The Serpins Are an Expanding Superfamily of Structurally Similar but Functionally Diverse Proteins

squamous cell carcinoma antigen 1 or 2 α1 antitrypsin (α1 proteinase inhibitor) α2 antiplasmin amyloid-β α1 antichymotrypsin antithrombin III monocyte-neutrophil elastase inhibitor ovalbumin plasminogen activator inhibitor type 1 or 2 pigment epithelium-derived factor vascular endothelial growth factor reactive site loop tissue plasminogen activator urokinase plasminogen activator The serpins (serineproteinase inhibitors) are a superfamily of proteins (350–500 amino acids in size) that fold into a conserved structure and employ a unique suicide substrate-like inhibitory mechanism. The serpins were last reviewed in 1994 (1Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Abstract Full Text PDF PubMed Google Scholar). More recent studies show: 1) an expanded distribution within the kingdoms of metazoa and plantae, as well as certain viruses, 2) a surprising effect on the covalently bound target proteinase, and 3) novel biochemical and biological functions. Most serpins inhibit serine proteinases of the chymotrypsin family. However, cross-class inhibitors have been identified. The viral serpin CrmA and, to a lesser extent, PI9 (SERPINB9) inhibit the cysteine proteinase, caspase 1 (2Komiyama T. Ray C.A. Pickup D.J. Howard A.D. Thornberry N.A. Peterson E.P. Salvesen G. J. Biol. Chem. 1994; 269: 19331-19337Abstract Full Text PDF PubMed Google Scholar), and SCCA11 (SERPINB3) neutralizes the potent papain-like cysteine proteinases, cathepsins L, K, and S (3Schick C. Pemberton P.A. Shi G.-P. Kamachi Y. Cataltepe S. Bartuski A.J. Gornstein E.R. Bromme D. Chapman H.A. Silverman G.A. Biochemistry. 1998; 37: 5258-5266Crossref PubMed Scopus (259) Google Scholar). In addition, several members no longer function as proteinase inhibitors but perform other roles such as hormone transport (thyroid-binding globulin (SERPINA6), corticosteroid-binding globulin (SERPINA7)), and blood pressure regulation (angiotensinogen (SERPINA8)) (1Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Abstract Full Text PDF PubMed Google Scholar). Data base searching provides evidence for ∼500 serpins, with full-length coding sequences known or predicted for about one-half of those (4Irving J.A. Pike R.N. Lesk A.M. Whisstock J.C. Genome Res. 2000; 10: 1845-1864Crossref PubMed Scopus (523) Google Scholar). A phylogenetic analysis divides serpins into 16 clades (see Supplemental Data, Table A) and 10 highly diverged “orphans” (4Irving J.A. Pike R.N. Lesk A.M. Whisstock J.C. Genome Res. 2000; 10: 1845-1864Crossref PubMed Scopus (523) Google Scholar). These data facilitate the construction of a consistent expandable nomenclature (see Supplemental Data for Serpin Nomenclature Guidelines, Table B). The completed DNA sequences of several organisms have yielded insight into the complexity of the family. The Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thalianagenomes encode for ∼20,000, 13,000, and 25,000 genes, respectively. However, these three species harbor ∼9, 32, and 13 serpin genes, respectively. The nonlinear relationship among the number of serpin genes, relative to the total gene number, suggests that at least a subset of serpins has evolved divergent functions despite a striking degree of sequence and structural conservation. Serpins adopt a metastable conformation that is required for their inhibitory activity (5Stein P.E. Carrell R.W. Nat. Struct. Biol. 1995; 2: 96-113Crossref PubMed Scopus (398) Google Scholar). This conformation consists of a conserved secondary structure comprised of β-sheets A, B, and C and at least 7 α-helices (most typically have 9, lettered A–I; Fig.1 A). The RSL, which contains the proteinase recognition site, is an exposed, flexible stretch of ∼17 residues tethered between β-sheets A and C. Serpins can undergo major structural rearrangements that involve alternative conformations for the RSL, β-sheet A, and the attached strand 1 of β-sheet C. Considering only intramolecular structural changes, serpins can convert to the more stable latent form (Fig. 1 B). The RSL inserts into the middle of β-sheet A to give a fully antiparallel β-sheet, and s1C is extracted from β-sheet C to provide an exposed “return” from the bottom of the serpin. Serpins in the latent conformation are noninhibitory but can be converted back to the active state by denaturation and refolding. The Tm for unfolding of latent PAI1 (SERPINE1) is 17 °C higher than that for the native state (reviewed in Ref. 6Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function and Biology, Molecular Biology Intelligence Unit. R. G. Landes Co., and Chapman 36: 5455-5464Crossref PubMed Scopus (104) Google Scholar). The most informative serpin structures, from a mechanistic viewpoint, are those of a Michaelis complex between Serpin 1 and trypsin (Fig.1 D) and of a covalent complex between α1AT (SERPINA1) and trypsin (8Huntington J.A. Read R.J. Carrell R.W. Nature. 2000; 407: 923-926Crossref PubMed Scopus (970) Google Scholar) (Fig. 1 E). This latter structure represents the proteinase after it has been kinetically trapped in the acyl-enzyme intermediate that forms normally along the peptide bond cleavage pathway. Whereas the bound serpin is almost indistinguishable from that of the RSL-cleaved form (Fig. 1 C), the proteinase is grossly distorted (see below). Serpins inhibit serine proteinases by an irreversible suicide substrate mechanism when the interaction proceeds down the inhibitory arm of a branched pathway (Fig. 2) (6Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function and Biology, Molecular Biology Intelligence Unit. R. G. Landes Co., and Chapman 8: 789-797Abstract Full Text Full Text PDF Scopus (55) Google Scholar), whereas the detection of an SDS-stable complex between SCCA1 and cathepsin S (a cysteine proteinase of the papain family) provides evidence for the formation of a stable, covalent thiol ester-type linkage (3Schick C. Pemberton P.A. Shi G.-P. Kamachi Y. Cataltepe S. Bartuski A.J. Gornstein E.R. Bromme D. Chapman H.A. Silverman G.A. Biochemistry. 1998; 37: 5258-5266Crossref PubMed Scopus (259) Google Scholar). The few convincing reports of reversible inhibition, such as of single-chain uPA by PCI (SERPINA5) (10Schwartz B.S. Espana F. J. Biol. Chem. 1999; 274: 15278-15283Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) or of chymotrypsin by α2AP (SERPINF2) (11Shieh B.H. Potempa J. Travis J. J. Biol. Chem. 1989; 264: 13420-13423Abstract Full Text PDF PubMed Google Scholar) may represent special cases in which unusual stabilization of the initial noncovalent Michaelis-like complex blocks progression to the substrate reaction. A negative consequence of the need for a metastable conformation in the active state is that natural mutations, either alone or in combination with environmental factors, can promote inappropriate loop insertion. When this occurs between the RSL of one molecule and the β-sheet of another, dimers and higher order oligomers can result. Either through depletion of active serpin or through pathological effects of the polymers themselves, such aggregate formation can lead to disease. The best characterized examples are the emphysema (serpin depletion) and cirrhosis (intracellular inclusions) associated with loop-sheet polymers of the Z or S variants of α1AT (12Elliott P.R. Lomas D.A. Carrell R.W. Abrahams J.P. Nat. Struct. Biol. 1996; 3: 676-681Crossref PubMed Scopus (249) Google Scholar) (see Supplemental Data, Fig. A) and the dementia associated with neuroserpin (SERPINI1) inclusion bodies (see below). Understanding the biologic function of serpins remains an ongoing challenge. For example, the biologic functions for many of the human serpins involved in the clotting and fibrinolytic cascades are well documented. However the role of human serpins in some other types of biologic processes awaits further validation (Fig.3). In 1993 amino acid similarities among chicken ovalbumin (ov), PAI2 (SERPINB2), and MNEI (SERPINB1) led to the identification of a subgroup of the serpin superfamily (13Remold-O'Donnell E. FEBS Lett. 1993; 315: 105-108Crossref PubMed Scopus (217) Google Scholar). The N and C termini of the ov-serpins are shorter than the prototypical serpin α1AT, and they also lack a classical secretory signal peptide. At present, there are 13 human ov-serpins (see Supplemental Data, Table B). They map to 6p25 and 18q21 and fall into two classes based on a single difference in gene structure (14Scott F.L. Eyre H.J. Lioumi M. Ragoussis J. Irving J.A. Sutherland G.A. Bird P.I. Genomics. 1999; 62: 490-499Crossref PubMed Scopus (37) Google Scholar). Like ovalbumin, many of the 18q21 serpin genes have an exon encoding a polypeptide loop between helices C and D (CD loop) that may contribute to accessory functions. Unlike ovalbumin itself, most ov-serpins reside intracellularly with a cytoplasmic or nucleocytoplasmic distribution. However, several ov-serpins (PAI2, megsin (SERPINB7), MNEI, maspin (SERPINB5), and the SCCAs (SERPINB3 and -4)) may function extracellularly as they are released from cells under certain conditions. Release may be facilitated by an embedded, noncleaved hydrophobic N-terminal signal sequence and appears to involve both conventional and non-endoplasmic reticulum-Golgi secretory pathways (15Belin D. Thromb. Haemostasis. 1993; 70: 144-147Crossref PubMed Scopus (40) Google Scholar). Regardless of how ov-serpins are released from cells, those with RSL cysteine or methionine residues are susceptible to oxidative inactivation and are likely to have a limited half-life in the extracellular milieu. With the possible exception of maspin, all human ov-serpins are functional, competitive inhibitors of serine or cysteine proteinases. Several members of the group inhibit more than one proteinase, and dual reactive sites (utilization of more than one P1 residue) have been described for PI6 (SERPINB6), PI8 (SERPINB8), PI9, SCCA1, SCCA2, and MNEI (for example see Ref. 16Riewald M. Schleef R.R. J. Biol. Chem. 1996; 271: 14526-14532Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). However, the CD loops of the ov-serpins have the potential to interact with other proteins. For example, the CD loop of PAI2 is required for its cell survival function (17Dickinson J.L. Bates E.J. Ferrante A. Antalis T.M. J. Biol. Chem. 1995; 270: 27894-27904Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar) and is a target for transglutamination (18Jensen P.H. Schuler E. Woodrow G. Richardson M. Goss N. Hojrup P. Petersen T.E. Rasmussen L.K. J. Biol. Chem. 1994; 269: 15394-15398Abstract Full Text PDF PubMed Google Scholar). Bomapin (SERPINB10; like the chicken ov-serpin, MENT, see below) carries a nuclear localization signal in its CD loop that presumably interacts with a nuclear importin (19Chuang T.L. Schleef R.R. J. Biol. Chem. 1999; 274: 11194-11198Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The physiological functions of ov-serpins are still emerging. PAI2 may play a role in the regulation of extracellular matrix remodeling through the inhibition of uPA, as high PAI2 and low uPA levels correlate with a positive prognosis in breast cancer (20Duggan C. Kennedy S. Kramer M.D. Barnes C. Elvin P. McDermott E. O'Higgins N. Duffy M.J. Br. J. Cancer. 1997; 76: 622-627Crossref PubMed Scopus (66) Google Scholar). Also, PAI2 may have a structural role inside some cells (perhaps keratinocytes) as suggested by its ability to spontaneously polymerize and undergo transglutamination (21Mikus P. Ny T. J. Biol. Chem. 1996; 271: 10048-10053Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Many ov-serpins reside in proteinase-secreting cells (22Bird P.I. Immunol. Cell Biol. 1999; 77: 47-57Crossref PubMed Scopus (58) Google Scholar). For example, PI9, a potent inhibitor of granzyme B, is also present in cytotoxic lymphocytes. Because PI9 can protect cells against granzyme B-mediated apoptosis, it probably protects cytotoxic lymphocytes from autodestruction due to misdirected granzyme B. A similar cytoprotective role can be envisaged for PI6, PI8, MNEI, PAI2, and the SCCAs. In addition, endogenous or exogenous ov-serpins may protect bystander cells and tissue from proteolytic damage. Studies in rats show that recombinant MNEI delivered to the airways prevents lung injury by neutrophil proteinases and point to its potential in treating inflammatory lung disease (23Rees D.D. Rogers R.A. Cooley J. Mandle R.J. Kenney D.M. Remold-O'Donnell E. Am. J. Respir. Cell Mol. Biol. 1999; 20: 69-78Crossref PubMed Scopus (43) Google Scholar). The ability of many ov-serpins to inhibit more than one proteinase and their presence in epithelial cells suggest that they play a role in barrier function or host defense against microbial or viral proteinases. For example, PI9 inhibits Bacillussubtilisin, and PI8 inhibits furin, a subtilisin-related enzyme (24Dahlen J.R. Foster D.C. Kisiel W. Biochem. Biophys. Res. Commun. 1997; 238: 329-333Crossref PubMed Scopus (19) Google Scholar,25Dahlen J.R. Jean F. Thomas G. Foster D.C. Kisiel W. J. Biol. Chem. 1998; 273: 1851Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Additional functions of ov-serpins include the regulation of: 1) cell growth or differentiation, as exemplified by the role of megsin in megakaryocyte differentiation (26Tsujimoto M. Tsuruoka N. Ishida N. Kurihara T. Iwasa F. Yamashiro K. Rogi T. Kodama S. Katsuragi N. Adachi M. Katayama T. Nakao M. Yamaichi K. Hashino J. Haruyama M. Miura K. Nakanishi T. Nakazato H. Teramura M. Mizoguchi H. Yamaguchi N. J. Biol. Chem. 1997; 272: 15373-15380Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), 2) tumor cell invasiveness and motility, as shown by the inhibitory role of maspin in breast and prostate tumors (27Zou Z. Anisowicz A. Hendrix M.J. Thor A. Neveu M. Sheng S. Rafidi K. Seftor E. Sager R. Science. 1994; 263: 526-529Crossref PubMed Scopus (842) Google Scholar), and 3) angiogenesis (see below). Grigoryev et al. (28Grigoryev S.A. Bednar J. Woodcock C.L. J. Biol. Chem. 1999; 274: 5626-5636Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) isolated a novel serpin, MENT, from the nuclei of terminally differentiated chicken hematopoietic cells. MENT is an ov-serpin with a CD loop that contains a nuclear localization signal, a lamin-like chromatin binding domain, and an A-T hook DNA binding motif. The molecule has a relatively high pI (9 versus 5–6.5 for that of other serpins) with the majority of positive charges clustering near the CD loop. Thus, MENT appears to utilize the CD loop to bind tightly to nucleosomes with an apparent stoichiometry of 2:1. MENT is the major non-histone chromatin protein in differentiated nuclei and is concentrated in the heterochromatin. MENT induces higher order chromatin compaction when it is expressed ectopically in cells or added to isolated nuclei in vitro. Although MENT contains a viable RSL, target proteinases have yet to be identified. which inhibits uPA, and growth factor in is from and cells G.A. S. E.P. R. S. M. D.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). may play a role in the from In a neuroserpin in within the and of the protein reduced the by and the number of cells by M. M. E. D.A. 2000; PubMed Google Scholar). In a form of dementia and neuroserpin Molecular analysis in two and in the C. D. P. J. F. D. M. D.A. B. P.R. Carrell R.W. Lomas D.A. Nature. 1999; PubMed Google Scholar). These are similar to that in α1AT in which an of β-sheet A and the formation of loop-sheet In these polymers and in the normal function is and, to a lesser extent, other serpins are within the of from with one of the most common forms of dementia (reviewed in Ref. S. 1998; 20: PubMed Scopus Google Scholar). Although the of this is the extracellular of may be by binding to low receptors and with appears to facilitate formation by as a for the The peptide inserts into A and C of in which it a conformation. Upon RSL is released into the extracellular in which the peptide is more to is a noninhibitory serpin that isolated from pigment epithelial cells but is also in and (6Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function and Biology, Molecular Biology Intelligence Unit. R. G. Landes Co., and Chapman PubMed Scopus Google Scholar) show that inhibits of the and endothelial cell vitro. In the cell as potent as other angiogenesis inhibitors such as and the effects of the angiogenesis growth growth and in the with the of Thus, and to blood growth in the by and angiogenesis and respectively. maspin, and RSL-cleaved have been shown to with angiogenesis in M. Shi N. Nat. 2000; PubMed Scopus Google S. E. G.A. D.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google S. J. Science. 1999; PubMed Scopus Google Scholar). However, it has yet to be of these are involved in the or regulation of blood A of function in serpin leads to the and of the E. C. D. M. J.A. Science. 1999; PubMed Scopus Google Scholar). of the pathway proteolytic cleavage of the In leads to an in both and the pathway. appears to in a negative loop by proteinases that Thus, the of and the to be secondary to proteolytic The function of these proteins remains Several studies show that serpins are of serine proteinase H. Rasmussen J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). However, with the exception of a proteinase in conventional serine proteinase are in of the sequence of trypsin as a to classical In studies by et al. K. Y. R.J. B. J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) show that of the with the of the to and on these a role for serpins in host Several of human serpins have been by in cells. of these have to an whereas show and structural as well as (see Supplemental Data, Table C). Serpins are within a number of within the of the and the (see Supplemental Data, Table of the serpins are required for growth in cell the and three highly conserved serpins, and types of proteinases The of the also three serpins, the contains serpin genes and lack serpin of encode serpins with P1 and the and all have a with a P1 residue the have a serpin with a at the P1 For more on serpins see Supplemental The serpins are a superfamily of genes that are the metazoa and Serpin members are by a conserved structure and a unique suicide substrate-like inhibitory mechanism. Serpins reside both intracellularly and extracellularly and are involved in a of biologic functions that the ability of these to irreversibly inhibit target proteinases. The of serpin function biological such as the Biochem. Full Text Full Text PDF PubMed Scopus Google Scholar) and the role that these play in and host