Hydrogen peroxide induced impairment of post-ischemic ventricular function is prevented by the sodium-hydrogen exchange inhibitor HOE 642 (cariporide)

Using highly degenerate, serine-protease-specific PCR primers on a midgut-specific cDNA library it was estimated that a minimum of 24 independent serine proteases were expressed in the midgut of Stomoxys calcitrans. The relative abundance of these 24 independent serine proteases has been estimated by restriction analysis of PCR products, showing that 69% fall into six almost equally abundant groups. Two highly abundant serine protease cDNAs (Ssp1 and Ssp2) were isolated and sequenced. They encode preproenzymes of 272 amino acids (Mr 28521) and 255 amino acids (Mr 27097) with putative signal peptides of 17 amino acids and 16 amino acids, putative activation peptides of 15 amino acids and 10 amino acids and mature enzymes of 239 amino acids (Mr 25322; pI 4.89) and 228 amino acids (Mr 24182; pI 7.59), respectively. Both deduced amino acid sequences contain the Asp/His/Ser catalytic triad and the highly conserved sequences surrounding it. Ssp2 also has the aspartate and two glycine residues in the specificity pocket, marking this as a typical trypsin. The positioning of the residues in the specificity pocket of Sspl is unusual; aspartate and glycine residues are present, which is typical of trypsin, but both are separated from surrounding conserved residues by additional amino acids; the second glycine found in the specificity pocket of trypsin is replaced by a serine, which is typical of chymotrypsin. Although a serine protease, the precise substrate specificity of Sspl remains to be determined. Northern analysis shows that both serine proteases are expressed constitutively with only a 20% change in the levels of expression of Ssp1 and Ssp2 through the digestive cycle, and that expression occurs predominantly in the opaque region of the midgut, the region responsible for secretion of digestive enzymes.     

cDNA cloning and expression of a novel serine protease, TLSP

A cDNA for a putative novel serine protease, TLSP, was cloned from human hippocampus cDNA with polymerase chain reaction based strategies. The putative amino acid sequence of TLSP is similar to the trypsin-type serine proteases. TLSP mRNA is expressed in keratinocytes. Overexpressed TLSP protein in neuro2a cells was detected in culture medium.     

A conserved element in the serine protease domain of complement factor B

Factor B and C2 are serine proteases that carry the catalytic sites of the complement C3 and C5 convertases. Their protease domains are activated by conformational changes that occur during convertase assembly and are deactivated upon convertase dissociation. Factor B and C2 share an 8-amino acid conserved sequence near their serine protease termini that is not seen in other serine proteases. To determine its importance, 24 factor B mutants were generated, each with a single amino acid substitution in this region. Whereas most mutants were functionally neutral, all five different substitutions of aspartic acid 715 and one phenylalanine 716 substitution severely reduced hemolytic activity. Several aspartic acid 715 mutants permitted the steps of convertase assembly including C3b-dependent factor D-mediated cleavage and activation of the high affinity C3b-binding site, but the resulting complexes did not cleave C3. Given that factor B and C2 share the same biological substrates and that part of the trypsin-like substrate specificity region is not apparent in either protein, we propose that the conserved region plays a critical role in the conformational regulation of the catalytic site and could offer a highly specific target for the therapeutic inhibition of complement.     

Substrate specificity of honeydew melon protease D, a plant serine endopeptidase

The substrate specificity of honeydew melon (Cucumis melo var. inodorus Naud) protease D was studied by the use of synthetic substrates and oligopeptides derived from a protein hydrolyzate. The hydrolysis rates of succinyl-(L-Ala)1-3-p-nitroanilide (Suc-(Ala)1-3-pNA) the hydrolysis rate progressively rose in proportion to the increased chain length. Benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester (Z-Tyr-ONp) and benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt) were cleaved by honeydew melon protease D, but benzoyl-L-arginine p-nitroanilide (Bz-Arg-pNA), benzyloxycarbonyl-L-lysine p-nitrophenyl ester (Z-Lys-ONp) and tosyl-L-arginine methyl ester (Tos-Arg-OMe) were not hydrolyzed. Contrary to the results obtained by using synthetic substrates, the carboxyl sides of charged amino acid residues were preferentially cleaved by the enzyme in the oligopeptide substrates. The substrates that had charged or polar amino acids at P2 positions were not cleaved. On the other hand, the non-polar amino acid or proline at P2 were favored for hydrolysis. The information concerning the subsite of protease D was obtained and is useful for synthesis of a good substrate. As it is distinct from molecular mass, the substrate specificity of honeydew melon protease D is most analogous to cucumisin [EC 3.4.21.25] among serine proteases from cucurbitaceous plants.     

Epstein-Barr virus (EBV) and Hodgkin''s disease in a multi-ethnic population in Malaysia

The plant pathogenic fungus Verticillium dahliae produced extracellular alkaline protease activity when grown in liquid medium supplemented with a protein source. A serine protease was purified 80-fold in a single step, using cation-exchange chromatography, from the filtrate of cultures grown with skim milk as a protein source. N-terminal amino acid sequence analysis of the 30-kDa protein (VDP30) that copurified with the serine protease activity suggested that VDP30 is a trypsin-like protein. The purified enzyme hydrolyzed the synthetic substrate N alpha-benzoyl-DL-arginine p-nitroanilide hydrochloride (BAPNA), and the activity on BAPNA was inhibited by leupeptin, further verifying the trypsin-like nature of the enzyme.     

A novel protease in the pupal yellow body of Sarcophaga peregrina (flesh fly). Its purification and cDNA cloning

We purified a novel serine protease with a molecular mass of 26 kDa from Sarcophaga pupae. This protease appeared almost exclusively in the yellow body, an organ that develops temporarily in the pupae of dipteran insects and expands to form the adult midgut by engulfing the larval midgut. cDNA analysis revealed that this protease consists of 239 amino acid residues and has significant structural similarity with bovine trypsin (about 40% sequence identity). The 26-kDa protease gene was transiently activated in 1-day-old pupae. The protease was found to cross-react immunologically with antibody against sarcotoxin IA, an antibacterial protein produced by this insect. It is suggested that this protease participates in the decomposition of the larval midgut in the yellow body during metamorphosis.     

Mutagenesis of the NS3 protease of dengue virus type 2

The flavivirus protease is composed of two viral proteins, NS2B and NS3. The amino-terminal portion of NS3 contains sequence and structural motifs characteristic of bacterial and cellular trypsin-like proteases. We have undertaken a mutational analysis of the region of NS3 which contains the catalytic serine, five putative substrate binding residues, and several residues that are highly conserved among flavivirus proteases and among all serine proteases. In all, 46 single-amino-acid substitutions were created in a cloned NS2B-NS3 cDNA fragment of dengue virus type 2, and the effect of each mutation on the extent of self-cleavage of the NS2B-NS3 precursor at the NS2B-NS3 junction was assayed in vivo. Twelve mutations almost completely or completely inhibited protease activity, 9 significantly reduced it, 14 decreased cleavage, and 11 yielded wild-type levels of activity. Substitution of alanine at ultraconserved residues abolished NS3 protease activity. Cleavage was also inhibited by substituting some residues that are conserved among flavivirus NS3 proteins. Two (Y150 and G153) of the five putative substrate binding residues could not be replaced by alanine, and only Y150 and N152 could be replaced by a conservative change. The two other putative substrate binding residues, D129 and F130, were more freely substitutable. By analogy with the trypsin model, it was proposed that D129 is located at the bottom of the substrate binding pocket so as to directly interact with the basic amino acid at the substrate cleavage site. Interestingly, we found that significant cleavage activity was displayed by mutants in which D129 was replaced by E, S, or A and that low but detectable protease activity was exhibited by mutants in which D129 was replaced by K, R, or L. Contrary to the proposed model, these results indicate that D129 is not a major determinant of substrate binding and that its interaction with the substrate, if it occurs at all, is not essential. This mutagenesis study provided us with an array of mutations that alter the cleavage efficiency of the dengue virus protease. Mutations that decrease protease activity without abolishing it are candidates for introduction into the dengue virus infectious full-length cDNA clone with the aim of creating potentially attenuated virus stocks.     

Dopamine D2 receptor mRNA is expressed in maturing neurons of the human hippocampal and subicular fields

Pyramidal neurons of the adult and fetal hippocampus and subicular fields were shown to express D2 mRNA using non-radioactive in situ hybridization histochemistry. At the earliest developmental stages examined (embryonic week (E) 13), cell packing within the CA1 region is dense and immature neuroblasts express D2 mRNA at high levels, as do more mature pyramid-like neurons in the deep aspect of the pyramidal cell layer. With development (E19 and E24), cell packing density is reduced, maturing neurons of the pyramidal layer are prominently D2 mRNA positive, while the majority of immature cells lining the superficial layer are D2 mRNA negative. In Layer II of the presubiculum there is a high density of immature D2 mRNA negative cells at E13 with D2 mRNA positive cells located on the periphery of the clusters. By E24, the cells in the layer II clusters are larger, express D2 mRNA, and D2 mRNA negative cells are rarely observed. Thus, expression of D2 mRNA in humans is an early and permanent feature of pyramidal neurons of these regions.     

cDNA sequence and chromosomal localization of human enterokinase, the proteolytic activator of trypsinogen

Enterokinase is a serine protease of the duodenal brush border membrane that cleaves trypsinogen and produces active trypsin, thereby leading to the activation of many pancreatic digestive enzymes. Overlapping cDNA clones that encode the complete human enterokinase amino acid sequence were isolated from a human intestine cDNA library. Starting from the first ATG codon, the composite 3696 nt cDNA sequence contains an open reading frame of 3057 nt that encodes a 784 amino acid heavy chain followed by a 235 amino acid light chain; the two chains are linked by at least one disulfide bond. The heavy chain contains a potential N-terminal myristoylation site, a potential signal anchor sequence near the amino terminus, and six structural motifs that are found in otherwise unrelated proteins. These domains resemble motifs of the LDL receptor (two copies), complement component Clr (two copies), the metalloprotease meprin (one copy), and the macrophage scavenger receptor (one copy). The enterokinase light chain is homologous to the trypsin-like serine proteinases. These structural features are conserved among human, bovine, and porcine enterokinase. By Northern blotting, a 4.4 kb enterokinase mRNA was detected only in small intestine. The enterokinase gene was localized to human chromosome 21q21 by fluorescence in situ hybridization.     

Two lineages of mannose-binding lectin-associated serine protease (MASP) in vertebrates

Mannose-binding lectin-associated serine protease (MASP) is a newly identified member of the serine protease superfamily. MASP is involved in host defense against pathogens through a novel system of complement activation, designated the lectin pathway. To elucidate the origin of the lectin pathway and the molecular evolution of MASP, we cloned six MASP cDNAs from five vertebrate species going from mammal to cyclostome. An alignment of the amino acid sequences deduced from the cDNAs revealed the presence of two different lineages of the MASP gene. This classification was supported by the precise correlation with two types of exon organization for the protease domain. One of the two lineages is unique in that a single exon encodes the protease domain, unlike most other serine proteases. All members of this group, termed the AGY type, have an AGY codon at the active site serine. A phylogenetic tree suggests that the AGY type diverged from another lineage, termed the TCN type, before the emergence of primitive vertebrates. Furthermore, the presence of MASP or MASP-like sequences in most vertebrate species suggests that the lectin pathway functions extensively in vertebrates and that its origin is traced back to the invertebrate stage.     

Isolation and characterization of immune-related genes from the fall webworm, Hyphantria cunea, using PCR-based differential display and subtractive cloning

Following injection of bacteria into the hemocoel of the fall webworm, Hyphantria cunea, several inducible genes were identified and characterized using PCR-based differential display (DD-PCR) and subtractive cloning. Ten immune-related cDNA clones (Hdd1, Hdd2, Hdd3, Hdd11, Hdd13, Hdd15, Hdd17, Hdd23, Hs106, Hs302) were isolated and characterized. The deduced amino acid sequence of Hdd2 was shown to be a member of the copper, zinc superoxide dismutase (Cu-Zn SOD) family. The H. cunea Cu-Zn SOD is novel in that it is up-regulated following a bacterial challenge and has a putative signal peptide suggesting its secretion and involvement in the insect immune response. Hdd3 was found to encode a new member of the serpin (serine protease inhibitor) family. The putative lectin corresponding to Hdd15 is of a different kind in that it has two lectin C domains in a single molecule. These two lectin C domains show significant homology to the lectin C domain of Periplaneta lipopolysaccharide binding protein (LPS-BP). Three cloned genes, Hdd17, Hs106 and Hs302, encode a homologue to Bombyx mori Gram negative binding protein, a hemolin-like protein and a attacin-like protein, respectively. The deduced amino acid sequences from Hdd11 showed weak homology with a Locusta migratoria hemolymph protein. On the contrary, Hdd1, Hdd13 and Hdd23 did not reveal any significant homology with known proteins. All of the 10 genes were clearly inducible by E. coli and M. luteus injection. Injection of distilled water only slightly induced mRNA levels. Comparison of temporal mRNA expression following E. coli injection showed three types of expression patterns.     

Age-related changes in the inner zone of the adrenal cortex of the rat--a morphologic and biochemical study

Despite an urgent medical need, a broadly effective anti-viral therapy for the treatment of infections with hepatitis C viruses (HCVs) has yet to be developed. One of the approaches to anti-HCV drug discovery is the design and development of specific small molecule drugs to inhibit the proteolytic processing of the HCV polyprotein. This proteolytic processing is catalyzed by a chymotrypsin-like serine protease which is located in the N-terminal region of non-structural protein 3 (NS3). This protease domain forms a tight, non-covalent complex with NS4A, a 54 amino acid activator of NS3 protease. The C-terminal two-thirds of the NS3 protein contain a helicase and a nucleic acid-stimulated nucleoside triphosphatase (NTPase) activities which are probably involved in viral replication. This review will focus on the structure and function of the serine protease activity of NS3/4A and the development of inhibitors of this activity.     

Isolation and identification of the cDNA encoding the pheromone biosynthesis activating neuropeptide and additional neuropeptides in the oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae)

The present study is concerned with cloning and characterizing Has-PBAN cDNA which is 756 nucleotides long, isolated from the brain and suboesophageal ganglion complex (Br-Sg) of Helicoverpa assulta adults. The 194-amino acid sequence deduced from this cDNA possessed the proteolytic endocleavage sites to generate multiple peptides. From the processing of the prepro-hormone, it can be predicted that the cDNA has a PBAN domain with 33 amino acids and four additional peptide domains: 24 amino acid-, 7 amino acid-, 18 amino acid- and 8 amino acid-long sequences, with FXPR (or K) L (X = G, T or S) amidated at their C-termini. The amino acid sequence of all five predicted peptides, including the PBAN, are identical to that of Helicoverpa zea (Raina, A.K., Jaffe, H., Kempe, T.G., Keim, P., Blacher, R.W., Fales, H.M., Riley, C.T., Klun, J.A., Ridgway, R.L., Hayes, D.K., 1989. Identification of a neuropeptide hormone that regulates sex pheromone production in female moths. Science 244, 796-798 and Ma, P.W.K., Knipple, D.C., Roelofs, W.L., 1994. Structural organization of the Helicoverpa zea gene encoding the precursor protein for pheromone biosynthesis-activating neuropeptide and other neuropeptides. Proc. Natl. Acad. Sci., U.S.A. 91, 506-510). A single mRNA species corresponding to the size of Has-PBAN cDNA was detected from the Br-Sg of 1-3-day old female and male adults, and their expression was also at a similar level. Pheromone production was induced upon injection of female or male Br-Sg extracts or synthetic PBAN into the haemocoel of decapitated 1-3-day old female adults during the photophase when they are not supposed to produce pheromone. From these results, H. assulta adult females seem to use their own PBAN for regulating sex pheromone biosynthesis. Functions of the four other peptides ending with FXPR (or K) L in the Has-PBAN cDNA and of the male PBAN remain to be elucidated.     

Site-specific cleavage of tRNA by imidazole and/or primary amine groups bound at the 5''-end of oligodeoxyribonucleotides

Thrombin, a serine protease, is a potent mitogen for vascular smooth muscle cells (SMCs), but its mechanism of action is not known. Since L-ornithine is metabolized to growth-stimulatory polyamines, we examined whether thrombin regulates the transcellular transport and metabolism of L-ornithine by vascular SMCs. Treatment of SMCs with thrombin initially (0 to 2 hours) decreased L-ornithine uptake, whereas longer exposures (6 to 24 hours) progressively increased transport. Kinetic studies indicated that thrombin-induced inhibition was associated with a decrease in affinity for L-ornithine, whereas stimulation was mediated by an increase in transport capacity. Thrombin induced the expression of both cationic amino acid transporter (CAT)-1 and CAT-2 mRNA. Furthermore, thrombin stimulated L-ornithine metabolism by inducing ornithine decarboxylase (ODC) mRNA expression and activity. The stimulatory effect of thrombin on both L-ornithine transport and ODC activity was reversed by hirudin, a thrombin inhibitor, and was mimicked by a 14-amino acid thrombin receptor-activating peptide. Thrombin also markedly increased the capacity of SMCs to generate putrescine, a polyamine, from extracellular L-ornithine. The thrombin-mediated increase in putrescine production was reversed by N(G)-methyl-L-arginine, a competitive inhibitor of cationic amino acid transport, or by alpha-difluoromethylornithine (DFMO), an ODC inhibitor. DFMO also inhibited thrombin-induced SMC proliferation. These results demonstrate that thrombin stimulates polyamine synthesis by inducing CAT and ODC gene expression and that thrombin-stimulated SMC proliferation is dependent on polyamine formation. The ability of thrombin to upregulate L-ornithine transport and direct its metabolism to growth-stimulatory polyamines may contribute to postangioplasty restenosis and atherosclerotic lesion formation.     

Characterization of the precursor of prostate-specific antigen. Activation by trypsin and by human glandular kallikrein

The precursor or zymogen form of prostate-specific antigen (pro-PSA) is composed of 244 amino acid residues including an amino-terminal propiece of 7 amino acids. Recombinant pro-PSA was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified. The zymogen was readily activated by trypsin at a weight ratio of 50:1 to generate PSA, a serine protease that cleaves the chromogenic chymotrypsin substrate 3-carbomethoxypropionyl-L-arginyl-L-prolyl-L-tyrosine-p-nitroanili ne- HCl (S-2586). In this activation, the amino-terminal propiece Ala-Pro-Leu-Ile-Leu-Ser-Arg was released by cleavage at the Arg-Ile peptide bond. The recombinant pro-PSA was also activated by recombinant human glandular kallikrein, another prostate-specific serine protease, as well as by a partially purified protease(s) from seminal plasma. The recombinant PSA was inhibited by alpha1-antichymotrypsin, forming an equimolar complex with a molecular mass of approximately 100 kDa. The recombinant PSA failed to activate single chain urokinase-type plasminogen activator, in contrast to the recombinant hK2, which readily activated single chain urokinase-type plasminogen activator. These results indicate that pro-PSA is converted to an active serine protease by minor proteolysis analogous to the activation of many of the proteases present in blood, pancreas, and other tissues. Furthermore, PSA is probably generated by a cascade system involving a series of precursor proteins. These proteins may interact in a stepwise manner similar to the generation of plasmin during fibrinolysis or thrombin during blood coagulation.     

Serine protease inhibitors expressed in the process of budding of tunicates as revealed by EST analysis

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.