Selective hydrolysis of triple-helical substrates by matrix metalloproteinase-2 and -9.
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.
Lauer-Fields
Janelle L
JL
Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, Florida 33431-0991, USA.
Sritharan
Thilaka
T
Stack
M Sharon
MS
Nagase
Hideaki
H
Fields
Gregg B
GB
D002942:Circular Dichroism; D003094:Collagen / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013696:Temperature; D014407:Tumor Cells, Cultured
D003094:Collagen; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9