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

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.. Moreover, the potential of the

Degradation of tropoelastin by matrix metalloproteinases – cleavage site specificities and release of matrikines

Andrea Heinz1, Michael C Jung1, Laurent Duca2, Wolfgang Sippl1, Samuel Taddese1,

Christian Ihling1, Anthony Rusciani2, Gu¨nther Jahreis3, Anthony S Weiss4, Reinhard H H Neubert1 and Christian E H Schmelzer1

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 P1¢ MMP-7 shows a strong preference for Leu at P1¢, 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 P1¢ All three MMPs showed a clear preference for Pro at P3 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 ) by protease assay ( MI:0435 )

l MINT-7709668 : MMP-9 (uniprotkb: P14780 ) cleaves ( MI:0194 ) Tropoelastin (uniprotkb:

P15502 ) by protease assay ( MI:0435 )

l MINT-7709289 : MMP-12 (uniprotkb: P39900 ) cleaves ( MI:0194 ) Tropoelastin (uniprotkb:

P15502 ) by protease 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.

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, inflammadifferentia-tion,

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 a2

-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 tropoelaselas-tin, 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 damdam-age, 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

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 P1–P4

and P1¢–P4¢ 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

P1¢ The P1¢–S1¢ 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 P1¢ [2,10,43,44] Only the charged amino acid Lys, which was found to be tolerated at P1¢ 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

P1¢ 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 P1¢

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 P1¢, 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 P1¢

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 P1¢, 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 P1¢ Another interesting differ-ence, however, can be found at x-Ala peptide bonds, which are almost resistant to hydrolysis by MMP-7

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.

(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 P1¢

(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 P1-P4and P1¢-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).

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 P1 With regard to the preferred amino acids at P1, 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 P1 (Fig 2B), which is in accordance with known P1 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 P1 in about 70%

of the cleavages, whereas in the other 30% small amino acids such as Pro and Val are mainly found

at P1 (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 S1¢ 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 P1¢ Leu with the MMP-7 binding site (B) Interaction of the peptide substrate PQGKAG containing a P1¢ Lys with the MMP-9 binding site (C) Interaction of the peptide substrate PQGKAG containing a P1¢ Lys with the MMP-12 binding site.

Mainly Gly and Ala are found at P2–P4and P2¢–P4¢

Table 2 shows that following digestion with MMP-7,

MMP-9 or MMP-12, predominantly Gly and Ala were

found at P2–P4 and P2¢–P4¢ Interestingly, a preference

for Pro at P3 was found for all three MMPs Pro

occurred at P3 at 18% (MMP-7) to 24% (MMP-12) of

all identified cleavage sites The preference for Pro at P3

was also confirmed by molecular modeling, in which the

interaction of the natural substrate LPYGYGPG

containing Pro at P3with the active site of MMP-12 was

investigated (Fig 4) Furthermore, it was observed that

MMP-7 tolerates Val at P3¢ 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‘

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).

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].

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.

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.