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

Abstract

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 ␣ 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.

Human kallikrein 2 (hK2), but not prostate‐specific antigen (PSA), rapidly complexes with protease inhibitor 6 (PI‐6) released from prostate carcinoma cells

“…PAI-2, having 49% amino acid identity with PI-6, has an apparent physiologic function both intracellularly and extracellularly. 16 The squamous cell carcinoma antigen (SCC-Ag) has 47% identity to PI-6 and is a tumor-associated, serologic marker for advanced SCC of the cervix and lung. 29 SCC-Ag release from SCC cells reflects the degree of histologic differentiation of the tumor.…”

“…PI-6 is expressed in platelets, kidney, heart and skeletal muscle in both endothelial and epithelial cells. 16,17 This serpin has dual inhibitory activity toward both trypsin and chymotrypsin. 18 PI-6 also inactivates thrombin, uPA, activated protein C and plasmin.…”

“…19 PI-6 has a potential role in differentiation and apoptosis. 20,21 PI-6 is exclusively expressed in the cytoplasm, 16 whereas hK2 is a secreted, extracellular protease. Thus, our study attempted to explore the nature and mechanism of complex formation between hK2 and PI-6.…”

The Aggregation-Prone Intracellular Serpin SRP-2 Fails to Transit the ER inCaenorhabditis elegans

“…The signal peptide directed sSRP-2::GFP to the ER where it appeared to form large inclusions, much like sGFP::ATZ, that also colocalized with the sDsRed::KDEL marker (Figure 2, P-T). This result was not surprising, as at least two members of the clade B serpin family, MASPIN/SERPINB5 and SERPINB6, appear to be obligate intracellular proteins and accumulate in the ER as endo-H-sensitive aggregates when they are forced into the secretory pathway by a synthetic signal peptide (Scott et al 1996;Teoh et al 2010). Moreover, SER-PINB6 no longer maintains its inhibitory activity when extracted from the ER.…”

“…Although some of these secreted intracellular serpins possess complex N-linked carbohydrates suggestive of Golgi processing, secretion still occurs in the presence of tunicamycin (Keller and Swank 1978) and may involve unconventional signal pathway(s) similar to those used by FGF-2 and IL-1b (Nickel and Rabouille 2009;Giuliani et al 2011;Malhotra 2013). Indeed, forced expression of wild-type protease inhibitor 6 (PI6)/SERPINB6 and MASPIN/SERPINB5 into the ERGolgi pathway by fusing an N-terminal signal peptide leads to overt polymerization and ER retention (Scott et al 1996;Teoh et al 2010). None of the 12 other human clade B serpins appear to be secreted.…”