Enhancing regional perinatal care: a clinical traineeship for perinatal nurses in a predominantly rural area

The gene nprM encoding the calcium-dependent extracellular proteinase from Bacillus megaterium ATCC 14581 was cloned in the vector pBR322 and expressed in Escherichia coli HB101. The DNA sequence of the cloned 3.7 kb fragment revealed only one open reading frame consisting of 1686 bp with a coding capacity of 562 amino acid residues. A predicted Shine-Dalgarno (SD) sequence was observed 9 bp upstream from the presumptive translation start site (ATG). A possible promoter sequence (TAGACG for the -35 region and TATAAT for the -10 region) was found about 69 bp upstream of the ATG start site. The deduced amino acid sequence exhibited a 24 amino acid residue signal peptide and an additional polypeptide 'pro' sequence of 221 amino acids preceding the putative mature protein of 317 amino acid residues. Amino acid sequence comparison revealed 84.5% homology between the mature protein and that of a thermolabile neutral protease from B. cereus. It also shares 73% homology with the thermostable neutral proteases of B. thermoproteolyticus and B. stearothermophilus. The zinc-binding sites and the catalytic residues are completely conserved in all four proteases. NprM has a temperature optimum of 58 degrees C, a pH optimum of between 6.4 and 7.2, and is stimulated by calcium ions and inhibited by EDTA. These results indicate that the enzyme is a neutral (metallo-) protease.     

Purification and biochemical analysis of WprA, a 52-kDa serine protease secreted by B. subtilis as an active complex with its 23-kDa propeptide

The Gram-positive bacterium Bacillus subtilis produces numerous proteases that are secreted to the extracellular milieu, and as strains are generated which lack the more prominent proteases, minor ones become detectable. We have isolated a 52-kDa secreted protease from the protease-deficient strain WB600. It is encoded by the wprA gene which encompasses a signal sequence, a 46-kDa propeptide further processed to 23 kDa, and the 52-kDa mature protease. The 52-kDa and 23-kDa polypeptides were previously detected in cell-wall preparations of a wild-type strain. We have co-purified these proteins from culture supernatant, and confirmed the same N-termini and molecular weights as the membrane-bound species. The WprA protease domain has 28.5% identity to subtilisin A, and like other subtilisins, it displays a broad substrate specificity. WprA and subtilisin A have similar pH profiles, showing optimal activity near pH 7.5 for substrates with Met, Gln, or Lys residues at P1. Using a substrate with Asp at P1, another peak of activity was observed for WprA at pH 5 and at pH 6 for subtilisin A. The pH dependence of some bacterial proteases in their interaction with substrates and inhibitors may be biologically relevant.     

Influence of selective opiate antagonists on striatal acetylcholine and dopamine release

Ty3 is a retroviruslike element found in Saccharomyces cerevisiae. It encodes GAG3 and GAG3-POL3 polyproteins which are processed into mature proteins found in the Ty3 viruslike particle. In this study, the region encoding a protease that is homologous to retroviral aspartyl proteases was identified and shown to be required for production of mature Ty3 proteins and transposition. The Ty3 protease has the Asp-Ser-Gly consensus sequence found in copia, Ty1, and Rous sarcoma virus proteases, rather than the Asp-Thr-Gly found in most retroviral proteases. The Asp-Ser-Gly consensus is flanked by residues similar to those which flank the active sites of cellular aspartyl proteases. Mutations were made in the Ty3 active-site sequence to examine the role of the protease in Ty3 particle maturation and to test the functional significance of the Ser active-site variant in the consensus sequence. Mutation of the active-site Asp blocked processing of Gag3 and Gag3-Pol3 and allowed identification of a GAG3-POL3 polyprotein. This protein was turned over rapidly in cells expressing the mutant Ty3. Changing the active-site Ser to Thr caused only a modest reduction in the levels of certain Ty3 proteins. Five putative cleavage sites of this protease in Ty3 GAG3 and GAG3-POL3 polyproteins were defined by amino-terminal sequence analysis. The existence of an additional protein(s) of unknown function, encoded downstream of the protease-coding region, was deduced from the positions of these amino termini and the sizes of known Ty3 proteins. Although Ty3 protease cleavage sites do not correspond exactly to known retroviral protease cleavage sites, there are similarities. Residues P3 through P2' in the regions encompassing each of the five sites are uncharged, and no P1 position is occupied by an amino acid with a branched beta carbon.     

Protease evolution in Streptomyces griseus. Discovery of a novel dimeric enzymes

This report describes the cloning and sequencing of a novel protease gene derived from Streptomyces griseus. Also described is the heterologous expression of the gene in Bacillus subtilis and characterization of the gene product. The sprD gene encodes a prepro mature protease of 392 amino acids tentatively named S. griseus protease D (SGPD). A significant component of the enzyme preregion was found to be homologous with the mitochondrial import signal of hsp60. The sprD gene was subcloned into an Escherichia coli/B. subtilis shuttle vector system such that the pro mature portion of SGPD was fused in frame with the promoter, ribosome binding site, and signal sequences of subtilisin. The gene fusion was subsequently expressed in B. subtilis DB104, and active protease was purified. SGPD has a high degree of sequence homology to previously described S. griseus proteases A, B, C, and E and the alpha-lytic protease of Lysobacter enzymogenes, but unlike all previously characterized members of the chymotrypsin superfamily, the recombinant SGPD forms a stable alpha 2 dimer. The amino acid sequence of the protein in the region of the specificity pocket is similar to that of S. griseus proteases A, B, and C. The purified enzyme was found to have a primary specificity for large aliphatic or aromatic amino acids. Nucleotide sequence data were used to construct a phylogenetic tree using a method of maximum parsimony which reflects the relationships and potentially the lineage of the chymotrypsin-like proteases of S. griseus.     

Human cathepsin F. Molecular cloning, functional expression, tissue localization, and enzymatic characterization

A cDNA for a novel human papain-like cysteine protease, designated cathepsin F, has been cloned from a lambdagt10-skeletal muscle cDNA library. The nucleotide sequence encoded a polypeptide of 302 amino acids composed of an 88-residue propeptide and a 214-residue mature protein. Protein sequence comparisons revealed 58% homology with cathepsin W; about 42-43% with cathepsins L, K, S, H, and O; and 38% with cathepsin B. Sequence comparisons of the propeptides indicated that cathepsin F and cathepsin W may form a new cathepsin subgroup. Northern blot analysis showed high expression levels in heart, skeletal muscle, brain, testis, and ovary; moderate levels in prostate, placenta, liver, and colon; and no detectable expression in peripheral leukocytes and thymus. The precursor polypeptide of human recombinant cathepsin F, produced in Pichia pastoris, was processed to its active mature form autocatalytically or by incubation with pepsin. Mature cathepsin F was highly active with comparable specific activities toward synthetic substrates as reported for cathepsin L. The protease had a broad pH optimum between 5.2 and 6.8. Similar to cathepsin L, its pH stability at cytosolic pH (7.2) was short, with a half-life of approximately 2 min. This may suggest a function in an acidic cellular compartment. Transient expression of T7-tagged cathepsin F in COS-7 cells revealed a vesicular distribution of the gene product in the juxtanuclear region of the cells. However, contrary to all known cathepsins, the open reading frame of the cathepsin F cDNA did not encode a signal sequence, thus suggesting that the protease is targeted to the lysosomal compartment via an N-terminal signal peptide-independent lysosomal targeting pathway.     

Serum ferritin, hemoglobin, and Helicobacter pylori infection: a seroepidemiologic survey comprising 2794 Danish adults

Subtilisin-like serine protease, which is associated with the dormant spores of Bacillus cereus, was solubilized by washing the spores with 2 M KCl and purified to homogeneity by carbobenzoxy-D-phenylalanine-liganded affinity column chromatography and hydrophobic interaction column chromatography. Enzyme activity was completely inhibited by reagents for sulfhydryl groups such as HgCl2 as well as by conventional subtilisin inhibitors, suggesting the enzyme to be cysteine-dependent. The enzyme retained activity in 5 M urea at 4 degrees C for at least 2 months, and the specific activity was 50 times that of subtilisin BPN when measured for a common chromogenic substrate, carbobenzoxy-glycyl-glycyl-L-leucine p-nitroanilide. The gene encoding this protease was cloned in Escherichia coli, and its nucleotide sequence was analyzed. The deduced amino acid sequence suggested that the protease is produced as a precursor comprising three portions; a signal sequence (28 amino acid residues), a prosequence (80 amino acid residues) and a mature enzyme (289 amino acid residues). The mature region of the enzyme had high similarity with a thermitase from Thermoactinomyces vulgaris (72% identity) and a thermostable alkaline protease from Thermoactinomyces sp. E79 (66% identity), which have the N-terminal sequence showing scarcely noticeable similarity with corresponding stretches of subtilisins and mercuric ion-sensitive free cysteine in the equivalent position of the primary structure.     

Molecular cloning, characterization and overexpression of two distinct cysteine protease cDNAs from Leishmania donovani chagasi

We have cloned and characterized two distinct cysteine protease cDNAs from Leishmania donovani chagasi. One of the cDNAs, Ldccy2, was isolated from a cDNA library prepared from total promastigote RNA while the other cDNA, Ldccys1, was isolated from a cDNA library prepared from total amastigote RNA. Ldccys2 has an open reading frame of 471 amino acids and Ldccys1 has an open reading frame of 447 amino acids. Comparison of the predicted protein sequences of the two distinct cysteine proteases with those of cysteine proteases from Leishmania pifanoi, a member of the L. mexicana complex, showed that the cysteine proteases from the two species of Leishmania are similar in their protein sequences. Each of the two cDNAs is distinct in genomic arrangement and chromosome location. Ldccys1 belongs to a family of cysteine proteases encoded by tandemly organized genes located on chromosome 7 while Ldccys2 appears to be a single cysteine protease gene located on chromosome 10. The organization of the two families of cysteine protease genes in L. donovani donovani was also found to be similar. In this species, the Lddcys1 genes are located on chromosome 5 while the Lddcys2 gene is located on chromosome 8. The Ldccys1 genes are expressed abundantly in the amastigotes recovered from infected hamsters, but at a very low level in the promastigote stage of development. On the other hand, the Ldccys2 gene is expressed both in the promastigote and amastigote stages. We have overexpressed the two cDNAs of cysteine proteases in Leishmania cells and the over-produced cysteine proteases are biologically active and are inhibited by cysteine protease inhibitors. Furthermore, the over-produced and indigenous amastigote specific cysteine protease, Ldccys1, reacted with polyclonal antibodies raised against this protein.     

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.     

The sequence of a subtilisin-type protease (aerolysin) from the hyperthermophilic archaeum Pyrobaculum aerophilum reveals sites important to thermostability

The hyperthermophilic archaeum Pyrobaculum aerophilum grows optimally at 100 degrees C and pH 7.0. Cell homogenates exhibit strong proteolytic activity within a temperature range of 80-130 degrees C. During an analysis of cDNA and genomic sequence tags, a genomic clone was recovered showing strong sequence homology to alkaline subtilisins of Bacillus sp. The total DNA sequence of the gene encoding the protease (named "aerolysin") was determined. Multiple sequence alignment with 15 different serine-type proteases showed greatest homology with subtilisins from gram-positive bacteria rather than archaeal or eukaryal serine proteases. Models of secondary and tertiary structure based on sequence alignments and the tertiary structures of subtilisin Carlsberg, BPN', thermitase, and protease K were generated for P. aerophilum subtilisin. This allowed identification of sites potentially contributing to the thermostability of the protein. One common transition put alanines at the beginning and end of surface alpha-helices. Aspartic acids were found at the N-terminus of several surface helices, possibly increasing stability by interacting with the helix dipole. Several of the substitutions in regions expected to form surface loops were adjacent to each other in the tertiary structure model.     

Engineering, characterization and phage display of hepatitis C virus NS3 protease and NS4A cofactor peptide as a single-chain protein

The polyprotein encoded by hepatitis C virus (HCV) genomic RNA is processed into functional polypeptides by both host- and virus-encoded proteases. The HCV-encoded NS3 protease and its cofactor peptide NS4A form a non-covalent complex, which participates in processing the viral polyprotein. This proteolytic activity is believed to be essential for virus proliferation and thus the NS3 protease is a prime target for developing anti-HCV pharmacological agents. Recent X-ray crystallography structural studies have revealed the nature of this non-covalent complex between NS3 protease and the 'active' central segment of NS4A, providing the opportunity to design a single-chain polypeptide. To this end, the DNA sequence encoding for the NS4A peptide (residues 21-34) was genetically fused via a short linker, capable of making a beta-turn, to the N-terminus of the NS3 protease domain. This engineered single-chain NS3-protease (scNS3) is fully active with kinetic parameters virtually identical with those of the NS3/ NS4A non-covalent complex. Moreover, the scNS3 protease can be displayed on filamentous phage and affinity selected using an immobilized specific inhibitor. The scNS3 expressed as a soluble protein and in a phage-display format facilitates enzyme engineering for further structural studies and in vitro selection of potential drug-resistant mutants. These are important steps towards developing effective anti-protease compounds.     

A polysomal ribonuclease involved in the destabilization of albumin mRNA is a novel member of the peroxidase gene family

We have purified an approximately 60 kDa endoribonuclease from Xenopus liver polysomes with properties expected for a messenger RNase involved in the estrogen-regulated destabilization of serum protein mRNAs (Dompenciel et al., 1995, J Biol Chem 270:6108-6118). The present report describes the cloning of this protein and its identification as a novel member of the peroxidase gene family. This novel enzyme, named polysomal RNase 1, or PMR-1 has 57% sequence identity with myeloperoxidase, and like that protein, appears to be processed from a larger precursor. Unlike myeloperoxidase, however, PMR-1 lacks N-linked oligosaccharide, heme, and peroxidase activity. Western blot and immunoprecipitation experiments using epitope-specific antibodies to the derived protein sequence confirm the identity of the cloned cDNA to the protein originally isolated from polysomes. The 80 kDa pre-PMR-1 expressed in a recombinant baculovirus was not processed to the 60 kDa form in Sf9 cells and lacks RNase activity. However, the baculovirus-expressed mature 60-kDa form of the enzyme has RNase activity. The recombinant protein is an endonuclease that shows selectivity for albumin versus ferritin mRNA. While it does not cleave at consensus APyrUGA elements, recombinant PMR-1 generates the same minor cleavage products from albumin mRNA as PMR-1 purified from liver. Finally, we show estrogen induces only a small increase in the amount of PMR-1. This result is consistent with earlier data suggesting estrogen activates mRNA decay through a posttranslational pathway.     

Pyrrolidone carboxyl peptidase from the hyperthermophilic Archaeon Pyrococcus furiosus: cloning and overexpression in Escherichia coli of the gene, and its application to protein sequence analysis

A gene for a pyrrolidone carboxyl peptidase (Pcp: EC 3.4.19.3, pyroglutamyl peptidase), which removes amino-terminal pyroglutamyl residues from peptides and proteins, has been cloned from the hyperthermophilic Archaeon Pyrococcus furiosus using its cosmid protein library, sequenced, and expressed in Escherichia coli. The DNA sequence encodes a protein containing 208 amino acid residues with methionine at the N-terminus. Analysis of the recombinant protein expressed in E. coli, including amino acid sequence analysis from the N-terminus by automated Edman degradation and ionspray mass spectrometric analysis of the peptides generated by enzymatic digestions with lysylendopeptidase and Staphylococcus aureus V8 protease, showed its primary structure to be completely identical with that deduced from its cDNA sequence. Comparison of the amino acid sequence of P. furiosus Pcp (P.f.Pcp) with those of bacterial Pcps revealed that a high degree of sequence identity (more than 40%) and conservation of the amino acid residues comprising the catalytic triad, Cys142, His166, and Glu79. On the other hand, a unique short stretch sequence (positions around 175-185) that is absent in bacterial Pcps was found in P.f.Pcp. A similar stretch has also been reported recently in the amino acid sequence of Pcp from the hyperthermophilic Archaeon Thermococcus litoralis [Littlechild et al., in abstracts of the "International Congress on Exthermophiles '98" p. 58 (1998)]. To elucidate their contribution to the hyperthermostability of these enzymes, further structural studies are required.     

The prosequence of thermolysin acts as an intramolecular chaperone when expressed in trans with the mature sequence in Escherichia coli

The zinc metalloendopeptidase, thermolysin (EC 3.4.24.27) produced by Bacillus thermoproteolyticus serves as a model of important physiological enzymes such as neprilysin, angiotensin converting enzyme and endothelin converting enzyme. Thermolysin is synthesised as a pre-proenzyme, with an N-terminal prosequence of 204 residues and a mature sequence of 316 residues. The prosequence facilitates the folding of the denatured mature sequence in vitro and the cleavage of the peptide bond linking the pro and mature sequences occurs by an autocatalytic, intramolecular process. With the aim to study the role of the prosequence in vivo and to produce active mutants for structural studies, the mature sequence of thermolysin has now been expressed in Escherichia coli, either alone or with the prosequence as an independent polypeptide, i.e. in trans form. In addition, the mature sequence of an inactive mutant in which Glu143 involved in the catalytic process was replaced by Ala has also been expressed in trans with the prosequence. The results show that the pro-sequence is required to obtain active thermolysin and that a covalent link with the mature sequence is not necessary for the correct folding of the protease in vivo. Moreover, when expressed in E. coli (in trans with the prosequence), the yield of correctly folded E143A mutant was similar to that of the wild-type protease, whereas no mature enzyme was detected when it was expressed as a pre-proenzyme in Bacillus subtilis. These results demonstrate that the thermolysin prosequence acts as an intramolecular chaperone in vivo and open the way to structural studies of catalytic site mutants produced in large quantities in E. coli.     

Molecular cloning of capillary permeability-increasing enzyme-2 from Agkistrodon caliginosus (Korean viper)

The gene of capillary permeability-increasing enzyme-2 (CPI enzyme-2) was cloned from the cDNA library of Agkistrodon caliginosus and its nucleotide sequence was determined. Its sequence indicates that CPI enzyme-2 is synthesized as a pre-zymogen of 258 amino acid residues, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acids. The amino terminal sequence deduced from the cDNA sequence was exactly consistent with that of CPI enzyme-2 except for the substitution of an amino acid (Gly27-->Ser). The open reading frame is very similar to those of plasminogen activator and thrombin-like proteases cloned from other snakes. The clone encoding CPI enzyme-2 belongs to the serine protease family. The active site of the enzyme is highly conserved at His41, Asp86 and Ser180. Its possible glycosylation sites, Asn-X-Thr/Ser, are located at amino acid residues 20-22, 55-57, 79-81 and 97-99.     

Identification of a new member of transforming growth factor-beta superfamily in Drosophila: the first invertebrate activin gene

Activins, a subgroup of the transforming growth factor-beta (TGF-beta) superfamily, have been extensively studied in vertebrates for their roles in growth and development. However, activins are not thought to be expressed in invertebrates. The identification of the first invertebrate activin gene is reported here. A genomic clone representing 102 F region of the Drosophila chromosome 4 is found to encode a putative activin beta. The predicted protein sequence has a multibasic protease site that would generate a mature C-terminal peptide containing 113 amino acids showing > 60% similarity to the vertebrate activin beta B (inhibin beta B) sequences. A TGF-beta family signature as well as all 9 cysteine residues conserved in the vertebrate activins are also present in this mature peptide sequence. Northern blot and RT-PCR analyses indicated that the activin beta gene is expressed in embryo, larva and adult stages of Drosophila.     

Cold-active serine alkaline protease from the psychrotrophic bacterium Shewanella strain ac10: gene cloning and enzyme purification and characterization

The gene encoding serine alkaline protease (SapSh) of the psychrotrophic bacterium Shewanella strain Ac10 was cloned in Escherichia coli. The amino acid sequence deduced from the 2,442-bp nucleotide sequence revealed that the protein was 814 amino acids long and had an estimated molecular weight of 85,113. SapSh exhibited sequence similarities with members of the subtilisin family of proteases, and there was a high level of conservation in the regions around a putative catalytic triad consisting of Asp-30, His-65, and Ser-369. The amino acid sequence contained the following regions which were assigned on the basis of homology to previously described sequences: a signal peptide (26 residues), a propeptide (117 residues), and an extension up to the C terminus (about 250 residues). Another feature of SapSh is the fact that the space between His-65 and Ser-369 is approximately 150 residues longer than the corresponding spaces in other proteases belonging to the subtilisin family. SapSh was purified to homogeneity from the culture supernatant of E. coli recombinant cells by affinity chromatography with a bacitracin-Sepharose column. The recombinant SapSh (rSapSh) was found to have a molecular weight of about 44,000 and to be highly active in the alkaline region (optimum pH, around 9.0) when azocasein and synthetic peptides were used as substrates. rSapSh was characterized by its high levels of activity at low temperatures; it was five times more active than subtilisin Carlsberg at temperatures ranging from 5 to 15 degreesC. The activation energy for hydrolysis of azocasein by rSapSh was much lower than the activation energy for hydrolysis of azocasein by the subtilisin. However, rSapSh was far less stable than the subtilisin.     

Partial purification and properties of enzymes involved in the processing of a chloroplast import protein from Chlamydomonas reinhardii

Two stromal peptidases (SPP-1 and SPP-2) were partially purified from chloroplasts of Chlamydomonas reinhardii. They specifically processed in vitro the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSS), which had been synthesized by using the cloned rbcS-2 gene of Chlamydomonas. SPP-1 shortened pSS to an intermediate-sized form (iSS), while SPP-2 cut pSS and iSS to the mature small subunit SS. N-terminal amino acid sequencing demonstrated that the reaction product obtained with SPP-2 had an N-terminus identical to natural SS, and that iSS derived from pSS by hydrolysis at the amino side of the methionine located within the transit sequence. By gel filtration, apparent molecular masses of 340 kDa and 90 kDa were determined for SPP-1 and SPP-2, respectively. The comparison of these molecular masses with the protein patterns obtained by SDS/PAGE of the partially purified enzymes suggested that at least SPP-1 was a multimeric protein. The enzymes differed also in their pH optima of about 8 (SPP-1) and 9 (SPP-2) and in their sensitivity to different inhibitors. However, both enzymes seem to be serine proteases as they were completely blocked by N-alpha-tosyl-L-lysinechloromethane or tosylphenylalaninechloromethane, respectively. Competition experiments, using either mature SS or a synthetic hexadecapeptide with 15 amino acids similar to the C-terminal end of the transit sequence of pSS, indicated that SPP-2 had some affinities not only to the transit sequence of pSS, but especially to sequences in the mature protein part. We conclude that SPP-2 in Chlamydomonas is the enzyme involved in import of pSS into chloroplasts and responsible for its processing by a one-step mechanism.