A New Family of 10 Murine Ovalbumin Serpins Includes Two Homologs of Proteinase Inhibitor 8 and Two Homologs of the Granzyme B Inhibitor (Proteinase Inhibitor 9)

Abstract

Serine proteinase inhibitors (serpins) are classically regulators of extracellular proteolysis, however, recent evidence suggests that some function intracellularly. Such "ovalbumin" serpins include the human proteinase inhibitors 6 (PI-6), 8 (PI-8), and 9 (PI-9), plasminogen activator inhibitor 2, and the monocyte/neutrophil elastase inhibitor. PI-9 is a potent granzyme B (graB) inhibitor that has an unusual P 1 Glu and is present primarily in lymphocytes. In a search for the murine equivalent of PI-9 we screened cDNA libraries, and performed reverse transcriptase-polymerase chain reaction on RNA isolated from leukocyte cell lines and from lymph nodes and spleens of allo-immunized mice. We identified 10 new ovalbumin serpin sequences: two resemble PI-8, two resemble PI-9, and the remaining six have no obvious human counterparts. By RNA analysis only one of the two sequences resembling PI-9 (designated SPI6) is present in mouse lymphocytes while the other (a partial clone designated mBM2A) is predominantly in testis. SPI6 comprises a 1.8-kilobase cDNA encoding a 374-amino acid polypeptide that is 68% identical to PI-9. mBM2A is 65% identical to PI-9 and over 80% identical to SPI6. Although the reactive loops of SPI6 and mBM2A differ from PI-9, both contain a Glu in a region likely to contain the P 1 -P 1 bond. SPI6 produced in vitro using a coupled transcription/translation system formed an SDS-stable complex with human graB and did not interact with trypsin, chymotrypsin, leukocyte elastase, pancreatic elastase, thrombin, or cathepsin G. Recombinant SPI6 produced in a yeast expression system was used to examine the interaction with human graB in more detail. The second-order rate constant for the interaction was estimated as 8 ؋ 10 4 M ؊1 s ؊1 , and inhibition depended on the Glu in the SPI6 reactive center. The SPI6 gene was mapped to the same region on mouse chromosome 13 as Spi3, which encodes the murine homolog of PI-6. We conclude that even though their reactive centers are not highly conserved, SPI6 is a functional homolog of PI-9, and that the regulation of graB in the mouse may involve a second serpin encoded by mBM2A. Our identification of multiple sequence homologs of PI-8 and PI-9, and six new ovalbumin serpins, is consonant with the idea that the larger set of granule and other proteinases known to exist in the mouse (compared with human) is balanced by a larger array of serpins.

A Novel Serpin Regulatory Mechanism

“…This may reflect the fact that the Cys pair in mouse Sb9 comprises the P1-P1Ј residues and that oxidation of the P1 Cys alone decreases efficiency of GzmB cleavage. Susceptibility to oxidation may explain our previous reports of relatively poor interaction between mouse GzmB and Sb9 in vitro, exemplified by a SI of 5 (7,8). If more effective antioxidants (e.g.…”

“…In vitro experiments also show that chemical modification can inactivate a serpin (4). Sb9 (SerpinB9) is an intracellular inhibitor of the mammalian cytotoxic lymphocyte (CL) serine protease, GzmB (granzyme B) (5)(6)(7). GzmB is mainly produced by CD8…”

Perforin deficiency and susceptibility to cancer

“…Granzymes and other toxic proteases are synthesized as zymogens and are typically activated only on reaching lysosome-like SGs; 24,25 in the cytosol of CLs, 'escaped' granzymes can be irreversibly inhibited by serpins (for example, human PI-9 or mouse SPI-6). [26][27][28][29] However, there does not seem to be any naturally occurring inhibitor for PRF. This paradox gave rise to various hypotheses, including receptor-dependent PRF activity.…”

Regulation of the plasminogen activator inhibitor-2 (PAI-2) gene in murine macrophages. Demonstration of a novel pattern of responsiveness to bacterial endotoxin

“…Nevertheless, the PAI-2 gene was recently mutated in the mouse genome without any clear phenotype, including endotoxin responsiveness, even when a PAI-2 null was combined with elimination of PAI-1 [24]. The explanation for the apparent redundancy of PAI-2 may lie in the discovery of 10 ovalbumin-like serpins in the mouse genome [25].…”