Purification and Functional Characterization of Recombinant Human ADAM8 protease
Xiangdi Yu
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.
Philipps-Universität Marburg
Medical sciences Medicine
urn:nbn:de:hebis:04-z2015-02722
opus:6103
https://doi.org/10.17192/z2015.0272
urn:nbn:de:hebis:04-z2015-02722
Publikationsserver der Universitätsbibliothek Marburg
Universitätsbibliothek Marburg
Medizin
BB-94
Xiangdi Yu
Philipps-Universität Marburg
opus:6103
ADAM8
Medical sciences Medicine
Medizin
ppn:359528848
Purification and Functional Characterization of Recombinant Human ADAM8 protease
English
In einer Reihe von klinischen Studien wurde nachgewiesen, dass die membranständige Metalloprotease ADAM8 eine Rolle bei der Tumorprogression spielt und eine erhöhte ADAM8-Expression mit einer verschlechterten Patientenprognose einhergeht. Es ist daher notwendig, die Rolle der ADAM8-Aktivität in vitro zu verstehen. In dieser Arbeit sollte daher die lösliche Variante von ADAM8, die Ektodomäne (hEctoA8), aufgereinigt und funktionell charakterisiert warden. Diese besteht aus der Prodomäne, der Metalloprotease-Domäne (MP), der Disintegrin-Domäne (DIS), der Cystein-reichen Domäne (Cys) and der EGF-artigen Domäne (EGF). Die autokatalytische Entfernung der Prodomäne führt zur aktiven Protease. Wie das gesamte ADAM8-Molekül auch, enthält das rekombinanten Protein hEctoA8 die für Metzinkine charakteristische Konsensus-Sequenz “HEXXHXXGXXHD” in der MP-Domäne und sollte daher proteolytische Aktivität besitzen. Bis heute war es nur schwer möglich, funktionelle Studien mit ADAM8 in vitro durchzuführen, da nicht genügend rekombinantes Protein mit biologischer Aktivität zur Verfügung stand. In dieser Arbeit wurde das rekombinante humane ADAM8 als hEctoA8 erfolgreich aus Überständen von stabil transfizierten und selektionierten HEK293 Zellen aufgereinigt. Das so gewonnene Protein hEctoA8 besitzt eine hohe katalytische Aktivität und kann in einem FRET-basierten Assay ein Fluoreszenz-gequenchtes Peptid spalten, das der ADAM8-Spaltstelle im niedrig-affinen IgE Rezeptor CD23 nachempfunden ist. Interessanterweise wird auch intaktes membranständiges CD23 von der Oberfläche stabiler CD23-exprimierender Zellen in Fragmente von 37, 33, 25 and 16 kDa abgespalten, womit erstmalig eine Susbtratspaltung in trans nachgewiesen wurde. Weiterhin wurden die ADAM8-Spaltstellen für das extrazelluläre Matrixprotein Fibronectin (FN) charakterisiert. Es entstanden dabei 9 FN-Fragmente von unterschiedlichem Molekulargewicht. Ein FN-Fragment mit einem Molekulargewicht von 38 kDa war dabei besonders interessant, da es so genannte Zellbindungsdomänen sowie ein RGD-Motiv enthält. Durch die ADAM8-abhängige Spaltung dieses Fragments kann man davon ausgehen, dass eine durch FN vermittelte Zelladhäsion verloren gehen sollte. Diese Hypothese wurde durch Pankreas-Tumorzellen Panc1 überprüft, bei denen eine ADAM8-abhängige Zelladhäsion auf FN mit und ohne vorherigen Verdau gemessen wurde. Es konnte gezeigt werden, dass der Verdau von Fibronektin durch ADAM8 die Zelladhäsion signifikant reduzierte. Die Expression des daran beteiligten Fibronektin-Rezeptors 51 wurde auf Proteinebene untersucht und dabei gezeigt, dass spezifisch die Integrin 5-Untereinheit vermindert ist, während die Integrin 1-Untereinheit nicht verändert ist. Auch die vom Fibronektin-Rezeptor abhängige Signalkaskade, an der unter anderem die Kinasen p-ERK1/2 und p-Akt beteiligt sind, zeigt keine Änderungen im Aktivierungsstatus, sodass man daraus schliessen kann, dass die von ADAM8 vermittelte Degradation von FN direkt über die Regulation der Integrin 5-Untereinheit zu einer verminderten Zelladhäsion führt.
CT1746
CD23
https://archiv.ub.uni-marburg.de/diss/z2015/0272/cover.png
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.
BB-94
CD23
ths
Prof. Dr.
Bartsch
Jörg W.
Bartsch, Jörg W. (Prof. Dr.)
doctoralThesis
application/pdf
Anderson IC, Shipp M, Docherty AJP, Teicher BA (1996) Combination therapy including a gelatinase inhibitor and cytotoxic agent reduces local invasion and metastasis of murine Lewis lung carcinoma. Cancer Res 56: 715-718.
Duhamel S, Hebert J, Gaboury L, Bouchard A, Simon R, Sauter G (2012) Sef downregulation by Ras causes MEK1/2 to become aberrantly nuclear localized leading to polyploidy and neoplastic transformation. Cancer Res 72: 626-35.
Brakebusch C, Fassler R (2005) Beta1 integrin function in vivo: adhesion, migration and more. Cancer Metastasis Rev 24: 403-411.
Zhang R, Yuan Y, Zuo J, Liu W (2012) Prognostic and clinical implication of a disintegrin and metalloprotease8 expression in pediatric medulloblastoma. J Neurol Sci 323: 46-51.
Henningsson F, Ding Z, Dahlin JS, Linkevicius M, Carlsson F, Grönvik KO, Hallgren J, Heyman B (2011) IgE-mediated enhancement of CD4+ T cell responses in mice requires antigen presentation by CD11c+ cells and not by B cells. PLoS ONE 6: e2176.
Ruoslahti E (1995) Fibronectin and its alpha5 beta1 integrin receptor in malignancy. Invasion Metastasis 14: 87-97.
Gomez-Gaviro M, Dominguez-Luis M, Canchado J, Calafat J, Janssen H, Lara-pezzi E, Fourie A, Tugores A, Valenzuela-Fernandez A, Mollinedo F, Sanchez-Madrid F, Diaz-Gonzalez F (2007) Expression and regulation of the metalloproteinase ADAM-8 during human neutrophil pathophysiological activation and its catalytic activity on L-selectin shedding. J. Immunol. 178: References 48 8053-8063.
Leiss M, Beckmann K, Girós A, Costell M, Fä ssler R (2008) The role of integrin binding sites in fibronectin matrix assembly in vivo. Curr Opin Cell Biol. 20: 502-7.
Pankov R, Yamada KM (2002) Fibronectin at a glance. Journal of Cell Science 115: 3861-3. References 49
Anderson DG, Li X, Balian G (2005) A fibronectin fragment alters the metabolism by rabbit intervertevral disc cells in vitro. Spine 30: 1242-6.
Kijimoto-Ochiai S (2002) CD23 (the low-affinity IgE receptor) as a C-type lectin: a multidomain and multifunctional molecule. Cellular and Molecular Life Sciences 59: 648-664.
Ghayouri M, Boulware D, Nasir A, Strosberg J, Kvols L, Coppola D (2010) Activation of the serine/theronine protein kinase Akt in enteropancreatic neuroendocrine tumors. Anticancer Res 30: 5063-5067.
Fourie AM, Coles F, Moreno V, Karlsson L (2003) Catalytic activity of ADAM8, ADAM15, and MDC-L (ADAM28) on synthetic peptide substrates and in ectodomain cleavage of CD23. J Biol Chem 278: 30469-30477.
Lichtman AH, Abbas AK (2003) Cellular and molecular immunology. Philadelphia: Saunders. pp: 324-325.
Naus S, Reipschlä ger S, Wildeboer D, Lichtenthaler SF, Mitterreiter S, Guan Z, Moss ML, Bartsch JW (2006) Identification of candidate substrate for ectodomain shedding by the metalloprotease-disintegrin ADAM8.J Biol Chem 387: 337-346.
Stöcker W, Bode W (1995) Structural features of a superfamily of zinc-endopeptidases: the metzincins. Curr Opin Struct Biol 5: 383-90.
Inoki K, Li Y, Zhu T, Wu J, Guan KL (2002) TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signaling. Nat. Cell Biol 4: 648-657.
T, Isshiki A, Erickson HP, Fassler R (2001) Plasma fibronectin supports neuronal survival and reduces brain injury following transient focal cerebral ischemia but is not essential for skin-wound healing and hemostasis. Nat. Med 7: 324-330.
Zack MD, Melton MA, Stock JL, Storer CE, Barve RA, Minnerly JC,Weiss DJ, Stejskal JA, Tortorella MD, Turk JR, Shevlin KM, Malfait AM (2009) Reduced incidence and severity of experimental autoimmune arthritis in mice expressing catalytically inactive A distingrin and metalloprotease 8 (ADAM8).
Delespesse G, Sarfati M, Wu CY, Fournier S, Letellier M (1992)The low-affinity receptor for IgE. Immunol 125: 77-97.
Amour A, Knight CG, English WR, Webster A, Slocombe PM, Knä uper V, Docherty AJ, Becherer JD, Blobel CP, Murphy G (2002) The enzymatic activity of ADAM8 and ADAM9 is not regulated by TIMPs. FEBS Lett. 524: 154-158.
Hoekstra R, Eskens FA, Verweij J (2001) Matrix metalloproteinase inhibitors: current developments and future perspectives. Oncologist 6: 415-27.
Thurnher D (2012) ADAM8 in squamous cell carcinoma of the head and neck: a retrospective study. BMC Cancer12: 76.
Corbett SA, Lee L, Wilson CL, Schwarzbauer JE (1997) Covalent cross-linking of fibronectin to fibrin is required for maximal cell adhesion to a fibronectin-fibrin matrix. J Biol Chem 272: 2499.
Buzza MS, Zamurs L, Sun J, Bird CH, Smith AI, Trapani JA, Froelich CJ, Nice EC, Bird PI (2005) Extracellular matrix remodeling by human granzyme B via cleavage of vitronectin, fibronectin, and lamin. J.Biol.Chem 280: 23549-23558.
Bartsch JW, Wildeboer D, Koller G, Naus S, Rittger A, Moss ML, Minai Y, Jockusch H (2010) Tumor Necrosis Factor-α (TNF-α) regulates shedding of TNF-a receptor 1 by the Metallopprotease-Disintegrin ADAM8: Evidence for a proteaseregulated feedback loop in neuroprotection. J. Neurosci. 30:12210-12218.
Wheeler DJ, Parveen S, Pollock K, Williams RJ (1998) Inhibition of sCD23 and immunoglobulin E release from human B cells by a metalloproteinase inhibitor, GI 129471.Immunology 95: 105-110.
Lloyd AC (2006) Distinct functions for ERKs? Journal of Biology 5:13.
Schwinn MK, Peters DM (2009) Functional properties of fibronectin in the trabecular meshwork. Exp Eye Res 88: 689-93.
Fibronectin expression modulates mammary epithelial cell proliferation during acinar differentiation. Cancer Research 68: 3185-92.
Ruel N, Markova DZ, Adams SL, Scanzello C, Cs-Szabo G, Gerard D, Shi P, Anderson G, Zack M, An HS, Chen D, Zhang YJ (2014) Fibronectin fragments and the cleaving enzyme ADAM-8 in the degenerative human intervertebral disc. Spine 39: 1274-9.
Bonnefoy JY, Plater-Zyberk C, Lecoanet-Henchoz S, Gauchat JF, Aubry JP, Graber P (1996) A new role for CD23 in inflammation. Immunol.17: 418-420.
Moss ML, Rasmussen FH (2007) Fluorescent substrates for the proteinases ADAM17, ADAM10, ADAM8, and ADAM12 useful for high-throughput inhibitor screening. Anal Biochem 366: 144-8.
Henry C, Hsia, Mohan R, Siobhan A, Corbett (2014) The fate of internalized α5 integrin is regulated by matrix-capable fibronectin. Journal of surgical research 191: 268-79.
Nishimura D, Sakai H, Sato F, Nishimura S, Toyama-Sorimachi N, Bartsch JW (2015) Sehara-Fujisawa A. Roles of ADAM8 in elimination of injured muscle fibers prior to skeletal muscle regeneration. Mech Dev. 135: 58-67.
Weskamp G, Ford JW, Sturgill J, Martin S, Docherty AJ, Blobel CP (2006) ADAM10 is a principal 'sheddase' of the low-affinity immunoglobulin E receptor Nat. Immunol. 7: 1293-1298.
monograph
Reinigung und funktionelle Charakterisierung von rekombinanter humaner Protease ADAM8
2015-05-28
2015
ADAM8
Medizin
https://doi.org/10.17192/z2015.0272
2015-05-12
Fibronectin
Fibronectin
CT1746
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