High-Throughput Multiplexed Peptide-Centric Profiling Illustrates Both Substrate Cleavage Redundancy and Specificity in the MMP Family

Abstract

Matrix metalloproteinases (MMPs) play incompletely understood roles in health and disease. Knowing the MMP cleavage preferences is essential for a better understanding of the MMP functions and design of selective inhibitors. To elucidate the cleavage preferences of MMPs, we employed a high-throughput multiplexed peptide-centric profiling technology involving the cleavage of 18,583 peptides by 18 proteinases from the main sub-groups of the MMP family. Our results enabled comparison of the MMP substrates on a global scale, leading to the most efficient and selective substrates. The data validated the accuracy of our cleavage prediction software. This software allows us and others to locate, with nearly 100% accuracy, the MMP cleavage sites in the peptide sequences. In addition to increasing our understanding of both the selectivity and the redundancy of the MMP family, our study generated a roadmap for the subsequent MMP structural-functional studies and efficient substrate and inhibitor design.

Fig. 1. Cleavage parameters of the catalytic domains versus the full-length enzymes of MMP-2 and MMP-9

The X-axis and Y-axis denote the Z-score of substrate cleavage by the catalytic domain and by the full-length proteinase, respectively. Black dots represent selected substrates (1,902 for MMP-2 and 2,167 for MMP-9) with raw sequencing counts >10. Red dotted line represents a linear regression fitted line. Pearson’s correlation coefficient and regression equation are shown within the panels. Assays were performed in triplicate. Average correlation among the replicates was 0.92 (not shown). Average correlation between the full-length enzyme and the catalytic domain was 0.72 (MMP-2) and 0.77 (MMP-9).

Fig. 2. Scatter density plot for the 18 MMPs

Density plots of Z-score versus raw sequencing counts of 18,583 peptide substrates. X-axis and Y-axis denote the Z-score of substrate cleavage and log₂ raw DNA counts in an MMP digest sample, respectively. Darker blue represents a higher density of substrates, with outlying substrates shown as dots (the Z-score >2.5; p-value <0.0062 by one tail test). All of the MMPs, except MMP-7 and MMP-19, exhibited >500 substrates with the Z-score >2.5. Because of a limited number of the efficiently cleaved peptide substrates, MMP-7 and MMP-19 were not analyzed further.

Fig. 3. Pearson’s correlation coefficient matrix of the Z-scores of 18,583 peptide substrates cleaved by MMPs

The bottom triangular matrix shows raw correlation coefficient. The top triangular matrix represents a degree of correlation in a form of an ellipse (a low minor axis denotes a high correlation). The color of the coefficient and ellipse also indicates the correlation strength to aid the eye. Cyan indicates low correlation (0 – 0.3), blue denotes medium correlation (0.3 – 0.8), while red denotes high correlation (0.8 – 1) among the MMP cleavage preferences.

Fig. 4. Frequency plot of the efficient cleavage sequences of MMPs in an IceLogo format

The height of a character is proportional to the frequency of the amino acid residue at the individual position of the cleaved peptide. 18,583 peptide substrates were cleaved by the individual MMPs. The scissile bond is between the P1 and P1′ residues. The Z-scores for the substrates were calculated and the substrates were ranked according to their Z-scores. The most efficient 100 substrates (Z-score >2; p-value <0.02) were selected for each MMP. Because the design of the peptide substrates was biased to the PXXL-containing sequences, the resulting position-specific matrix of the top 100 substrates was normalized at each position by using the amino acid residue frequency at this particular position in the entire 18,583 peptide library. The five most frequently occurring sequence motifs for each MMP are also shown.

Fig. 5. Frequency plot of the selective cleavage sequences of MMPs in an IceLogo format

The height of a character is proportional to the frequency of the amino acid residue at the individual position of the cleaved peptide. Substrates were ranked according to the difference between their Z-score for the particular MMP and Z-score for all of other MMPs. Top 50 peptide substrates were selected for each MMP (Z-score >1.65; p-value <0.05). Because the design of the peptide substrates was biased to the PXXL-containing sequences, the resulting position-specific matrix of the top 50 substrates was normalized at each position by using the amino acid residue frequency at this particular position in the entire 18,583 peptide library. The three most frequent sequence motifs for each MMP are also shown.

Fig. 6. Supervised clustering of the ten most selective peptide substrates for each of MMPs in a heatmap format

Z-scores indicating cleavage activity of all 18,583 peptide substrates were calculated for each MMP, all substrates were ranked according to their Z-score difference relative to all other MMPs. Top 10 most selective substrates were chosen for each MMP (Z-score >1.65). X-axis shows MMPs. Y-axis shows 160 peptides (top 10 specific peptides for each MMP). Red, black and blue designate high, medium and low Z-scores, respectively.