorganische Chemie, Am Klopferspitz 18A, 82152 Martinsried, Germany

关键词: Collagen peptide;    Synthesis;    Heterotrimer;    Triple helix;    Enzymatic digestion;    Collagenase;    Catalytic domain;    Hemopexin domain;   

DOI  :  10.1016/S0014-5793(96)01212-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A heterotrimeric collagen peptide was designed and synthesized which contains the collagenase cleavage site () of type I collagen linked to a C-terminal cystine-knot, and N-terminally extended with (Gly-Pro-Hyp)₅ triplets for stabilization of the triple-helical conformation. By employing a newly developed regioselective cysteine pairing strategy based exclusively on thiol disulfide exchange reactions, we succeeded in assembling in high yields and in a reproducible manner the triple-stranded cystine peptide. While the single chains showed no tendency to self-association into triple helices, the heterotrimer (α1α2α1′) was found to exhibit a typical collagen-like CD spectrum at room temperature and a melting temperature (T _m ) of 33°C. This triple-helical collagen-like peptide is cleaved by the full-length human neutrophil collagenase (MMP-8) at a single locus fully confirming the correct raster of the heterotrimer. Its digestion proceeds at rates markedly higher than that of a single α1′ chain. In contrast, opposite digestion rates were measured with the catalytic Phe⁷⁹-MMP-8 domain of HNC. Moreover, the full-length enzyme exhibits K _m values of 5 μM and 1 mM for the heterotrimer and the single α1′ chain, respectively, which compare well with those reported for collagen type I (∼ 1 μM), gelatine (∼ 10 μM) and for octapeptides of the cleavage sequence (≥1 mM). The high affinity of the MMP-8 for the triple-helical heterotrimer and the fast digestion of this collagenous peptide confirm the decisive role of the hemopexin domain in recognition and possibly, partial unfolding of collagen.

【 授权许可】

Unknown