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Retinol and retinaldehyde specifically increase alpha1-proteinase inhibitor in the human cornea.

ABSTRACT: alpha1-Proteinase inhibitor is a serpin and can inhibit most serine proteinases. The cornea is one of several extrahepatic tissues that synthesizes this inhibitor. In the presence of retinol, corneal alpha1-proteinase inhibitor levels were increased 3.8-fold. The maximal response was achieved 2 h after the addition of retinol (1 microM final concentration) to the culture medium. A similar increase in alpha1-proteinase inhibitor was observed with retinaldehyde (1 nM final concentration). Concentrations of alpha1-proteinase inhibitor in other tested cells (Hep G2, CaCo 2, MCF-7, monocytes and macrophages) remained unchanged in the presence of retinol. Retinoic acid did not affect alpha1-proteinase inhibitor levels in the cornea or the other cells tested. The acute-phase cytokine, interleukin-6, increased alpha1-proteinase inhibitor levels in all tested tissues/cells except the cornea. These results demonstrate that alpha1-proteinase inhibitor levels are controlled differently in the cornea compared with other tissues/cells. alpha1-Proteinase inhibitor is the first protein identified whose levels are regulated by a mechanism supported by retinol and retinaldehyde but not retinoic acid.

SUBMITTER: Boskovic G

PROVIDER: S-EPMC1218251 | biostudies-other | 1997 Mar

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Human alpha1-proteinase inhibitor (alpha1-PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, alpha1-PI is available for replacement therapy as an FDA licensed plasma-derived (pd) product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant alpha1-PI (r-alpha1-PI) could provide an attractive alternative. Although r-alpha1-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation. RESULTS: We have explored the possibility of expressing the gene for human alpha1-PI in the filamentous fungus Aspergillus niger (A. niger), a system reported to be capable of providing more "mammalian-like" glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of alpha1-PI with a strongly expressed, secreted leader protein (glucoamylase G2), separated by dibasic processing site (N-V-I-S-K-R) that provides in vivo cleavage. SDS-PAGE, Western blot, ELISA, and alpha1-PI activity assays enabled us to select the transformant(s) secreting a biologically active glycosylated r-alpha1-PI with yields of up to 12 mg/L. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis further confirmed that molecular mass of the r-alpha1-PI was similar to that of the pd-alpha1-PI. In vitro stability of the r-alpha1-PI from A. niger was tested in comparison with pd-alpha1-PI reference and non-glycosylated human r-alpha1-PI from E. coli. CONCLUSION: We examined the suitability of the filamentous fungus A. niger for the expression of the human gene for alpha1-PI, a medium size glycoprotein of high therapeutic value. The heterologous expression of the human gene for alpha1-PI in A. niger was successfully achieved to produce the secreted mature human r-alpha1-PI in A. niger as a biologically active glycosylated protein with improved stability and with yields of up to 12 mg/L in shake-flask growth.

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Retinol and retinaldehyde specifically increase alpha1-proteinase inhibitor in the human cornea.

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