Proteinase inhibitor 6 cannot be secreted, which suggests it is a new type of cellular serpin.
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 alpha 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.
Scott
F L
FL
Department of Medicine, Monash Medical School, Clive Ward Centre, Box Hill Hospital, Australia.
Coughlin
P B
PB
Bird
C
C
Cerruti
L
L
Hayman
J A
JA
Bird
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D019084:Fluorescent Antibody Technique, Indirect; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D015539:Platelet Activation / Q000187:drug effects; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D015843:Serpins / Q000032:analysis / Q000096:biosynthesis* / Q000494:pharmacology; D012867:Skin / Q000166:cytology* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D015843:Serpins; D014409:Tumor Necrosis Factor-alpha; C094472:serpin B6; D003994:Bucladesine; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate