Active site specificity profiling of the matrix metalloproteinase family: Proteomic identification of 4300 cleavage sites by nine MMPs explored with structural and synthetic peptide cleavage analyses

Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6′ in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1′ (with a ~ 32–93% preference for leucine depending on the MMP), and basic and small residues in P2′ and P3′, respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1′ leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1′-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with leucine locked in S1′. Similar negative cooperativity between P3 proline and the novel preference for asparagine in P1 cements our conclusion that non-prime side flexibility greatly impacts MMP binding affinity and cleavage efficiency. Thus, unexpected sequence cooperativity consequences were revealed by PICS that uniquely encompasses both the non-prime and prime sides flanking the proteomic-pinpointed scissile bond

The Peri-islet Basement Membrane, a Barrier to Infiltrating Leukocytes in Type 1 Diabetes in Mouse and Human

We provide the first comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet basement membrane (BM) and subjacent interstitial matrix (IM), in development of type 1 diabetes in NOD mice and in human type 1 diabetes. Our data demonstrate global loss of peri-islet BM and IM components only at sites of leukocyte infiltration into the islet. Stereological analyses reveal a correlation between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte penetration of the peri-islet BM is a critical step. Protease- and protease inhibitor–specific microarray analyses (CLIP-CHIP) of laser-dissected leukocyte infiltrated and noninfiltrated pancreatic islets and confirmatory quantitative real time PCR and protein analyses identified cathepsin S, W, and C activity at sites of leukocyte penetration of the peri-islet BM in association with a macrophage subpopulation in NOD mice and human type 1 diabetic samples and, hence, potentially a novel therapeutic target specifically acting at the islet penetration stage. Interestingly, the peri-islet BM and underlying IM are reconstituted once inflammation subsides, indicating that the peri-islet BM-producing cells are not lost due to the inflammation, which has important ramifications to islet transplantation studies

Cleavage Specificity Analysis of Six Type II Transmembrane Serine Proteases (TTSPs) Using PICS with Proteome-Derived Peptide Libraries

<div><p>Background</p><p>Type II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.</p><p>Methodology/Principal Finding</p><p>To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS). Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin) to simultaneously determine sequence preferences on the N-terminal non-prime (P) and C-terminal prime (P’) sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1′ position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.</p><p>Conclusions</p><p>Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1′ positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.</p></div

Cleavage Specificity Analysis of Six Type II Transmembrane Serine Proteases (TTSPs) Using PICS with Proteome-Derived Peptide Libraries - Figure 4

<p><b>A. <i>Upper panel</i>.</b> MALDI-TOF spectrum of the synthetic peptide AVIGRKFGDP. The sample contained a minor synthesis contaminant of AVIGRKFGD. The experimental determined [M+H]⁺ is 1059.52 Da (predicted m/z is 1059.22 Da). The sequence is presented by the spectra. The two asterix peaks represent the MALDI matrix peaks which are found in all the spectra in this m/z range. <b><i>Second panel.</i></b> MALDI-TOF spectrum of matriptase-2. <b><i>Third panel.</i></b> MALDI-TOF spectrum of the synthetic peptide added to matriptase-2 at 0 h. <b><i>Lower panel.</i></b> MALDI-TOF spectrum of the assay reaction products generated after incubation of the synthetic peptide with matriptase-2 for 18 h. The spectral peak at 1059.52 m/z disappeared and a new peak at 515.31 Da appeared corresponding to the cleavage product [AVIGR+H]⁺ (predicted m/z is 514.96 Da). <b>B. </b><b><i>Upper panel.</i></b> MALDI-TOF spectrum of the dimethylated (dm) synthetic peptide (dm)AVIGR(dm)KFGDP. The experimental determined [M+H]⁺ is 1115.52 Da. The sequence is presented on the spectral peak. The two asterix peaks represent the MALDI matrix peaks and they can be found in all the spectra. <b><i>Second panel.</i></b> MALDI-TOF spectrum of matriptase-2. <b><i>Third panel.</i></b> MALDI-TOF spectrum of the reaction products after incubation of the dimethylated synthetic peptide with matriptase-2 added at 0 h. <b><i>Lower panel.</i></b> MALDI-TOF spectrum of the reaction products after incubation of the dimethylated synthetic peptide with matriptase-2 for 18 h. The peak at 1115.52 Da disappeared and a new peak at 543.30 Da appeared, corresponding to [dAVIGR+H]⁺. Red (dm) is dimethylation.</p

Known protein substrates obtained from MEROPS [32] and UniProt [66] showing the P4-P4′ cleavage specificity in natural substrates of matriptase, matriptase-2, matriptase-3, HAT, hepsin and corin where cleaved as native proteins <i>versus</i> denatured peptides in PICS.

<p>Known protein substrates obtained from MEROPS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105984#pone.0105984-Rawlings1" target="_blank">[32]</a> and UniProt <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105984#pone.0105984-Magrane1" target="_blank">[66]</a> showing the P4-P4′ cleavage specificity in natural substrates of matriptase, matriptase-2, matriptase-3, HAT, hepsin and corin where cleaved as native proteins <i>versus</i> denatured peptides in PICS.</p

A. Schematic representation of the protein structures and arrangements of the four TTSP subfamilies: HAT/DESC, hepsin/TMPRSS, matriptase and corin.

<p>All TTSPs contain a N-terminal transmembrane signal anchor domain (<b>TM</b>) and a C-terminal serine protease domain (<b>H D S</b>). In case of polyserase-1 one of the 3 catalytic domains is inactive (<b>H D A</b>). The stem region of the TTSPs contains 1–6 different domains: the <u>s</u>ea urchin sperm protein/<u>e</u>nteropeptidase/<u>a</u>grin domain (<b>SEA</b>), group A <u>s</u>cavenger <u>r</u>eceptor domain (<b>SR</b>), low-density lipoprotein receptor class A domain (<b>L</b>), <u>C</u>ls/CLr, <u>u</u>rchin embryonic growth factor, <u>b</u>one morphogenetic protein-1 domain (<b>CUB</b>) and Corin contains two frizzled (FRIZ) domain. <b>B.</b> Murine CLIP-CHIP RNA expression profile and its distribution of 19 members of the TTSP family in 10 murine tissues in duplicates according to their average signal intensity.</p

A Schematic representation of the dodecapeptide (AEAALR↓KLLEVA) used for active site docking.

<p>The deep TTSP S1 subsite, accommodating the P1-Arg, is shown. <b>B</b>. Structural model of matriptase-2 with the peptide substrate (AEAALRKLLEVA) identified by PICS docked into the active site. Matriptase-2 was colored according to its surface charge distribution, and is shown in standard orientation with the modeled peptide occupying subsites S6 to S6′ (from left to right). <b>C</b>. Close-up views of the active site illustrating the deep and negatively charged S1 pocket (left) and key stabilizing interaction between the protease and the modeled peptide (right).</p

A. Multiple sequence alignment of the catalytic domains of six TTSPs was performed with Clustal Omega [59] and displayed using ESPript 2.2 (http://esprit.ibcp.fr/ESPript/cgi-bin/ESPript.cgi[63].

<p>Green stars indicate the residues of the catalytic triad; conserved residues are shaded in red. Blue boxes indicate amino acid with similar physico-chemical properties. Secondary structure elements are represented with flat arrows for β sheets and helices for α- and 3₁₀- (η) helices. Three conserved disulfide bridges are indicated in orange numerals below the alignment. Sequence numbering is according to the respective UniProt entries. <b>B.</b> Topology diagram of a TTSP catalytic domain. Residues of the catalytic triad are indicated as green stars, disulfide bonds (DSB) are shown in yellow. Loops (L-) are labeled corresponding to the thrombin nomenclature suggested by Bode et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105984#pone.0105984-Bode1" target="_blank">[64]</a>. <b>C.</b> Superposition of the catalytic domains of matriptase, matriptase-2, matriptase-3, hepsin, DESC1 and HAT, including both known and modeled structures, in traditional serine protease standard orientation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105984#pone.0105984-Huber1" target="_blank">[65]</a>. Catalytic triad and conserved disulfide bridges are shown in ball and stick representation. Important loops and exosites are indicated and labeled.</p

Network Analyses Reveal Pervasive Functional Regulation Between Proteases in the Human Protease Web

<div><p>Proteolytic processing is an irreversible posttranslational modification affecting a large portion of the proteome. Protease-cleaved mediators frequently exhibit altered activity, and biological pathways are often regulated by proteolytic processing. Many of these mechanisms have not been appreciated as being protease-dependent, and the potential in unraveling a complex new dimension of biological control is increasingly recognized. Proteases are currently believed to act individually or in isolated cascades. However, conclusive but scattered biochemical evidence indicates broader regulation of proteases by protease and inhibitor interactions. Therefore, to systematically study such interactions, we assembled curated protease cleavage and inhibition data into a global, computational representation, termed the protease web. This revealed that proteases pervasively influence the activity of other proteases directly or by cleaving intermediate proteases or protease inhibitors. The protease web spans four classes of proteases and inhibitors and so links both recently and classically described protease groups and cascades, which can no longer be viewed as operating in isolation <i>in vivo</i>. We demonstrated that this observation, termed reachability, is robust to alterations in the data and will only increase in the future as additional data are added. We further show how subnetworks of the web are operational in 23 different tissues reflecting different phenotypes. We applied our network to develop novel insights into biologically relevant protease interactions using cell-specific proteases of the polymorphonuclear leukocyte as a system. Predictions from the protease web on the activity of matrix metalloproteinase 8 (MMP8) and neutrophil elastase being linked by an inactivating cleavage of serpinA1 by MMP8 were validated and explain perplexing <i>Mmp8</i>^−/− versus wild-type polymorphonuclear chemokine cleavages <i>in vivo</i>. Our findings supply systematically derived and validated evidence for the existence of the protease web, a network that affects the activity of most proteases and thereby influences the functional state of the proteome and cell activity.</p></div