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Conversion of human interferon-β from a secreted to a phosphatidylinositol anchored protein by fusion of a 17 amino acid sequence to its carboxyl terminus

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Abstract

A number of cell-surface proteins are anchored in plasma membranes by a glycosylated phosphatidylinositol (PI) moiety that is covalently attached to the carboxyl-terminal amino acid of the mature protein. We have previously reported the construction of a cDNA clone of a truncated Platelet-derived growth factor (PDGF) receptor that consists of the extracellular domain without the transmembrane and cytoplasmic domains. In the construction of the vector, a sequence of 51 base pairs (bp) from the 3′-untranslated region of the receptor cDNA was linked in frame with the external domain coding sequence. The truncated receptor protein with the peptide VTSGHCHEERVDRHDGE fused to its carboxyl terminus was covalently attached to the membrane by a PI linkage and it was released by phosphatidylinositol specific-phospholipase C (PI-PLC). When the 51 bp sequence was deleted, the external domain receptor protein was secreted into the media. To determine whether the PI linkage of the protein was due to the 17 amino acids added, the peptide was fused to the carboxyl terminus of the secreted protein human Interferon-β (hu-IFN-β). Chinese hamster ovary (CHO) cells transfected with the hu-IFN-β cDNA secreted the protein to theconditioned media, whereas CHO cells transfected with the carboxyl terminus modified-hu-IFN-β cDNA did not secrete detectable levels of protein. CHO cells expressing the carboxyl terminus modified-hu-IFN-β were treated with PI-PLC, the media and cell lysates were analyzed by SDS-PAGE after immunoprecipitation with antibodies against hu-IFN-β. The modified protein is anchored to the plasma membrane by a PI linkage and it is specifically released by PI-PLC, whereas a control preparation of CHO cells expressing wild type hu-IFN-β does not show the same pattern. The 17 amino acid peptide fused to the carboxyl terminus of IFN-β directs attachment of a PI anchor and targets the fusion protein to the plasma membrane.

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Authors and Affiliations

  1. Instituto de Investigaciones Bioquímicas de Bahía Blanca INIBIBB, C.C. 857, 8000, Bahía Blanca, Argentina

    Graciela E. Santillán, Marisa J. Sandoval & Patricia L. Orchansky

  2. The Charing Cross Sunley Research Centre, 1 Lurgan Avenue, Hammersmith, W6 8LW, London, UK

    Yuti Chernajovsky

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  1. Graciela E. Santillán
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  2. Marisa J. Sandoval
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  3. Yuti Chernajovsky
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  4. Patricia L. Orchansky
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Santillán, G.E., Sandoval, M.J., Chernajovsky, Y. et al. Conversion of human interferon-β from a secreted to a phosphatidylinositol anchored protein by fusion of a 17 amino acid sequence to its carboxyl terminus. Mol Cell Biochem 110, 181–191 (1992). https://doi.org/10.1007/BF02454197

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  • DOI: https://doi.org/10.1007/BF02454197

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