Proteinase Inhibitor 6 Cannot Be Secreted, Which Suggests It Is a New Type of Cellular Serpin (∗)

• Published in: The Journal of biological chemistry 1996-01, Vol.271 (3), p.1605-1612
• Main Authors: Scott, Fiona L., Coughlin, Paul B., Bird, Catherina, Cerruti, Loretta, Hayman, John A., Bird, Phillip
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Blood Platelets - drug effects; Blood Platelets - physiology; Bucladesine - pharmacology; Cell Line; Chlorocebus aethiops; Cloning, Molecular; DNA Primers; DNA, Complementary; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum - ultrastructure; Fluorescent Antibody Technique, Indirect; Glycosylation; Humans; Immunohistochemistry; In Vitro Techniques; Kinetics; Molecular Sequence Data; Platelet Activation - drug effects; Recombinant Proteins - analysis; Recombinant Proteins - biosynthesis; Recombinant Proteins - pharmacology; Sequence Homology, Amino Acid; Serpins - analysis; Serpins - biosynthesis; Serpins - pharmacology; Skin - cytology; Skin - metabolism; Tetradecanoylphorbol Acetate - pharmacology; Thrombin - metabolism; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.3.1605

We have recently described a new serine proteinase inhibitor, proteinase inhibitor 6 (PI-6). This serpin has features that suggest it may function intracellularly, but its close resemblance to ovalbumin serpins like plasminogen activator inhibitor 2 (PAI-2) raises the possibility that it is secreted to regulate an extracellular proteinase. To determine whether PI-6 is secreted, we have examined its cellular distribution by immunohistochemistry and have attempted to induce its release from platelets and from cultured cells. We find that PI-6 is present in endothelial and epithelial cells, but it is apparently cytoplasmic and it is not released from cells in response to phorbol ester, dibutyryl cAMP or tumor necrosis factor α treatment. It is also not released from activated platelets. The addition of a conventional signal peptide to the amino terminus of PI-6 directed its translocation into the endoplasmic reticulum (ER), resulting in glycosylation but not secretion of the molecule. By contrast, the addition of the same signal peptide to PAI-2 markedly enhanced its translocation and secretion. Glycosylated PI-6 was sequestered in the ER and was incapable of interacting with thrombin. The failure of PI-6 to move along the secretory pathway, and the loss of inhibitory function of ER-localized PI-6, demonstrates that unlike PAI-2, PI-6 is not naturally secreted. Taken together, these results suggest that PI-6 has evolved to fulfil an intracellular role and that it represents a new type of cellular serpin.