Tài liệu Báo cáo khoa học: Degradation of tropoelastin by matrix metalloproteinases – cleavage site

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: [email protected]

(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.

FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS 1939