Purification and Characterization of α1-Proteinase Inhibitor from Carp (Cyprinus carpio) Serum

α₁-Proteinase inhibitor was purified to homogeneity from carp serum with an increase in specific inhibitory activity of 17-fold and a 3% recovery rate. The inhibitor was estimated to have molecular weight of 55,000 under reducing and nonreducing conditions, indicating its composition of a single polypeptide. The inhibitor immunologically crossreacted faintly with carp muscular serine proteinase inhibitor but had no crossreactivity with serine proteinase inhibitors from other species. Carp serum inhibitor exhibited marked stability over broad pH ranges of 4.0 to 10.0 and temperatures below 55°C. The inhibitor potently inhibited the activities of carp intestinal and fish myofibril-binding proteinases, and its respective inhibitions of trypsin-type and carp muscular proteinases were more severe than those of chymotrypsin-type and white croaker muscular proteinases. Its inhibitions were similar to those of bovine pancreatic trypsin and α-chymotrypsin, and the amount required to completely inactivate 0.2 μg of each of these two proteinases was evaluated as 0.43 to 0.45 μg. This indicates a molar ratio close to 1:1 during combination of the inhibitor with each proteinase. In addition, its ability to form irreversible complexes with the proteinases was observed electrophoretically and immunologically under denaturing and reducing conditions. Received April 3, 1998; accepted June 30, 1998.     

New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro

Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1 inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons (Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human α₁-antitrypsin in double immunodiffusion. PI-1 corresponding to α₁ - antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin, kallikrein and plasmin weakly. It had higher molecular weight (200–300 Kd) than that of PI-1, and did not crossreact with antisera against human α₁-antitrypsin, α₂-macroglobulin, α₁-antichymotrypsin, α₂-plasmin inhibitor, inter-α-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma cell lines that secrete functionally active trypsin inhibitors, including α₁-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors.     

Interactions of α1–proteinase inhibitor with small ligands of therapeutic potential: binding with retinoic acid

Human α₁–proteinase inhibitor (α₁–PI), also known as α₁-antitrypsin, is the most abundant plasma serine protease inhibitor (serpin). It is best recognized for inhibition of neutrophil elastase. The α₁–PI interactions with non-protease ligands were investigated mainly in regards to those molecules that may block the aggregation of α₁–PI Z mutant. The objective of this study was to evaluate the potential of α₁–PI to bind small non-peptide ligands of pharmaceutical interest that may attain additional properties to currently available α₁–PI therapeutic preparations. Among putative ligands of bio-medical interest examined in this study, all-trans retinoic acid (RA) was selected due to its recently proposed roles in the lungs, and as an efficient optical probe. The results of this study, including absorption spectroscopy data, fluorescence quenching and the protein-induced chirality of the visible circular dichroism strongly suggest that α₁–PI does bind RA in vitro to non-covalent complexes of up to two moles of RA per one mole of the protein. To our knowledge, this is the first report that provides experimental evidence of the α₁–PI potential towards bi-functional drugs via a combination with RA, or potentially other molecules of pharmaceutical interest, that ultimately, may enhance currently available α₁–PI therapies.     

Localization of a serine proteinase inhibitor, B-43, in the bovine pancreas

 B-43, a serine proteinase inhibitor belonging to the ovalbumin branch of the serpin superfamily, was purified and cloned from bovine brain. Since [³⁵S]-labeled B-43 forms SDS-stable complexes with pancreatic serine proteinases, trypsin, α-chymotrypsin, and kallikrein, it has been suggested that B-43 is capable of inhibiting these serine proteinases and that B-43 may be present in the pancreas. In the present study, we investigated the localization of B-43 in the bovine pancreas immunohistochemically and examined the effect of B-43 on the amidolytic activities of pancreatic serine proteinases. Strong B-43-like immunoreactivity was localized in acinar cells, especially in the basal sides of the cells where the rough endoplasmic reticulum is located. The nuclei of the subpopulation of acinar cells were also immunoreactive for B-43. The recombinant glutathione S-transferase–B-43 fusion protein inhibited the amidolytic activity of trypsin and, to a lesser extent, α-chymotrypsin and kallikrein, but not elastase. These results suggest a role of B-43 in regulating serine proteinases both in the cytoplasm and the nucleus. Accepted: 13 January 1998     

Concentration-dependent effects of native and polymerised α1-antitrypsin on primary human monocytes, <Emphasis Type="Italic">in vitro</Emphasis>

Background  

α1-antitrypsin (AAT) is one of the major serine proteinase inhibitors controlling proteinases in many biological pathways. There is increasing evidence that AAT is able to exert other than antiproteolytic effects. To further examine this question we compared how various doses of the native (inhibitory) and the polymerised (non-inhibitory) molecular form of AAT affect pro-inflammatory responses in human monocytes, in vitro. Human monocytes isolated from different donors were exposed to the native or polymerised form of AAT at concentrations of 0.01, 0.02, 0.05, 0.1, 0.5 and 1 mg/ml for 18 h, and analysed to determine the release of cytokines and to detect the activity of NF-κB.     

Expression,Purification, and Characterization of Recombinant Human α<Subscript>1</Subscript>-Antitrypsin Produced Using Silkworm–Baculovirus Expression System

Human α₁-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α₁-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~?15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use.     

A novel serine protease complexed with α2-macroglobulin from skeletal muscle of lizard fish (Saurida undosquamis)

A novel fish muscle serine protease named muscle soluble serine protease (MSSP) was purified from the soluble fraction of lizard fish (Saurida undosquamis: Synodontidae) muscle by ammonium sulfate fractionation followed by four steps of column chromatographies. In native-PAGE, the purified enzyme appeared as a single band with an estimated mol. mass of approximately 380 kDa by gel filtration. In SDS-PAGE under reducing conditions, the purified enzyme migrated as two protein bands at 110 and 100 kDa, named subunits A and B, respectively. The 20 residues of N-terminal amino acid sequence of subunit B showed 70% of homology to β-chain of carp α₂-macroglobulin-1. Moreover, both subunits A and B showed immunoreactivity with anti carp α₂-macroglobulin antibody. Purified MSSP was inactivated by Pefabloc SC, aprotinin, benzamidine and TLCK, but not by α₁-antitrypsin. After acid treatment (pH 2, 24 h), however, the enzyme activity eluted at 14 kDa from Sephacryl S-200 carried out under acidic conditions was inhibited by α₁-antitrypsin. Lizard fish MSSP most rapidly hydrolyzed Boc-Val-Pro-Arg-MCA and Boc-Gln-Arg-Arg-MCA, but did not hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA and Suc-Ala-Ala-Pro-Phe-MCA, and was not suppressed either by E-64, pepstatin A and ethylenediaminetetraacetic acid (EDTA). These results indicate that the purified MSSP is a serine protease complexed with α₂-macroglobulin, and the entrapped protease was dissociated by the acid treatment. Purified and free MSSPs were most active at pH 10.0 and 9.0, respectively. Purified MSSP degraded myofibrillar proteins and casein but time courses of degradation of these substrates by the enzyme differed.     

Engineering the serpin α1‐antitrypsin: A diversity of goals and techniques

α₁‐Antitrypsin (α₁‐AT) serves as an archetypal example for the serine proteinase inhibitor (serpin) protein family and has been used as a scaffold for protein engineering for >35 years. Techniques used to engineer α₁‐AT include targeted mutagenesis, protein fusions, phage display, glycoengineering, and consensus protein design. The goals of engineering have also been diverse, ranging from understanding serpin structure–function relationships, to the design of more potent or more specific proteinase inhibitors with potential therapeutic relevance. Here we summarize the history of these protein engineering efforts, describing the techniques applied to engineer α₁‐AT, specific mutants of interest, and providing an appended catalog of the >200 α₁‐AT mutants published to date.     

Isolation and characterization of four serine proteinase inhibitors (serpins) from hemolymph of Manduca sexta

Four serine proteinase inhibitors have been isolated from hemolymph of fifth instar larvae of Manduca sexta. One of these, an inhibitor specific for elastase, has been previously shown to be a member of the serpin family of serine proteinase inhibitors. Of the three remaining inhibitors, two are specific for chymotrypsin and one for trypsin. The four inhibitors have molecular weights of approx. 47,000 and isoelectric points between 4.4 and 4.8. The four proteins have very similar amino acid compositions, and NH₂-terminal sequence analysis suggests that they represent members of a gene family.     

Expression of human α<Subscript>1</Subscript>-proteinase inhibitor in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Background  

Human α₁-proteinase inhibitor (α₁-PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, α₁-PI is available for replacement therapy as an FDA licensed plasma-derived (pd) product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant α₁-PI (r-α₁-PI) could provide an attractive alternative. Although r-α₁-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation.     

Molecular linkage of the human αl-antitrypsin and corticosteroid-binding globulin genes on Chromosome 14q32.1

The genes encoding α1-antitrypsin (α1AT; gene symbol PI) and corticosteroid-binding globulin (CBG) are part of a cluster of structurally related serine protease inhibitor (serpin) genes on human Chromosome (Chr) 14q32.1. This cluster also includes the genes encoding α1-antichymotrypsin (AACT) and protein C inhibitor (PCI), as well as an α1-antitrypsin-related sequence (ATR; gene symbol PIL). In this report we present a detailed restriction map of a 110-kb region of genomic DNA that includes the α1AT, ATR, and CBG genes. Gene order in this interval is tel–α1AT–ATR–CBG–cen, and all three genes are transcribed in a distal-to-proximal orientation. Within the gene cluster, ATR is approximately 12 kb downstream of α1AT, and CBG is about 57 kb downstream of α1AT. Repetitive DNA sequences have been mapped throughout the interval, and several new restriction site polymorphisms in the region are described. Received: 25 May 1997 / Accepted: 23 July 1997     

On hypervariability at the reactive center of proteolytic enzymes and their inhibitors

The pattern of synonymous and nonsynonymous substitutions at the reactive center of proteases (kallikrein) and their inhibitors (₁-antitrypsin and serpin) was examined. In the case of ₁-antitrypsin, the proportion of different nonsynonymous sites exceeds that of different synonymous sites at the reactive center for sequence pairs of recent duplication. The result indicates that the positive selection has operated after duplication to increase functional diversity. In the cases of kallikrein, serpin, and remote sequence pairs of ₁-antitrypsin, the proportion of different synonymous sites at the reactive center exceeds that of different synonymous sites at the remaining region. The result indicates that gene conversion followed by natural selection is working. On the whole, it is concluded that hypervariability of amino acids at the reactive center is generated by an interaction among natural selection, random genetic drift, point mutation, and gene conversion. Gene duplication may provide potential for them to interact.     

Isolation, characterization, and cDNA sequencing of alpha-1-antiproteinase-like protein from rainbow trout seminal plasma

Seminal plasma of teleost fish contains serine proteinase inhibitors related to those present in blood. These inhibitors can be bound to Q-Sepharose and sequentially eluted with a NaCl gradient. In the present study, using a two-step procedure, we purified (73-fold to homogeneity) and characterized the inhibitor eluted as the second fraction of antitrypsin activity (inhibitor II) from Q-Sepharose. The molecular weight of this inhibitor was estimated to be 56 kDa with an isoelectric point of 5.4. It effectively inhibited trypsin and chymotrypsin but was less effective against elastase. It formed SDS-stable complexes with cod and bovine trypsin. Inhibitor II appeared to be a glycoprotein. Carbohydrate content was determined to be 16%. N-terminal Edman sequencing allowed identification of the first 30 N-terminal amino acids HDGDHAGHTEDHHHHLHHIAGEAHPQHSHG and 25 amino acids within the reactive loop IMPMSLPDTIMLNRPFLLFILEDST. The N-terminal sequence did not match any known sequence, however, the sequence within the reactive loop was significantly similar to carp and mammalian alpha1-antiproteinases. Both sequences were used to construct primers and obtain a cDNA sequence from liver. The mRNA coding the protein is 1675 nt in length including a single open reading frame of 1281 nt that encodes 426 amino acid residues. Analysis of this sequence indicated the presence of putative conserved serpin domains and confirmed the similarity to carp alpha1-antiproteinase and mammalian alpha1-antiproteinase. Our results indicate that inhibitor II belongs to the serpin superfamily and is similar to alpha1-antiproteinase.     

Subtilisin-like proteinase secreted by the <Emphasis Type="Italic">Bacillus pumilus</Emphasis> KMM 62 strain at different growth stages

Proteinase secreted in the environment by bacilli on different growth stages was isolated by ion chromatography from the culture medium of Bacillus pumilus KMM 62. According to the hydrolysis character of specific chromogenic substrates and inhibition type, the enzyme belongs to subtilisin-like serine proteinases. The isolated proteinase with the molecular mass of 30 kDa displays maximum activity on hydrolysis of the peptide substrate Z-Ala-Ala-Leu-pNA at pH 8.0–8.5 and temperature 30°C. The protein is stable in the range of pH 7.5–10.0. It was shown that subtilisin-like serine proteinase from B. pumilus KMM 62 possessed thrombolytic activity.     

Anticoagulant and anticomplement effects of an endogenous inhibitor from hepatopancreas of red king crab (<Emphasis Type="Italic">Paralithosed camtschaticus</Emphasis>) on human blood

Serpins are the superfamily of serine and cysteine protease inhibitors (SERine Protease Inhibitors) acting by an irreversible suicide mechanism. A novel serpin from hepatopancreas of red king crab (Paralithosed camtschaticus) was isolated and its effect on the process of human blood plasma coagulation was investigated. The investigated serpin exhibited a significant anticoagulant effect, which dramatically increased in the combination with heparin. The study of the crab serpin on C1s (C1 esterase) revealed its competition with the C1 inhibitor from blood plasma. Although the inhibitor weakly influenced thrombin activity, inhibition constant for C1s was (2.02 ± 0.71) 10⁻⁷ M. Unlike the C1 inhibitor the novel red king crab serpin does not inhibit fibrinolysis but inhibits blood coagulation. This creates certain clinical perspectives.     

Evolution and Regulation of the Downstream Gene of Hypoxia-Inducible Factor-1α in Naked Carp (Gymnocypris przewalskii) from Lake Qinghai, China

The naked carp (Gymnocypris przewalskii) is a native teleost of Lake Qinghai (altitude, 3.2 km) on the Qinghai-Tibet Plateau in China. Hypoxia-inducible factor (HIF)-1α of Gymnocypris przewalskii was cloned and a phylogenetic tree for vertebrate HIF-1α was constructed. By analysis of maximum likelihood models of codon substitutions for HIF-1α, three positive sites in the branch lineages of crucian carp, eelpout, and flounder, and a higher proportion of neutral sites in naked carp, antarctic eelpout, rainbow trout, and grayling, were detected among all teleosts. It seems that low habitat temperatures relax the purifying selection of HIF-1α in these stenothermal coldwater fish, and both cold and hypoxic lake water contributed to the evolution of the HIF-1α gene in the naked carp. Furthermore, Glut 1 mRNA, a gene downstream from HIF-1α, has a time-course- and tissue-specific dependent response to hypoxic challenge.     

A positively charged loop on the surface of kallistatin functions to enhance tissue kallikrein inhibition by acting as a secondary binding site for kallikrein

Kallistatin is a serine proteinase inhibitor (serpin) that specifically inhibits tissue kallikrein. The inhibitory activity of kallistatin is abolished upon heparin binding. The loop between the H helix and C2 sheet of kallistatin containing clusters of basic amino acid residues has been identified as a heparin-binding site. In this study, we investigated the role of the basic residues in this region in tissue kallikrein inhibition. Kallistatin mutants containing double Ala substitutions for these basic residues displayed a 70-80% reduction of association rate constants, indicating the importance of these basic residues in tissue kallikrein inhibition. A synthetic peptide derived from the sequence between the H helix and C2 sheet of kallistatin was shown to suppress the kallistatin-kallikrein interaction through competition for tissue kallikrein binding. To further evaluate the function of this loop, we used alpha1-antitrypsin, a non-heparin-binding serpin and slow tissue kallikrein inhibitor as a scaffold to engineer kallikrein inhibitors. An alpha1-antitrypsin chimera harboring the P3-P2' residues and a sequence homologous to the positively charged region between the H helix and C2 sheet of kallistatin acquired heparin-suppressed inhibitory activity toward tissue kallikrein and exhibited an inhibitory activity 20-fold higher than that of the other chimera, which contained only kallistatin's P3-P2' sequence, and 2300-fold higher than that of wild-type alpha1-antitrypsin. The alpha1-antitrypsin chimera with inhibitory characteristics similar to those of kallistatin demonstrates that the loop between the H helix and C2 sheet of kallistatin is crucial in tissue kallikrein inhibition, and this functional loop can be used as a module to enhance the inhibitory activity of a serpin toward tissue kallikrein. In conclusion, our results indicate that a positively charged loop between the H helix and C2 sheet of a serpin can accelerate the association of a serpin with tissue kallikrein by acting as a secondary binding site.     

The role of conformational change in serpin structure and function

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.     

Purification of a novel myofibril-bound serine proteinase inhibitor (MBSPI) from the skeletal muscle of lizard fish

A novel myofibril-bound serine proteinase inhibitor (MBSPI) was purified to homogeneity from the skeletal muscle of lizard fish (Saurida wanieso). Purification was carried out by ammonium sulfate fractionation, followed by column chromatographies on DEAE-Sephacel, SP-Sepharose and Sephadex G-150. MBSPI was purified 7.7-fold starting from the DEAE-Sephacel fraction, with a yield of 0.2%. It is a monomeric protein with the molecular mass of 50 kDa as estimated by SDS-PAGE and gel filtration. MBSPI reveals high inhibition specificity toward a myofibril-bound serine proteinase (MBSP) purified from lizard fish muscle. No inhibition is detected toward bovine trypsin, bovine chymotrypsin, two trypsins from carp hepatopancreas and a serine proteinase isolated from the sarcoplasmic fraction of white croaker muscle. It does not exert any inhibitory activity toward a myofibril-bound serine proteinase from carp muscle.     

Study on a prolyl endopeptidase from the skeletal muscle of common carp (Cyprinus carpio)

A prolyl endopeptidase (PEP) was purified to homogeneity from the skeletal muscle of common carp using a procedure involving ammonium sulfate fractionation and column chromatography involving DEAE-Sephacel, Phenyl-Sepharose, DEAE-Sepharose Fast Flow, and hydroxyapatite. The molecular weight of the PEP was 82 kDa as determined by SDS-PAGE. Using Suc-Gly-Pro-MCA as a substrate, the optimal pH and temperature of the purified enzyme were pH 6.0 and 35 °C, respectively, and the K_m and k_cat were 8.33 μM and 1.71 S^?1, respectively. The activity of the PEP was inhibited by SUAM-14746, a specific inhibitor of prolyl endopeptidases, and was partially inhibited by the serine proteinase inhibitors PMSF and Pefabloc SC. According to peptide mass fingerprinting, 12 peptide fragments with a total of 134 amino acid residues were obtained, which were highly identical to prolyl endopeptidases from zebrafish (Danio rerio) and sponge (Amphimedon queenslandica), confirming the purified enzyme was a prolyl endopeptidase. Our present study for the first time reported the existence of a prolyl endopeptidase in fish muscle.