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NORMAL GENOMIC

• Hejgaard J
• Inhibitory serpins from rye grain with glutamine as P1 and P2 residues in the reactive center.
• FEBS Lett. 2001; 488: 149-53
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• Six of seven serpins detected in grains of rye (Secale cereale) were purified and characterized. The amino acid sequence close to the blocked N-terminus, the reactive center loop sequence and the second order association rate constant (k(a)') for irreversible complex formation with chymotrypsin were determined for each serpin. Three of four serpins containing the unusual reactive center P2-P1' QQ/S and one with P2-P1' PQ/M were equally efficient inhibitors of chymotrypsin (k(a)' approximately 10(5) M(-1) s(-1)). One serpin with P2-P1' PY/M was a faster inhibitor (k(a)' approximately 10(6) M(-1) s(-1)). Similar but differently organized glutamine-rich reactive centers were recently found in grain serpins cloned from wheat [Ostergaard et al. (2000) J. Biol. Chem. 275, 33272] but not from barley. The prolamin storage proteins of cereal grains contain similar sequences in their glutamine-rich repeats. A possible adaption of hypervariable serpin reactive centers late in Triticeae cereal evolution as defence against insects feeding on cereal grains is discussed.

• Gan H, Wang Y, Jiang H, Mita K, Kanost MR
• A bacteria-induced, intracellular serpin in granular hemocytes of Manduca sexta.
• Insect Biochem Mol Biol. 2001; 31: 887-98
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• Serine proteinase inhibitors from the serpin superfamily have been identified as hemolymph proteins from several groups of arthropods, including horseshoe crabs, crayfish, and insects. In the tobacco hornworm, Manduca sexta, one group of serpins present in plasma is generated by alternate exon splicing from serpin gene-1. We have identified a second serpin gene from this insect, M. sexta serpin-2. A serpin-2 DNA clone was isolated from a fifth instar larval cDNA library. The full-length cDNA is 1.5 kb long and encodes a protein of 381 amino acid residues. Amino acid sequence comparisons with other invertebrate serpins reveal approximately 25-40% identity with serpin-2. An expressed sequence tag from Bombyx mori, which is very similar to M. sexta serpin-2, was identified, and the corresponding full-length cDNA sequence was determined. This silkworm homolog of serpin-2 is 57% identical to M. sexta serpin-2. Recombinant M. sexta serpin-2 was used as an antigen to generate a rabbit polyclonal antiserum. This antiserum recognized a 43 kDa protein present in hemocytes but absent from plasma. Western and Northern blot results revealed that serpin-2 gene expression increased dramatically after larvae were injected with bacteria. In situ hybridization showed that the serpin-2 mRNA is present in granular hemocytes of immune-stimulated larvae. Serpin-2 purified from hemocytes obtained 24 h after injection of larvae with bacteria lacked inhibitory activity for all proteinases tested except for human cathepsin G. The intracellular location of serpin-2 suggests a function for serpin-2 different from the plasma serpin-1 proteins.

• Zang X, Maizels RM
• Serine proteinase inhibitors from nematodes and the arms race between host and pathogen.
• Trends Biochem Sci. 2001; 26: 191-7
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• Serine proteinase inhibitors are encoded by a large gene family of long evolutionary standing. Recent discoveries of parasite proteins that inhibit human serine proteinases, together with the complete genomic sequence from Caenorhabditis elegans, have provided a set of new serine proteinase inhibitors from more primitive metazoan animals such as nematodes. The structural features (e.g. reactive centre residues), gene organization (including intron arrangements) and inhibitory function and targets (e.g. inflammatory and coagulation pathway proteinase) all contribute important new insights into proteinase inhibitor evolution. Some parasite products have evolved that block enzymes in the mammalian host, but the human host responds with a significant immune response to the parasite inhibitors. Thus, infection produces a finely balanced conflict between host and pathogen at the molecular level, and this might have accelerated the evolution of these proteins in parasitic species as well as their hosts.

• Hengst U, Albrecht H, Hess D, Monard D
• The phosphatidylethanolamine-binding protein is the prototype of a novel family of serine protease inhibitors.
• J Biol Chem. 2001; 276: 535-40
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• Serine proteases are involved in many processes in the nervous system and specific inhibitors tightly control their proteolytic activity. Thrombin is thought to play a role in tissue development and homeostasis. To date, protease nexin-1 is the only known endogenous protease inhibitor that specifically interferes with thrombotic activity and is expressed in the brain. In this study, we report the detection of a novel thrombin inhibitory activity in the brain of protease nexin-1(-/-) mice. Purification and subsequent analysis by tandem mass spectrometry identified this protein as the phosphatidylethanolamine-binding protein (PEBP). We demonstrate that PEBP exerts inhibitory activity against several serine proteases including thrombin, neuropsin, and chymotrypsin, whereas trypsin, tissue type plasminogen activator, and elastase are not affected. Since PEBP does not share significant homology with other serine protease inhibitors, our results define it as the prototype of a novel class of serine protease inhibitors. PEBP immunoreactivity is found on the surface of Rat-1 fibroblast cells and although its sequence contains no secretion signal, PEBP-H(6) can be purified from the conditioned medium upon recombinant expression.

• Chang WS, Chang NT, Lin SC, Wu CW, Wu FY
• Tissue-specific cancer-related serpin gene cluster at human chromosome band 3q26.
• Genes Chromosomes Cancer. 2000; 29: 240-55
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• Approximately one quarter of the identified human serpin genes are cancer-related and clustered mainly at two distinct loci: 6p25 and 18q21. We have studied a novel serpin gene cluster at 3q26 containing at least two recently identified members: the pancreas-specific protease inhibitor, pancpin (PI14), and the brain-associated protease inhibitor, neuroserpin (PI12). In this, unlike a previous study, both PI14 and PI12 at 3q26 were found to consist of 9 exons and 8 introns and to share a perfectly conserved gene organization whose pattern is very different from that of the ov-serpin family. This distinct pattern appears identical in the genomic structures of human plasminogen activator inhibitor-1 (PAI1) at 7q21 and protease nexin 1 (PI7) at 2q33-35, confirming that these four genes in three different chromosomes form a discrete subset within the serpin superfamily. As in the other three members whose gene expression is altered during tumorigenesis, PI12 expression was found to be down-regulated in tumor brain tissues and in two brain cancer cell lines: U-87 MG and H4. By screening genomic libraries, we isolated two overlapping clones showing that the marker SGC32223 (centromere) is located within intron F of PI12 and the marker WI-10077 (telomere) is located downstream of the 3'-flanking region of PI14. This finding indicates that the distance between human PI14 and PI12 is approximately 100 kb, and hence we speculate that other tissue-specific cancer-related serpin genes are likely to reside within this 3q26.1 cluster region.

• Simonovic M, Gettins PGW, Volz K
• Crystal structure of viral serpin crmA provides insights into its mechanism of cysteine proteinase inhibition.
• Protein Sci. 2000; 9: 1423-7
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• CrmA is an unusual viral serpin that inhibits both cysteine and serine proteinases involved in the regulation of host inflammatory and apoptosis processes. It differs from other members of the serpin superfamily by having a reactive center loop that is one residue shorter, and by its apparent inability to form SDS-stable covalent complexes with cysteine proteinases. To obtain insight into the inhibitory mechanism of crmA, we determined the crystal structure of reactive center loop-cleaved crmA to 2.9 A resolution. The structure, which is the first of a viral serpin, suggests that crmA can inhibit cysteine proteinases by a mechanism analogous to that used by other serpins against serine proteinases. However, one striking difference from other serpins, which may be significant for in vivo function, is an additional highly charged antiparallel strand for b sheet A, whose sequence and length are unique to crmA.

• Boigegrain RA, Pugniere M, Paroutaud P, Castro B, Brehelin M
• Low molecular weight serine protease inhibitors from insects are proteins with highly conserved sequences.
• Insect Biochem Mol Biol. 2000; 30: 145-52
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• A low molecular weight protease inhibitor peptide found in ovaries of the desert locust Schistocerca gregaria (SGPI-2), was purified from plasma of the same locust and sequenced. It was named SGCI. It was found active towards chymotrypsin and human leukocyte elastase. SGCI was synthesized using a solid-phase procedure and the sequence of its reactive site for chymotrypsin was determined. Compared with an inhibitor purified earlier from another locust species, the total sequence of SGCI showed 88% identity. In particular, the sequence of the reactive site of these inhibitors was identical. Our search for a closely related peptide in an insect species far removed from locusts, the lepidopteran Spodoptera littoralis, was unfruitful but a different chymotrypsin inhibitor, belonging to the Kazal family, was found whose mass is greater than that of SGCI (20 vs 3.6 kDa). Its N-terminal sequence shares 80% identity with that of a chymotrypsin inhibitor purified earlier from the haemolymph of another lepidopteran. Conservation of the amino acid sequence in the reactive site seems to be an exception among protease inhibitors.

• Pszenny V et al.
• Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii.
• Mol Biochem Parasitol. 2000; 107: 241-9
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• A cDNA clone from a Toxoplasma gondii tachyzoite cDNA library encoding a serine proteinase inhibitor (serpin) was isolated. The 1376 bp cDNA sequence encodes a 294 amino acid protein with a putative signal peptide of 23 amino acids resulting in a mature protein with a predicted mass of 30,190 Da and a pI of 4.86. This protein has internal sequence similarity of residues 30-66, 114-150, 181-217 and 247-283 indicating a four-domain structure. The four domains exhibit high identity to serine proteinase inhibitors belonging to the non-classical Kazal-type family. The gene is single copy in the tachyzoite haploid genome of RH strain and was amplified by polymerase chain reaction (PCR). Several introns were identified. The sequence encoding the mature protein was amplified by PCR, cloned into the pQE30 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay and two bands of 38 and 42 kDa were detected in a whole parasite homogenate. The recombinant protein showed trypsin-inhibitory activity, one of the two potential specificities. We discuss the possible roles that T. gondii serpin(s) may play in the survival of the tachyzoites in the host.

• Leung D, Abbenante G, Fairlie DP
• Protease inhibitors: current status and future prospects.
• J Med Chem. 2000; 43: 305-41

• Jiang H, Kanost MR
• The clip-domain family of serine proteinases in arthropods.
• Insect Biochem Mol Biol. 2000; 30: 95-105

• Patston PA
• Serpins and other serine protease inhibitors.
• Immunol Today. 2000; 21: 354-354

• Strukelj B et al.
• Equistatin, a protease inhibitor from the sea anemone actinia equina, is composed of three structural and functional domains.
• Biochem Biophys Res Commun. 2000; 269: 732-6
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• A cDNA encoding a precursor of equistatin, a potent cysteine and aspartic proteinase inhibitor, was isolated from the sea anemone Actinia equina. The deduced amino acid sequence of a 199-amino-acid residue mature protein with 20 cysteine residues, forming three structurally similar thyroglobulin type-1 domains, is preceded by a typical eukaryotic signal peptide. The mature protein region and those coding for each of the domains were expressed in the periplasmic space of Escherichia coli, isolated, and characterized. The whole recombinant equistatin and its first domain, but not the second and third domains, inhibited the cysteine proteinase papain (K(i) 0.60 nM) comparably to natural equistatin. Preliminary results on inhibition of cathepsin D, supported by structural comparison, show that the second domain is likely to be involved in activity against aspartic proteinases.

• Maass N, Hojo T, Zhang M, Sager R, Jonat W, Nagasaki K
• Maspin--a novel protease inhibitor with tumor-suppressing activity in breast cancer.
• Acta Oncol. 2000; 39: 931-4
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• Maspin (mammary serpin) is a novel serine protease inhibitor related to the serpin family with a tumor-suppressing function in breast cancer. Maspin was originally identified from normal mammary epithelium by subtractive hybridization and might function as a class II tumor-suppressor gene. Maspin's decreased expression with increased level of malignancy and its loss in metastatic cells is regulated at the transcriptional level. Cytosin methylation and heterochromatinization in the promoter region might account for this down-regulation of maspin. Transfection of tumor cells with maspin cDNA inhibits invasion and motility and decreases tumor growth and metastatic ability in nude mice. Maspin interacts with the p53 tumor-suppressor pathway and function as an inhibitor of angiogenesis in vitro and in vivo. The progressive loss of expression of maspin during tumor progression makes this new protein an interesting diagnostic and prognostic marker. The re-expression of maspin by pharmacological intervention potentially offers a promising approach as a therapeutic option in breast cancer therapy.

• Milstone AM, Harrison LM, Bungiro RD, Kuzmic P, Cappello M
• A broad spectrum Kunitz type serine protease inhibitor secreted by the hookworm Ancylostoma ceylanicum.
• J Biol Chem. 2000; 275: 29391-9
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• Although blood-feeding hookworms infect over a billion people worldwide, little is known about the molecular mechanisms through which these parasitic nematodes cause gastrointestinal hemorrhage and iron deficiency anemia. A cDNA corresponding to a secreted Kunitz type serine protease inhibitor has been cloned from adult Ancylostoma ceylanicum hookworm RNA. The translated sequence of the A. ceylanicum Kunitz type inhibitor 1 (AceKI-1) cDNA predicts a 16-amino acid secretory signal sequence, followed by a 68-amino acid mature protein with a molecular mass of 7889 daltons. Recombinant protein (rAceKI-1) was purified from induced lysates of Escherichia coli transformed with the rAceKI-1/pET 28a plasmid, and in vitro studies demonstrate that rAceKI-1 is a tight binding inhibitor of the serine proteases chymotrypsin, pancreatic elastase, neutrophil elastase, and trypsin. AceKI-1 inhibitory activity is present in soluble protein extracts and excretory/secretory products of adult hookworms but not the infective third stage larvae. The native AceKI-1 inhibitor has been purified to homogeneity from soluble extracts of adult A. ceylanicum using size exclusion and reverse-phase high pressure liquid chromatography. As a potent inhibitor of mammalian intestinal proteases, AceKI-1 may play a role in parasite survival and the pathogenesis of hookworm anemia.

• Jackson RM, Russell RB
• The serine protease inhibitor canonical loop conformation: examples found in extracellular hydrolases, toxins, cytokines and viral proteins.
• J Mol Biol. 2000; 296: 325-34
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• Methods for the prediction of protein function from structure are of growing importance in the age of structural genomics. Here, we focus on the problem of identifying sites of potential serine protease inhibitor interactions on the surface of proteins of known structure. Given that there is no sequence conservation within canonical loops from different inhibitor families, we first compare representative loops to all fragments of equal length among proteins of known structure by calculating main-chain RMS deviation. Fragments with RMS deviation below a certain threshold (hits) are removed if residues have solvent accessibilities appreciably lower than those observed in the search structure. These remaining hits are further filtered to remove those occurring largely within secondary structure elements. Likely functional significance is restricted further by considering only extracellular protein domains. By comparing different canonical loop structures to the protein structure database, we show that the method is able to detect previously known inhibitors. In addition, we discuss potentially new canonical loop structures found in secreted hydrolases, toxins, viral proteins, cytokines and other proteins. We discuss the possible functional significance of several of the examples found, and comment on implications for the prediction of function from protein 3D structure.

• Araujo MS et al.
• Preliminary characterization of a Kazal-type serine protease inhibitor from Caiman crocodilus yacare plasma.
• Immunopharmacology. 1999; 45: 179-83
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• Blood serine protease inhibitors are becoming better understood and increasingly applied in blood clotting, cancer and other diseases. Reptiles are suitable models for blood coagulation and related processes, moreover, caiman is a good comparative model of a non-poisonous reptile. Recently, we reported the purification of a kininogen, the presence of proteases involved in blood clotting, and a serine protease inhibitor in Caiman crocodilus yacare plasma. In this paper, we described the partial sequence of an inhibitor (CcTI). The inhibitor is an 80-kDa protein, and it inactivates trypsin and chymotrypsin the hydrolysis of specific chromogenic substrates and in the degradation of gelatin. The inhibitor is member of Kazal-type inhibitor family and consists of several domains, its putative reactive site is Arg-His.

• Lopuska A, Polanowska J, Wilusz T, Polanowski A
• Purification of two low-molecular-mass serine proteinase inhibitors from chicken liver.
• J Chromatogr A. 1999; 852: 207-16
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• Two serine proteinase inhibitors, designated clTI-1 and clTI-2 were purified from livers of chickens to apparent homogeneity by a combination of ethanol-acetone fractionation, gel filtration and ion-exchange chromatography on CM-cellulose and Mono S columns. The inhibitor clTI-1 is a single polypeptide chain, low-molecular-mass protein (Mr about 6200), very stable to heat and ethanol. It inhibits chicken, porcine and bovine trypsins as well as human plasmin. The second protein, clTI-2 of Mr 17,000 was shown to be a very effective inhibitor of both trypsins and human cathepsin G. Since both inhibitors are sensitive to arginine modification with phenylglyoxal it is assumed that this amino acid residue is present at the P1 position of the reactive site peptide bond. The N-terminal amino acid sequence of 28 residues of clTI-2 (SVDVSKYPSTVSKDGRTLVACPRILSPV) revealed a high homology of this protein to the third domain of the chicken ovoinhibitor, whereas, the clTI-1 (APPAAEKYYSLPPGAPRYYSPVV) has some sequence identity to a fragment of the human inter-alpha-trypsin inhibitor.

• Bird PI
• Regulation of pro-apoptotic leucocyte granule serine proteinases by intracellular serpins.
• Immunol Cell Biol. 1999; 77: 47-57
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• Caspase activation and apoptosis can be initiated by the introduction of serine proteinases into the cytoplasm of a cell. Cytotoxic lymphocytes have evolved at least one serine proteinase with specific pro-apoptotic activity (granzyme B), as well as the mechanisms to deliver it into a target cell, and recent evidence suggests that other leucocyte granule proteinases may also have the capacity to kill if released into the interior of cells. For example, the monocyte/granulocyte proteinase cathepsin G can activate caspases in vitro, and will induce apoptosis if its entry into cells is mediated by a bacterial pore-forming protein. The potent pro-apoptotic activity of granzyme B and cathepsin G suggests that cells producing these (or other) proteinases would be at risk from self-induced death if the systems involved in packaging, degranulation or targeting fail and allow proteinases to enter the host cell cytoplasm. The purpose of the present review is to describe recent work on a group of intracellular serine proteinase inhibitors (serpins) which may function in leucocytes to prevent autolysis induced by the granule serine proteinases.

• Abts HF, Welss T, Mirmohammadsadegh A, Kohrer K, Michel G, Ruzicka T
• Cloning and characterization of hurpin (protease inhibitor 13): A new skin-specific, UV-repressible serine proteinase inhibitor of the ovalbumin serpin family.
• J Mol Biol. 1999; 293: 29-39
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• Epidermal keratinocytes are the primary target of the midrange ultraviolet part (UVB, 280-320 nm) of terrestrial sunlight. Analysis of the resulting UV response at the transcriptional level by differential display PCR identified a formerly unrecognized large group of repressed genes. Among those UV-repressible genes, a novel serine proteinase inhibitor (serpin) termed hurpin (HaCaT UV-repressible serpin) has been identified. The isolated full-length cDNAs harbour a 1176 bp open reading frame encoding a potential protein with 391 amino acid residues and a predicted molecular mass of approximately 44 kDa. The novel serpin has nearly 59 % amino acid identity with the squamous cell carcinoma antigen 1 (SCCA1) and squamous cell carcinoma antigen 2 (SCCA2). In addition, it displays all of the structural features unique to the ovalbumin family of serpins (ov-serpins). The amino acid sequence of the hinge region in the reactive site loop suggests that hurpin has the potential for protease inhibition. The putative reactive center P1-P1'residues were identified as Thr356-Ser357 by alignment with other ov-serpins. The physiological target protease is unknown and the in vitro translated hurpin does not form SDS-stable complexes with a variety of known serine proteases. Expression of hurpin is restricted to epidermal cells where two distinct transcripts of 3.0 and 3.4 kb are detectable. Furthermore, expression of hurpin appears to be related to the activation or proliferation state of keratinocytes, since hurpin transcripts are more abundant in immortalized keratinocytes (HaCaT) and in cultured normal human keratinocytes, compared to the expression in normal skin. Moreover, in psoriasis, a skin disease characterized by hyperproliferation of keratinocytes and responsive to therapeutic UV irradiation, overexpression of hurpin is noted in psoriatic skin lesions compared to non-lesional skin.

• Kojima S, Deguchi M, Miura K
• Involvement of the C-terminal region of yeast proteinase B inhibitor 2 in its inhibitory action.
• J Mol Biol. 1999; 286: 775-85
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• Yeast proteinase B inhibitor 2 (YIB2), which is composed of 74 amino acid residues, is an unusual serine protease inhibitor, since it lacks disulfide bonds. To identify its reactive site for proteases, we constructed an expression system for a synthetic YIB2 gene and then attempted to change the inhibitory properties of YIB2 by amino acid replacements. The purified wild-type YIB2 inhibited the activity of subtilisin BPN', a protein homologous to yeast proteinase B, although its binding ability was not strong, and a time-dependent decrease in its inhibitory activity was observed, demonstrating that wild-type YIB2 behaves as a temporary inhibitor when subtilisin BPN' is the target protease. Since YIB2 exhibits sequence homology to the propeptide of subtilisin, which inhibits a cognate protease using its C-terminal region, we replaced the six C-termi nal residues of YIB2 with those of the propeptide of subtilisin BPN' to make the mutant YIB2m1. This mutant exhibited markedly increased inhibitory activity toward subtilisin BPN' without a time-dependent decrease in its inhibitory activity. Replacement of only the C-terminal Asn of YIB2 by Tyr, or deletion of the C-terminal Tyr of YIB2m1, inhibited subtilisin, but the ability of these mutants to bind subtilisin and their resistance to proteolytic attack were weaker than those of YIB2m1, indicating that the C-terminal residue contributes to the interaction with the protease to a greater extent than the preceding five residues and that the resistance of YIB2 to proteolyic attack is closely related to its ability to bind a protease. These results demonstrate that YIB2 is a unique protease inhibitor that involves its C-terminal region in the interaction with the protease.

• Berger P, Kozlov SV, Cinelli P, Kruger SR, Vogt L, Sonderegger P
• Neuronal depolarization enhances the transcription of the neuronal serine protease inhibitor neuroserpin.
• Mol Cell Neurosci. 1999; 14: 455-67
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• Neuroserpin is an axonally secreted neuronal serine protease inhibitor. Based on its inhibitory activity towards tissue plasminogen activator (tPA) and its predominant expression in the cerebral cortex, the hippocampus, and the amygdala, a role for neuroserpin in the regulation of neural plasticity has been suggested. We recently found that neuroserpin mRNA is increased in cultured hippocampal neurons upon depolarization with elevated extracellular KCl. Using luciferase reporter constructs containing segments of the promoter region of the neuroserpin gene, we identified a 200-bp segment near the transcription initiation site that is responsible for both the neuron-specific expression of the neuroserpin gene and the enhanced transcription resulting from depolarization. Nerve growth factor, which alone had no effect on the expression of neuroserpin mRNA in hippocampal neurons, had a marked potentiating effect when supplied in combination with elevated extracellular KCl. In contrast, the transcription factor zif/268 blocked neuroserpin transcription. These results implicate neuroserpin as an activity-regulated modulator of tPA activity at the synapse and provide further support for the occurrence of activity-regulated proteolytic processes at the synapse.

• Takeno H, Yamamoto S, Tanaka T, Sakano Y, Kikuchi Y
• Selection of an RNA molecule that specifically inhibits the protease activity of subtilisin.
• J Biochem (Tokyo). 1999; 125: 1115-9
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• RNA ligands (RNA aptamers) to a protease subtilisin were selected from pools of random RNA by SELEX (systematic evolution of ligands by exponential enrichment) and by use of a subtilisin-immobilized Sepharose column. After eight rounds of selection, RNA aptamers were isolated by cloning to a plasmid vector. We characterized one of the selected RNA molecules. This RNA aptamer displayed specific inhibition toward the subtilisin activity, even when the assay for subtilisin was performed using the chromogenic small peptide as substrate, and almost no inhibitory activity toward trypsin and chymotrypsin, although these enzymes are serine proteases similar to subtilisin. These findings indicate that this RNA can differentially recognize the surfaces of similar proteases. Kinetic analysis of the RNA aptamer revealed that the inhibition constant (Ki) toward subtilisin was 2.5 microM.

• Xu Y, Carr PD, Guss JM, Ollis DL
• The crystal structure of bikunin from the inter-alpha-inhibitor complex: a serine protease inhibitor with two Kunitz domains.
• J Mol Biol. 1998; 276: 955-66
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• Bikunin is a serine protease inhibitor found in the blood serum and urine of humans and other animals. Its sequence shows internal repetition, suggesting that it contains two domains that resemble bovine pancreatic trypsin inhibitor (BPTI). A fragment of bikunin has been crystallised, its structure solved and subsequently refined against 2.5 A data. The two BPTI-like domains pack closely together and are related by an approximate 60 degrees rotation combined with a translation. These domains are very similar to each other and other proteins with this fold. The largest variations occur in the loops responsible for protease recognition. The loops of the first domain are unobstructed by the remaining protein. However, the loops of the second domain are close to the first domain and it is possible that protease binding may be affected or, in some cases, abolished by the presence of the first domain. Thus, cleavage of the two domains could alter the substrate specificity of domain II. Bikunin has a hydrophobic patch close to the N terminus of domain I, which is the most likely site for cell-surface receptor binding. In addition, there is a basic patch at one end of domain II that may be responsible for the inhibition of calcium oxalate crystallization in urine.

• Kakiuchi N et al.
• Non-peptide inhibitors of HCV serine proteinase.
• FEBS Lett. 1998; 421: 217-20
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• We screened a chemical library of 2000 compounds for inhibitors of hepatitis C virus (HCV) serine proteinase using an in vitro screening method measuring the hydrolysis of the peptide substrate. Three compounds were found to be the most potent inhibitors (IC50 < 10(-5) M). Two of them had a similar structure, that of halogenated benzanilide, and were not inhibitory for common serine proteinases. They inhibited the enzyme non-competitively with the substrate. Together with the result of the analogous compounds in the chemical library, the presumed structural requirements of the inhibition are pointed out.

• Roch P, Ville P, Cooper EL
• Characterization of a 14 kDa plant-related serine protease inhibitor and regulation of cytotoxic activity in earthworm coelomic fluid.
• Dev Comp Immunol. 1998; 22: 1-12
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• We have purified and characterized the serine protease inhibitor activity contained in the coelomic fluid of the earthworms, Eisenia. Serine protease inhibitor activity was stable between pH3 and 9.5, not flocculable by pH 3.0 and resistant to 100 degrees C for 15 min. or to 4 degrees C for 24 h. Ten microL of coelomic fluid was sufficient to inhibit in vitro the protease activity of 0.12 microgram of trypsin. Injection of living bacteria into earthworms resulted in increased serine protease activity 1-2 days post-injection, and increased serine protease inhibitor activity on day 4, suggesting that serine protease inhibitor is responsible for serine protease neutralization. Purified to homogeneity by affinity chromatography on trypsin, the serine protease inhibitor of Eisenia is a monomer of 14 kDa. Its partial NH2 amino acid sequence revealed a basic hydrophobic fragment which shared 68-75% homologies and 47-60% identities with several plant serine protease inhibitors. Eisenia cytotoxic activity due to the two fetidins of 40 and 45 kDa was stimulable in vitro by several serine proteases. Incubation with soybean trypsin inhibitor variant a (STIa) resulted in less cytotoxicity. The inhibitory effect occurred only when STIa was added before cell disruption. Interpretative cytotoxic scheme involving the release of intracellular cytotoxic proteins, intracellular trypsin-like activator and extracellular serine protease inhibitor suggests regulatory mechanisms for cellular/humoral immune system of earthworms.

• Robson P, Li F, Youson JH, Keeley FW
• Identification and characterization of a serpin with differential expression during the life cycle of the sea lamprey.
• Comp Biochem Physiol B Biochem Mol Biol. 1998; 120: 253-63
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• We have cloned a member of the serine proteinase inhibitor gene superfamily from the sea lamprey, Petromyzon marinus. The predicted translation product contains a putative signal peptide and mRNA expression is localized mainly to the liver. Northern blot analysis indicates that the mRNA increases in the larvae and peaks in late larval life. At the onset of metamorphosis there is a approximately 10-fold drop after which it remains low. These changes correspond with levels of circulating thyroid hormone suggesting that this serpin is involved in or regulated by molecular signals that induce metamorphosis in the lamprey. Use of alignments and structural information from other serpins indicates that the lamprey serpin has the potential to be inhibitory. In addition the lamprey serpin contains methionine and serine at the P1 and P1' positions, respectively. Appropriate residues at positions important in allowing the insertion of strand s4A into beta-sheet A that occurs upon cleavage in inhibitory serpins are also found in the lamprey serpin.

• Berger P, Kozlov SV, Krueger SR, Sonderegger P
• Structure of the mouse gene for the serine protease inhibitor neuroserpin (PI12).
• Gene. 1998; 214: 25-33
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• Neuroserpin (PI12), initially identified as an axonally secreted protein in cultured chicken dorsal root ganglion neurons, belongs to the serpin family of the serine protease inhibitors and is mainly expressed by neurons of both the developing and the adult nervous system. Here we report on the cloning and structural characterization of the neuroserpin gene of the mouse. The murine neuroserpin gene spans over more than 55kb and consists of nine exons. The positions and phases of the exonintron borders are completely conserved between neuroserpin and its nearest homologues, protease nexin-1 and plasminogen activator inhibitor-1. A single transcription initiation site, which is colocalized with a potential initiation (Inr) sequence, has been determined by primer extension and RNase protection. Sequence analysis revealed a TATA-less promoter with a CAAT box and several sites for the general transcription factor Sp1 and the neuron-specific transcription factor AP-2.

• Ozaki K et al.
• Isolation and characterization of a novel human pancreas-specific gene, pancpin, that is down-regulated in pancreatic cancer cells.
• Genes Chromosomes Cancer. 1998; 22: 179-85
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• By means of the differential display method, we isolated a novel human gene that is expressed specifically in pancreas. The cDNA, designated "pancpin," contained an open reading frame of 1,215 nucleotides encoding a 405 amino acid protein, showing a high degree of similarity to serine protease inhibitors belonging to the serpin superfamily. To investigate its possible role in pancreatic carcinogenesis, we looked for genetic alterations of this gene in pancreatic cancer cell lines and primary pancreatic cancer tissues. Expression of pancpin was barely detectable in any of the four pancreatic cancer cell lines examined, and very weak also in 10 of 13 pancreatic cancer tissues. A somatic missense mutation at codon 221 was found in two of 16 primary pancreatic cancers. These findings indicate that down-regulation of pancpin expression may play a significant role in development or progression of pancreatic cancer.

• Joubert AM, Louw AI, Joubert F, Neitz AW
• Cloning, nucleotide sequence and expression of the gene encoding factor Xa inhibitor from the salivary glands of the tick, Ornithodoros savignyi.
• Exp Appl Acarol. 1998; 22: 603-19
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• The N-terminal sequence of the competitive and slow tight-binding factor Xa inhibitor (fXaI; Ki = 0.83 +/- 0.10 nM) isolated from the salivary glands of Ornithodoros savignyi ticks (Acari: Argasidae) was employed to design a degenerate gene-specific primer (GSP) for 3'-rapid amplification of cDNA ends (3'-RACE). The primer consisted of a sequence encoding for amino acid residues 5-11. A full-length gene was next constructed from the 3'-RACE product in a two-step PCR procedure and successfully expressed by the BAC-TO-BAC baculovirus expression system. The deduced amino acid sequence of the gene showed 46% identity and 78% homology to an fXaI (TAP) from Ornithodoros moubata. Recombinant fXaI (rfXaI) consists of 60 amino acid residues, has a molecular mass of approximately 7 kDa and inhibited fXa by approximately 91%. The availability of the rfXaI will aid further investigations of its potential for therapeutic applications and as vaccine against tick infestation. The authentic nucleotide sequence of the gene encoding tick fXaI furthermore enables studies at the genetic level and probing of other tick species for similar and related genes.

• Kawamura K, Hayata D, Fujiwara S, Yubisui T
• Serine protease inhibitors expressed in the process of budding of tunicates as revealed by EST analysis.
• J Biochem (Tokyo). 1998; 124: 1004-12
• Display abstract

• To identify genes expressed during budding of the tunicate Polyandrocarpa misakiensis, we isolated and sequenced 624 clones from a directionally constructed cDNA library to prepare a catalog of expressed sequence tags (ESTs). A total of 233 ESTs matched genes of known sequence in the SwissProt database. About 24% out of them showed high similarity to ribosomal proteins, twice the value (12%) of pre-budding animals. ESTs involved in the respiratory chain also appeared with significant redundancy, suggesting that tunicate budding is accompanied by the enhancement of energy conversion as well as protein synthesis. Serine protease inhibitor (serpin) afforded another striking example of a gene that was highly expressed in the process of budding. The deduced amino acid sequences of five serpin cDNAs all had two consensus signatures of the Kazal's type of secretory protease inhibitor, one of which had an active site for trypsin and the other for elastase. In line with this, recombinant GST-fusion protein showed both trypsin and elastase inhibitor activities. In accordance with the EST analysis, the hemolymph taken from the budding stage showed the highest activity of trypsin inhibitor. We discuss a possible role that Polyandrocarpa serpins may play in bud development by counteracting trypsin-like serine protease, which could facilitate dedifferentiation of formative tissues.

• Moser M, Auerswald E, Mentele R, Eckerskorn C, Fritz H, Fink E
• Bdellastasin, a serine protease inhibitor of the antistasin family from the medical leech (Hirudo medicinalis)--primary structure, expression in yeast, and characterisation of native and recombinant inhibitor.
• Eur J Biochem. 1998; 253: 212-20
• Display abstract

• We have reported earlier the isolation and amino acid composition of bdellin A from medical leech, and characterised it as an inhibitor of trypsin, plasmin and acrosin [Fritz, H., Gebhardt, M., Meister, R. & Fink, E. (1971) in Proceedings of the international research conference on proteinase inhibitors (Fritz, H. & Tschesche, H., eds) pp. 271-280, Walter de Gruyter, Berlin]. In the present study, one of several chromatographic forms of this inhibitor was isolated from a semi-pure preparation. Elucidation of its amino acid sequence revealed that bdellin A is a member of the antistasin family. Therefore, it was renamed bdellastasin to avoid confusion with bdellin B, which is another trypsin-plasmin inhibitor from the medical leech, but of the Kazal type. Furthermore, a synthetic gene of bdellastasin was constructed, and the protein expressed in Saccharomyces cerevisiae with yields of 29 mg/l. The recombinant bdellastasin was purified by hydrophobic interaction and anion-exchange chromatography. Comparison by mass spectroscopy, far-ultraviolet circular dichroism studies, sequence determination, and inhibition characteristics demonstrated the identity of recombinant and native bdellastasin. The Ki values of bdellastasin for inhibition of bovine trypsin and human plasmin are in the nanomolar range; no inhibition was detected for factor Xa, thrombin, tissue kallikrein, plasma kallikrein and chymotrypsin. Circular dichroism analyses indicated that bdellastasin is devoid of secondary-structural elements.

• Bottomley SP, Stone SR
• Protein engineering of chimeric Serpins: an investigation into effects of the serpin scaffold and reactive centre loop length.
• Protein Eng. 1998; 11: 1243-7
• Display abstract

• The exposed Serpin reactive centre loop controls the specificity of the serpin proteinase interaction. Mutations within this region have been used to generate novel potentially therapeutic inhibitors. In this study we examine the effect of the serpin scaffold and reactive centre loop length upon the generation of such inhibitors. The reactive centre loop regions, P7-P3', of alpha1-antitrypsin and alpha1-antichymotrypsin were replaced by the corresponding residues of the viral serpin, Serp1, to form AT/Serp1 and ACT/Serp1, respectively. AT/Serp1 formed SDS stable complexes with a range of proteinases with association rate constants for plasmin, tissue plasminogen activator, urokinase, thrombin and factor Xa of approximately 10(4) M(-1)s(-1) and a stoichiometry of inhibition of approximately 1 for all of them. ACT/Serp1, however, formed SDS-stable complexes with only plasmin and thrombin with association rate constant 100-fold slower than AT/Serp1 and an increased stoichiometry of inhibition. The reactive centre loop of ACT/Serp1 is four amino acid residues longer than AT/Serp1. These four additional residues (VETR) were inserted into AT/Serp1 to form AT/Serp1(VETR). AT/Serp1(VETR) formed SDS stable complexes with plasmin, thrombin and tissue plasminogen activator similar to AT/Serp1, however, the association rate constants were 10-fold slower than those observed with AT/Serp1, while the stoichiometry of inhibition remained around 1. These results suggest that the additional reactive centre loop residues effect the rate of initial complex formation by placing the reactive centre loop in a non-ideal conformation. This study demonstrates that both reactive centre loop length and serpin scaffold are important in defining the inhibitory characteristics of a serpin.

• Lu CC, Nguyen T, Morris S, Hill D, Sakanari JA
• Anisakis simplex: mutational bursts in the reactive site centers of serine protease inhibitors from an ascarid nematode.
• Exp Parasitol. 1998; 89: 257-61

• Hill RM et al.
• A new intracellular serine protease inhibitor expressed in the rat pituitary gland complexes with granzyme B.
• FEBS Lett. 1998; 440: 361-4
• Display abstract

• We have cloned a novel serpin (raPIT5a) from a rat pituitary cDNA library which is structurally related to members of the ovalbumin subfamily of serine protease inhibitors. This new cDNA encodes a 374-amino acid protein, designated raPIT5a. raPIT5a was expressed in specific cells in the intermediate and anterior lobes of the pituitary. Recombinant raPIT5a was not secreted suggesting raPIT5a functions to inhibit intracellular proteases. Recombinant raPIT5a formed an SDS-stable complex with human granzyme B, a serine protease which induces apoptosis by activating members of the caspase enzyme family. These data suggest raPIT5a may have a role in regulating granzyme B or related enzymes and apoptosis in the pituitary gland.

• Schrimpf SP et al.
• Human neuroserpin (PI12): cDNA cloning and chromosomal localization to 3q26.
• Genomics. 1997; 40: 55-62
• Display abstract

• Neuroserpin is a novel serine protease inhibitor of the serpin family. It has been reported as a 55-kDa glycoprotein that is secreted from the axons of cultured central and peripheral nervous system neurons. In situ hybridization and Northern blot analyses at different developmental stages of the chicken revealed that neuroserpin is predominantly expressed in the nervous system and that most cells expressing neuroserpin can be qualified as bona fide neurons. We have isolated the full-length cDNA for human neuroserpin from a fetal retina cDNA library. The open reading frame of the cDNA of human neuroserpin, like that of its chicken counterpart, encodes a protein of 410 amino acids. The human and the chicken neuroserpin exhibit an amino acid sequence identity of 80%. Northern blot analysis of human organs demonstrated predominant expression of neuroserpin in the brain. By fluorescence in situ hybridization the human neuroserpin gene (HGMW-approved symbol PI12) was mapped to region q26 of chromosome 3.

• Lawrence DA
• The serpin-proteinase complex revealed.
• Nat Struct Biol. 1997; 4: 339-41

• Wilczynska M, Fa M, Karolin J, Ohlsson PI, Johansson LB, Ny T
• Structural insights into serpin-protease complexes reveal the inhibitory mechanism of serpins.
• Nat Struct Biol. 1997; 4: 354-7

• Luisetti M
• Mapping the activities of secretory leukoprotease inhibitor.
• Monaldi Arch Chest Dis. 1997; 52: 496-7

• Groutas WC, Ruan S, Kuang R, Hook JB, Sands H
• Inhibition of human leukocyte proteinase 3 by a novel recombinant serine proteinase inhibitor (LEX032).
• Biochem Biophys Res Commun. 1997; 233: 697-9
• Display abstract

• The interaction of a bioengineered serpin (LEX032) with human leukocyte proteinase 3 (PR 3) has been investigated. LEX032 was found to be a time-dependent inhibitor of PR 3, forming a highly-stable enzyme-inhibitor complex (Ki 12 nM).

• Kawaguchi T et al.
• Purification and cloning of hepatocyte growth factor activator inhibitor type 2, a Kunitz-type serine protease inhibitor.
• J Biol Chem. 1997; 272: 27558-64
• Display abstract

• Hepatocyte growth factor (HGF) activator is a serine protease responsible for proteolytic activation of HGF in response to tissue injury and thus plays an important role in the regulation of biological functions of HGF in regenerating tissue. We previously purified an inhibitor of HGF activator (HGF activator inhibitor type 1, HAI-1) from the conditioned medium of a human stomach carcinoma cell line MKN45 and cloned its cDNA. HAI-1 is a novel member of the Kunitz family of serine protease inhibitors. In the present study, we purified a second type of HGF activator inhibitor (HAI-2) from the conditioned medium of MKN45 cells and molecularly cloned its cDNA. The cDNA sequence revealed that HAI-2 is derived from a precursor protein of 252 amino acids and contains two Kunitz domains, indicating that HAI-2 is also a member of the Kunitz family of serine protease inhibitors. The primary translation product of HAI-2 has a hydrophobic sequence in the COOH-terminal region, suggesting that, like HAI-1, HAI-2 is produced in a membrane-associated form and secreted in a proteolytically truncated form. Because HAI-2 and HAI-1 are potent inhibitors specific for HGF activator, they may be involved in regulation of proteolytic activation of HGF in injured tissues.

• Zitnik RJ et al.
• The cloning and characterization of a murine secretory leukocyte protease inhibitor cDNA.
• Biochem Biophys Res Commun. 1997; 232: 687-97
• Display abstract

• Human secretory leukocyte protease inhibitor (hSLPI) is produced by epithelial cells at mucosal surfaces, where it regulates both the neutrophil-mediated inflammation that characterizes inflammatory diseases, and pathogens themselves via both antiprotease and "defensin-like" activities. Additionally, hSLPI may regulate other processes such as cutaneous desquamation and placental invasiveness. To better understand the primary physiologic roles of SLPI, it will be important to establish a genetically tractable animal model, the most attractive candidate being the mouse. In this report, the cloning and characterization of murine (m) SLPI is described. mSLPI is encoded by a single copy gene, and appears structurally highly similar to hSLPI. At the same time, significant differences between mSLPI and hSLPI are presented, notably a difference in expression pattern, and a structural difference in the protease binding site that correlates with a difference in the spectrum of protease inhibiton. Such species-specific evolution of this protease inhibitor is notable given that species-specific structure-function differences have previously been reported for the alpha-1 antitrypsin family.

• Rubin H
• Serine protease inhibitors (SERPINS): where mechanism meets medicine.
• Nat Med. 1996; 2: 632-3

• Lundwall A, Ulvsback M
• The gene of the protease inhibitor SKALP/elafin is a member of the REST gene family.
• Biochem Biophys Res Commun. 1996; 221: 323-7
• Display abstract

• Members of the REST gene family characteristically have a transcription unit consisting of three exons. The first and the last exon are conserved among members, while the second exon--encoding almost all of the mature protein--differs considerably. The so far known REST genes are highly, and almost exclusively, expressed in the seminal vesicles. By sequence analysis we have now identified the gene for the protease inhibitor SKALP/elafin as a new member of the REST gene family. The protein is expressed in the epidermis and serves, like the product of several REST genes, as substrate for transglutaminase. We have also found what seems to be a locus encompassing both transglutaminases and REST genes centered around the region q12 on the human chromosome 20, raising the question whether the enzymes and substrates have evolved in parallel.

• Tsuda M, Kitagawa K, Imaizumi K, Wanaka A, Tohyama M, Takagi T
• Induction of SPI-3 mRNA, encoding a serine protease inhibitor, in gerbil hippocampus after transient forebrain ischemia.
• Brain Res Mol Brain Res. 1996; 35: 314-8
• Display abstract

• We cloned genes the expression of which is induced in the Mongolian gerbil (Meriones unguiculatus) hippocampus after transient forebrain ischemia by a differential display technique. Among these genes, a rat serine protease inhibitor SPI-3 homologue was isolated. Present analyses suggested that the expression of gerbil SPI-3 mRNA was closely associated with delayed neuronal death and may block activities of proteases leaking from degenerating neurons or may support neuronal survival.

• Wright HT
• The structural puzzle of how serpin serine proteinase inhibitors work.
• Bioessays. 1996; 18: 453-64
• Display abstract

• Serine proteinase cleavage of proteins is essential to a wide variety of biological processes and is primarily regulated by protein inhibitors. Many inhibitors are conformationally rigid simulations of optimal serine proteinase substrates, which makes them highly efficient competitive inhibitors of target proteinases. In contrast, members of the serpin family of serine proteinase inhibitors display extensive flexibility and polymorphism, particularly in their reactive site segments and in beta-sheet secondary structure, which can take up and expel strands. Reactive site and beta-sheet polymorphism appear to be coupled in the serpins and may account for the extreme stability of serpin-proteinase complexes through the insertion of the reactive site strand into a beta-sheet. These unusual properties may have opened an adaptive pathway of proteinase regulation that was unavailable to the conformationally rigid proteinase inhibitors.

• Rasmussen SK, Dahl SW, Norgard A, Hejgaard J
• A recombinant wheat serpin with inhibitory activity.
• Plant Mol Biol. 1996; 30: 673-7
• Display abstract

• A full-length clone encoding the wheat (Triticum aestivum L.) serpin WSZ1 was isolated from a cDNA library based on mRNA from immature grain. The 398 amino acid sequence deduced from the cDNA was corroborated by sequencing CNBr peptides of WSZ1 purified from resting grain. WSZ1 belongs to the subfamily of protein Z-type serpins and the amino acid sequence is 70% identical with the barley serpins BSZ4 and BSZx and 27-33% identical with human serpins such as alpha 1-proteinase inhibitor, antithrombin III, and plasminogen activator inhibitor. The cDNA was subcloned in the pET3d expression vector, equipped with a histidine affinity tag at the N-terminus and expressed in Escherichia coli BL(21) DE3 pLysS. Recombinant WSZ1 from the soluble fraction was partially purified on Ni-NTA agarose and MonoQ columns and shown to form SDS-stable complexes with alpha-chymotrypsin. Southern blots and amino acid sequencing indicated that only few serpins are encoded by wheat, but at least three distinct genes are expressed in the grain. Cleavage experiments on a chymotrypsin column suggested a Gln-Gln reactive site bond not previously observed in inhibitory serpins.

• Elliott PR, Lomas DA, Carrell RW, Abrahams JP
• Inhibitory conformation of the reactive loop of alpha 1-antitrypsin.
• Nat Struct Biol. 1996; 3: 676-81
• Display abstract

• The reactive site loop of the serpin family of serine proteinase inhibitors is flexible and can adopt a number of diverse conformations. A 2.9 A resolution structure of alpha 1-antitrypsin-the principal proteinase inhibitor in human plasma-shows the loop in a stable canonical conformation matching that found in all other families of serine proteinase inhibitors. This unexpected finding in the absence of loop insertion into the body of the molecule favours a two-stage mechanism of inhibition and provides a model for the heparin activation of antithrombin. The beta-pleated strand conformation of the loop also accounts for the polymerization of the serpins in disease and for their association with other beta-sheet structures, most notably the beta-amyloid of Alzheimer's disease.

• New L, Liu K, Kamali V, Plowman G, Naughton BA, Purchio AF
• cDNA cloning of rasp-1, a novel gene encoding a plasma protein associated with liver regeneration.
• Biochem Biophys Res Commun. 1996; 223: 404-12
• Display abstract

• We constructed a cDNA library using mRNA isolated from liver 48 hr after hepatectomy (HX) and screened it by differential hybridization using cDNA from normal and regenerating rat liver. We isolated one clone termed regeneration-associated serpin-1 (rasp-1) that was expressed in normal liver but was upregulated approximately 3-4 fold by 48 hr after HX. DNA sequence analysis of rasp-1 indicated that it encoded a novel 436 amino acid secreted protein. Moderate homology was found with several members of the serpin family of serine-protease inhibitors. The 1.7 kb raps-1 mRNA was highly expressed in liver but not in brain, heart, kidney, lung, testis or spleen. We also found the RASP-1 protein in normal and HX rat plasma using a polyclonal antibody generated against a deduced peptide of rasp-1. Rasp-1 may encode a novel serine-protease inhibitor associated with liver regeneration.

• Fitzpatrick PA, Wong DT, Barr PJ, Pemberton PA
• Functional implications of the modeled structure of maspin.
• Protein Eng. 1996; 9: 585-9
• Display abstract

• The tumor suppressor maspin (mammary-specific serpin) is an unstable serpin that does not undergo the stressed to relaxed transition typical of proteinase inhibitory serpins and, consequently, is not likely to function as a serine proteinase inhibitor. This suggests that the positioning and configuration of the reactive site loop (RSL) of maspin are likely to resemble those of ovalbumin, the best studied non-inhibitory serpin. Accordingly, the tertiary structure of maspin has been modeled on the crystal structure of native ovalbumin. Biochemical data and the modeled theoretical structure of maspin reveal the absence of disulfide bonds in the molecule and the presence of an unstable RSL that adopts a distorted helical structure. We confirm that the RSL is extremely sensitive to limited proteolysis and suggest that this may provide a structural basis for the proteolytic inactivation of maspin, a process that is likely to modulate the activity of maspin in biological systems.

• Hirose S et al.
• Discovery of a new type of proteinase inhibitor family whose members have an anchoring sequence.
• Adv Exp Med Biol. 1996; 389: 43-9

• Tamechika I et al.
• Accelerated evolution in inhibitor domains of porcine elafin family members.
• J Biol Chem. 1996; 271: 7012-8
• Display abstract

• Through the analysis of the porcine gene encoding the elastase inhibitor elafin, we demonstrated that there are at least three closely related members of the elafin family, and their genes have arisen by accelerated evolution. A porcine genomic DNA library was screened with a previously cloned human elafin cDNA probe, and several positive clones were obtained that can be distinguished by a combination of restriction enzymes. Sequence analysis of these clones revealed the presence of three homologous members whose genes, all consisting of three exons and two introns, are almost identical except the exon 2 sequences encoding the inhibitor domain called "WAP motif"; the intron sequences are related to each other with sequence similarities of 93-98%, whereas the exon 2 sequences exhibited only 60-77% similarities among the three members. The extreme divergence in the exon 2 sequences compared to the highly conserved intron sequences may be generated by accelerated mutations confined in a short stretch of the genes following recent duplication events of a single ancestral gene. An RNase protection assay indicated that the messages of the elafin family members are abundantly expressed in the trachea and intestine, suggesting that the most likely selective forces for the accelerated evolution are extrinsic proteinases produced by invasive microorganisms.

• Ascenzi P, Amiconi G, Bode W, Bolognesi M, Coletta M, Menegatti E
• Proteinase inhibitors from the European medicinal leech Hirudo medicinalis: structural, functional and biomedical aspects.
• Mol Aspects Med. 1995; 16: 215-313

• Huang CJ, Lee MS, Huang FL, Chang GD
• A protease inhibitor of the serpin family is a major protein in carp perimeningeal fluid: II. cDNA cloning, sequence analysis, and Escherichia coli expression.
• J Neurochem. 1995; 64: 1721-7
• Display abstract

• A cDNA clone, pCP9, has been isolated from a common carp liver cDNA library by immunoscreening with polyclonal antiserum raised against purified bighead carp alpha 1-antitrypsin. This clone is 1,396 bp in length and has an open reading frame encoding a protein of 410 amino acid residues. The deduced amino acid sequence shows moderate homology to human alpha 1-antitrypsin (38%), guinea pig contrapsin (35%), human alpha 1-antichymotrypsin (34%), and human proteinase C inhibitor (31%), all members of the serine protease inhibitor (serpin) family. To confirm further that the cDNA clone was derived from the authentic carp alpha 1-antitrypsin gene, the presumptive mature protein of pCP9 was expressed in Escherichia coli. The molecular mass of the recombinant protein matched that predicted from the nucleotide sequence. This recombinant protein, which was recognized by antiserum against native alpha 1-antitrypsin, was capable of formation of serpin-enzyme complexes with trypsin, chymotrypsin, and elastase. Therefore, we conclude that the protein encoded by the pCP9 clone is indeed carp alpha 1-antitrypsin. Expression of alpha 1-antitrypsin in brain was confirmed by reverse transcription and polymerase chain reaction performed on mRNA derived from both common carp and bighead carp brain.

• Jiang H, Mulnix AB, Kanost MR
• Expression and characterization of recombinant Manduca sexta serpin-1B and site-directed mutants that change its inhibitory selectivity.
• Insect Biochem Mol Biol. 1995; 25: 1093-100
• Display abstract

• Hemolymph of Manduca sexta contains a number of serine proteinase inhibitors from the serpin superfamily. During formation of a stable complex between a serpin and a serine proteinase, the enzyme cleaves a specific peptide bond in an exposed loop (the reactive-site region) at the surface of the serpin. The amino acid residue on the amino-terminal side of this scissile bond, the P1 residue, is important in defining the selectivity of a serpin for inhibiting different types of serine proteinases. M. sexta serpin-1B, with alanine at the position predicted from sequence alignments to be the P1 residue, was previously named alaserpin. This alanyl residue was changed by site-directed mutagenesis to lysine (A343K) and phenylalanine (A343F). The serpin-1B cDNA and its mutants were inserted into an expression vector, H6pQE-60, and the serpin proteins were expressed in Escherichia coli. Affinity-purified recombinant serpins selectively inhibited mammalian serine proteinases: serpin-1B inhibited elastase; serpin-1B(A343K) inhibited trypsin, plasmin, and thrombin; serpin-1B(A343F) inhibited chymotrypsin as well as trypsin. All three serpins inhibited human cathepsin G. This insect serpin and its site-directed mutants associated with mammalian serine proteinases at rates similar to those reported for mammalian serpins. Serpin-1B and its mutants formed SDS-stable complexes with the enzymes they inhibited. The scissile bond was determined to be between residues 343 and 344 in wild-type serpin-1B and in serpin-1B with mutations at residue 343. These results demonstrate that the P1 alanine residue defines the primary selectivity of serpin-1B for elastase-like enzymes, and that this selectivity can be altered by mutations at this position.

• Perry DJ
• Antithrombin and its inherited deficiencies.
• Blood Rev. 1994; 8: 37-55
• Display abstract

• Human antithrombin is the major inhibitor of the coagulation serine proteases accounting for approximately 80% of the thrombin inhibitory activity of plasma. It is a member of the serpin family of serine protease inhibitors and in common with some other members of this family it undergoes a dramatic increase in its inhibitory activity in the presence of heparin and other sulphated glycosaminoglycans. Two functional domains in antithrombin are recognised, the reactive site domain which interacts with the active site serine residue of the protease and the heparin binding domain. The gene for antithrombin has been cloned and its entire nucleotide sequence determined. A deficiency or functional abnormality of antithrombin may result in an increased risk of thromboembolic disease. Such deficiencies are estimated to affect as many as 1:300 of the general population and 3 to 5% of patients with thrombotic disease. On the basis of functional and immunological antithrombin assays, antithrombin deficiency may be subdivided into Types I and II. Type I disease is due to a wide variety of heterogeneous DNA mutations whilst in Type II disease missense mutations leading to single amino acid substitutions have been identified in all cases. Clinically, Type I antithrombin deficiency is associated with recurrent thromboembolic disease whereas in Type II deficiency the risk of thrombosis is closely related to the position of the mutation within the protein. Thus, heterozygotes with mutations within the heparin binding domain of antithrombin have a relatively low risk of thrombosis compared to those with mutations at or close to the reactive site of the molecule.

• Potempa J, Korzus E, Travis J
• The serpin superfamily of proteinase inhibitors: structure, function, and regulation.
• J Biol Chem. 1994; 269: 15957-60

• Sager R et al.
• RNA genetics of breast cancer: maspin as paradigm.
• Cold Spring Harb Symp Quant Biol. 1994; 59: 537-46

• Sheng S, Pemberton PA, Sager R
• Production, purification, and characterization of recombinant maspin proteins.
• J Biol Chem. 1994; 269: 30988-93
• Display abstract

• Maspin, a novel mammary serine protease inhibitor, was shown to have tumor suppressing activity (Zou, Z., Anisowicz, A., Hendrix, M. J. C., Thor, A., Neveu, M., Sheng, S., Rafidi, K., Seftor, E., and Sager, R. (1994) Science 263, 526-529). In this paper, we report the production of recombinant glutathione S-transferase-maspin fusion protein, expressed in the bacterium Escherichia coli, and recombinant maspin, expressed in the insect Spodoptera frugiperda cells. The fusion protein was purified by glutathione affinity chromatography. Maspin expressed in insect cells was purified by a combination of Bio-Rad AG1-2X anion exchange chromatography and heparin affinity chromatography. The recombinant maspin from insect cells was cleaved at the putative reactive center, as confirmed by protein sequencing. Both recombinant proteins demonstrated strong inhibitory effects on the invasion by two breast tumor cell lines across reconstituted basement membranes and such inhibitory effect was abolished in the presence of the polyclonal antibody made against the reactive center region of maspin. The trypsin-cleaved recombinant maspin did not inhibit invasion, indicating that the inhibitory activity requires the intact putative reactive center. This paper provides evidence that recombinant maspin protein itself inhibits invasion, and supports the role of maspin as a tumor suppressor.

• Teschauer WF, Mentele R, Sommerhoff CP
• Primary structure of a porcine leukocyte serpin.
• Eur J Biochem. 1993; 217: 519-26
• Display abstract

• Inhibitors of neutral serine proteinases (serpins) have been shown to be colocalized with their target enzymes in leukocytes of several mammalian species. Here we report the purification and complete primary structure of a cytosolic inhibitor from porcine granulocytes which is directed against neutrophil elastase. Two molecular mass forms of the leukocyte neutral proteinase inhibitor (LNPI) were isolated by affinity and ion-exchange chromatography followed by gel filtration, and identified as the inhibitorily active monomer and homodimer of the inhibitor protein. According to the amino acid sequence the molecular mass of the non-glycosylated inhibitor was calculated to 42,597 Da (378 amino acid residues). A sequence identity of 81% was found between LNPI and the homologous elastase inhibitors from both human and equine leukocytes, whereas only 50% of the positions are identical in LNPI and human plasminogen activator inhibitor 2. These data suggest that LNPI is a member of a new group of cytosolic serpins closely related to the ovalbumin branch of the superfamily.

• Sallenave JM, Silva A
• Characterization and gene sequence of the precursor of elafin, an elastase-specific inhibitor in bronchial secretions.
• Am J Respir Cell Mol Biol. 1993; 8: 439-45
• Display abstract

• Human bronchial mucous secretions have been shown to contain inhibitors of serine proteinases secreted by neutrophils. The role of these inhibitors is probably to control the enzymes secreted in the airways and in the lung interstitium. Three of these inhibitors have been identified and characterized: alpha 1-proteinase inhibitor, mucus proteinase inhibitor, and elafin. The elafin molecule, a 6.0 kD inhibitor of serine proteinases shows homology with mucus proteinase inhibitor. We recently isolated both molecules in bronchial secretions. In this report, we present evidence for the existence of a precursor of the elafin molecule. We have cloned and sequenced the gene for this precursor and show that it is composed of three exons. The coding information for a 117 amino acid precursor protein of elafin (inclusive of the signal peptide) is contained in the first two exons. This was confirmed at the mRNA and protein levels. By Northern Blot analysis we detected a 800 bp long product, and by immunoaffinity we detected in sputum and in cultured epithelial cell supernatant (NCI-H322 cell line) a 12 kD protein species cross-reacting with anti-elafin IgG. The finding of possible cross-linking function for the precursor in addition to its antiproteinase activity indicates a possible role for this molecule as a cross-linker agent in the extracellular matrix.

• Chai KX, Chen LM, Chao J, Chao L
• Kallistatin: a novel human serine proteinase inhibitor. Molecular cloning, tissue distribution, and expression in Escherichia coli.
• J Biol Chem. 1993; 268: 24498-505
• Display abstract

• We have recently purified a novel human serine proteinase inhibitor (serpin), designated as kallistatin, which binds to tissue kallikrein and inhibits kallikrein's kininogenase and amidolytic activities. In the present studies, we have cloned a full-length cDNA encoding kallistatin from human liver RNA by the polymerase chain reaction. The cDNA is 1284 base pairs in length and encodes 427 amino acid residues, including a 26-residue signal peptide and a 401-residue mature peptide. The translated amino acid sequence of kallistatin matches with the protein sequence and shares 44-46% sequence identity with human alpha 1-antichymotrypsin, protein C inhibitor, corticosteroid-binding globulin, alpha 1-antitrypsin, thyroxin-binding globulin, and rat kallikrein-binding protein. Kallistatin is a new member of the serpin superfamily with a unique reactive site P1-P1' of Phe-Ser. Four potential glycosylation sites are found in the translated amino acid sequence of kallistatin. In a Southern blot analysis following reverse transcription and polymerase chain reaction, kallistatin was found to be expressed in human liver, stomach, pancreas, kidney, aorta, testes, prostate, artery, atrium, ventricle, lung, renal proximal tubular cell, and a colonic carcinoma cell line T84. A genomic Southern blot using the full-length kallistatin cDNA probe revealed simple banding patterns suggesting the gene encoding kallistatin is single-copied. The kallistatin cDNA encoding the mature peptide was expressed in Escherichia coli. The recombinant kallistatin forms an SDS-stable complex with 125I-human tissue kallikrein and has a molecular mass of 40 kDa. The cloning of human kallistatin cDNA established the identity of the novel kallikrein inhibitor and its expression in a functional form in E. coli provides means for studying its structure-function relationship through protein engineering.

• Gettins P, Patston PA, Schapira M
• The role of conformational change in serpin structure and function.
• Bioessays. 1993; 15: 461-7
• Display abstract

• Serpins are members of a family of structurally related protein inhibitors of serine proteinases, with molecular masses between 40 and 100kDa. In contrast to other, simpler, proteinase inhibitors, they may interact with proteinases as inhibitors, as substrates, or as both. They undergo conformational interconversions upon complex formation with proteinase, upon binding of some members to heparin, upon proteolytic cleavage at the reactive center, and under mild denaturing conditions. These conformational changes appear to be critical in determining the properties of the serpin. The structures and stabilities of these various forms may differ significantly. Although the detailed structural changes required for inhibition of proteinase have yet to be worked out, it is clear that the serpin does undergo a major conformational change. This is in contrast to other, simpler, families of protein inhibitors of serine proteinases, which bind in a substrate-like or product-like manner. Proteolytic cleavage of the serpin can result in a much more stable protein with new biological properties such as chemo-attractant behaviour. These structural transformations in serpins provide opportunities for regulation of the activity and properties of the inhibitor and are likely be important in vivo, where serpins are involved in blood coagulation, fibrinolysis, complement activation and inflammation.

• Coughlin PB, Tetaz T, Salem HH
• Identification and purification of a novel serine proteinase inhibitor.
• J Biol Chem. 1993; 268: 9541-7
• Display abstract

• We report the identification, purification, and partial amino acid sequence of a novel serine proteinase inhibitor which is present in extracts from human placentas and in the cytosolic fraction of the leukemic cell line K562. Extracts from these tissues exhibited time-dependent inhibition of the serine proteinase thrombin. This activity was not accelerated by heparin and corresponded to a protein which formed a 67-kDa complex with 125I-thrombin. The complex was stable on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A cleaved and functionally inactive form of the protein was purified from placental tissue by chromatography on DEAE-Sepharose, followed by affinity chromatography on thrombin-Sepharose. Antibodies raised against the placental protein recognized the inhibitor from K562 cells and placental extract. Western blotting experiments using the antibody showed that the uncleaved inhibitor has a molecular mass of 38 kDa. Amino acid sequencing was performed on the purified protein. Sequences of peptides resulting from digestion with cyanogen bromide followed by Endoproteinase Lys-c confirmed that this is a novel inhibitor with significant homology to the serpin family.

• Pignolo RJ, Cristofalo VJ, Rotenberg MO
• Senescent WI-38 cells fail to express EPC-1, a gene induced in young cells upon entry into the G0 state.
• J Biol Chem. 1993; 268: 8949-57
• Display abstract

• Recently we reported the isolation of cDNAs for a number of genes that are differentially expressed between nonproliferating early (young) and late (senescent) population doubling level (PDL) WI-38 human, fetal lung-derived, fibroblast-like cells. We now demonstrate that one of these isolates, EPC-1 (early PDL cDNA-1), derives from an approximately 1.4-kilobase mRNA species that is expressed at a > or = 100-fold higher level in serum-starved, confluent, young versus similarly treated senescent WI-38 cells. Complete nucleotide sequence analysis of this cDNA confirms its identity with that of a cDNA encoding a secreted, retinal pigmented epithelium differentiation factor as well as similarity of the encoded protein with a number of mammalian serine protease inhibitors. We show that EPC-1 expression is associated with G0 growth arrest in WI-38 cells. The mRNA readily accumulates in density-arrested and/or serum-starved young cells but not in log phase, low density young cells. In contrast, EPC-1 transcript abundance and expression of the encoded, secreted protein are both negligible in senescent WI-38 cells under all culture conditions tested. These findings support the hypothesis that senescent WI-38 cells exhibit a state of growth arrest fundamentally distinct from that of quiescent (G0) young cells.

• Coughlin P, Sun J, Cerruti L, Salem HH, Bird P
• Cloning and molecular characterization of a human intracellular serine proteinase inhibitor.
• Proc Natl Acad Sci U S A. 1993; 90: 9417-21
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• We describe a cDNA encoding a serine proteinase inhibitor present in placental tissue and the cytosolic fraction of K562 cells. On the basis of its interaction with thrombin, through which it was discovered, the inhibitor has been operationally named the placental thrombin inhibitor (PTI). Amino acid sequence comparisons suggest that its reactive center is located at Arg-341 and Cys-342, that it lacks a classical N-terminal signal sequence, and that it has the highest degree of similarity to intracellular serine proteinase inhibitors (serpins), such as the human monocyte/neutrophil elastase inhibitor and the equine leukocyte elastase inhibitor. PTI also resembles these inhibitors in that it contains oxidation-sensitive residues adjacent to the reactive site. The PTI cDNA was expressed in rabbit reticulocyte lysate and in COS-7 cells and a 42-kDa protein was produced. Recombinant PTI formed a 67-kDa complex when incubated with thrombin. The ability of native PTI to bind thrombin was destroyed by incubation with iodoacetamide. Analysis of human tissue mRNA indicated that PTI is expressed widely with the highest levels in cardiac and skeletal muscle and placenta. We conclude that PTI is a member of an emerging class of intracellular serpins.

• Hanneman EH, Kanost MR
• Differential alaserpin expression during development of the antennae in the tobacco hawkmoth, Manduca sexta.
• Arch Insect Biochem Physiol. 1992; 19: 39-52
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• The expression of alaserpin, a serine protease inhibitor, was studied in the developing adult olfactory system of Manduca sexta. Alaserpin mRNA in the antenna was identical to alaserpin from larval fat body, by restriction mapping and partial sequencing of clones isolated from a male pupal stage 6 antennal cDNA library. Northern and primer extension analysis showed that alaserpin transcripts were the same length at all stages and were present at stages 3 and 6, declined at stages 9-15, reappeared at stage 18, and persisted in adults. Western blotting revealed that alaserpin was present at stages 3-12, declined at stage 15, and disappeared at stage 18 and in adults. Immunocytochemistry at stage 6 revealed labeled cells closely associated with neurite bundles and scattered throughout the receptor cell layer. An occasional cell was seen in the antennal lumen. Labeled cells were segmentally distributed along the length of the antenna. The results suggest that alaserpin may have a role in antennal morphogenesis and olfactory neuron axon outgrowth.

• Crowther DC, Evans DL, Carrell RW
• Serpins: implications of a mobile reactive centre.
• Curr Opin Biotechnol. 1992; 3: 399-407
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• The serpins are unique among the families of serine proteinase inhibitors in having a reactive centre that is situated on a mobile loop. The structures of three alternative conformations are now known, and it can be deduced that the active form involves the partial insertion of the loop into the A sheet of the molecule. The ability of the loop to move in and out of this sheet has been adapted by evolution to allow the modulation of inhibitory activity. Manipulation of the structure of the loop and of other functional domains in the serpin superfamily enables the production of serpins with tailor-made activities. The ability of the loop to lock in latent conformations or to take part in intermolecular polymerization has implications for the production and stabilization of recombinant serpins. This review has been adapted from Current Opinion in Structural Biology 1992, 2:438-446.

• Beck BL et al.
• pNiXa, a Ni(2+)-binding protein in Xenopus oocytes and embryos, shows identity to Ep45, an estrogen-regulated hepatic serpin.
• Res Commun Chem Pathol Pharmacol. 1992; 77: 3-16
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• A Ni(2+)-binding protein (pNiXa, 45 kD, pI 8.5) discovered in Xenopus embryos, was isolated from oocytes. Based on amino acid sequences, pNiXa belongs to the serpin superfamily and shows identity to the cDNA sequence of Ep45, an estrogen-regulated hepatic serpin that contains an (HX)n-motif found in eukaryotic transcription factors. Nondenatured pNiXa, purified by Ni-affinity chromatography, inhibited bovine alpha-chymotrypsin. The presence of pNiXa in embryos when they are susceptible to Ni2+, the high avidity of pNiXa for Ni2+, and the (HX)n-motif point to pNiXa as a molecular target of Ni(2+)-teratogenesis.

• Simmen RC, Michel FJ, Fliss AE, Smith LC, Fliss MF
• Ontogeny, immunocytochemical localization, and biochemical properties of the pregnancy-associated uterine elastase/cathepsin-G protease inhibitor, antileukoproteinase (ALP): monospecific antibodies to a synthetic peptide recognize native ALP.
• Endocrinology. 1992; 130: 1957-65
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• Expression of the mRNA encoding the elastase/cathepsin-G protease inhibitor, antileukoproteinase (ALP), is highest in pig uterus during mid- and late pregnancy, suggesting a stage of pregnancy-dependent role for ALP in feto-maternal interactions. To elucidate a function for ALP in these events, immunogenic probes were developed to localize sites of ALP expression in the environment of the developing fetus. Monospecific antibodies raised against a 16-mer synthetic peptide corresponding to residues 21-36 (ALP 16P) of the deduced amino acid sequence of pig uterine ALP were generated by active immunization of sheep. ALP 16P conjugated to keyhole limpet hemocyanin elicited high titer antibodies that were specific to ALP. The antipeptide antibodies were used to characterize pig uterine ALP from allantoic fluids. Uterine ALP has an approximate mol wt of 14,000 and a pI of 8.2 and exhibits elastase inhibitor activity. Amino-terminal amino acid sequencing of uterine ALP indicated the sequence AENALKGGACPPRKIVQC, which has 44% identity with the corresponding region in human bronchial ALP. RIA for ALP, developed using ALP 16P as standard and iodinated tracer, demonstrated the presence of immunoreactive ALP in early, mid-, and late pregnant endometrium and myometrium, placenta, allantoic fluids, fetal cord blood, and fetal liver. ALP was undetectable in the maternal circulation. The ALP levels in endometrium, allantoic fluids, and fetal cord blood changed with the stage of pregnancy; however, ALP content in placenta, myometrium, and fetal liver, although different among tissues, remained invariant during gestation. By immunocytochemical analyses, ALP was localized in the glandular epithelium of the uterus, in placenta, and in fetal liver, consistent with the presence of immunoreactive ALP as measured by RIA. The localization of uterine ALP in placenta and its corresponding transport to fetal circulation provide strong evidence to support a physiological function for the protease inhibitor in the biological mechanisms controlling fetal development in utero.

• Kawanomoto M, Motojima K, Sasaki M, Hattori H, Goto S
• cDNA cloning and sequence of rat ribonuclease inhibitor, and tissue distribution of the mRNA.
• Biochim Biophys Acta. 1992; 1129: 335-8
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• A cDNA clone encoding ribonuclease inhibitor was isolated from a rat lung cDNA library. The deduced amino acid sequence revealed a high degree of conservation of a repeated structure. The mRNA was detected in all seven tissues of rat examined, the amount being highest in the lung and lowest in the heart.

• Nastruzzi C, Feriotto G, Gambari R
• DNA-binding activity of the proteinase inhibitor TAPP.
• Thromb Res. 1991; 63: 557-61

• Carrell RW, Evans DL, Stein PE
• Mobile reactive centre of serpins and the control of thrombosis.
• Nature. 1991; 353: 576-8
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• Two protease inhibitors in human plasma play a key part in the control of thrombosis: antithrombin inhibits coagulation and the plasminogen activator inhibitor PAI-1 inhibits fibrinolysis, the dissolving of clots. Both inhibitors are members of the serpin family and both exist in the plasma in latent or inactive forms. We show here that the reactive centre of the serpins can adopt varying conformations and that mobility of the reactive centre is necessary for the function of antithrombin and its binding and activation by heparin; the identification of a new locked conformation explains the latent inactive state of PAI-1. This ability to vary conformation not only allows the modulation of inhibitory activity but also protects the circulating inhibitor against proteolytic attack. Together these findings explain the retention by the serpins of a large and unconstrained reactive centre as compared to the small fixed peptide loop of other families of serine protease inhibitors.

• Borriello F, Krauter KS
• Multiple murine alpha 1-protease inhibitor genes show unusual evolutionary divergence.
• Proc Natl Acad Sci U S A. 1991; 88: 9417-21
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• The murine alpha 1-protease inhibitors (alpha 1-PI) are encoded by a small gene family on chromosome 12. Studies of alpha 1-PI and other serine protease inhibitor genes have revealed an unusually high rate of mutation of the reactive centers of the inhibitors. Using a modification of the PCR technique, we have previously identified five distinct alpha 1 PI reactive site sequences present in the genome of C57BL/6 mice. In this report, we use cDNA cloning techniques to demonstrate that all five genes are expressed in the adult mouse liver. DNA sequence analysis shows that three of the five mRNAs expressed have a substitution for methionine-353, which is essential for normal activity of the homologous human protein, alpha 1-antitrypsin (alpha 1-AT). Comparison of the DNA sequences of the five cDNAs indicates a higher degree of polymorphism in the carboxyl-terminal half of the protein and an extraordinarily replacement/silent ratio of nucleotide changes in a narrow region surrounding the reactive site. The clustering of polymorphisms near the reactive site combined with the high replacement/silent ratio suggest an evolutionary mechanism that apparently selects for functional diversity of the alpha 1-PI genes. Finally, modeling of the three-dimensional positions of the alpha 1-PI polymorphic residues into the homologous positions of the crystallographic structure of ovalbumin, a member of the alpha 1-PI supergene family, predicts that many of these amino acids are on the surfaces, which are likely to interact with the protease targets.

• Suminami Y, Kishi F, Sekiguchi K, Kato H
• Squamous cell carcinoma antigen is a new member of the serine protease inhibitors.
• Biochem Biophys Res Commun. 1991; 181: 51-8
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• We have cloned cDNA of squamous cell carcinoma antigen. Sequence analysis of the complete 1711 basepairs SCC antigen cDNA revealed that it coded 390 amino acids and no typical signal sequence in the NH2-terminus. Northern blot analysis of human squamous cell poly(A)+ RNA using this cDNA insert as the probe showed a single RNA species of about 1.7 kilobases. The cDNA was expressed in Escherichia coli and the product was detected by immunological methods using antibodies against SCC antigen, indicating that this cDNA encodes the entire SCC antigen sequence. The amino acid homology search revealed that SCC antigen was a member of the serine protease inhibitors family.

• Patterson SD, Bell K, Manton VJ
• Equus przewalskii plasma protease inhibitor (Pi) system.
• Anim Genet. 1990; 21: 129-39
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• A detailed biochemical characterization of four of the five previously described alleles of the plasma protease inhibitor (Pi) system of Equus przewalskii was performed using both one- and two-dimensional electrophoretic techniques. The proteins have been characterized in terms of isoelectric point, relative molecular mass, inhibitory activity to bovine trypsin and chymotrypsin, immunochemical cross-reactivity, terminal sialic acid content and enzyme:inhibitor complex formation and the oxidation sensitivity of this interaction. Using these functional criteria, only three loci (Spi 1, 2 and 3) were found to control the plasma Pi proteins of the E. przewalskii haplotypes. In contrast a fourth locus, Spi 4, was found in some E. caballus haplotypes. The significance of these results with respect to the complexity of the protein pattern exhibited by the equine Pi multigene family is discussed.

• Kuhn LA et al.
• Elucidating the structural chemistry of glycosaminoglycan recognition by protein C inhibitor.
• Proc Natl Acad Sci U S A. 1990; 87: 8506-10
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• Glycosaminoglycans (GAGs) including heparin accelerate the inhibition of serine proteases by serine protease inhibitors (serpins), an essential process in regulating blood coagulation. to analyze the molecular basis for GAG recognition by the plasma serpin protein C inhibitor (PCI; also known as plasminogen activator inhibitor 3), we have constructed a complete, energy-minimized, three-dimensional model of PCI by using the structure of homologous alpha 1-antitrypsin as a template. Sequence analysis, hydrogen-bonding environment, and shape complementarity suggested that the N-terminal residues of PCI, which are not homologous to those of alpha 1-antitrypsin, form an amphipathic alpha-helix, here designated A+ since it precedes the alpha 1-antitrypsin A helix. Electrostatic calculations revealed a single, highly positive surface region arising from both the A+ and H helices, suggesting that this two-helix motif is required for GAG binding by PCI. The dominant role of electrostatic interactions in PCI-heparin binding was confirmed by the strong ionic strength dependence of heparin stimulation. The involvement of the A+ helix in heparin binding was verified by demonstrating that an anti-PCI antibody that specifically binds the A+ peptide blocks heparin binding.

• Pages G et al.
• Primary structure and assignment to chromosome 6 of three related rat genes encoding liver serine protease inhibitors.
• Gene. 1990; 94: 273-82
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• Three closely related SPI genes which encode highly homologous proteins of the serine protease inhibitor family secreted by rat liver (SPI-1, SPI-2 and SPI-3), were isolated from genomic libraries and sequenced, totally (SPI-2) or partially (SPI-1 and SPI-3). These genes all map on rat chromosome 6. Each of them spans about 10 kb and contains five exons separated by four introns, located at equivalent positions. S1 mapping analysis indicated that initiation of transcription occurs at the same position (tsp) in each of the three genes. In vitro transcription experiments demonstrated the presence of promoter elements upstream from the putative tsp. Detailed analysis of 5'-flanking sequences in the three SPI revealed major differences. A high degree of identity (70%) was found within a 350-bp region preceding the 'cap' site, with the exception of a 42-bp spacer, which was only found in SPI-3. Upstream from that point, SPI-1 and SPI-2 sequences remain largely homologous over at least 1 kb but completely diverge from the corresponding sequence in SPI-3. This may, at least partly, account for the differential regulation of the three SPI observed during acute inflammation and upon hypophysectomy.

• Pages G, Rouayrenc JF, Le Cam G, Mariller M, Le Cam A
• Molecular characterization of three rat liver serine-protease inhibitors affected by inflammation and hypophysectomy. Protein and mRNA analysis and cDNA cloning.
• Eur J Biochem. 1990; 190: 385-91
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• Rat hepatocytes have the potential to secrete three similar acidic glycoproteins, serine protease inhibitors 1, 2 and 3 (SPI-1, SPI-2, SPI-3), recognized by the same antibodies. They were synthesized as precursors of comparable sizes (45 kDa), which were post-translationally modified by N-glycosylation at three (SPI-3) or four (SPI-1 and SPI-2) sites. This appeared to account for the size difference of mature proteins. The mRNA sequences, derived from cDNA clones, displayed a high degree of similarity (70-90%), except the sequence of the antiprotease-reactive centers which were completely divergent. SPI-1 and SPI-2 mRNAs were of similar sizes (1.8 kb), and were smaller than that of SPI-3 (2.2 kb); the difference corresponded to a longer, 3'-end untranslated sequence. Production of SPI-1 and SPI-2, which was constitutive in the normal animal, could be abolished by hypophysectomy and was strongly decreased during acute inflammation. In contrast, production of SPI-3, which was barely detectable in normal rats, was transiently induced during inflammation.

• Ing NH, Roberts RM
• The major progesterone-modulated proteins secreted into the sheep uterus are members of the serpin superfamily of serine protease inhibitors.
• J Biol Chem. 1989; 264: 3372-9
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• The uterine milk proteins (UTMP) are a pair of structurally related basic glycoproteins that when newly synthesized carry phosphorylated mannosyl residues on their carbohydrate chains. They are the major proteins secreted by ovine endometrium under the influence of progesterone. RNA from a late pregnant ewe endometrium was isolated for use in in vitro translation assays and for constructing cDNA libraries. Translation experiments, initially with total cellular RNA and subsequently with RNA selected by hybridization with a specific cDNA, demonstrated the production of two polypeptides (Mr = 47,000 and 55,000) that were precipitated with antiserum to the UTMP. With microsomal membranes in the translation assay, there was increased production of an Mr = 57,000 form that was protected from protease digestion. Antibody screening of a cDNA library in lambda gt11 identified a short clone representing the 3' terminus of the mRNA that was shown by epitope selection experiments to be UTMP specific. This clone was then used to screen a lambda gt10 library. A longer clone (1.3 kilobases) was isolated and sequenced but lacked the 5' terminus to the mRNA. The latter sequence was obtained directly from the mRNA. Interesting features of the UTMP mRNA sequence, which was 1,352 bases long and contained a 1,287-base open reading frame, were two strong start codons, two potential sites for N-glycosylation and a repeat of 21 bases, six bases apart, that resulted in a repeat of seven amino acids. The inferred amino acid sequence agreed closely with the NH2-terminal amino acid sequence obtained directly from the UTMP and clearly placed the UTMP in the serpin superfamily of protease inhibitors. However, we have been unable to demonstrate inhibitory activity toward any serine protease so far tested.

• Kanost MR, Prasad SV, Wells MA
• Primary structure of a member of the serpin superfamily of proteinase inhibitors from an insect, Manduca sexta.
• J Biol Chem. 1989; 264: 965-72
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• A cDNA clone isolated from a fat body cDNA library from an insect, Manduca sexta, has been sequenced and shown to code for a member of the serpin family of proteinase inhibitors. The cDNA has an open reading frame which codes for a 392-residue polypeptide of Mr = 43,500 with a hydrophobic NH2-terminal sequence which appears to be a signal peptide. An alignment of this amino acid sequence with 11 members of the serpin superfamily reveals that the insect protein is 25-30% identical with most members of the superfamily. The alignment was used to construct an evolutionary tree of the serpin sequences analyzed, which indicates that the progenitor of the M. sexta serpin and the human serpins most closely related to it diverged from other serpin genes prior to the divergence of the vertebrates and invertebrates. The M. sexta serpin is predicted to inhibit elastase due to the presence of alanine at the P1 position of its reactive center and is classified as an alaserpin. A glycoprotein of Mr = 47,000 isolated from hemolymph of M. sexta larvae has an NH2-terminal sequence identical to that deduced from the alaserpin cDNA clone and inhibits porcine pancreatic elastase and bovine chymotrypsin.

• Yoon JB, Towle HC, Seelig S
• Growth hormone induces two mRNA species of the serine protease inhibitor gene family in rat liver.
• J Biol Chem. 1987; 262: 4284-9
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• In order to study the molecular actions of growth hormone on gene expression, we have cloned and characterized two unique, but related, cDNA sequences from rat liver, lambda Spi-1 and lambda Spi-2. These two cDNA sequences are complementary to rat hepatic mRNA species previously designated as Spots 3 and 20 when assayed by in vitro translation and two-dimensional gel electrophoresis. By Northern blot, the two mRNAs are both 1900 bases in length and growth hormone administered to hypophysectomized rats increases the levels of both of these mRNAs. In contrast, the combined administration of thyroxine, corticosterone, and dihydrotestosterone to hypophysectomized rats did not augment these mRNAs. The simultaneous administration of all four hormones resulted in a level greater than that observed for animals treated with growth hormone alone. Analysis of genomic DNA suggests the presence of two similar, but not identical, genes. DNA sequencing of lambda Spi-1 and lambda Spi-2 revealed that they were 90% homologous at the nucleotide level and 87% homologous at the amino acid sequence level. lambda Spi-2 has 78% homology with mouse contrapsin, 60% with human alpha 1-antichymotrypsin, and 51-55% with alpha 1-antitrypsins, all members of the serine protease inhibitor gene family. The nucleotide and deduced amino acid sequences of lambda Spi-1 and lambda Spi-2 which align with the reactive centers of known members of this family differ substantially from each other and from other members of the family. The difference in the reactive center suggests that the specificity or function of these proteins may differ from other members of serine protease inhibitor gene family.

• Hill RE, Hastie ND
• Accelerated evolution in the reactive centre regions of serine protease inhibitors.
• Nature. 1987; 326: 96-9
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• The serine protease inhibitors (serpins) are a family of proteins that function to control the action of serine proteases in many diverse physiological processes. The functional region or reactive centre of these inhibitors is near the C-terminal end and is an exposed site that acts as a bait for the appropriate serine protease to recognize and covalently bind. The specificity of the inhibitor is determined, at least in part, by a single amino acid that resides in this region at the P1 position. We show here that following a gene duplication event the reactive centres of three related rodent protease inhibitors have diverged from each other at unprecedented rates. This has resulted in proteins with different predicted specificities and we postulate that these changes were fixed by positive darwinian selection and that the most likely selective forces are extrinsic proteases, namely those used by parasites to facilitate their spread throughout the host.

• Suzuki K et al.
• Characterization of a cDNA for human protein C inhibitor. A new member of the plasma serine protease inhibitor superfamily.
• J Biol Chem. 1987; 262: 611-6
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• A cDNA library in lambda-phage lambda gt11 containing DNA inserts prepared from human liver mRNA was screened with monoclonal antibodies to human protein C inhibitor. Six positive clones were isolated from 6 X 10(6) phages and plaque purified. The cDNA in the phage containing the largest insert, which hybridized to a DNA probe prepared on the basis of the amino-terminal amino acid sequence of the mature inhibitor, was sequenced. This cDNA insert contained 2106 base pairs coding for a 5'-noncoding region, a 19-amino acid signal peptide, a 387-amino acid mature protein, a stop codon, and a long 3'-noncoding region of 839 base pairs. Based on the amino acid sequence of the carboxyl-terminal peptide released by cleavage of protein C inhibitor by activated protein C as well as by thrombin, the reactive site peptide bond of protein C inhibitor is Arg354-Ser355. Five potential carbohydrate-binding sites were found in the mature protein. The high homology of the amino acid sequence of protein C inhibitor to the other known inhibitors clearly demonstrates that protein C inhibitor is a member of the superfamily of serine protease inhibitors including alpha 1-antichymotrypsin, alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. Based on the difference matrices for these proteins, we present possible phylogenetic trees for these proteins.

• Que BG, Petra PH
• Isolation and analysis of a cDNA coding for human C1 inhibitor.
• Biochem Biophys Res Commun. 1986; 137: 620-5
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• A cDNA coding for C1 inhibitor was isolated from a human liver lambda gt11 expression library and sequenced by the dideoxy method. The amino acid sequence deduced from the cDNA indicated that the insert was a partial clone coding for 310 amino acids including the reactive site present at the carboxyl end of the molecule. The reactive site corresponds to that previously reported by Salvesen et al. (J. Biol. Chem. 260, 2432, 1985). The cDNA also contained a stop codon of TGA, 264 nucleotides at the 3' noncoding region, and a polyadenylation signal sequence of AATAAA 15 nucleotides upstream from the poly(A) tail. The amino acid sequence flanking the reactive site of the inhibitor is homologous to other members of the superfamily of plasma serine protease inhibitors.

• Upton C, Carrell RW, McFadden G
• A novel member of the serpin superfamily is encoded on a circular plasmid-like DNA species isolated from rabbit cells.
• FEBS Lett. 1986; 207: 115-20
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• A novel member of the serpin family of serine protease inhibitors is presented. A plasmid-like DNA was isolated from rabbit cells by its homology to the genome of Shope fibroma virus (SFV), a tumorigenic poxvirus of rabbits, and was shown elsewhere to encode a serpin-like protein [(1986) Mol. Cell. Biol. 6, 265-276]. Although significant DNA homology exists between the rabbit plasmid serpin open reading frame and the SFV terminal inverted repeat DNA there is no intact serpin counterpart encoded by this region of the SFV genome. The alignment of the novel plasmid-borne polypeptide with the serpin family of proteins confirms its status within this group.

• McPhalen CA, Evans C, Hayakawa K, Jonassen I, Svendsen I, James MN
• Preliminary crystallographic data for the serine protease inhibitor CI-2 from barley seeds.
• J Mol Biol. 1983; 168: 445-7

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