Hydroxyproline-containing collagen analogs trigger the release and activation of collagen-sequestered proMMP-2 by competition with prodomain-derived peptide P33-42

Abstract

BackgroundFibrolytic and profibrotic activities of the matrix metalloproteinases (MMPs)-2 and -9 play a central role in liver fibrosis. Since binding to the extracellular matrix influences the activity of both gelatinases, here the role of fibrillar collagens as the most abundant matrix components in fibrotic tissue was investigated.ResultsIn situ zymography and immunohistology showed association of enzymatically inactive prodomain-containing proMMP-2 and proMMP-9 but not of their activated forms to fibrillar collagen structures, which are not substrates of these gelatinases. In solid-phase binding studies with human collagens and collagen fragments, up to 45% of [125I]-labeled proMMP-2 and proMMP-9 but not of active (act)MMP-2 and actMMP-9 were retained by natural collagenous molecules and by synthetic analogs containing repeated Gly-Pro-Hyp triplets (GPO). Surface plasmon resonance yielded binding constants for the interaction of collagen type I (CI) with proMMP-2 and proMMP-9 in a nanomolar range. Values for actMMP-2 and actMMP-9 were 30-40 times higher. Tenfold molar excesses of (GPO)10 reduced the interaction of CI with pro- and actMMP-2 by 22- or 380-fold and resulted in prodomain release accompanied by high enzymatic activation and activity. Pointing to gelatine substrate displacement, higher (GPO)10 concentrations blocked the enzymatic activity. The MMP-2 prodomain-derived collagen-binding domain peptide (P33-42) binds to the collagen-binding domain of MMP-2, thereby preserving enzymatic inactivity. Synthetic P33-42 peptide competed with proMMP-2 binding to CI and prevented (GPO)10-mediated proMMP-2 activation. In contrast to (GPO)10, P33-42 did not activate proMMP-2, making triple helical and hydroxyproline-containing (GPO)10 unique in modulating gelatinase availability and activity.ConclusionsThese findings suggest novel strategies using collagen analogs for the resolution of liver fibrosis via fibrotic matrix-sequestered gelatinases.

The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)10promotes enzymatic activity of matrixmetalloproteinase-2in vitro

“…HSC treated with (GPO) 10 showed a proliferation of ~66% of the maximum (Fig. 1) whereas treatment with the linear controlpeptide GAP [17] showed no stimulatory effects on HSC proliferation as compared to control (not shown).…”

“…ProMMP-2 was activated by incubation with 4-aminophenylmercuric acetate (APMA) as described [17]. To exclude APMA-specific effects, the respective controls contained APMA alone.…”

“…The triple helical collagen analogue (GPO) 10 -H-Gly-CysHyp-(Gly-Pro-Hyp) 10 -Gly-Cys-Hyp-Gly-NH 2 -and the linear control peptide GAP -H-Gly-Ala-Cys-(Gly-AlaPro) 5 -Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Ala-Pro) 5 -NH 2 -were synthesized and characterized as described previously [17,19]. The lyophilized peptides were dissolved and stored in stock solutions of 2 mg/ml in 150 mM acetic acid at −20 °C.…”

“…Using (GPO) 10 as a binding competitor for the interaction of the gelatinase proMMP-2 with collagen, a dissociation of the proMMP-2 prodomain accompanied by high enzymatic activation of MMP-2 could be achieved [17].…”

“…In previous studies, we identified the α2 chain of collagen type VI as the main binding structure for sequestration of collagenases and stromelysin-1 proforms in fibrotic tissue [16], and showed that gelatinase binding sites are located on fibrillar collagen structures and the synthetic collagen analogue (Gly-Pro-Hyp) 10 ((GPO) 10 ) [17]. Using (GPO) 10 as a binding competitor for the interaction of the gelatinase proMMP-2 with collagen, a dissociation of the proMMP-2 prodomain accompanied by high enzymatic activation of MMP-2 could be achieved [17].…”