Abstract
Matrix metalloproteinases (MMP) 2 and 9, the gelatinases, have consistently been associated with tumor progression. The development of gelatinase-specific probes will be critical for identifying in vivo gelatinoic activity to understand the molecular role of the gelatinases in tumor development. Recently, a self-assembling homotrimeric triple-helical peptide (THP), incorporating a sequence from type V collagen, with high substrate specificity to the gelatinases has been developed. To determine whether this THP would be suitable for imaging protease activity, 5-carboxyfluorescein (5FAM) was conjugated, resulting in 5FAM3-THP and 5FAM6-THP, which were quenched up to 50%. 5FAM6-THP hydrolysis by MMP-2 and MMP-9 displayed kcat/KM values of 1.5 × 104 and 5.4 × 103 M−1 s−1, respectively. Additionally 5FAM6-THP visualized gelatinase activity in gelatinase positive HT-1080 cells, but not in gelatinase negative MCF-7 cells. Furthermore, the fluorescence in the HT-1080 cells was greatly attenuated by the addition of a MMP-2 and MMP-9 inhibitor, SB-3CT, indicating that the observed fluorescence release was mediated by gelatinase proteolysis and not non-specific proteolysis of the THPs. These results demonstrate that THPs fully substituted with fluorophores maintain their substrate specificity to the gelatinases in human cancer cells and may be useful in in vivo molecular imaging of gelatinase activity.
Highlights
Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases that are capable of degrading extracellular matrix (ECM) components such as collagen, elastin, gelatin, and proteoglycans
Single-stranded collagen peptides were synthesized via solid phase peptide synthesis with a pair of
The goal of the study was to synthesize and evaluate homotrimeric triple-helical peptides composed of single-stranded peptides bearing 5FAM fluorescent dyes flanking the hydrolysis site for their ability to detect gelatinase activity in cell culture
Summary
Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases that are capable of degrading extracellular matrix (ECM) components such as collagen, elastin, gelatin, and proteoglycans. The MMPs can be classified into different groups. One such group is the gelatinases, which consists of the type IV collagenases MMP-2 and MMP-9. MMP levels are low; they are elevated during cell proliferation, cell migration, and tissue remodeling events [1]. MMPs are initially synthesized as inactive zymogens (pro-MMP) and are secreted into the extracellular space, anchored to the cell membrane, or remain localized inside of the cells [2,3]. Upon cleavage of the pro-peptide domain, the active site is exposed within the catalytic domain [4]
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