Design and synthesis of heterotrimeric collagen peptides with a built-in cystine-knot. Models for collagen catabolism by matrix-metalloproteases.
A heterotrimeric collagen peptide was designed and synthesized which contains the collagenase cleavage site (P4-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 (alpha1 alpha2 alpha1') was found to exhibit a typical collagen-like CD spectrum at room temperature and a melting temperature (Tm) of 33 degrees 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 alpha1' chain. In contrast, opposite digestion rates were measured with the catalytic Phe79-MMP-8 domain of HNC. Moreover, the full-length enzyme exhibits Km values of 5 microM and 1 mM for the heterotrimer and the single alpha1' chain, respectively, which compare well with those reported for collagen type I (approximately 1 microM), gelatine (approximately 10 microM) and for octapeptides of the cleavage sequence (> or = 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.
Ottl
J
J
Max-Planck-Institut für Biochemie, AG Bioorganische Chemie, Martinsried, Germany.
Battistuta
R
R
Pieper
M
M
Tschesche
H
H
Bode
W
W
Kühn
K
K
Moroder
L
L
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003094:Collagen / Q000737:chemistry*; D017364:Collagenases / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity
D004220:Disulfides; D010446:Peptide Fragments; D003553:Cystine; D003094:Collagen; D017364:Collagenases; D003545:Cysteine