Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi

Definitions

• This invention relates to a novel Pneumocystis carinii protease and to nucleic acids encoding it.
• the invention also relates to vectors containing the nucleic acids, to cells transformed with the vectors and to antibodies specific for the protease.
• the invention describes uses of all of the above.
• the fungal pathogen Pneumocystis carinii causes potentially fatal pneumonia in the immunocompromised, including those receiving immunosuppressive therapy for organ transplantation, those with advanced malignancy and in particular those with HIV infection.
• the lack of an effective in vitro culture system still remains a major obstacle in the understanding of the biology of P. carinii and its interactions with its host.
• Molecular techniques have been employed in the study of the organism, and a number of genes have now been cloned. Among these is the multi- gene family encoding the major surface glycoprotein, (MSG or gpA) of the parasite.
• the P.cannii major surface glycoprotein is highly mannosylated and is antigenically distinct in organisms isolated from different mammalian host species (Lundgren et al., 1991 ; Gigliotti, 1992).
• the MSG multi-gene family has been identified in the genome of P.carinii sp. f. carinii (rat-derived P.carinii) Kovacs et al., 1993; Wada et al., 1993; Sunkin et al., 1994), P.carinii sp. f. mustelae (ferret-derived P.carinii) (Haidaris et al., 1992; Wright et al., 1995), P.carinii sp. f. hominis (human- derived P.carinii) (Stringer et al., 1993) Garbe & Stringer, 1994) and
• P.carinii sp. f. muris (mouse-derived P.carinii) (Wright et al., 1994).
• the different copies of P.carinii sp. f. carinii MSG genes are of similar size but heterogeneous in sequence. They have been found on multiple chromosomes and often organised in tandem arrays. The majority of MSG genes are located in the subtelomeric regions of the P.carinii sp. f. carinii chromosomes (Underwood et al, 1996; Sunkin & Stringer, 1996).
• MSG genes have been shown to be mediated by the upstream conserved sequence (UCS) which is found on a single chromosome situated in the subtelomeric region. Different copies of MSG have been shown to be linked to the UCS. It has been postulated that this differential expression of MSG may occur in a strategy to evade the immune response of the host by antigenic variation (Wada et al., 1995; Sunkin & Stringer, 1996).
• UCS upstream conserved sequence
• Pneumocystis pneumonia there are two standard treatments for Pneumocystis pneumonia, namely pentamidine or cotrimoxazole. These drugs were originally used because it was thought that Pneumocystis was a protozoan; only recently has genetic sequence analysis placed it in the fungal kingdom. Despite its classification as a fungus, Pneumocystis does not respond to the usual anti-fungal drugs and hence the drug regimes have remained all but unchanged. These regimes are particularly unpleasant with many patients reacting adversely, thus requiring a switch in treatment. Thus AIDS patients in particular would benefit from the development of new anti-PneL/mocys. s therapies since a high proportion of AIDS patients suffer adverse side effects, and many have multiple episodes of P. carinii pneumonia due to their decreasing CD4+ lymphocyte count and persistence of immune suppression.
• Wada and Nakumura (1994) describes the discovery of an open reading frame (designated ORF-3) encoding a protein of unknown function in P.carinii sp. f. carinii and located close to the MSG genes.
• ORF-3 open reading frame
• the sequence given corresponds to a portion of the genes discussed above (Lugli and Wakefield 1996).
• PPT7 Protease 1
• the novel multi-gene family is known as PPT7 (Protease 1); the genes show high levels of homology with the subtilisin-like serine proteases.
• the subtilisin-like serine proteases are a group of endoproteases which have been characterised from a wide variety of organisms including bacteria, fungi and higher eukaryotes.
• pro- hormone processing enzyme kexin encoded by the KEX2 gene of Saccharomyces cerevisiae has been characterised and found to cleave the precursors of the ⁇ -mating factor and the killer toxin (Fuller et al., 1989).
• Mammalian homologues have also been identified incluidng the human fur gene (fes upstream region) in the region upstream of the fes proto- oncogene, encoding the enzyme furin (van den Ouweland et al., 1990).
• the genes Dfurl and Dfur2 from the insect Drosophila melanogaster encoding furin-like proteins (Roebroek et al., 1992) and the bli-4 gene from the nematode Caenorhabditis elegans have also been studied.
• Other members of the subtilisin-like serine protease family have been identified and the specific endoproteolytic activity of some of them has been elucidated. However for many others, the precise biological function has not yet been determined.
• the PRT1 gene product may be a specific endoproteolytic processing enzyme, such as is seen in other subtilisin-like serine proteases. Given that in genetic organisation some copies of PRT1 are generally found in the subtelomeric region, just downstream from the MSG gene, the PRT1 protein encoded by these genes may be involved in the processing of MSG to its mature form. The multicopy nature of the PRT1 gene may reflect the need for processing of enzymes of different specificity for the different types of MSG. Whatever its precise role, the activity of the PRT1 protein is undoubtedly essential to the viability and therefore the pathogenesis of P.carinii.
• the Invention Part of the catalytic domain of a PRT1 gene has been cloned, sequenced and characterised from three types of the host specific fungal pathogen P.carinii, namely P.carinii sp. f. rattus (rat variant), P.carinii sp. f. muris (mouse) and P.carinii sp. f. hominis (human).
• the newly discovered human-infecting P.carinii PRT1 catalytic domain sequence is shown in figure 1 and nucleotide sequence alignments for rat P. carinii, rat variant P. carinii, mouse P. carinii and human-infecting P.carinii PRT1 clones are shown in figure 2. These will enable the sequencing of the remaining parts of a PRT1, using techniques known to those skilled in the art of molecular biology.
• the invention therefore provides in one aspect an isolated DNA comprising part or all of a PRT1 gene of a non-rat infecting species of Pneumocystis carinii.
• the invention also provides an isolated DNA comprising a sequence shown in figure 1 , or a non-rat P. carinii sequence shown in figure 2, or a sequence which hybridises to either of these under stringent conditions.
• the invention provides recombinant vectors containing PPT7 DNA sequences as described herein, and recombinant polypeptides which are part or all of a PRT1 gene product, encoded by the vectors.
• the invention provides synthetic peptides corresponding to antigenic portions of a PRT1 gene product.
• the invention provides a method of producing antibodies specifically immunoreactive with a P.carinii protease, which method comprises using a recombinant polypeptide or a synthetic peptide as described herein to generate an immune response; and antibodies produced by the method.
• the invention provides a method of screening for anti-P ⁇ et/mocys /s carinii compounds, which method comprises providing a source of a recombinant polypeptide expressed by part or all of a PRT7 gene or cDNA, and contacting the compound with the recombinant polypeptide.
• the invention provides an engineered cell transfected with a recombinant vector containing PRT1 DNA sequences as described herein.
• the invention provides an engineered cell line expressing a recombinant polypeptide from part or all of a PPT7 gene or cDNA, useful in a method of screening for anti-P carinii compounds such as protease inhibitors effective against P.carinii.
• the invention provides a P.carinii protease isolated using an antibody specifically immunoreactive with a P.carinii protease, as described herein.
• the invention provides PPT7 clones for part or all of a human-infecting P.carinii PRT1 gene from the PRT1 multi- gene family.
• a part of the PRT1 gene as referred to herein may be for example a fragment of the gene which codes for a specific domain such as the catalytic domain, or it may be a shorter sequence such as a sequence not less than 15 nucleotides in length or not less than 20 nucleotides in length. Sequences of about 15 or about 20 nucleotides in length are generally the shortest practical length of oligonucleotide useful as a sequence specific primer or probe. That is, these are generally the shortest lengths of sequence that will hybridise specifically to a gene sequence under stringent conditions.
• PRT1 multi-gene family will be related genes which will be easily identifiable as such by those skilled in the art, but which may nevertheless differ in location, function and sequence. It will be evident that all members of the PRT1 multi-gene family, which members may variously be described as different genes in the family or as different copies of the PRT1 gene, are included within the scope of the invention.
• mutate or modify nucleic acid sequences can be used in conjunction with this invention to generate useful PRT1 mutant sequences.
• Such methods include but are not limited to point mutations, site directed mutagenesis, deletion mutations, insertion mutations, mutations obtainable from homologous recombination, and mutations obtainable from chemical or radiation treatment.
• Figure 1 shows the genomic DNA sequence of part of the catalytic domain of PRT1 from P.carinii sp. f. hominis. (SEQ ID NO: 22)
• Figure 2 shows DNA sequence alignments for part of the catalytic domain of PRT1 from P.carinii. (Found in GenBank AF001305, GenBank
• Figure 3 shows amino acid sequence alignments of part of the catalytic domain of PRT1, translated from the nucleotide sequences in figure 2.
• Figure 4 shows alignment of P.carinii PRT1 derived amino acid sequences from P.carinii sp. f. carinii clones. (Found in GenBank AF001305,
• Figure 5 shows DNA sequence alignments for P.carinii sp.f. carinii PRT1 clones. (Found in GenBank AF001305, GenBank AF001304 and SEQ ID NOS: 30, 31 , 33 - 47, 32, 48 - 50).
• Figure 5 shows DNA sequence alignments for P.carinii sp.f. carinii PRT1 clones. (Found in GenBank AF001305, GenBank AF001304 and SEQ ID NOS: 30, 31 , 33 - 47, 32, 48 - 50).
• Figure 6 shows a schematic representation of the P.carinii sp. f. carinii
• FIG. 7 shows expressed recombinant PRT1 fragments.
• Some of these copies may be significantly different and form a number of different sub-types. They will all, however, be classed as members of the PRT1 multi-gene family by virtue of homology at some domains of the gene, for example the catalytic domain.
• P.carinii sp. f. carinii PRT1 amino acid sequence namely: i) N-terminal hydrophobic domain ii) Pro-domain iii) Catalytic domain iv) P-domain v) Proline-rich domain vi) Serine-threonine rich domain vii) C-terminal hydrophobic domain
• the P.carinii sp. f. hominis homologues may have fewer, the same number or more domains. Although some domains in some members of P.carinii sp. f. hominis PRT1 gene family may be absent or some extra domains may be present, these genes will still be considered to be members of the PR7 ⁇ 7 multi-gene family.
• the proteins encoded by different copies of this gene family may have a variety of different functions, including: i) as a constituent of the outer cell surface of the parasite, and attached to the cell membrane by a glycosyl- phosphatidylinositol (GPI) anchor ii) the proteolytic processing within a P. carinii sub-cellular organelle of the P.carinii major surface glycoprotein (MSG) to its mature form, possibly at a conserved dibasic amino acid site in the upstream conserved sequence of MSG iii) in the interaction of the parasite with its host, forming a specific ligand on the parasite cell surface which binds to a host receptor molecule
• GPI glycosyl- phosphatidylinositol
• protease is a surface protease.
• Therapeutic intervention The PRT1 protein presents a target for a variety of different therapeutic interventions, which may include: i) Inhibitors of protease activity
• PRT1 may comprise a major surface antigen and therefore provide a potential target for vaccine development.
• Passive immunisation with antibodies to PRT1 may be protective, iv) Analogues Analogues designed to imitate PRT1 may be active in blocking the adherence of P.carinii organisms to a receptor on the human cells.
• P.carinii infection was induced in Sprague Dawley rats by steroid immunosuppression.
• the organisms were isolated and purified from infected rat lung tissue by the method described by Peters et al., (1992).
• Genomic P.carinii DNA was extracted by digestion with proteinase K (1 mg/ml) in the presence of 0.5% SDS and 10mM EDTA, pH8.0, at 50°C for 16h, followed by phenokchloroform extraction and ethanol precipitation.
• P.carinii DNA for use in PFGE experiments was prepared in SeaPlaque GTG agarose as described by Banerji et al., (1993).
• a copy of the PPT7 gene was isolated from an unamplified genomic library from P.carinii sp. f. carinii constructed in ⁇ EMBL3 (Banerji et al., 1993). The library was screened with a cDNA clone containing a region of a P.carinii sp. f. carinii MSG gene (GenBank Accession number GBPLN:PMCANTIA, donated by Dr C J Delves and Dr F Volpe).
• a relatively high number of recombinant plaques gave positive hybridization signals compared to the positive recombinant plaques when the library was screened with a probe derived from the single copy arom locus (Banerji et al., 1993). Five recombinant phages were isolated from the tertiary screen and the DNA was subcloned into the plasmid vector pBluescriptl I .
• a P. carinii sp. f. carinii cDNA library constructed in ⁇ ZAPII was screened with PCR products derived from amplification of the 5' end of the gene with oligonucleotide primer pair pcprot9 and prp4r (9/4r product), and of the 3' end of the gene with pcprot13/RI and pcprot12/RI (13/12 product).
• the primary screening was carried out using both probes, and the secondary and tertiary screens were carried out using only the 9/4r product.
• Oligonucleotide primers were designed to various regions of the P.carinii PRTI nucleotide sequences. Some oligonucleotides had an EcoRI restriction endonuclease site incorporated at the 5' end to facilitate cloning of the amplification products into EcoRI-digested plasmid vectors pBluescript SK(-) (Stratagene) or pUC18 (Pharmacia).
• the final concentration of the amplification reaction mix was 50mM KCI, 10mM Tris (pH8.0), 0.1 % Triton X-100, 3mM MgCI 2 , 400 ⁇ M (each) deoxynucleoside triphosphate, 1 ⁇ M oligonucleotide primer and 0.025 U Taq polymerase ml "1 (Promega, UK).
• primer pair pcprot ⁇ and pcprotI O forty cycles of amplification was performed at 94°C for 1.5 min., 53°C for 1.5 min., and 72°C for 2.0 min.
• primer pair pcpro.9 and pcprot4r the same conditions were used, except an annealing temperature of 50°C was used.
• Prp ⁇ e 1626 bp
• M14 (1279 bp)
• Prp2g 251 bp
• Oligonucleotide primer pairs pcprot ⁇ with pcprotI O, followed by pcprot ⁇ /RI with pcprot4/RI were used in a nested PCR to amplify the 5' fragment, designated Prp5e, of length 1626 base pairs (bp).
• the second portion spanning 1279 bp of the central region oi PRTI, was amplified using a nested PCR with primer pairs pcprot2/RI with pcprotl4/RI, followed by pcprot7/RI with pcprot12/RI.
• the third fragment, Prp2g encompassing the 3' end of the sequence (251 bp), was amplified using oligonucleotides primers pcprot13/RI and pcprot14/RI (Table 1 and Lugli et al 1997).
• the first region amplified with primer pair pcprot1/RI and pcprot3/RI spanned approximately half of the subtilisin-like catalytic domain
• the second region amplified with primer pair pcprot2/RI and pcprot4/RI spanned the end of the subtilisin-like catalytic domain and the start of the P-domain
• the third region amplified with primer pair pcprot7/RI and pcprot8/RI spanned the P-domain
• the fourth region amplified with primer pair 36ex/RI and P.3/RI spanned the proline-rich domain
• the fifth region amplified with primer pair pcprot13/RI and pcprot 14/RI spanned the C-terminal hydrophobic domain.
• the amplification products were gel-purified (GeneCleanl l , BIO101) and labelled with [ ⁇ - 32 P]-dCTP by random priming (Megaprime, Amersham). Hybridisation was carried out at 45°C and stringency washing at 60°C in 0.2xSSC and 0.1 % SDS. Southern blots of genomic P.carinii DNA digested with restriction endonuclease Ps l or SamHI were probed with oligonucleotide probes pcprot3/RI, pcprot5/Rl, pctel2, and msgterm, labelled with [ ⁇ - 32 P]- dATP using polynucleotide kinase. Hybridisation was carried out at 46°C and stringency washing at 52°C in 5xSSC and 0.5% SDS.
• the base composition of the 5' upstream sequence was 74% A+T, and the 3' downstream sequence was 75% A+T.
• PPT7(Paga), the cDNA clone PRT1(73 ⁇ ), the three fragments obtained by PCR amplification of the cDNA library and the other recombinant clones generated by DNA amplification were compared ( Figure 4). Several regions of homology were found and also a number of regions in which significant divergence was observed. These data suggested that the sequences were derived from different copies of the PPT7 gene. Comparison with other subtilisin-like serine proteases
• the deduced amino acid sequence of the cDNA clone PRT1(73 ) was aligned with nine other subtilisin-like serine proteases including fungal, mammalian, insect and nematode sequences.
• the PRT1 sequences showed homology with all the other sequences, with a high level of homology in the subtilisin-like catalytic domain.
• the three essential residues of the catalytic active site, aspartic acid (Asp 21 ), histidine (His 252 ) and serine (Ser 423 ) were conserved in all the PRT1 sequences. The highest levels of homology between all the sequences were around these residues.
• the structural organisation of the fungal sequences showed domains characteristic of this class of processing endoproteases, a hydrophobic signal sequence, a pro domain that may be cleaved by autoproteolysis, a subtilisin-like catalytic domain, a P-domain which is known as such because it is essential for proteolytic activity, a serine/threonine-rich domain which may potentially be modified by O-linked glycosylation, a carboxy-terminal hydrophobic trans-membrane domain and a C-terminal tail with acidic residues (Van de Ven et al., 1993)
• the P.carinii PRT1 sequences showed a putative similar structural organisation but unlike the nine other subtilisin-like serine proteases, they also had a proline-rich domain preceeding the serine-threonine rich domain and the C- terminal hydrophobic domain ( Figure 6).
• the P.carinii PRT1 (73j) sequence had a hydrophobic signal sequence at the N-terminus, followed by a putative pro-domain, a subtilisin-like catalytic domain from Ser 171 to His 474 , a P-domain from residue Tyr 475 to Ser 631 , a proline-rich domain from residue Pro 641 to Pro 707 , a serine-threonine rich domain from residues Thr 708 to Ser 765 , and a carboxy-terminal hydrophobic domain from residues His 771 to Phe 790 .
• subtilisin-like catalytic domain from Ser 171 to His 474
• a P-domain from residue Tyr 475 to Ser 631
• a proline-rich domain from residue Pro 641 to Pro 707
• serine-threonine rich domain from residues Thr 708 to Ser 765
• carboxy-terminal hydrophobic domain from residues His 771 to Phe 790 .
• subtilisin BPNVNovo from Bacillus amyloliquefaciens (Hirono et al., 1984; Bott et al., 1988), subtilisin Carlsberg from ⁇ . licheniformis (McPhalen & James, 1988), thermitase from Thermoactinomyces vulgaris (Gros et al., 1989; Teplyakov et al., 1990) and proteinase K from Titirachium album (Betzel et al., 1988).
• proteases The amino acid sequence of these four proteases has been compared to that of 31 other subtilisin-like serine proteases isolated from bacteria, fungi and higher eukaryotes and the essential core structure of the catalytic domain of this group of molecules has been identified (Siezen et al., 1991).
• subtilase family Nineteen variable regions, generally located in loops on the surface of the molecule, have been identified in the subtilase family, of which 14 were found in the P.carinii PRT1 sequences. Three positions have been identified at which charge is totally conserved in all the subtilisin-like proteases examined, and these were also conserved in the P.carinii PRT1 sequences, the positive charge on Arg 282 and the negative charges on residue Asp 214 (active site) and Asp 223 .
• the putative domains of the PRT1 (73j) polypeptide are summarised in Figure 6.
• a hydrophobicity plot of the PRT1 (73j) sequence revealed a hydrophobic region at the N-terminus suggesting that this may be a signal sequence.
• Residues 1 to 23 of the N-terminus of the sequence showed a high level of homology to the N-terminus of the P.carinii sp.f. carinii multifunctional folic acid synthesis fas gene which encodes dihydroneopterin aldolase, hydroxymethyldihydropterin pyrophosphokinase and dihydropteroate synthase (Volpe er a/., 1992, 1993).
• the PRT1 sequences showed homology with the other subtilisin-like serine proteases in the region of the P-domain, the highest homology being with the derived amino acid sequence of the S. pombe krp gene.
• Four potential sites for N-linked glycosylation were observed in all the PRT1 sequences, three in the subtilisin-like catalytic domain (Asn 194 , Asn 277 , Asn 442 ), and one in the P-domain (Asn 603 ).
• Nucleotide sequences encoding polyproline were found in all the sequences, and also the dipeptides Pro- Glu and Pro-Gin and the tetrapeptides Pro-Glu-Pro-GIn and Pro-Glu-Thr- Gln. The order and number of tandem repeats varied in each sequence. The overall length of this region varied from approximately 67 amino acid residues in the shortest sequence, PRT1 (73j), to 233 residues in the longest sequence, PRT1(M14).
• P.carinii sp. f. carinii chromosomes were analysed by hybridisation with three probes derived from different domains of PRT1. All three probes showed similar patterns of hybridization, anealing at high stringency to all the chromosome bands except for one, the third smallest in size, approximatey 350Kbp. This provided further evidence that the P.carinii sp. f. carinii genome contained many copies of the PPT7 gene, which were present on most of the P.carinii sp. f. carinii chromosomes.
• the sequences of the PPT7 gene family showed high levels of homology with ORF3, which has been demonstrated to be contiguous with a copy of the gene encoding the major surface glycoprotein MSG100 (Wada & Nakamura, 1994). This gene arrangement was reported in 15 other ⁇ clones, in which a gene showing high homology to ORF3 was located downstream of a copy of MSG (Wada & Nakamura, 1994). Most copies of the MSG genes have been demonstrated to be located in the P.carinii sp. f. car//.// subtelomeric regions (Underwood et al., 1996; Sunkin & Stringer, 1996).
• the copy of the PRT1 gene encoded by the PRTI (Paga) sequence was cloned from a ⁇ EMBL3 genomic library as a single 14kb fragment and was approximately 1150bp downstream of a copy of MSG.
• Four other ⁇ clones isolated from the same library contained a copy of PPT7 contiguous with a copy of MSG.
• P.carinii sp. f. carinii genomic DNA was digested with either restriction endonuclease Pstl or ⁇ amHI and probed sequentially with four oligonucleotide probes, derived from the 5' end of PPT7 gene (pcprot5/RI), from the catalytic domain of the gene (pcprot3/RI), an MSG probe (msgterm) and a subtelomeric probe (Pctel2). All probes hybridised to multiple bands. The hybridisation pattern of some of the bands, ranging in size from 7kb to greater than 12kb, were the same for all four probes. However, hybridisation to other fragments was not coincident, with the PRT7 probes alone hybridising to some high molecular weight fragments and also low molecular weight fragments of less than 7kb.
• the domain organisation of the PPT7 gene strongly resembled that of the fungal prohormone processing proteases, with the exception of the proline-rich domain. This proline-rich region is very uncommon in the subtilisin-like serine protease superfamily, although the KRP6 gene from Y.
• lipolytica is reported to contain a short region of a tetrapeptide repeat, the consensus sequence of the four amino acids being Glu (Asp/Glu) Lys Pro (Enderlin and Ogrydziak, 1994).
• a proline-rich region has also been found in the carboxy-terminal tail domain of the mammalian serine protease acrosin, a proteolytic enzyme of sperm cells, located in the acrosome at the apical end of the spermatozoan (Klemm et al., 1991).
• PARPs procyclic acidic repetitive proteins
• carinii DNA probed with PRT1 sequences also confirmed the presence of many copies of the gene. Analysis of sequence data generated by the amplification of the locus showed heterogeneity, suggesting that a variety of different copies of the gene were present in the P.carinii genome. Some domains, including the subtilisin-like catalytic domain and the P-domain, were highly conserved between gene copies, whereas the highest levels of divergence were observed in the proline-rich domain, which varied both in length and in sequence.
• the P.carinii PRT1 gene family shows some striking similarities to that of MSG. Both are composed of many genes, copies of which are found on most P.carinii chromosomes and show sequence heterogeneity. Some copies of PPT7 are contiguous with MSG and are located in the subtelomeric regions of the P.carinii chromosomes.
• Leishmania major surface protease msp or gp63
• msp or gp63 a zinc endoprotease
• the krp gene product from S.pombe which cleaves the pheromone precursor pro-P-factor to its active form, is thought to also function in the processing of other regulatory proteins, since its activity is essential for cell viability (Davey et al., 1994).
• the XPR6 gene product from Y. lipolytica although not essential for cell viability, when disrupted was found to cause aberrant growth and morphology (Enderlin and Ogrydziak, 1994).
• the function of the products of the P.carinii PRT1 gene family is not yet understood but it is likely to play an important role in the life cycle and possibly also the pathogenicity of the organism.
• PCR strategies using degenerate primers designed using P.carinii sp. f. carinii PRT1 sequence information failed to isolate any P.carinii sp. f. hominis PRT1 clones.
• the strategies employed included single round PCR and nested PCR, on post mortem samples from infected patients.
• Samples of Pneumocystis carinii sp. f. hominis were derived from HIV positive patients by fibreoptic bronchoscopy, an aliquot of this bronchoscopic alveolar lavage (BAL) sample being immediately frozen, stored at -20°C and transported to the Institute of Molecular Medicine for DNA extraction (samples D503B and D122B).
• BAL bronchoscopic alveolar lavage
• One sample (C180) was derived from a post mortem lung from an HIV-negative patient; the parasites were first enriched by successive filtration through 70 ⁇ m, 12 ⁇ m and 8 ⁇ m filters.
• Pneumocystis from the infected lungs of four other mammalian hosts were used. These were Pneumocystis carinii sp. f. muris (mouse derived), Pneumocystis carinii sp. f. mustelae (ferret derived), Pneumocystis carinii sp. f. suis (pig derived), Pneumocystis carinii sp. f. carinii (rat-derived) and Pneumocystis carinii sp. f. rattus (rat derived). These were enriched for parasites prior to DNA extraction. DNA Extraction
• the final concentration of the reaction mix was 50mM KCI, 10mM Tris (pH 8.0), 0.1 % Triton X-100, 3mM MgCI 2 , 400 ⁇ M of each deoxynucleoside triphosphate, 1 ⁇ M of each oligonucleotide primer and 0.025U of Taq polymerase (Promega) per ml.
• a total of forty cycles was used with 10 cycles at 94°C for 1.5 min (denaturation), annealing at a temperature between 48°C and 55°C dependant on primer Tm and required stringency of reaction for 1.5min and 72°C for 2min (extension), followed by 30 cycles at 94°C for 1.5min, 63°C for 1.5min and 72°C for 2min (the increased temperature at annealing now including the EcoRI site at the 5' end of the primers). Where there was no EcoRI site in the primer or where particularly low stringency was required all 40 cycles were carried out at the lower annealing temperature.
• a positive control of rat Pneumocyctis DNA (rat 1458 or rat 1189) was included in each PCR reaction.
• Negative controls of no added template DNA were included after each sample to monitor for cross contamination.
• a negative control of human DNA Sigma
• 0.8ng/ ⁇ l was included to monitor for non-specific amplification of human DNA, which was unavoidably co- extracted with all human Pneumocystis DNA samples.
• the primers used are shown in Table 1 herein (and Table 1 of Lugli er a/ 1997).
• DNA and RNA is prepared from P.carinii sp. f. hominis organisms, obtained from either bronchoalveolar lavage samples from P.carinii infected patients or from post-mortem lung samples, i) P.carinii sp. f. hominis genomic library
• a P.carinii sp. f. carinii genomic library is constructed in ⁇ FIX and this is screened with the cloned fragment of PRT1. Positive recombinant phage are analysed by further rounds of screening, and full length clones selected for analysis. The arrangement of introns within the gene sequence is determined.
• the genomic organisation of copies of PRT1 is elucidated, and in particular the relationship with gene copies of MSG.
• the chromosomal organisation of different PRT7 copies is examined, including the analysis of copies which are in the subtelomeric regions and others which are at an internal location, i) Expressed copies of PPT7
• Amplification of cDNA Ends is used to extend 5'- and 3'- of the cloned fragment of PPT7, using total RNA or poly A + RNA from the enriched parasite preparation. Primers are designed to the sequence of the cloned fragment for use in this technique.
• the second approach is the construction of a cDNA library in ⁇ ZAP from P.carinii sp. f. hominis, which is then screened with the cloned fragment. Different recombinant clones are compared for variation in sequence and used for expression studies. Expression i) Expression of cloned fragment of P.carinii sp. f. hominis
• the known portion of the catalytic domain is subcloned into the pET32a expression vector and expressed in an E. coli expression system. Recombinant protein is purified and used to raise polyclonal antiserum in rabbits. In addition, synthetic peptides designed to the PRT1 derived amino acid sequence are used in the production of antibodies. ii) Expression of the complete gene sequence and fragments of the gene spanning different domains. Recombinant protein is expressed and purified from different domains and from the complete sequence, for use in the production of antibodies, and in biochemical and immunohistochemical studies. Biochemical studies
• Biochemical studies are performed to determine the substrate specificity of the protease and the optimum conditions (e.g. pH, metal cofactors) for proteolytic activity. This provides an in vitro system for the testing of inhibitors to the PR77 protease. Crystallisation of the recombinant protein is carried out and the 3-D structure of the protein determined by X-ray crystallography and compared with the 3D structure of the four other subtilisin-like serine proteases whose structure has previously been determined. These structural data can used for purposes including the design of specific inhibitors of PRT7, and the prediction of antigenically important epitopes.
• optimum conditions e.g. pH, metal cofactors
• Antibodies raised to the recombinant PRT7 protein or to synthetic peptides can be used in the analysis of the subcellular localisation of PRT7 in P.carinii organisms, using both light microscopy and electron microscopy with immunogold.
• the oligonucleotides above have SEQ ID NOS: 1-15, according to the order in which they appear in the above table.
• New primers were designed based on regions of homology of the newly obtained rat variant P. carinii and mouse P. carinii PRT1 sequences with the rat prototype P. carinii sequence at both the DNA level and amino acid level. These were not fully degenerate, given that Pneumocystis DNA shows a high AT bias (60-70%); unless the sequence data suggested otherwise only A or T was used at potentially degenerate sites (as seen in the amino acid sequences). These new primers were used in reactions with one another and previously used primers. Of these reactions, only Pcprot16d/R1 and Pcprot26d/R1 gave a clear positive product at the expected Mr, close to that of the rat P.
• the primers used were Pcprot25d/R1 + Pcprot26d/R1 ; Pcprot1d/R1 + Pcprot26d/R1 ; Pcprot16d/R1 + Pcprot26d/R1 ; Pcprot25d/R1 + Pcprotl 7d/R1 ; Pcprot25d/R1 + Pcprotl 8d/R1 ;
• Table showing percentage divergence of prototype rat-derived Pneumocystis (P.carinii sp. f. carinii). mt LSU rRNA - mitochondrial large subunit rRNA; mt SSU rRNA - mitochondrial small subunit rRNA. Values for Variant rat P. carinii from two clones; values for Mouse P. cahnii from three clones. DNA divergence calculated with Jukes-Cantor correction method. Protein divergence calculated using Kimura protein distance.
• the homology of the amino acid sequences from all three types of Pneumocystis to the subtilisin-like serine proteases is high. Of the known conserved residues, most can be seen to be conserved in the PRT7 sequences (where the data are available). Certainly in the P.carinii sp. f. hominis PRT1 amino acid sequence there is greater conservation of the negatively charged amino acids at the substrate-binding face than is seen in the P.carinii sp. f. carinii sequence. Although the homology to the subtilases is unmistakable, there is considerable variation to be seen between the PRT7 sequences.
• subtilisin-like serine proteases so far studied is in the specific endoproteolytic processing of precursor proteins to their active form. Although the precise function of many subtilases is yet to be determined, some fungal homologues have been shown to be vital to cell viability or normal function. Thus krp in S. pombe has been shown to be vital to cell viability and disruption of XPR6 in Y. lipolytica causes aberrant growth and morphology. Parallels may also be drawn between Gp63 in Leishmania and PRT7 in Pneumocystis, as discussed in the introduction.
• Polyclonal antiserum was generated in rabbits to synthetic peptides, designed to the Pneumocystis carinii sp. f. carinii PRT1 sequence. Regions of the protein which were likely to be immunogenic were predicted using the appropriate software, and peptides (15 mers) to six different regions were synthesized. A mixture of six synthetic peptides was administered by subcutaneous injection to rabbits (New Zealand white). An antibody response was elicited by standard procedures, using Freunds complete adjuvant for the first injection and Freunds incomplete adjuvant for subsequent injections.
• the resulting polyclonal antisera were tested against the peptides.
• the greatest cross-reactivity of the antisera was found with Peptide 7, designed to a region of the catalytic domain (amino acid residues 424 - 438 of the PRT1 (73j) sequence) and with Peptide 9, designed to the pro-domain (amino acid residues 64 - 78 of the PRT1(73j) sequence).
• ITSPSGVTSVLAHRR (4) (SEQ ID NO: 17)
• the E. coli expression vector pET32a (Novagen, Madison, WI) was used. This vector contains an inducible T7lac promotor, a 6-His tag, a multiple cloning site and the recombinant protein is expressed as fusion protein with the Trx-tag thioredoxin protein (109 amino acids). Recombinant thioredoxin fusion proteins are generally more soluble and remain in the E. coli cytoplasmic fraction.
• the specific fragments were amplified by PCR from the PRT1 (73j) sequence as follows - i) Cat2f1 using primers Pcprot39/R1 and 73j Ex4; ii) F1a1j using primers Pcprot31/RI and Pcprot32/RI; iii) G1 b1c using primers Pcprot33/RI and 73jEx5/RI (see Table 1). All primers included an EcoRI site the 5' end to facilitate cloning. The fragments were initially cloned into the plasmid vector pUC, linearized with EcoRI and treated with alkaline phosphatase, to produce a stable, high copy number, recombinant plasmid. The recombinant DNA was then subcloned into the EcoRI site of the expression vector pET32a.
• E. coli DH5 ⁇ competent cells were transformed with the recombinant plasmids.
• the cells were transformed with recombinant pUC plasmids, and also recombinant pET32a plasmids.
• the recombinant expression vector pET32a constructs were also transferred into E. coli DE3 (BL21) cells, for expression of the recombinant peptides.
• the recombinant pET32a constructs, transformed into E. coli DE3(BL21) were induced with IPTG, and the bacteria were grown for 3 to 4 hours.
• the cells were collected by centrifugation and disrupted by sonication.
• the bacterial proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose filter.
• the immobilised proteins were cross-reacted with anti-thioredoxin antibody (Sigma), and the bound antibody was visualised with a swine anti-rabbit immunoglobulins secondary antibody, conjugated to alkaline phosphatase.
• P.carinii sp. f. carinii organisms were extracted and enriched from infected rat lungs. Organisms were disrupted by heating to 95°C in denaturing solution and the proteins separated by SDS-PAGE, followed by transfer to nitocellulose filters. The immolbilised proteins were cross- reacted with the anti-Peptide 7 and the anti-Peptide 9 antibody. Bound antibody was detected using an anti-rabbit secondary antibody, conjugated to alkaline phosphatase. A single, major band, at 40 kDa, was seen with each of the mono-specific antibodies. In addition, another major band at 38 kDa was seen with anti-Peptide 7 antibody and minor bands at 98 kDa and 16 kDa.
• the predicted size of the full length PRT1 protein ranges from 87 to 102 kDa.
• the proteins detected with the mono-specific antibodies are assumed to be the products of autocatalysis at a number of dibasic residues found in the PRT1 sequence.
• the E.coli expression vector pET32a (Novagen, Madison, WI) was used. This vector contains an inducible T7lac promotor, a 6-His tag, a multiple cloning site and recombinant protein is expressed as fusion protein with the Trx-tag thioredoxin protein (109 amino acids). Thioredoxin fusion proteins are generally more soluble and remain in the E.coli cytoplasmic fraction. A 367bp portion of the cloned P. carinii sp. f. hominis
• PRT1(H13) sequence was amplified using PCR with the primers PcprotH34/RI and PcprotH35/RI, corresponding to position 111 to position 478 on the PRT1 (H13) sequence, in the catalytic domain of the gene (see Table 1).
• the primers included an EcoRI site at the 5' end to facilitate cloning.
• the resulting fragment (H1a1a) was initially cloned into the EcoRI site of the plasmid vector pUC, and then subcloned into the EcoRI site of the expression vector pET32a.
• E. coli DH5 ⁇ competent cells were transformed with the recombinant plasmid.
• the cells were transformed with the recombinant pUC plasmid, and also the recombinant pET32a plasmid.
• the recombinant expression vector pET32a construct was also transferred into E. coli DE3 (BL21) cells, for expression of the recombinant peptide.
• the recombinant pET32a construct (H1a1a), transformed into E. coli DE3(BL21) was induced with IPTG, and the bacteria were grown for 3 to 4 hours.
• the cells were collected by centrifugation and disrupted by sonication.
• the bacterial proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose filter.
• the immobilised proteins were cross-reacted with anti-thioredoxin antibody (Sigma), and the bound antibody was visualised with a swine anti-rabbit immunoglobulins secondary antibody, conjugated to alkaline phosphatase.
• P.carinii sp. f. hominis organisms were extracted from bronchoalveolar lavage fluid from a patient with P. carinii pneumonia. The organisms were disrupted by heating to 95°C in denaturing solution and the proteins separated by SDS-PAGE, followed by transfer to nitrocellulose filters. The immobilised proteins were cross-reacted with the anti-Peptide 7 and the anti-Peptide 9 antibody. Bound antibody was detected using an anti-rabbit secondary antibody, conjugated to alkaline phosphatase. Two major bands, at 56 kDa and 49 kDa was seen with each of the mono- specific antibodies.
• the major surface glycoprotein (GP63) is present in both life stages of Leishmania. Mol & Biochem Paras 38, 25-32.
• Yeast prohormone processing enzyme (KEX2 gene product) is a Ca 2+ -dependent serine protease. Proc. Natl. Acad. Sci. USA 86, 1434-1438.
• Lugli, E.B. and Wakefield, A.E. (1996). A novel subtelomeric multi- gene family in Pneumocystis carinii . 4th International Workshop on Opportunistic Protists, Tuscon, Arizona, USA, June 1996. Lugli, E.B..Allen, A.G. and Wakefield, A.E. (1997) A Pneumocystis carinii multi-gene family with homology to subtilisin-like serine proteases. Microbiology 143: 2223-2236.
• the multifolate folic acid synthesis fas gene of Pneumocystis carinii appears to encode dihydropteroate synthase and hydroxymethyldihydropterin pyrophosphokinase. Gene 112, 213-218. Volpe, F., Ballantine, S. P., and Delves, C. J. (1993).
• the multifunctional folic acid synthesis fas gene of Pneumocystis carinii encodes dihydroneopterin aldolase, hydroxymethyldihydropterin pyrophosphokinase and dihydropteroate synthase.
• MSG gene cluster encoding major cell surface glycoproteins of rat Pneumocystis carinii. DNA Research 1 , 163-168.
• Figure 3 Amino acid sequence alignments of part of the catalytic domain of PRT1, translated from the nucleotide sequences ( Figure 2).
• Patterned boxes represent different domains; small dots represent hydrophobic regions (HR), diagonal lines indicate the catalytic domain
• CAT woven pattern indicates the P-domain (P)
• vertical lines indicate the proline-rich region
• squares indicate the serine-threonine rich region (STR).
• Boxes that are defined by a shaded line indicate length and sequence variation in these regions.
• Diamonds indicate potential glycosylation sites; (t) catalytic active site residues D214, H252- S423; (
• FIG. 7 Recombinant PRT1 polypeptides, expressed in E. coli as thioredoxin fusion proteins, separated by SDS-PAGE and cross-reacted with an anti-thioredoxin antibody.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Genetics & Genomics (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Wood Science & Technology (AREA)
• Molecular Biology (AREA)
• General Health & Medical Sciences (AREA)
• Microbiology (AREA)
• Biomedical Technology (AREA)
• Medicinal Chemistry (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Mycology (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Enzymes And Modification Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Peptides Or Proteins (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=10808742

Family Applications (1)

Country Status (4)

Cited By (1)

Families Citing this family (2)

Family Cites Families (4)

• 1997
  • 1997-03-05 GB GBGB9704559.5A patent/GB9704559D0/en active Pending
• 1998
  • 1998-03-05 WO PCT/GB1998/000704 patent/WO1998039424A1/en not_active Application Discontinuation
  • 1998-03-05 EP EP98910855A patent/EP0973876A1/de not_active Withdrawn
  • 1998-03-05 JP JP53829498A patent/JP2001514505A/ja active Pending

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events