Abstract
Human apolipoprotein (apo) A-IV, a 46 kd protein, is capable of serving as a cofactor for the enzyme lecithin: cholesterol acyltransferase (LCAT), to bind to apo A-I/A-II receptor sites on mouse adipose cells and thereby promote cellular cholesterol efflux. These observations together with the appearence of apo A-IV in human interstitial fluid argue for a role of apo A-IV in reverse cholesterol transport. In order to obtain large amounts of this protein and to perform site-directed mutagenesis, we expressed apo A-IV in E. coli. The recombinant protein was soluble and could be isolated avoiding lipid extraction or other denaturating procedures. Characterization of the recombinant protein showed an additional methionine residue at the N-terminus and exhibited otherwise identical physicochemical properties when compared to apo A-IV isolated from human plasma. The cofactor function for LCAT, the binding to mouse adipose cells and the promotion of cellular cholesterol efflux were indistinguishable between recombinant apo A-IV and the human plasma counterpart. The expression of apo A-IV in E. coli provides a powerful tool to study the physiologic function of apo A-IV by sitedirected mutagenesis.
Zusammenfassung
Menschliches Apolipoprotein (Apo) A-IV, 46 kD, ist in vitro in der Lage, das Enzym Lecithin-Cholesterin-Acyltransferase (LCAT) zu aktivieren, an Apo A-I/A-IIRezeptoren von Maus-Adipozyten zu binden und Cholesterinausstrom aus diesen Zellen zu vermitteln. Diese Beobachtungen, zusammen mit dem Vorkommen von Apo A-IV in der interstitiellen Flüssigkeit, sprechen für die Mitwirkung von Apo A-IV beim Cholesterinrücktransport. Um große Mengen dieses Proteins zu erhalten und gezielte Mutanten herzustellen, wurde menschliches Apo A-IV in Escherichia coli exprimiert. Das rekombinante Protein war löslich und konnte ohne Lipidextraktion oder Denaturierung isoliert werden. Die Charakterisierung des exprimierten Proteins zeigte einen zusätzlichen Methioninrest N-terminal, war jedoch ansonsten in seinen physikochemischen Eigenschaften identisch mit Apo A-IV aus menschlichem Plasma. In bezug auf die Funktion zeigte das rekombinante Apo A-IV im Vergleich mit Apo A-IV aus menschlichem Plasma gleiche Kofaktorfunktionen für die LCAT, war ebenso in der Lage, an Adipozyten der Maus zu binden und Cholesterinausstrom aus diesen Zellen zu vermitteln. Die Expression von Apo A-IV in Escherichia coli bietet die Möglichkeit, zum Beispiel durch gezielte Mutationen die physiologische Rolle von Apo A-IV zu untersuchen.
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© 1992 Springer Fachmedien Wiesbaden
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Steinmetz, A., Kaffarnik, H., Fruchart, JC., Denefle, P. (1992). The function of recombinant human apolipoprotein A-IV from E. coli. In: Heinle, H., Schulte, H., Schaefer, H.E. (eds) Arteriosklerotische Gefäßerkrankungen. Vieweg+Teubner Verlag, Wiesbaden. https://doi.org/10.1007/978-3-663-19646-4_8
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DOI: https://doi.org/10.1007/978-3-663-19646-4_8
Publisher Name: Vieweg+Teubner Verlag, Wiesbaden
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