Neutrophil elastase cleavage of the gC1q domain impairs the EMILIN1-α4β1 integrin interaction, cell adhesion and anti-proliferative activity
Identifieur interne : 008B98 ( Ncbi/Merge ); précédent : 008B97; suivant : 008B99Neutrophil elastase cleavage of the gC1q domain impairs the EMILIN1-α4β1 integrin interaction, cell adhesion and anti-proliferative activity
Auteurs : Orlando Maiorani [Italie] ; Eliana Pivetta [Italie] ; Alessandra Capuano [Italie] ; Teresa Maria Elisa Modica [Italie] ; Bruna Wassermann [Italie] ; Francesco Bucciotti [Italie] ; Alfonso Colombatti [Italie] ; Roberto Doliana [Italie] ; Paola Spessotto [Italie]Source :
- Scientific Reports [ 2045-2322 ] ; 2017.
Abstract
The extracellular matrix glycoprotein EMILIN1 exerts a wide range of functions mainly associated with its gC1q domain. Besides providing functional significance for adhesion and migration, the direct interaction between α4β1 integrin and EMILIN1-gC1q regulates cell proliferation, transducing net anti-proliferative effects. We have previously demonstrated that EMILIN1 degradation by neutrophil elastase (NE) is a specific mechanism leading to the loss of functions disabling its regulatory properties. In this study we further analysed the proteolytic activity of NE, MMP-3, MMP-9, and MT1-MMP on EMILIN1 and found that MMP-3 and MT1-MMP partially cleaved EMILIN1 but without affecting the functional properties associated with the gC1q domain, whereas NE was able to fully impair the interaction of gC1q with the α4β1 integrin by cleaving this domain outside of the E933 integrin binding site. By a site direct mutagenesis approach we mapped the bond between S913 and R914 residues and selected the NE-resistant R914W mutant still able to interact with the α4β1 integrin after NE treatment. Functional studies showed that NE impaired the EMILIN1-α4β1 integrin interaction by cleaving the gC1q domain in a region crucial for its proper structural conformation, paving the way to better understand NE effects on EMILIN1-cell interaction in pathological context.
Url:
DOI: 10.1038/srep39974
PubMed: 28074935
PubMed Central: 5225433
Links toward previous steps (curation, corpus...)
- to stream Pmc, to step Corpus: 000977
- to stream Pmc, to step Curation: 000977
- to stream Pmc, to step Checkpoint: 000169
Links to Exploration step
PMC:5225433Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Neutrophil elastase cleavage of the gC1q domain impairs the EMILIN1-α4β1 integrin interaction, cell adhesion and anti-proliferative activity</title><author><name sortKey="Maiorani, Orlando" sort="Maiorani, Orlando" uniqKey="Maiorani O" first="Orlando" last="Maiorani">Orlando Maiorani</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Pivetta, Eliana" sort="Pivetta, Eliana" uniqKey="Pivetta E" first="Eliana" last="Pivetta">Eliana Pivetta</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Capuano, Alessandra" sort="Capuano, Alessandra" uniqKey="Capuano A" first="Alessandra" last="Capuano">Alessandra Capuano</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Modica, Teresa Maria Elisa" sort="Modica, Teresa Maria Elisa" uniqKey="Modica T" first="Teresa Maria Elisa" last="Modica">Teresa Maria Elisa Modica</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Wassermann, Bruna" sort="Wassermann, Bruna" uniqKey="Wassermann B" first="Bruna" last="Wassermann">Bruna Wassermann</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Bucciotti, Francesco" sort="Bucciotti, Francesco" uniqKey="Bucciotti F" first="Francesco" last="Bucciotti">Francesco Bucciotti</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Colombatti, Alfonso" sort="Colombatti, Alfonso" uniqKey="Colombatti A" first="Alfonso" last="Colombatti">Alfonso Colombatti</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Doliana, Roberto" sort="Doliana, Roberto" uniqKey="Doliana R" first="Roberto" last="Doliana">Roberto Doliana</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Spessotto, Paola" sort="Spessotto, Paola" uniqKey="Spessotto P" first="Paola" last="Spessotto">Paola Spessotto</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">28074935</idno><idno type="pmc">5225433</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225433</idno><idno type="RBID">PMC:5225433</idno><idno type="doi">10.1038/srep39974</idno><date when="2017">2017</date><idno type="wicri:Area/Pmc/Corpus">000977</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000977</idno><idno type="wicri:Area/Pmc/Curation">000977</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000977</idno><idno type="wicri:Area/Pmc/Checkpoint">000169</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">000169</idno><idno type="wicri:Area/Ncbi/Merge">008B98</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Neutrophil elastase cleavage of the gC1q domain impairs the EMILIN1-α4β1 integrin interaction, cell adhesion and anti-proliferative activity</title><author><name sortKey="Maiorani, Orlando" sort="Maiorani, Orlando" uniqKey="Maiorani O" first="Orlando" last="Maiorani">Orlando Maiorani</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Pivetta, Eliana" sort="Pivetta, Eliana" uniqKey="Pivetta E" first="Eliana" last="Pivetta">Eliana Pivetta</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Capuano, Alessandra" sort="Capuano, Alessandra" uniqKey="Capuano A" first="Alessandra" last="Capuano">Alessandra Capuano</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Modica, Teresa Maria Elisa" sort="Modica, Teresa Maria Elisa" uniqKey="Modica T" first="Teresa Maria Elisa" last="Modica">Teresa Maria Elisa Modica</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Wassermann, Bruna" sort="Wassermann, Bruna" uniqKey="Wassermann B" first="Bruna" last="Wassermann">Bruna Wassermann</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Bucciotti, Francesco" sort="Bucciotti, Francesco" uniqKey="Bucciotti F" first="Francesco" last="Bucciotti">Francesco Bucciotti</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Colombatti, Alfonso" sort="Colombatti, Alfonso" uniqKey="Colombatti A" first="Alfonso" last="Colombatti">Alfonso Colombatti</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Doliana, Roberto" sort="Doliana, Roberto" uniqKey="Doliana R" first="Roberto" last="Doliana">Roberto Doliana</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Spessotto, Paola" sort="Spessotto, Paola" uniqKey="Spessotto P" first="Paola" last="Spessotto">Paola Spessotto</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Scientific Reports</title><idno type="eISSN">2045-2322</idno><imprint><date when="2017">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The extracellular matrix glycoprotein EMILIN1 exerts a wide range of functions mainly associated with its gC1q domain. Besides providing functional significance for adhesion and migration, the direct interaction between α4β1 integrin and EMILIN1-gC1q regulates cell proliferation, transducing net anti-proliferative effects. We have previously demonstrated that EMILIN1 degradation by neutrophil elastase (NE) is a specific mechanism leading to the loss of functions disabling its regulatory properties. In this study we further analysed the proteolytic activity of NE, MMP-3, MMP-9, and MT1-MMP on EMILIN1 and found that MMP-3 and MT1-MMP partially cleaved EMILIN1 but without affecting the functional properties associated with the gC1q domain, whereas NE was able to fully impair the interaction of gC1q with the α4β1 integrin by cleaving this domain outside of the E933 integrin binding site. By a site direct mutagenesis approach we mapped the bond between S913 and R914 residues and selected the NE-resistant R914W mutant still able to interact with the α4β1 integrin after NE treatment. Functional studies showed that NE impaired the EMILIN1-α4β1 integrin interaction by cleaving the gC1q domain in a region crucial for its proper structural conformation, paving the way to better understand NE effects on EMILIN1-cell interaction in pathological context.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Hynes, R O" uniqKey="Hynes R">R. O. Hynes</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Watt, F M" uniqKey="Watt F">F. M. Watt</name></author><author><name sortKey="Fujiwara, H" uniqKey="Fujiwara H">H. Fujiwara</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Streuli, C H" uniqKey="Streuli C">C. H. Streuli</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Moreno Layseca, P" uniqKey="Moreno Layseca P">P. Moreno-Layseca</name></author><author><name sortKey="Streuli, C H" uniqKey="Streuli C">C. H. Streuli</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Doliana, R" uniqKey="Doliana R">R. Doliana</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Colombatti, A" uniqKey="Colombatti A">A. Colombatti</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Colombatti, A" uniqKey="Colombatti A">A. Colombatti</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ghai, R" uniqKey="Ghai R">R. Ghai</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kishore, U" uniqKey="Kishore U">U. Kishore</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Colombatti, A" uniqKey="Colombatti A">A. Colombatti</name></author><author><name sortKey="Bressan, G M" uniqKey="Bressan G">G. M. Bressan</name></author><author><name sortKey="Castellani, I" uniqKey="Castellani I">I. Castellani</name></author><author><name sortKey="Volpin, D" uniqKey="Volpin D">D. Volpin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Colombatti, A" uniqKey="Colombatti A">A. Colombatti</name></author><author><name sortKey="Poletti, A" uniqKey="Poletti A">A. Poletti</name></author><author><name sortKey="Bressan, G M" uniqKey="Bressan G">G. M. Bressan</name></author><author><name sortKey="Carbone, A" uniqKey="Carbone A">A. Carbone</name></author><author><name sortKey="Volpin, D" uniqKey="Volpin D">D. Volpin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Danussi, C" uniqKey="Danussi C">C. Danussi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Danussi, C" uniqKey="Danussi C">C. Danussi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zacchigna, L" uniqKey="Zacchigna L">L. Zacchigna</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Spessotto, P" uniqKey="Spessotto P">P. Spessotto</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Spessotto, P" uniqKey="Spessotto P">P. Spessotto</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Danussi, C" uniqKey="Danussi C">C. Danussi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Verdone, G" uniqKey="Verdone G">G. Verdone</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rose, D M" uniqKey="Rose D">D. M. Rose</name></author><author><name sortKey="Alon, R" uniqKey="Alon R">R. Alon</name></author><author><name sortKey="Ginsberg, M H" uniqKey="Ginsberg M">M. H. Ginsberg</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hauzenberger, D" uniqKey="Hauzenberger D">D. Hauzenberger</name></author><author><name sortKey="Klominek, J" uniqKey="Klominek J">J. Klominek</name></author><author><name sortKey="Bergstrom, S E" uniqKey="Bergstrom S">S. E. Bergstrom</name></author><author><name sortKey="Sundqvist, K G" uniqKey="Sundqvist K">K. G. Sundqvist</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Humphries, J D" uniqKey="Humphries J">J. D. Humphries</name></author><author><name sortKey="Byron, A" uniqKey="Byron A">A. Byron</name></author><author><name sortKey="Humphries, M J" uniqKey="Humphries M">M. J. Humphries</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Humphries, M J" uniqKey="Humphries M">M. J. Humphries</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Danussi, C" uniqKey="Danussi C">C. Danussi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kamarajan, P" uniqKey="Kamarajan P">P. Kamarajan</name></author><author><name sortKey="Garcia Pardo, A" uniqKey="Garcia Pardo A">A. Garcia-Pardo</name></author><author><name sortKey="D Ilva, N J" uniqKey="D Ilva N">N. J. D’Silva</name></author><author><name sortKey="Kapila, Y L" uniqKey="Kapila Y">Y. L. Kapila</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zucchetto, A" uniqKey="Zucchetto A">A. Zucchetto</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lund, S A" uniqKey="Lund S">S. A. Lund</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pivetta, E" uniqKey="Pivetta E">E. Pivetta</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pivetta, E" uniqKey="Pivetta E">E. Pivetta</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Stegemann, C" uniqKey="Stegemann C">C. Stegemann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mongiat, M" uniqKey="Mongiat M">M. Mongiat</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Schilling, O" uniqKey="Schilling O">O. Schilling</name></author><author><name sortKey="Overall, C M" uniqKey="Overall C">C. M. Overall</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hedstrom, L" uniqKey="Hedstrom L">L. Hedstrom</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bank, U" uniqKey="Bank U">U. Bank</name></author><author><name sortKey="Ansorge, S" uniqKey="Ansorge S">S. Ansorge</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pham, C T" uniqKey="Pham C">C. T. Pham</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mittendorf, E A" uniqKey="Mittendorf E">E. A. Mittendorf</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Houghton, A M" uniqKey="Houghton A">A. M. Houghton</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gregory, A D" uniqKey="Gregory A">A. D. Gregory</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gregory, A D" uniqKey="Gregory A">A. D. Gregory</name></author><author><name sortKey="Hale, P" uniqKey="Hale P">P. Hale</name></author><author><name sortKey="Perlmutter, D H" uniqKey="Perlmutter D">D. H. Perlmutter</name></author><author><name sortKey="Houghton, A M" uniqKey="Houghton A">A. M. Houghton</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wei, Z" uniqKey="Wei Z">Z. Wei</name></author><author><name sortKey="Lei, X" uniqKey="Lei X">X. Lei</name></author><author><name sortKey="Seldin, M M" uniqKey="Seldin M">M. M. Seldin</name></author><author><name sortKey="Wong, G W" uniqKey="Wong G">G. W. Wong</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Waki, H" uniqKey="Waki H">H. Waki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yuan, Y" uniqKey="Yuan Y">Y. Yuan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ruiz, S" uniqKey="Ruiz S">S. Ruiz</name></author><author><name sortKey="Henschen Edman, A H" uniqKey="Henschen Edman A">A. H. Henschen-Edman</name></author><author><name sortKey="Nagase, H" uniqKey="Nagase H">H. Nagase</name></author><author><name sortKey="Tenner, A J" uniqKey="Tenner A">A. J. Tenner</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tissot, B" uniqKey="Tissot B">B. Tissot</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Casasnovas, J M" uniqKey="Casasnovas J">J. M. Casasnovas</name></author><author><name sortKey="Pieroni, C" uniqKey="Pieroni C">C. Pieroni</name></author><author><name sortKey="Springer, T A" uniqKey="Springer T">T. A. Springer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cannizzaro, R" uniqKey="Cannizzaro R">R. Cannizzaro</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Spessotto, P" uniqKey="Spessotto P">P. Spessotto</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Xing, J Z" uniqKey="Xing J">J. Z. Xing</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Sci Rep</journal-id><journal-id journal-id-type="iso-abbrev">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type="epub">2045-2322</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">28074935</article-id><article-id pub-id-type="pmc">5225433</article-id><article-id pub-id-type="pii">srep39974</article-id><article-id pub-id-type="doi">10.1038/srep39974</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Neutrophil elastase cleavage of the gC1q domain impairs the EMILIN1-α4β1 integrin interaction, cell adhesion and anti-proliferative activity</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Maiorani</surname><given-names>Orlando</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Pivetta</surname><given-names>Eliana</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Capuano</surname><given-names>Alessandra</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Modica</surname><given-names>Teresa Maria Elisa</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Wassermann</surname><given-names>Bruna</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Bucciotti</surname><given-names>Francesco</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Colombatti</surname><given-names>Alfonso</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Doliana</surname><given-names>Roberto</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Spessotto</surname><given-names>Paola</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><aff id="a1"><label>1</label><institution>Experimental Oncology 2, Department of Translational Research, CRO-IRCCS, National Cancer Institute</institution>, Aviano 33081,<country>Italy</country></aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>pspessotto@cro.it</email></corresp><fn id="n1"><label>*</label><p>These authors jointly supervised this work.</p></fn></author-notes><pub-date pub-type="epub"><day>11</day><month>01</month><year>2017</year></pub-date><pub-date pub-type="collection"><year>2017</year></pub-date><volume>7</volume><elocation-id>39974</elocation-id><history><date date-type="received"><day>11</day><month>08</month><year>2016</year></date><date date-type="accepted"><day>29</day><month>11</month><year>2016</year></date></history><permissions><copyright-statement>Copyright © 2017, The Author(s)</copyright-statement><copyright-year>2017</copyright-year><copyright-holder>The Author(s)</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link></license-p></license></permissions><abstract><p>The extracellular matrix glycoprotein EMILIN1 exerts a wide range of functions mainly associated with its gC1q domain. Besides providing functional significance for adhesion and migration, the direct interaction between α4β1 integrin and EMILIN1-gC1q regulates cell proliferation, transducing net anti-proliferative effects. We have previously demonstrated that EMILIN1 degradation by neutrophil elastase (NE) is a specific mechanism leading to the loss of functions disabling its regulatory properties. In this study we further analysed the proteolytic activity of NE, MMP-3, MMP-9, and MT1-MMP on EMILIN1 and found that MMP-3 and MT1-MMP partially cleaved EMILIN1 but without affecting the functional properties associated with the gC1q domain, whereas NE was able to fully impair the interaction of gC1q with the α4β1 integrin by cleaving this domain outside of the E933 integrin binding site. By a site direct mutagenesis approach we mapped the bond between S913 and R914 residues and selected the NE-resistant R914W mutant still able to interact with the α4β1 integrin after NE treatment. Functional studies showed that NE impaired the EMILIN1-α4β1 integrin interaction by cleaving the gC1q domain in a region crucial for its proper structural conformation, paving the way to better understand NE effects on EMILIN1-cell interaction in pathological context.</p></abstract></article-meta></front><floats-group><fig id="f1"><label>Figure 1</label><caption><title>Enzymatic action of MMPs and NE on EMILIN1.</title><p>(<bold>a</bold>) EMILIN1 was incubated with the recombinant catalytic domain of MMP-3, MMP-9 or MT1-MMP (MT1) at a 1:4 (E:S) molar ratio and with the full length (FL) form of the recombinant MMP-9 at a 1:20 molar ratio at 37 °C for 30 min, 2 h or 24 h. The cleavage products were detected by Coomassie staining. FN was used as a positive control of cleavage. (<bold>b</bold>) Coomassie staining of EMILIN1 samples incubated with NE (1:50, 1 h) or MT1-MMP (1:4, 24 h). Black arrows and smaller black arrows indicate the intact band and fragmented band of EMILIN1, respectively. (<bold>c</bold>) Western blotting analysis of EMILIN1 samples incubated with MMP-3 and MT1-MMP (MT1) or NE at times and doses as indicated. Membranes were probed with polyclonal antibodies against EMILIN1 (As556). (<bold>d</bold>) Western blotting analysis of dose and time-dependent EMILIN1 fragmentation by NE. Reactivity for As556 (left) and AP As556 (right) is displayed.</p></caption><graphic xlink:href="srep39974-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>MT1-MMP treatment does not impair EMILIN1 functional properties.</title><p>(<bold>a</bold>) Dynamic monitoring of SKLMS1 cell attachment in response to the effect of MT1-MMP on EMILIN1 (E1) cleavage measured with the XCELLigence instrument and expressed as the cell index. A 1:25 (E:S) molar ratio was used for a 24 h incubation. The wells were finally coated with 10 μg/ml of EMILIN1 (incubated or not with MT1-MMP). The data shown is the mean ± SD from n = 3 experiments with n = 6 replicates. (<bold>b</bold>) SKLMS1 cell proliferation 48 h after plating. The cell index of dynamic monitoring calculated as the mean ± SD from n = 3 experiments with n = 6 replicates is reported. The wells were coated with EMILIN1 (10 μg/ml) treated with MMP-3, MT1-MMP (1:25–E:S) or NE (1:200–E:S) or incubated in the corresponding (M3, MT1, NE) buffer for 24 h. *<italic>P</italic> < 0.05.</p></caption><graphic xlink:href="srep39974-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>NE cleaves EMILIN1 functional domain.</title><p>Coomassie staining of gC1q digestion curves by MMPs, collagenase, PR3, and NE. The resulting mixtures were loaded on SDS PAGE gels without boiling to prevent the dissociation of the trimeric gC1q into monomers. Representative full length gels (<bold>a</bold> and <bold>b</bold>) or the lower part (<bold>c</bold>) are shown. (<bold>a</bold>) gC1q is not cleaved by MMPs even at high enzyme concentration. Incubations at different times (2, 4 and 24 h) were performed with a molar ratio of 1:37 (E:S). FN was used as positive control of cleavage. (<bold>b</bold>) Collagenase and PR3 were added to gC1q for 1 h at different doses as indicated. Collagen type I (coll) and FN were used as positive control of cleavage. No digestion fragments are detected. Vertical lines indicated cropped gels derived from independent runs. (<bold>c</bold>) Dose- and time-dependent digestion curves of gC1q by NE show that increasing concentration of NE and prolonged time of incubation produce the appearance of cleaved fragments and the formation of a slower migrating form of gC1q trimer, as indicated by arrow heads. Note that above all at very low doses of the enzyme the stability of the gC1q homotrimer is highly compromised.</p></caption><graphic xlink:href="srep39974-f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><title>Identification of the possible NE cleavage site on gC1q.</title><p>(<bold>a</bold>) Schematic representation of gC1q primary structure with the two possible cleavage sites emerged from MALDI TOF analysis, one placed between S913 and R914 (8651calculated MW, blue arrow) and a second between L912 and S913 (8647 calculated MW, red arrow). (<bold>b</bold>) NMR model of the gC1q domain where the cleavage site is highlighted in yellow to indicate its good exposure to the solvent to favor the interaction with NE. (<bold>c</bold>) gC1q mutants produced with a site-directed mutagenesis approach. Soluble mutants are indicated in green whereas the insoluble in red. (<bold>d</bold>) Coomassie staining of the melting transition profile of the gC1q domain. Ten μg of WT and mutants of gC1q domain were incubated in sample buffer for 1 h at the indicated temperature and then separated in a 4–20% gradient SDS-PAGE. Mutants show a melting pattern comparable to that of the WT domain.</p></caption><graphic xlink:href="srep39974-f4"></graphic></fig><fig id="f5"><label>Figure 5</label><caption><title>R914W gC1q mutant is resistant to NE enzymatic degradation.</title><p>Degradation pattern (Coomassie staining of representative gels, lower part) of gC1q mutants (<bold>a)</bold> R914W and S913T; (<bold>b</bold>) R914K, R914H and R914V; (<bold>c</bold>) E976A and E933A) after incubation with NE at 1:200 (E:S) molar ratios at different times as indicated. Note that all the mutants except R914W are well fragmented as well as WT gC1q after very short times of incubation.</p></caption><graphic xlink:href="srep39974-f5"></graphic></fig><fig id="f6"><label>Figure 6</label><caption><title>R914W gC1q mutant retains functional properties after NE treatment.</title><p>(<bold>a</bold>) Adhesion (CAFCA assay) of Jurkat cells on WT or R914W gC1q preincubated (with NE) or not (w/o NE) with NE. The E:S molar ratios and incubation times are “boxed” in the corresponding gel at the bottom of the graphic indicating the status (fragmented or not) of the resulting gC1q used to coat the wells (10 μg/ml). (<bold>b</bold>) Differential centrifugal forces applied in the reverse direction during the second centrifugal step of CAFCA assay (see Materials and Methods) to allow the removal of the unbound/weakly bound cells under controlled conditions. The relative Jurkat cell adhesion strengths to WT and R914W gC1q preincubated or not with NE at the same conditions applied in (<bold>a</bold>) are reported. R914W gC1q incubated with NE allowed Jurkat cell adhesion at each reverse centrifugal force applied. (<bold>c</bold>) Dynamic monitoring of SKLMS1 cell attachment in response to the effect of NE on WT and R914W gC1q cleavage measured with the XCELLigence instrument and expressed as cell index. Dose and incubation time are the same as used in (<bold>a</bold>). The wells were coated with 10 μg/ml of gC1q (incubated or not with NE). (<bold>d</bold>) Morphological appearance of SKLMS1 adhering (30 min) on WT or gC1q mutants treated or not with NE (1:25–E:S, 1 h). BSA was used as control. (<bold>e</bold>) Proliferation of SKLMS1 cells plated on gC1q (WT or R914W mutant) (10 μg/ml) treated with NE (1:25–E:S, 1 h). The cell index after 48 h of dynamic monitoring calculated as the mean ± SD from n = 3 experiments with n = 6 replicates is reported. *<italic>P</italic> < 0.05. (<bold>f</bold>) Adhesion of Jurkat cells on WT gC1q preincubated or not with NE. The E:S molar ratio and incubation time are “boxed” in the corresponding gel next to the graphic. Data are expressed as the means ± SD of n = 3 independent experiments with n = 6 replicates. **<italic>P</italic> < 0.001.</p></caption><graphic xlink:href="srep39974-f6"></graphic></fig><fig id="f7"><label>Figure 7</label><caption><title>R914W gC1q mutant is able to fully compete in cell adhesion and proliferation assays when used as agonist.</title><p>In adhesion (Jurkat cells, CAFCA assay, (<bold>a</bold>) and proliferation (SKLMS1 cells, dynamic monitoring by XCelligence instrument, (<bold>b</bold>) assays R914W gC1q mutant was present as an agonist at different doses as indicated. The mixtures (R914W gC1q mutant at different doses and EMILIN1, E1 at 5 μg/ml) were preincubated for 1 h with NE or not (w/o NE) and the resulting products used to coat the wells. Data are expressed as the means ± SD of n = 3 independent experiments with n = 6 replicates. **<italic>P</italic> < 0.001; NS, not significant.</p></caption><graphic xlink:href="srep39974-f7"></graphic></fig></floats-group></pmc><affiliations><list><country><li>Italie</li></country></list><tree><country name="Italie"><noRegion><name sortKey="Maiorani, Orlando" sort="Maiorani, Orlando" uniqKey="Maiorani O" first="Orlando" last="Maiorani">Orlando Maiorani</name></noRegion><name sortKey="Bucciotti, Francesco" sort="Bucciotti, Francesco" uniqKey="Bucciotti F" first="Francesco" last="Bucciotti">Francesco Bucciotti</name><name sortKey="Capuano, Alessandra" sort="Capuano, Alessandra" uniqKey="Capuano A" first="Alessandra" last="Capuano">Alessandra Capuano</name><name sortKey="Colombatti, Alfonso" sort="Colombatti, Alfonso" uniqKey="Colombatti A" first="Alfonso" last="Colombatti">Alfonso Colombatti</name><name sortKey="Doliana, Roberto" sort="Doliana, Roberto" uniqKey="Doliana R" first="Roberto" last="Doliana">Roberto Doliana</name><name sortKey="Modica, Teresa Maria Elisa" sort="Modica, Teresa Maria Elisa" uniqKey="Modica T" first="Teresa Maria Elisa" last="Modica">Teresa Maria Elisa Modica</name><name sortKey="Pivetta, Eliana" sort="Pivetta, Eliana" uniqKey="Pivetta E" first="Eliana" last="Pivetta">Eliana Pivetta</name><name sortKey="Spessotto, Paola" sort="Spessotto, Paola" uniqKey="Spessotto P" first="Paola" last="Spessotto">Paola Spessotto</name><name sortKey="Wassermann, Bruna" sort="Wassermann, Bruna" uniqKey="Wassermann B" first="Bruna" last="Wassermann">Bruna Wassermann</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Sante/explor/LymphedemaV1/Data/Ncbi/Merge
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 008B98 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Ncbi/Merge/biblio.hfd -nk 008B98 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Sante
|area= LymphedemaV1
|flux= Ncbi
|étape= Merge
|type= RBID
|clé= PMC:5225433
|texte= Neutrophil elastase cleavage of the gC1q domain impairs the EMILIN1-α4β1 integrin interaction, cell adhesion and anti-proliferative activity
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Ncbi/Merge/RBID.i -Sk "pubmed:28074935" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Ncbi/Merge/biblio.hfd \
| NlmPubMed2Wicri -a LymphedemaV1
| This area was generated with Dilib version V0.6.31. |  |