Abstract
The role of proteases in the tumor cell invasion process is multifaceted. Members of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and degradation of extracellular matrix (ECM) components. Differentiating between the up-regulation of MMP production and the presence of activated MMPs can be difficult but may well dictate which MMPs are critical to invasion. Because the hydrolysis of collagens is one of the committed steps in ECM turnover, we have investigated selective MMP action on collagenous substrates as a means to evaluate active MMPs. Two triple-helical peptide (THP) models of the MMP-9 cleavage site in type V collagen, alpha1(V)436-450 THP and alpha1(V)436-447 fTHP, were hydrolyzed by MMP-2 and MMP-9 at the Gly-Val bond, analogous to the bond cleaved by MMP-9 in the corresponding native collagen. Kinetic analyses showed k(cat)/K(m) values of 14,002 and 5,449 s(-1)m(-1) for MMP-2 and -9 hydrolysis of alpha1(V)436-447 fTHP, respectively. These values, along with individual k(cat) and K(m) values, are comparable with collagen hydrolysis by MMP-2 and -9. Neither THP was hydrolyzed by MMP-1, -3, -13, or -14. alpha1(V)436-447 fTHP and a general fluorogenic THP were used to screen for triple-helical peptidase activity in alpha(2)beta(1) integrin-stimulated melanoma cells. Binding of the alpha(2)beta(1) integrin resulted in the production of substantial triple-helical peptidase activity, the majority (>95%) of which was non-MMP-2/-9. THPs were found to provide highly selective substrates for members of the MMP family and can be used to evaluate active MMP production in cellular systems.
Highlights
The role of proteases, metalloproteases, in the tumor cell invasion process is multifaceted
The first triple-helical peptide (THP) constructed as a possible matrix metalloproteinase (MMP)-2 and -9 substrate was based upon the MMP-9 cleavage site in types V and XI collagen
The ␣1(V)436 – 450 THP had a Tm value of 49.5 °C in 1.0% (v/v) fluorometric assay buffer (Fig. 1), which is a desirable thermal stability for an MMP substrate
Summary
Materials—All standard peptide synthesis chemicals were analytical reagent grade or better and purchased from Novabiochem (San Diego, CA) or Fisher Scientific. 9-Fluorenylmethoxycarbonyl-amino acid derivatives were obtained from Novabiochem. ProMMP-1 was activated by reacting with 1 mM 4-aminophenylmercuric acetate and a 0.1 molar amount of MMP-3 at 37 °C for 6 h. MMP assays were carried out in assay buffer by incubating a range of substrate concentrations (1–50 M) with 40 nM enzyme at 30 °C. Cellular MMP Assay—Melanoma cell adhesion to substrate-coated non-tissue culture-treated plates (BD Biosciences) was performed as described previously [35]. The plate was incubated at 30 °C in a humidified atmosphere for 30 min, and one volume of conditioned media, adhesion medium (as a negative control), or MMP (as a positive control) was added to each well.
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