Use of Edman degradation sequence analysis and matrix-assisted laser desorption/ionization mass spectrometry in designing substrates for matrix metalloproteinases.
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.
Lauer-Fields
J L
JL
Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton 33431-0991, USA.
Nagase
H
H
Fields
G B
GB
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000737:chemistry; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D020539:Sequence Analysis, Protein / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity
C043517:2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinene 2-oxide; D009943:Organophosphorus Compounds; D010455:Peptides; D020782:Matrix Metalloproteinases