Purification and Functional Characterization of Recombinant Human ADAM8 protease
From recent studies in different tumor entities it became apparent that the membrane-anchored metalloprotease-disintegrin ADAM8 plays an active role in tumor progression and consequently in patient prognosis. It is therefore desirable to understand the functional role of ADAM8 in vitro by characteri...
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| Format: | Dissertation |
| Sprache: | Englisch |
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Philipps-Universität Marburg
2015
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| Zusammenfassung: | From recent studies in different tumor entities it became apparent that the membrane-anchored metalloprotease-disintegrin ADAM8 plays an active role in tumor progression and consequently in patient prognosis. It is therefore desirable to understand the functional role of ADAM8 in vitro by characterizing the soluble extracellular part of ADAM8, which is called the ADAM8 ectodomain. The human ectodomain ADAM8 (hEctoA8) protein consists of the prodomain, metalloprotease domain (MP), a disintegrin domain (DIS), a cysteine-rich domain (Cys) and a EGF-like domain (EGF), whilst removal of the prodomain by autocatalysis leads to the active form of hEctoA8. Like the full-length ADAM8, hEctoA8 contains the catalytic consensus sequence HEXXHXXGXXHD in the metalloprotease domain and is therefore predicted to be proteolytically active. Up to now, functional studies on ADAM8 in vitro were hampered by the lack of sufficient quantities of folded, biologically active, and purified recombinant forms of hEctoA8. In our study, we successfully purified the recombinant hEctoA8 protein from supernatants of transfected HEK cells. Purified recombinant hEctoA8 containing the catalytic function of ADAM8 can cleave CD23 in cells in trans and FN in vitro; CD23 cleavage resulted in fragments of 37, 33, 25 and 16 kDa and FN cleavage by recombinant hEctoA8 resulted in 9 fragments, one fragment of 38 kDa contains a RGD motif essential for cell adhesion. Functionally, ADAM8-dependent cell adhesion of pancreatic tumor cells Panc1 was affected by FN cleavage and resulted in reduced cell adhesion. Concomitantly, a decreased expression of integrin α5; was found, whereas cleavage of FN by recombinant hEctoA8 had no effect on the expression levels of integrin β1, and on the activation of p-ERK1/2 and p-Akt. |
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| DOI: | 10.17192/z2015.0272 |