Tài liệu Báo cáo khoa học: Degradation of tropoelastin by matrix metalloproteinases – cleavage site specificities and release of matrikines pptx

Degradation of tropoelastin by matrix metalloproteinases – cleavage site specificities and release of matrikines Andrea Heinz 1 , Michael C. Jung 1 , Laurent Duca 2 , Wolfgang Sippl 1 , Samuel Taddese 1 , Christian Ihling 1 , Anthony Rusciani 2 ,Gu ¨ nther Jahreis 3 , Anthony S. Weiss 4 , Reinhard H. H. Neubert 1 and Christian E. H. Schmelzer 1 1 Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany 2 Faculte ´ des Sciences, Laboratoire de Biochimie, Reims, France 3 Max Planck Research Unit for Enzymology of Protein Folding, Halle (Saale), Germany 4 School of Molecular and Microbial Biosciences, University of Sydney, Australia Keywords gelatinase B; GxxPG; macrophage elastase; matrilysin; mass spectrometry Correspondence Christian E. H. Schmelzer, Martin Luther University Halle-Wittenberg, Institute of Pharmacy, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany Fax: +49 345 5527292 Tel: +49 345 5525215 E-mail: schmelzer@pharmazie.uni-halle.de (Received 7 January 2010, revised 3 February 2010, accepted 12 February 2010) doi:10.1111/j.1742-4658.2010.07616.x To provide a basis for the development of approaches to treat elastin- degrading diseases, the aim of this study was to investigate the degradation of the natural substrate tropoelastin by the elastinolytic matrix metallopro- teinases MMP-7, MMP-9, and MMP-12 and to compare the cleavage site specificities of the enzymes using complementary MS techniques and molec- ular modeling. Furthermore, the ability of the three proteases to release bioactive peptides was studied. Tropoelastin was readily degraded by all three MMPs. Eighty-nine cleavage sites in tropoelastin were identified for MMP-12, whereas MMP-7 and MMP-9 were found to cleave at only 58 and 63 sites, respectively. Cleavages occurred predominantly in the N-terminal and C-terminal regions of tropoelastin. With respect to the cleavage site specificities, the study revealed that all three MMPs similarly tolerate hydrophobic and ⁄ or aliphatic amino acids, including Pro, Gly, Ile, and Val, at P 1 ¢. MMP-7 shows a strong preference for Leu at P 1 ¢, which is also well accepted by MMP-9 and MMP-12. Of all three MMPs, MMP-12 best tolerates bulky charged and aromatic amino acids at P 1 ¢. All three MMPs showed a clear preference for Pro at P 3 that could be structurally explained by molecular modeling. Analysis of the generated peptides revealed that all three MMPs show a similar ability to release bioactive sequences, with MMP-12 producing the highest number of these peptides. Furthermore, the generated peptides YTTGKLPYGYGPGG, YGARPGVGVGGIP, and PGFGAVPGA, containing GxxPG motifs that have not yet been proven to be bioactive, were identified as new matrikines upon biological activity testing. Structured digital abstract l MINT-7709630: MMP-7 (uniprotkb:P09237) cleaves (MI:0194) Tropoelastin (uniprotkb: P15502)byprotease assay (MI:0435) l MINT-7709668: MMP-9 (uniprotkb:P14780) cleaves (MI:0194) Tropoelastin (uniprotkb: P15502)byprotease assay ( MI:0435) l MINT-7709289: MMP-12 (uniprotkb:P39900) cleaves (MI:0194) Tropoelastin (uniprotkb: P15502)byprotease assay ( MI:0435) Abbreviations ACN, acetonitrile; EBP, elastin-binding protein; ECM, extracellular matrix; EDP, elastin-derived peptide; i.d., internal diameter; MMP, matrix metalloproteinase; qTOF, quadrupole time-of-flight; TFA, trifluoroacetic acid. FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS 1939 Introduction Matrix metalloproteinases (MMPs) form a large family of multidomain zinc-dependent and calcium-dependent endopeptidases that are known to cleave various com- ponents of the extracellular matrix (ECM). MMPs play a central role in connective tissue remodeling pro- cesses and regulation of cell matrix composition through their effects on cell migration, cell differentia- tion, cell growth, wound healing, inflammation, angio- genesis, and apoptosis. The disruption of the physiological balance between MMP activation and deactivation is connected with severe diseases such as atherosclerosis, arthritis, pulmonary emphysema, myo- cardial infarction, and tumor growth and metastasis [1–5]. Three of the most widely studied MMPs with respect to their biological actions are MMP-7 (EC 3.4.24.23), MMP-9 (EC 3.4.24.35), and MMP-12 (EC 3.4.24.65). MMP-7, which is also referred to as matrilysin 1, is mainly expressed by epithelial cells and processes various ECM constituents, such as collagens, gelatin, and laminin, and also non-ECM proteins, including pro-tumor necrosis factor-a and a 2 -macro- globulin [2]. MMP-9 (gelatinase B) is secreted by neu- trophils and macrophages and has also been found in various malignant cells, ras-transformed murine cells, and chemically stimulated fibroblasts. It has, for instance, been shown to cleave native type IV and VII collagens, gelatin, laminin, and plasminogen [2]. MMP-12 (macrophage elastase) is expressed mainly by macrophages. The enzyme cleaves a variety of sub- strates, including collagens, gelatin, laminin, pro-tumor necrosis factor-a, and plasminogen [2]. Natural sub- strates that are known to be degraded by MMP-7, MMP-9, MMP-12, and a further member of the MMP family, MMP-2, are the connective tissue pro- tein elastin and its monomeric precursor tropoelastin [6–11]. The biopolymer elastin, which provides elasticity and resilience to several tissues, including lungs, arter- ies, and skin, shows a unique chemical composition characterized by the presence of large amounts of the four hydrophobic amino acids Gly, Val, Ala, and Pro. The protein consists of molecules of its soluble precur- sor tropoelastin that are cross-linked at Lys residues. Owing to its hydrophobicity and extensive cross-link- ing, elastin is insoluble and highly resistant to proteo- lytic degradation. Moreover, elastin does not undergo substantial turnover in healthy tissue [12–16]. In the last two decades, studies have revealed that elastin is not only a structural protein influencing the architecture and biomechanical properties of the ECM but also plays an active role in various physiological processes [16]. Some elastin-derived peptides (EDPs), which may occur upon proteolytic degradation of elas- tin and tropoelastin, promote angiogenesis [17] and are associated with the regulation of various cell activities, including cell adhesion, chemotaxis, migration, prolif- eration, protease activation, and apoptosis [18–21]. Such EDPs are matrikines; this name generally denotes bioactive ligands that exist as part of an ECM protein. The results of different studies suggest that EDPs con- taining the GxxPG motif, in particular, are biologically active, as these are able to interact with the elastin- binding protein (EBP) [18,19,22–25]. In light of the diverse and complex biological func- tions of elastin, EDPs, and MMPs, it is clear that the previously mentioned aberrant expression of elastin- degrading enzymes such as MMPs often leads to dam- age to elastic fibers. This damage, together with other biological processes triggered by EDPs and MMPs, may support the development and progression of vari- ous pathological conditions. It has, for instance, been found that aortic stenosis is associated with increased activity of MMP-2 and MMP-9 [26], atherosclerosis is influenced by MMP-12, which promotes athero- sclerotic plaque instability [27], and the development of aortic aneurysms is enhanced by MMP-2, MMP-9, and MMP-12 [28,29]. Studies have also indicated that MMP-9 is involved in processes such as cardiac rup- ture after myocardial infarction [30] and photoaging of the skin [11,31]. Furthermore, overexpression of MMP-12 has been found to be associated with the development and progression of pulmonary emphy- sema [32], photoaging of the skin [33], and granuloma- tous skin diseases [34]. MMP-7 is strongly expressed in tumors of almost every organ in the body and seems to play a vital role in tumor progression and angiogen- esis [35,36]. Taken together, these examples show that it is of utmost importance to understand and charac- terize elastin-degrading processes, including the cleav- age behavior of elastinolytic MMPs and the nature of the peptides released on degradation. This approach may aid in the development of directed therapies to treat pathologies related to elastin degradation, the overexpression of MMPs, and the consequent release of bioactive peptides. Few studies have investigated the enzymatic degra- dation of elastin or its precursor tropoelastin [10,37,38] and the release of bioactive peptides upon enzymatic degradation of elastin, tropoelastin or synthesized domains derived from tropoelastin [39–41]. The aim of the present study was to obtain detailed information Tropoelastin degradation by elastinolytic MMPs A. Heinz et al. 1940 FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS on the cleavage site specificities of MMP-7, MMP-9, and MMP-12 in tropoelastin using complementary MS techniques and to characterize and compare the cleav- age behavior of the three enzymes using molecular modeling. Tropoelastin was chosen as substrate in this study because of its biological relevance during elastin turnover and matrix remodeling and to increase the number of identifiable peptides resulting from proteo- lytic digestion. Mature elastin, in contrast, shows only limited suitability for characterization of the cleavage site specificity of proteolytic enzymes, owing to its extensive cross-linking which restricts MS fragmenta- tion and sequencing approaches [16,37]. In contrast to previous studies, the present work, for the first time, sought to obtain a comprehensive insight into the pre- ferred amino acids at the cleavage site positions P 1 –P 4 and P 1 ¢–P 4 ¢ of the substrate and to give a structural explanation of the amino acid preferences of MMP-7, MMP-9, and MMP-12, which have not been described to date. Moreover, the potential of the three MMPs to produce bioactive peptides upon proteolytic digestion was investigated, and peptides containing the GxxPG motif resulting from the digestion of tropoelastin by MMP-7, MMP-9, and MMP-12 were tested for their bioactivity. Results Highest number of cleavages and highest sequence coverage obtained for MMP-12 Sequence coverages of 71.1, 59.5, and 80.7% were determined for degradation by MMP-7, MMP-9, and MMP-12, respectively (Fig. 1). The cleavage sites iden- tified for all three MMPs occurred mainly in amino or carboxyl regions of the tropoelastin sequence, in agree- ment with previous studies on bovine and human elas- tin [10,37]. Altogether, for MMP-12, 89 cleavage sites and 132 peptides were identified in almost all domains of tropoelastin with the exception of domains 8, 9, and 11. In contrast, for MMP-7 and MMP-9, only 58 (84 peptides) and 63 (74 peptides) cleavage sites could be determined, respectively. For MMP-7, no cleavages were observed in domains 8–11, 17, 19–21, 23, and 36. MMP-9 showed a similar cleavage behavior and did not degrade domains 8–11, 16–20, and 36. Altogether, 23 cleavage sites and 20 peptides were found that were common for all three MMPs. It is worth mentioning that in MALDI-TOF experiments several unidentified higher-mass peptides of between 10 kDa and 20 kDa were observed for the three MMPs, underlining the finding that some domains resisted proteolysis (data not shown). Aliphatic and ⁄ or hydrophobic residues favored at P 1 ¢ The P 1 ¢–S 1 ¢ interaction has been identified as the main determinant of the cleavage position of MMPs in pep- tide substrates [5,10,42,43]. The results of this work are in agreement with previous studies proposing that the three enzymes can accept a variety of amino acids with hydrophobic and ⁄ or aliphatic residues including Ala, Gly, Val, Leu, Ile, Tyr, and Phe at P 1 ¢ [2,10,43,44]. Only the charged amino acid Lys, which was found to be tolerated at P 1 ¢ by MMP-12, has been reported to be an exception to the previously men- tioned preferences [43]. The present study appears to confirm that MMP-12 shows a preference for x-Lys, as the enzyme cleaved at 11 of 35 (31%) of such cleavage sites (Fig. 2A; Table 1). It was also found that MMP-7 and MMP-9 cleaved N-terminal to Lys; however, this was to a lesser extent than MMP-12, with MMP-9 cleaving 7 of 35 (20%), and MMP-7 cleaving only 1 of the 35 (3%) possible cleavage sites. The interaction of the hexapeptide substrate PQGKAG containing Lys at P 1 ¢ with the active sites of MMP-9 and MMP-12 as investigated by molecular modeling is shown in Fig. 3 and confirms that both enzymes are able to accept Lys at P 1 ¢. Another difference in the cleavage behavior of the three MMPs was found at possible cleavage sites with Leu. Figure 2A and Table 1 show that MMP-7 has a strong preference for Leu at P 1 ¢, which has also been described previously [44,45]. MMP-7 cut at 29 of 40 (73%) possible cleavage sites with Leu, whereas MMP-9 and MMP-12 only cleaved at 14 (35%) and 19 (48%) sites, respectively. The preference of MMP-7 is also shown in Fig. 3, where the interaction of the hexapeptide substrate PQGLAG containing Leu at P 1 ¢ is modeled. Small differences in the cleavage site speci- ficities of the three MMPs were observed at cleavage sites with bulky aromatic amino acids such as Tyr and Phe at P 1 ¢, which were cut, in particular, by MMP-12. While MMP-12 hydrolyzed 53% (8 of 15) of the possi- ble x-Tyr peptide bonds, MMP-7 and MMP-9 showed similar cleavage behavior and only cut at 3 and 4 of 15 possible x-Tyr cleavage sites, respectively. x-Phe bonds were found to be hydrolyzed by MMP-7 (2 of 16) and MMP-12 (4 of 16) but not by MMP-9. The cleavage behavior of MMP-7, MMP-9, and MMP-12 at x-Pro, x-Gly, x-Ile, and x-Val peptide bonds is very similar. All three MMPs similarly toler- ate amino acids with relatively small aliphatic and ⁄ or hydrophobic residues at P 1 ¢. Another interesting differ- ence, however, can be found at x-Ala peptide bonds, which are almost resistant to hydrolysis by MMP-7 A. Heinz et al. Tropoelastin degradation by elastinolytic MMPs FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS 1941 Fig. 1. Cleavage sites identified after digestion of human tropoelastin isoform 2 (SwissProt accession number: P15502-2) with MMP-7, MMP-9, and MMP-12. Cleavage sites are indicated by triangles (MMP-7, red; MMP-9, green; MMP-12, black), and all regions covered by peptides are labeled with solid lines (MMP-7, red; MMP-9, green; MMP-12, black). Bioactive sequences [17,19,46–65] are shown in blue. The sequence of the octapeptide 226–233 used to model the interaction between a natural substrate and the active site of MMP-12 (Fig. 4) is marked with a blue bar. The sequences of three peptides containing GxxPG motifs (YTTGKLPYGYGPGG, YGARPGVGVGGIP, and PGFGAVPGA) used for bioactivity tests are labeled with asterisks and orange arrows. Tropoelastin degradation by elastinolytic MMPs A. Heinz et al. 1942 FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS (2 of 157 possible x-Ala bonds) but are cleaved to a small extent by MMP-9 (12 of 157) and even more by MMP-12 (24 of 157). The determined cleavage site preference of MMP-7 was based on the number of cleavages occurring N-ter- minal to the respective amino acid and follows A B Fig. 2. Number of amino acids found in P 1 ¢ (A) and P 1 (B) in peptides of tropoelastin after digestion with either MMP-7, MMP-9 or MMP-12. Table 1. Occurrence of different amino acids at the substrate positions P 1 -P 4 and P 1 ¢-P 4 ¢ after digestion with MMP-7, MMP-9, and MMP-12. Values are based on the total amounts of each of the amino acids in tropoelastin (isoform 2). A. Heinz et al. Tropoelastin degradation by elastinolytic MMPs FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS 1943 the order Leu (48% of all identified cleavage sites) >> Val ⁄ Gly (each 12%) > Pro (10%) > Tyr (5%), with Leu being clearly preferred over other aliphatic and ⁄ or hydrophobic amino acids (Table 2). The cleavage site specificity of MMP-9 follows the order Leu (22% of all identified cleavage sites) > Ala (19%) > Gly (14%) > Lys (11%) > Val (9%). MMP-12, which is the most active of the three enzymes, shows a cleavage site specificity according to the order Ala (26% of all identified cleavage site- s) > Leu (20%) > Lys (12%) > Val ⁄ Tyr (each 9%) > Gly (7%). With respect to the charged or polar amino acids Arg, Gln, Ser, Asp, Cys, and Glu, which together con- stitute only 3.6% of the tropoelastin sequence, this study revealed that hardly any cleavage occurred N-terminal to these amino acids upon digestion with MMP-7, MMP-9, and MMP-12. An exception is a sin- gle cleavage between Ala and Cys, which was found for MMP-12. Mainly Gly and Ala are found at P 1 With regard to the preferred amino acids at P 1 , the present study revealed strong similarities between the three MMPs. After hydrolysis by MMP-7, MMP-9, and MMP-12, Ala and Gly, which constitute 52% of the tropoelastin sequence, were predominant at P 1 (Fig. 2B), which is in accordance with known P 1 specificities for MMPs [10,44]. In detail, the experi- ments with MMP-7 showed that Gly occurred N-ter- minal to 33 (55%) of the 60 identified cleavage sites and Ala occurred N-terminal to 9 (15%) (Table 2; Fig. 2B). After digestion with MMP-9, Gly was found N-terminal to 24 (38%) of the 64 identified cleavage sites, and Ala was found N-terminal to 21 (33%). Similar results were obtained for MMP-12, where Gly was found N-terminal of 30 (33%) and Ala N-terminal of 36 (39%) of the 92 identified cleavage sites, respectively. In summary, it can be stated that Gly and Ala occur at P 1 in about 70% of the cleavages, whereas in the other 30% small amino acids such as Pro and Val are mainly found at P 1 (Table 2). A B C Fig. 3. Interaction of hexapeptide substrates with the binding sites of MMP-7, MMP-9, and MMP-12. For clarity, only non- conserved residues of the S 1 ¢ pocket within the three studied MMPs are shown. The zinc ion at the catalytic site is shown as a yellow ball. (A) Interaction of the peptide substrate PQGLAG containing a P 1 ¢ Leu with the MMP-7 binding site. (B) Interaction of the peptide substrate PQGKAG containing a P 1 ¢ Lys with the MMP-9 binding site. (C) Interaction of the peptide substrate PQGKAG containing a P 1 ¢ Lys with the MMP-12 binding site. Tropoelastin degradation by elastinolytic MMPs A. Heinz et al. 1944 FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS Mainly Gly and Ala are found at P 2 –P 4 and P 2 ¢–P 4 ¢ Table 2 shows that following digestion with MMP-7, MMP-9 or MMP-12, predominantly Gly and Ala were found at P 2 –P 4 and P 2 ¢–P 4 ¢. Interestingly, a preference for Pro at P 3 was found for all three MMPs. Pro occurred at P 3 at 18% (MMP-7) to 24% (MMP-12) of all identified cleavage sites. The preference for Pro at P 3 was also confirmed by molecular modeling, in which the interaction of the natural substrate LPYGYGPG containing Pro at P 3 with the active site of MMP-12 was investigated (Fig. 4). Furthermore, it was observed that MMP-7 tolerates Val at P 3 ¢ better than MMP-9 and MMP-12. Peptides with bioactive sequences released upon proteolytic digestion of tropoelastin with MMP-7, MMP-9 or MMP-12 Table 3 and Fig. 1 show that some of the 42 bioactive sequences [17,19,46–65] partly overlap in tropoelastin. Altogether, 35 of these sequences were found: 15 sequences, of which 11 were nonrepetitive, were found in 27 peptides of different lengths released by MMP-7; 22 sequences, of which 11 were nonrepetitive, were found in 23 peptides released by MMP-9; and 20 sequences, of which 13 were nonrepetitive, were found in 41 peptides released by MMP-12 (Tables 3 and 4). Table 2. Occurrence of different amino acids at the substrate positions P 1 -P 4 and P 1 ¢ -P 4 ¢ after digestion with MMP-7, MMP-9, and MMP-12. Values are based on the number of cleavage sites identified on MS analysis of the digests. P3‘ P4‘ A B Fig. 4. Interaction of the natural substrate LPYGYGPG (residues 226–233 from tropoelastin isoform 2; see Fig. 1) and the MMP-12 active site. (A) The molecular surface of the binding pocket is colored according to electrostatic potential (red indicates negative electrostatic poten- tial; blue indicates positive electrostatic potential). (B) The hydrogen bonds between the backbone residues of the substrate and the residues of the MMP-12 binding site are highlighted (yellow dashed lines). A. Heinz et al. Tropoelastin degradation by elastinolytic MMPs FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS 1945 Bioactive sequences were predominantly released from the N-terminal and C-terminal parts of tropoela- stin, where most cleavages occurred (Fig. 1). From the central part of the tropoelastin molecule, only three peptides containing the matrikines VPGVG (341–345), VGVPG (344–348), and GARPG (384–388) were released by MMP-12 exclusively (Tables 3 and 4). The smallest peptides found with bioactive sequences Table 3. Bioactive sequences within tropoelastin isoform 2 and those that were identified as parts of peptides of different lengths after digestion with MMP-7, MMP-9, and MMP-12. The bioactive sequences were selected on the basis of several publications [17,19,46–65]. Moreover, all sequences containing the GxxPG motif found in tropoelastin isoform 2 were listed, except for the peptide GGVPG, which is known to show no bioactivity [19,47]. Tropoelastin degradation by elastinolytic MMPs A. Heinz et al. 1946 FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS Table 4. Peptides containing bioactive sequences that were identified after digestion of recombinant tropoelastin with MMP-7, MMP-9, or MMP-12. Bioactive sequences are in bold red letters. A. Heinz et al. Tropoelastin degradation by elastinolytic MMPs FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS 1947 included eight peptides of lengths between 9 and 18 amino acids (Table 4). The shortest peptide was identi- fied after digestion with MMP-9 and MMP-12, and displayed the sequence PGFGAVPGA (578–586). In addition to the 8 relatively short peptides, 53 longer peptides of lengths up to 105 amino acids were identi- fied. The longest peptide (103–207) was released by MMP-12 and contained 9 partly overlapping bioactive sequences (Tables 3 and 4). It is worth mentioning that domain 24, which can be considered as one huge matrikine encompassing 15 par- tially overlapping bioactive peptides, remained intact upon proteolytic digestion with MMP-7, MMP-9, and MMP-12 (Fig. 1). An interesting finding is a peptide that was released after digestion with MMP-9 and spans the sequence 442–523 and so contains all the 15 bioactive sequences within domain 24 (Table 4). This includes GLVPG and repeats of VGVAPG and GVAPGV, which have been reported to show biologi- cal effects [46–60], as well as further GxxPG sequences (GIGPG and GLAPG). Because none of the three MMPs was capable of cleaving within domain 24, it is also likely that treatment with MMP-7 and MMP-12 resulted in additional peptides comprising the whole of domain 24; however, these could not be identified upon MS analysis. Among the different peptides released by MMP-7, MMP-9, and MMP-12, three contain GxxPG motifs for which no biological activity has yet been described: YTTGKLPYGYGPGG (residues 221-234, released by MMP-7, MMP-9, and MMP-12), YGARPGVGVG- GIP (residues 383–395, released by MMP-12), and Table 4. (continued) Fig. 5. Zymography analysis of pro-MMP-2 secretion. Cells were stimulated for 24 h with or without elastin peptides, and cell culture media were subjected to gelatin zymography. Lower panel: densitometric analysis. The statistical test compares control and elastin peptides. **P < 0.01. Differences observed in biological activities between the three peptides are not significant. Tropoelastin degradation by elastinolytic MMPs A. Heinz et al. 1948 FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... structures of MMP-7 (2ddy.pdb), MMP-9 (2ovx.pdb), and MMP-12 (2oxw.pdb), were taken from the Protein Data Bank and prepared using moe The structures of the substrate peptides LPYGYGPG (residues 22 6–2 33 from tropoelastin isoform 2; Fig 1), PQGLAG, and PQGKAG were generated by taking the coordinates of the prodomain (YPFALAPT, residues 9 6–1 03) of the crystallized proform of MMP-9 (1lj6.pdb) and the coordinates... TFA at a flow rate of 1 mLÆmin)1 over 30 min; and detection at k = 220 nm The molecular masses of the peptides were confirmed by ESI or MALDI-TOF MS FEBS Journal 277 (2010) 193 9–1 956 ª 2010 The Authors Journal compilation ª 2010 FEBS 1951 Tropoelastin degradation by elastinolytic MMPs A Heinz et al MALDI-LIFT-TOF ⁄ TOF MS ⁄ MS experiments by applying 2000 laser shots Proteolysis of tropoelastin For proteolysis... acids such as Lys, Tyr, and Phe (Tables 1 and 2; Fig 2A) result from differences in the secondary structures and amino acid compositions of the active sites of the three MMPs (Fig 3) As described above, MMP-12 has a more polar and larger active site than MMP-7 and MMP-9 and hence tolerates such residues better The low number or lack of cleavages by all three MMPs N-terminal to large and polar amino acids,... system of solvent A and solvent B (80% ACN ⁄ 20% H2O containing 0.04% TFA): linear gradient of 5–5 0% solvent B in 60 min, linear gradient of 5 0–1 00% solvent B in 2 min, maintenance at 100% solvent B for 6 min, and linear gradient of 10 0–5 % solvent B in 2 min The column was maintained at 30 °C and the flow rate was 300 nLÆ min)1 Detection was performed by UV absorption at 214 nm and 280 nm Between 8 min and. .. correlate with the total number of molecules of the respective amino acid in tropoelastin and may also be influenced by the amino acid at P1¢ As Ala and Gly constitute more than 50% of the tropoelastin sequence, it seems likely that the nature of the amino acid at P1 is governed not only by the amino acid preferences of the elastinolytic enzyme at this position, but also by the tropoelastin sequence itself... Elastin degradation by matrix metalloproteinases Cleavage site specificity and mechanisms of elastolysis J Biol Chem 272, 1807 1–1 8076 Herouy Y, Vanscheidt W, Schopf E & Norgauer J ¨ (1999) Gen- und Proteinfamilie der Matrix- Metalloproteinasen Struktur, Funktion und Expression in der Haut Der Hautarzt 50, 82 5–8 36 Vrhovski B & Weiss AS (1998) Biochemistry of tropoelastin Eur J Biochem 258, 1–1 8 Mithieux SM... Mapping of macrophage elastase cleavage sites in insoluble human skin elastin Matrix Biol 27, 42 0– 428 38 Barroso B, Abello N & Bischoff R (2006) Study of human lung elastin degradation by different elastases using high-performance liquid chromatography ⁄ mass spectrometry Anal Biochem 358, 21 6–2 24 39 Taddese S, Weiss AS, Jahreis G, Neubert RHH & Schmelzer CEH (2009) In vitro degradation of human tropoelastin. .. automated de novo sequencing of the nanoESI–qTOF and MALDI-TOF ⁄ TOF MS data followed by database matching (peaks protein id and spider) [86] was FEBS Journal 277 (2010) 193 9–1 956 ª 2010 The Authors Journal compilation ª 2010 FEBS A Heinz et al performed using the in-chorus search of the software peaks studio (version 4.5; Bioinformatics Solutions, Waterloo, Canada) [87] with precursor and fragment mass error... degraded and the structure of Lys is significantly altered upon cross-link- FEBS Journal 277 (2010) 193 9–1 956 ª 2010 The Authors Journal compilation ª 2010 FEBS 1949 Tropoelastin degradation by elastinolytic MMPs A Heinz et al ing, Lys is not commonly found in high amounts at P1¢ upon analysis of the cleavage site specificity of MMPs in elastin The results of this study show that predominantly Gly and Ala... Australian Research Council and Heart Research Foundation References 1 Maskos K (2005) Crystal structures of MMPs in complex with physiological and pharmacological inhibitors Biochimie 87, 24 9–2 63 2 Visse R & Nagase H (2003) Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry Circ Res 92, 82 7–8 39 3 Sternlicht MD & Werb Z (2001) How matrix metalloproteases . Degradation of tropoelastin by matrix metalloproteinases – cleavage site specificities and release of matrikines Andrea Heinz 1 , Michael. cleavage site positions P 1 –P 4 and P 1 ¢–P 4 ¢ of the substrate and to give a structural explanation of the amino acid preferences of MMP-7, MMP-9, and