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

Abstract

A heterotrimeric collagen peptide was designed and synthesized which contains the collagenase cleavage site ( P 4- P′ 9 10 ) 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 Phe 79-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.