Use of Edman degradation sequence analysis and matrix-assisted laser desorption/ionization mass spectrometry in designing substrates for matrix metalloproteinases.
Lauer-Fields JL., Nagase H., Fields GB.
The matrix metalloproteinase (MMP) family has been implicated in the process of a variety of diseases such as arthritis, atherosclerosis, and tumor cell metastasis. We have been designing single-stranded peptides (SSPs) and triple-helical peptides (THPs) as potential discriminatory MMP substrates. Edman degradation sequence and matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analyses of proteolytic activity have been utilized to aid in further substrate design. THP models of the alpha1(I)772-786 sequence from type I collagen were synthesized to examine the triple-helical substrate specificity of MMP family members. Sequence and MALDI-MS analyses were used in conjunction with a fluorometric assay to determine the exact point of cleavage by each MMP. MMP-1 (interstitial collagenase) cleaved the substrates at a single Gly-Ile bond, analogous to the cleavage site in type I collagen. MMP-2 (Mr 72 000 type IV collagenase; gelatinase A) was found to cleave the substrates at two sites, a Gly-Ile bond and a Gly-Gln bond. MMP-3 (stromelysin 1) was found to cleave only one of the substrates after reaction for 48 h. Ultimately, sequence and MALDI-MS analyses allowed us to detect an additional cleavage site for MMP-2 in comparison to MMP-1, while MMP-3 was found to cleave a substrate after an extended time period. The second cleavage site would cause the kinetic parameters for MMP-2 to be overestimated by the fluorometric assay. Further design variations for these substrates need to consider the presence of more stable triple-helical conformation (to eliminate MMP-3 binding) and the removal of Gly-Gln bonds that may be susceptible to MMP-2.