EP1078077A1 - Nucleic acids and proteins from pineapple stem - Google Patents

Nucleic acids and proteins from pineapple stem

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Publication number
EP1078077A1
EP1078077A1 EP99940397A EP99940397A EP1078077A1 EP 1078077 A1 EP1078077 A1 EP 1078077A1 EP 99940397 A EP99940397 A EP 99940397A EP 99940397 A EP99940397 A EP 99940397A EP 1078077 A1 EP1078077 A1 EP 1078077A1
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Prior art keywords
seq
protein
nucleic acid
ddbj
embl
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German (de)
French (fr)
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Tracey Lehanne Wolfson Laboratories MYNOTT
Ben Wolfson Laboratories CROSSETT
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Cortecs Ltd
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Cortecs Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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)
    • C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to proteins which are components of bromelain and to genes encoding those proteins.
  • the present invention relates to proteins which have anti-cancer and immunostimulant activity and the nucleic acid encoding these proteins.
  • Bromelain is the collective name for the proteolytic enzymes found in the tissues of the plants of the Bromeliaceae family. Although fruit bromelain is known, the most common form of bromelain is a mixture of various moieties derived from the stem of the pineapple plant (Ananas comosus). Stem bromelain (hereafter called bromelain) is known to contain at least five proteolytic enzymes but also non-proteolytic enzymes, including an acid phosphatase and a peroxidase; it may also contain amylase and cellulase activity. In addition, various other components are present.
  • Bromelain has previously been used in the treatment of a variety of conditions including inflammation and, in particular, it has been used in the treatment of diarrhoea.
  • the use of bromelain in the treatment of infectious diarrhoea is described in WO-A-9301800, where it is suggested that bromelain works by destroying intestinal receptors for pathogens by proteolysis, and in WO-A-8801506, which teaches that bromelain detaches pathogens from intestinal receptors.
  • bromelain can reduce toxin binding activity and can inhibit the secretory effect of toxins such as heat labile toxin (LT) and cholera toxin (CT) and also toxins such as heat stable toxin (ST).
  • toxins such as heat labile toxin (LT) and cholera toxin (CT) and also toxins such as heat stable toxin (ST).
  • WO-A-9600082 also relates to bromelain and discloses that crude bromelain is capable of interfering with signalling pathways which are important for growth, in particular, signalling pathways which lead to the production of growth factors such as interleukin- 2 (IL-2), platelet derived growth factor (PDGF) and insulin like growth factor (IGF).
  • IL-2 interleukin- 2
  • PDGF platelet derived growth factor
  • IGF insulin like growth factor
  • bromelain is capable of acting as an anti-cancer agent.
  • bromelain can be used either as an immunosuppressive agent or an immunostimulant depending on the type of cell being treated and whether the cell has previously been activated.
  • Chem 17 (4) 351-361(1998) which have a molecular mass of 23,550 and 23,560 respectively, and are glycosylated.
  • WO-A-98/38320 relates to a fraction of bromelain designated CCX. This fraction contains two components termed CCXl and CCX2.
  • the stem bromelain-like proteinase, CCX2 inhibits the growth of human ovarian tumours in a nude mouse model.
  • stem bromelain, CCXl and other stem bromelain-like proteinases do not affect tumour growth.
  • eleven peptide sequences belonging to CCX2 protein are disclosed. These are as follows:
  • bracketed amino acids represent alterntives to the preceding amino acid and a "-" represents an unidentified amino acid.
  • RNA from a pineapple stem was extracted RNA from a pineapple stem and used a reverse transcriptase polymerase chain reaction (RT-PCR) to isolate and sequence novel cDNA sequences encoding proteins similar to CCX2 and having the same activity.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • primers Two pairs of primers were used in the first instance; one set was made against conserved regions of the mature peptide, whereas the other pair was designed to amplify the full length open reading frame. An additional pair of primers was also designed for use in Example 3 below. All the primers used were derived from the nucleic acid sequence having accession number D 14059 in the
  • nucleic acid sequence comprising one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ
  • SEQ ID NO: 11 SEQ ID NO: 13 or SEQ ID NO: 15 or a sequence differing from one of the above by not more than forty point mutations when aligned in the manner shown in Figure 1, or an RNA equivalent of one of these, provided that the sequence does not include the sequences having accession numbers D14059(SEQ ID NO: 17) and D38534 (SEQ ID NO: 19) in the DDBJ/EMBL/GenBank database.
  • sequence of the invention differs from SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 or 15 by fewer than 30 point mutations, more preferably fewer than 20 point mutations and most preferably fewer than 10 point mutations from the sequences given above.
  • the present invention also provides a sequence which is complementary to one of the sequences of the first aspect or its RNA equivalent.
  • RNA equivalent when used above indicates that a given RNA molecule has a sequence which is complementary to that of a given DNA molecule (allowing for the fact that in RNA, "U” replaces "T” in the genetic code).
  • BESTFIT When comparing nucleic acid sequences for the purposes of determining the degree of homology or identity one can use programs such as BESTFIT and GAP (both from the Wisconsin Genetics Computer Group (GCG) software package), BESTFIT, for example, compares two sequences and produces an optimal alignment of the most similar segments. GAP enables sequences to be aligned along their whole length and finds the optimal alignment by inserting spaces in either sequence as appropriate.
  • GCG Wisconsin Genetics Computer Group
  • sequences listed above encode proteins which are extremely similar to the CCX2 protein disclosed in WO-A-98/38320 and which have the same activity.
  • a protein comprising one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16 or a sequence differing from one of the above by not more than 15 amino acid substitutions, provided that the sequence is not CCX2 as disclosed in WO-A-98/38320 or the protein encoded by the nucleic acid sequence having accession number D14059 or D38534 in the DDBJ/EMBL/GenBank database.
  • the protein of SEQ ID NO: 2 forms the mature peptide region of a longer protein, SEQ ID NO: 14
  • SEQ ID NO: 14 or a sequence differing from it by not more than 15 amino acid residues, provided that the sequence is not CCX2 as disclosed in WO-A-98/38320 or the protein encoded by the nucleic acid sequence having accession number D14059 in the DDBJ/EMBL/GenBank database.
  • the protein sequence of the present invention has not more than 10, and more preferably, not more than five amino acid substitutions compared with the sequences given above.
  • proteins of the invention have anti-cancer and immunostimulant activity and, therefore, in a further aspect of the invention, there is provided a protein of the invention for use in human or veterinary medicine, in particular the treatment or prevention of cancer.
  • the protein of the invention When used in medicine, the protein of the invention will preferably be isolated and purified to remove the possibility of side effects arising from the presence of impurities.
  • the protein of the invention may be used in a method for the treatment of cancer, the method comprising administering to a patient an effective amount of the isolated and purified protein of the first aspect of the invention.
  • bromelain mixture has anti-cancer activity and, as discussed in our earlier application WO-A-9500169, this appears to arise from bromelain' s ability to affect intracellular signalling pathways, in particular, pathways which are modulated by MAP kinases. It is therefore possible that this is the mechanism of action of the protein of the present invention. However, the present invention is not dependent upon the correctness or otherwise of this theory.
  • Ras proteins help relay signals from growth- factor receptors on the surface of cells to transducer molecules to stimulate cell proliferation or differentiation.
  • Oncogenic (or mutant) ras genes produce defective ras proteins that have acquired independence from externally supplied growth factors and, at the same time, may no longer respond to external growth-inhibitory signals. Mutant ras proteins are thus persistently hyperactive and their unbridled catalytic activity has a detrimental effect on the control of cell growth. Oncogenic ras genes therefore promote cancer and tumour formation by disrupting the normal controls on cell proliferation and differentiation. Approximately 30% of human cancers have mutations in a ras gene.
  • MAP mitogen-activated protein
  • ERKs extracellular-signal regulated kinases
  • the protein of the present invention acts by activating the innate immune system.
  • the immune response has two functional divisions: the innate immune system and the adaptive immune system.
  • the innate immune response is mediated by macrophages, natural killer cells and neutrophils.
  • the adaptive immune response is mediated by B and T cells.
  • Innate immunity provides the first line of defence against infectious agents and most potential pathogens before they establish infection.
  • the adaptive immune response is developing. If the first defences are breached, the adaptive immune system should be sufficiently developed to produce a specific reaction to the infectious agent, which normally eradicates this agent.
  • the innate immune system is also critically important in killing tumour cells.
  • the protein of the present invention has been shown to activate macrophages and natural killer cells (NK), critical mediators of the innate immune system important for controlling tumour growth.
  • the protein has also been shown to increase interferon- ⁇ - mediated nitric oxide (NO) production.
  • NO interferon- ⁇ - mediated nitric oxide
  • Hibbs 119, Res. Immunol, 142, 565-569 has shown that when macrophages produce NO, they kill tumour cells in vitro.
  • increased NO production and activation of innate immunity may be the mechanism by which the protein of the present invention acts against tumours. Again, however, the effectiveness of this protein as an anti-tumour agent is not dependent upon the correctness of this proposition.
  • the protein of the present invention is useful for treating many different types of cancer including solid cancers such as ovarian, colon, breast or lung cancer and melanoma as well as non-solid tumours and leukaemia.
  • CCX2 is able to activate natural killer (NK) cells.
  • NK cells are lymphocytes which can recognise and destroy cells infected with various viral, bacterial or parasitic pathogens. They are also able to kill tumour cells by specifically recognising the expression of virus-induced molecules on tumour cells or other molecules associated with tumours. Therefore, because CCX2 is able to activate NK cells, it will also be of use in the treatment or prevention of virus-induced tumours.
  • tumours examples include hepatocellular carcinoma (which may result from hepatitis B virus); non-Hodgkin's lymphoma, nasopharyngeal carcinoma or Burkitt's lymphoma (resulting from Epstein-Barr virus); Kaposi's sarcoma (resulting from cytomegalovirus in HIV-infected patients); T-cell leukaemia (resulting from human T cell lympho tropic virus); and cervical carcinoma (resulting from human papilloma viruses such as HPV16 and HPV18).
  • hepatocellular carcinoma which may result from hepatitis B virus
  • Kaposi's sarcoma resulting from cytomegalovirus in HIV-infected patients
  • T-cell leukaemia resulting from human T cell lympho tropic virus
  • cervical carcinoma resulting from human
  • the ability of the protein of the invention to activate the innate immune response suggests that it would also be of use in situations where the adaptive immune response, such as B or T cell responses, are not fully functional. This may occur in many secondary immunodeficiencies which may arise because of malnutrition, infection (for example HIV and malaria), tumours (for example lymphoid, myeloma and other), trauma (for example burns, wounds and surgery), medical treatment (for example with drugs such as steriods, cyclosporin and cyclophosphamide), protein loss (such as in diarrhoea and burns), genetic abnormalities (such as those found in combined immunodeficiency patients who lack T and/or B cells), diabetes and old age.
  • infections for example HIV and malaria
  • tumours for example lymphoid, myeloma and other
  • trauma for example burns, wounds and surgery
  • medical treatment for example with drugs such as steriods, cyclosporin and cyclophosphamide
  • protein loss such
  • the protein of the present invention is capable of increasing interferon-mediated NO production. Therefore, the protein of the invention may be used to treat diseases or conditions which respond to increased NO production.
  • NO has a critical role in host defence against infection.
  • NO and its derivatives have potent anti-microbial activity against many pathogens including fungi, bacteria and viruses. Therefore, the protein may be administered to patients receiving chemotherapy to protect against opportunisic infections. It may also be used to treat pathogenic infections including parasites, such as Babesia, Brugia, Cryptosporidium, Encephalitoxoon, Entamoeba, Leishmania, Naegleria, Ochocerca, Opisthorchis, Plasmodium, Schistosoma, Toxoplasma and Trypanosoma.
  • NO has activity against fungi such as Aspergillus, Candida, Cryptococcus, Histoplasma, Pneumocystis and Saccharomyces and against viruses, for example, Coxsackievirus, Ectomelia virus,
  • Encephalomyocarditis virus Epstein-Barr virus, Herpes simplex virus, Human immunodeficiency virus type 1, Japanese encephalitis virus, mouse hepatitis virus, parvovirus, poliovirus, rabies virus, simian virus 40, vaccinia virus and vesicular stomatitus virus (Fang, 1997, ASM News, 63, 668-673).
  • the activity of the protein of the present invention in increasing NO production complements its immunostimmulant activity and means that it can be used as an antimicrobial agent against parasites, bacteria, fungi and viruses such as those listed above.
  • the invention provides the protein of the present invention for use as an antimicrobial agent and the use of the protein of the present invention in the preparation of an antimicrobial agent.
  • the protein will usually be formulated before administration to patients and so, in a further aspect of the invention there is provided a pharmaceutical or veterinary composition comprising the isolated and purified protein of the first aspect of the invention together with a pharmaceutically or veterinarily acceptable excipient.
  • the protein may be administered by a variety of routes including enteral, for example oral, nasal, topical, buccal, or anal administration or parenteral administration, for example by the intravenous, subcutaneous, intramuscular or intraperitoneal routes.
  • enteral for example oral, nasal, topical, buccal, or anal administration or parenteral administration, for example by the intravenous, subcutaneous, intramuscular or intraperitoneal routes.
  • the oral route may be preferred as this is often the route which patients find most acceptable.
  • the oral route may be particularly useful if many doses of the protein are required as will often be the case in the treatment of cancer.
  • another orally administrable dosage form may be used, for example a syrup, elixir or a hard or soft gelatin capsule, either of which may be enteric coated.
  • the protein may be formulated in distilled water or another pharmaceutically acceptable solvent or suspending agent.
  • a suitable dose of the protein to be administered to a patient may be determined by the clinician. However, as a guide, a suitable dose may be from about 0.5 to 100 mg per kg of body weight. It is expected that in most cases, the dose will be from about 1 to 50 mg per kg of body weight and preferably from 1 to 20 mg per kg of body weight. For a man having a weight of about 70 kg, a typical dose would therefore be from about 70 to 7000 mg.
  • the nucleic acid of the invention may be used in recombinant DNA technology and, for that purpose, may be inserted into a vector.
  • the vector may be, for example, a plasmid, cosmid or phage.
  • the nucleic acid in the vector may be under the control of a suitable control sequence and may be operatively linked to a suitable promoter.
  • the promoter may be either constitutive or induceable.
  • the vector may be used to transform cells, which may be single celled organisms, such as bacteria or yeast cells.
  • the nucleic acid may be used in gene therapy, in which case, the transformed cells will be cells of a patient being treated using gene therapy.
  • FIGURE 1 shows a pile up of NF 36/NF 37 RT-PCR product sequences in which the sequence for the PCR products has been aligned with the published sequence of D 14059.
  • An asterisk denotes a nucleotide substitution which does not affect the translation of the reading frame, whereas a dollar ($) denotes a substitution which does alter the translation.
  • FIGURE 2 shows an alignment of two NF 43/NF 44 RT-PCR product sequences, one NF 76/NF 77 RT-PCR product sequences, D14059 and D38534. The sequences from cDNAs have been aligned and the translation of the open reading frame is also shown.
  • FIGURE 3 shows the predicted amino acid sequence for cDNA sequences pCRScript 569 (SEQ ID NO: 2), pCRScript 571 (SEQ ID NO: 4), 573 (SEQ ID NO: 8) and 574 (SEQ ID NO: 10) shown aligned to the known sequences of stem bromelain-like proteinases D38534 (SEQ ID NO:18) and D38534 (SEQ ID NO: 20).
  • a full stop (.) represents a match to D38534 and a dash (-) indicates that there was no cDNA sequence data available for that section. Where the amino acid sequences differ, the single letter code for the amino acid is shown.
  • Methods Plant Material A pineapple plant, which was a Jamaica Queen cultivar, was generously donated by the Lost Gardens of Heligan, St Austell, UK. The plant was 3 years old and had fruited in the previous year. After being stripped of offsets and leaves, the stem weighed 256 g and measured approximately 20 cm in length and 7 cm in diameter. The stem was immediately cut into 1 cm cubes and snap frozen in liquid nitrogen, and stored in liquid nitrogen until required.
  • RNA extraction was performed using a RNeasyTM plant mini kit (Qiagen Ltd, Crawley,
  • IPTG isopropyl ⁇ -D-thioglactopyranoside.
  • the first step is to prepare the complementary DNA (cDNA) from the messenger RNA (mRNA).
  • cDNA complementary DNA
  • mRNA messenger RNA
  • an oligo d(T), 6 primer is bound to the poly A tail at the 3' end of the mRNA, this enables the enzyme reverse transcriptase to synthesise the first strand of cDNA.
  • the cDNA will cover the entire open reading frame as well as the 5' and 3' untranslated regions at each end. Assuming that CCX2 is similar to the other bromelain-like proteinases, the open reading frame will consist of 3 regions.
  • the first region will encode the prepeptide, or signal peptide, a short sequence of approximately 20 amino acids which is involved in determining the cellular localisation of the protein once it is synthesised.
  • the second region encodes for the propeptide, which consists of almost 100 amino acids that must be removed before the proteinase becomes active.
  • the third region is the section which encodes for the active protein. Ten of the eleven sequences from WO-A-9838320are derived from this region and the first set of PCR primers were designed to amplify this region.
  • RNA and 50 pmol oligo d(T) 16 primer where mixed with 10 X PCR buffer, dNTP's and 50 U MuLV Reverse Transcriptase (all reagents from Perkin-Elmer, Beaconsfield, UK).
  • the total reaction volume was 20 ⁇ l, and reactions were incubated at room temperature for 10 min, followed by 15 min at 42°C, and finally, 5 min at 99°C.
  • the first strand cDNA was stored on ice or at -20°C until required.
  • the first set (NF 36: 5' TGC CTC AAA GTA TTG ATT GG 3' (SEQ ID NO: 32)/NF 37: 5' ATA CGG ACG TAT CCA CCC TC 3' (SEQ ID NO: 33)); see Figure 1) of primers were made to regions where the bromelain proteinases' sequences are conserved and where the amino acid sequence of D 14059 is identical to
  • NF 36 and NF 37 were based on the D 14059 cDNA sequence, but the primers, in theory, would amplify CCX2 as well as D14059. It was expected that the primers would amplify a short 574 bp fragment of the cDNA corresponding to the mature peptide.
  • the second set of primers (NF 43: 5' GCA TGG CTT CCA AAG TTC AAC TC 3' (SEQ ID NO: 34)/ NF 44: 5' TTG CTA AAT CAT TTG CTT CTT CCG AC 3' (SEQ ID NO: 35)) were made to less well conserved regions of bromelain proteinases, but when used in the PCR reaction they would amplify the cDNA corresponding to the full length of the open reading frame, which was expected to be 1092 bp.
  • the third set of primers (NF 76: 5' CCC CCC GCT AGC CGT GAC GAA CCC AGT GAT C 3' (SEQ ID NO: 36) and NF 88: 5' CCC CCC GAG CTC TTA TGA TTG TAG AGT GGG AAA GAG AG 3' (SEQ ID NO: 37))were designed to remove the prepeptide and the C-terminal thirteen residues, which were thought to be removed during post-translational modifications in vivo. Although these primers contained the Nhel and Sad restriction sites, respectively, which enables the PCR product to be inserted in frame into certain expression vectors, the RT-PCR products were blunt end ligated into pCRScript as before.
  • Each PCR consisted of 5 ⁇ l cDNA template, lOOng of each primer, lOx PCR buffer, 2.5 mM dNTPs, AmpliTaqTM DNA Polymerase (all from Perkin-Elmer, Beaconsfield, UK) and water to a total volume of 25 ⁇ l.
  • the amplification took place on a thermal cycling block under the following conditions: an initial denaturation of 94°C for 4 min, followed by 30 cycles of 94°C for 30 seconds, annealing temp of 56°C for 30 seconds and extension temperature of 72°C for 30 seconds.
  • PCR When the PCR resulted in multiple products, they were size fractionated on a 1% agarose/TAE gel.
  • One PCR product was then excised from the gel and purified using a QIAquick gel extraction kit (Qiagen, Crawley, UK). The PCR product was then incubated with 1 unit Pfu DNA polymerase at 72 °C for 30 minutes to remove 5' overhangs and then blunt-end ligated into pCRScript Amp SK (+) cloning vector (Stratagene Ltd, Cambridge, UK). Escherichia coli strain XL-1 transformants were plated onto agar plates containing the antibiotic ampicillin, X-gal and IPTG. E.
  • coli which contain the pCRScript vector are resistant to ampicillin, and so are able to grow on the agar plates.
  • the PCR product When the PCR product is inserted into the pCRScript vector it disrupts the ⁇ -galactosidase gene, an enzyme which processes X-gal into an insoluble blue product. It is therefore possible to select E. coli transformants which have pCRScript containing a PCR product (white colonies) and avoid those which contain pCRScript without a PCR product (blue colonies).
  • the colonies containing the plasmids with a PCR product were grown up in L-broth and 10 ⁇ g of the plasmid was purified using a QIAprepTM miniprep (Qiagen Ltd, Crawley, UK).
  • the plasmid was digested with restriction enzyme EcoRl and Notl (Boehringer-Mannheim, Lewes, UK). The restriction enzymes cleave the plasmid either side of the PCR product, allowing the size of the PCR product to be estimated by gel electrophoresis. Plasmids containing a PCR product of the expected size were then sequenced using an automated sequencer. After the sequencing reaction, each pCRScript construct was given an identity number as indicated in Figure 1.
  • the oligonucleotides ⁇ F 36/ ⁇ F 37 gave a single band of approximately 600 bp. This is comparable to the expected size of 574 bp, assuming that CCX2 is similar to D14059 and D38534. 24 "white" bacterial colonies that contained the ampicillin resistance marker were selected and the plasmids were analysed by restriction enzyme digest. 17 of the plasmids had a 600 bp PCR product, 6 plasmids had a shorter PCR product of approximate 350 bp, and 1 plasmid contained no detectable PCR product.
  • Plasmid pCRScript 571 (SEQ ID NO: 3) has an additional mutation ($1 in Figure 1) which would have caused a histidine to be substituted for a tyrosine.
  • Plasmid pCRScript 573 (SEQ ID NO: 7) also has one additional mutation ($5 in Figure 1) which would cause a serine for pro line substitution and pCRScript 574 (SEQ ID NO: 9) has two additional mutations ($2 and $4 in Figure 1) which would cause a glycine for alanine and serine for glycine substitutions respectively.
  • pCRScript 574 also as an ambiguous base, marked by $3 in
  • Figure 1 which may be G or A and so could cause aspartic acid to be substituted for glycine.
  • all of the sequences obtained are extremely similar to D14059, but they all contain a number of unique amino acid substitutions making them different from D 14059. They also differ from other previously published bromelain proteinase sequences, including D38534. Moreover, they also differ from the 11 peptides from CCX2 at various positions.
  • the oligonucleotides NF 43/NF 44 resulted in two major PCR products, one of approximately 500 bp and one of approximately 1100 bp, as well as 3 minor PCR products between 600 and 1600 bp.
  • the expected size of the open reading frame for the full length pre-propeptide is 1092 bp. Therefore the 1100 bp PCR product was purified on an agarose gel and ligated into pCRScript. Of the 11 white colonies that were selected and analysed by restriction enzyme digestion, 9 contained inserts of 1100 bp, and of those, two, designated pCR Script 673 and pCR Script 674, were sequenced ( Figure 2).
  • Plasmid pCRScript 673 has only been partially sequenced, although over the known sequence it appears to be identical to plasmid pCRScript 674 (SEQ ID NO: 13), with the exception of 1 nucleotide at position 725. This mutation, however, does not affect the translation of the reading frame. Apart from the substitution at position 725, both plasmids match pCRScript 569 in the region which corresponds to the mature peptide. However, in the pro-region, the two sequences have 5 substitutions when compared to D 14059 and they are marked by an asterisk above the sequence. The resultant change to the theoretical translation of the reading frame is shown in bold. Although only two PCR products were sequenced it appears that the sequence obtained, is very similar to D 14059 and the CCX2 peptides, especially in the region corresponding to the mature peptide. There are however, a number of amino acid substitutions.
  • pCRScript 673 and 674 encode the entire open reading frame and consists of a 351 amino acid peptide, which by homology to other proteinases, contains a 24 amino acid signal peptide, a 98 amino acid pro-peptide and a 229 amino acid peptide v/hich would form an active proteinase. However, it is likely that a portion of the C-terminal would be removed during processing of the mature peptide this would result in a loss of 13 amino acids leaving 216 amino acids, with a predicted molecular mass of 23,490 Da.
  • the first cDNA's were obtained from the Jamaica Queen cultivar. Of these one plasmid, pCRScript 570, contained a deletion of 198 bp, this is probably due to alternative splicing of the mRNA, although its is not known what effect such a deletion would have on the protein.
  • the other 4 plasmids (pCRScript 569, 571, 473 and 574) all gave theoretical protein sequences that were all unique, i.e. they had one or more amino acid substitutions that have not been previously reported.
  • D14059 and D38534 are extremely similar, for example over the region that encodes for the mature peptide, the sequences differ by only four amino acid residues. Moreover, in the region which corresponds to the section between primers NF 36 and NF 37 the amino acid sequence only differs by two amino acids (see positions 27 and 88 as numbered in Figure 3). Neither of these amino acids is covered by the peptide sequences which characterise CCX2. However, peptide sequence (ID NO 28) shows that CCX2 is similar to D38534 at the C-terminal.
  • the present inventors have identified six new proteinases in the stem of the pineapple plant. They have high similarity to two bromelain-like proteinases

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Abstract

Nucleic acids encode peptides which are found in crude stem bromelain and which have anti-cancer and immunostimulant activity.

Description

NUCLEIC ACIDS AND PROTEINS FROM PINEAPPLE STEM
The present invention relates to proteins which are components of bromelain and to genes encoding those proteins. In particular, the present invention relates to proteins which have anti-cancer and immunostimulant activity and the nucleic acid encoding these proteins.
Bromelain is the collective name for the proteolytic enzymes found in the tissues of the plants of the Bromeliaceae family. Although fruit bromelain is known, the most common form of bromelain is a mixture of various moieties derived from the stem of the pineapple plant (Ananas comosus). Stem bromelain (hereafter called bromelain) is known to contain at least five proteolytic enzymes but also non-proteolytic enzymes, including an acid phosphatase and a peroxidase; it may also contain amylase and cellulase activity. In addition, various other components are present.
Bromelain has previously been used in the treatment of a variety of conditions including inflammation and, in particular, it has been used in the treatment of diarrhoea. The use of bromelain in the treatment of infectious diarrhoea is described in WO-A-9301800, where it is suggested that bromelain works by destroying intestinal receptors for pathogens by proteolysis, and in WO-A-8801506, which teaches that bromelain detaches pathogens from intestinal receptors.
Taussig et al, Planta Medica, 1985, 538-539 and Maurer et al, Planta Medica, 1988, 377-381 both suggest that bromelain may be of use in inhibiting tumour growth. US 5,223,406, DE-A-4302060 and JP-A-59225122 also teach the use of bromelain in the treatment of cancer. US 5,223,406 teaches that bromelain is capable of inducing tumour necrosis factor (TNF) while DE-A-4302060 teaches that bromelain can prevent metastasis by the structural modification of the tumour surface protein CD44.
In WO-A-9400147, various experiments were described which demonstrate that proteolytic enzymes and, in particular, bromelain, are capable of inhibiting secretion. The application also discloses that bromelain can reduce toxin binding activity and can inhibit the secretory effect of toxins such as heat labile toxin (LT) and cholera toxin (CT) and also toxins such as heat stable toxin (ST). These observations were explained by the fact that one component of the bromelain mixture, stem bromelain protease, appears to be capable of modulating cyclic nucleotide pathways and this is discussed further in WO-A-9500169. In addition, bromelain has also been demonstrated to inhibit secretion caused by the calcium dependent pathway.
WO-A-9600082 also relates to bromelain and discloses that crude bromelain is capable of interfering with signalling pathways which are important for growth, in particular, signalling pathways which lead to the production of growth factors such as interleukin- 2 (IL-2), platelet derived growth factor (PDGF) and insulin like growth factor (IGF). This document teaches that, as a consequence of its ability to block signalling pathways, bromelain is capable of acting as an anti-cancer agent. In addition, bromelain can be used either as an immunosuppressive agent or an immunostimulant depending on the type of cell being treated and whether the cell has previously been activated.
Components of bromelain have also been studied. However, although there is a wealth of literature about pineapple proteinases, there has been some confusion as to the number and identity of the cysteine proteinases present. To date, most studies have concentrated on the proteinases isolated from the stem of the pineapple plant. This is probably due to the fact that crude stem bromelain is more readily available than crude fruit bromelain. Using conventional protein biochemical techniques and the application of molecular biological techniques, it has been possible to isolate and classify the various components found in crude bromelain. The proteinase component can be split into acidic and basic cysteine proteinases. Stem bromelain (EC.3.4.22.32; Rowan A. D., et. al, Arch. Biochem Biophys 267 (1) 262-270 (1988)) is the predominant proteinase and best characterised of the basic cysteine proteinases. When the amino acid sequence was determined by Ritonja et al in 1989 (FEBS 247 (2) 419- 424), stem bromelain was shown to be a member of the Cl clan of cysteine proteinases, a group that includes papain, chymopapin and cathepsin B. Stem bromelain is believed to occur as two isomers which are thought to be the product of the same gene (Rowan, et al 1988, supra; Harrach T. et. al, Journal of Protein Chem 14 (1) 41-50 (1995)). Further analysis of the basic stem bromelain proteinases identified three additional proteinases namely, ananain, comosain and fruit bromelain. Ananain was first described in 1988 (Rowan, et al 1988, supra) and its amino acid and cDNA sequences were subsequently published in 1997 (Lee, K.L., et. al, Biochem
Journal 327 (1) 199-202 (1997), Robertson and Goodenough, database submission, 1997, respectively). Comosain was identified two years later (Rowan A. D., et. al, Biochemistry Journal 266 869-875 (1990)). At the time of writing only the N-terminal sequence of comosain has been published that, in conjunction with other data, indicates that comosain is distinct from the other basic proteinases within the pineapple stem (Napper AD et. al, Biochem Journal 301 (3) 727-735 (1994)). Finally the stem also contains detectable amounts of fruit bromelain (EC 3.4.22.33) which is the predominant proteinase found in the fruit and is distinct from stem bromelain (Rowan, et al 1990, supra).
Several acidic cysteine proteinases have been identified in crude stem bromelain. One of the first, a glycosylated protein of molecular mass 23,000 Da, was described in 1985 (Ota S., et. al, Journal of Biochemistry (Tokyo) 98 (1) 219-228 (1985)). It is probable that this acidic proteinase is closely related to two forms of stem bromelain proteinase (SBA/a and SBA/b) described by T. Harrach et al, (Journal of Protein
Chem 17 (4) 351-361(1998)) which have a molecular mass of 23,550 and 23,560 respectively, and are glycosylated.
Far less well described are the fruit bromelains. Apart from ascertaining that stem and fruit bromelain are distinct enzymes and that stem bromelain is also found in the fruit, little is know about the heterogeneity of cysteine proteinases found in the fruit (Rowan, et al 1990, supra). More recently, seven cysteine proteinases sequences derived from a pineapple fruit cDNA library have been submitted to the databases. Four of the seven sequences are very closely related, to the extent that three are identical over the region coding for the mature peptide. One of the sequences (D38532) is closely related to stem bromelain (94% identity), whereas the two remaining sequences are closely related to each other (98% identity), but are less related to the other proteinases (68-82% identity).
WO-A-98/38320 relates to a fraction of bromelain designated CCX. This fraction contains two components termed CCXl and CCX2. The stem bromelain-like proteinase, CCX2, inhibits the growth of human ovarian tumours in a nude mouse model. However, stem bromelain, CCXl and other stem bromelain-like proteinases do not affect tumour growth. In WO-A-98/38320, eleven peptide sequences belonging to CCX2 protein are disclosed. These are as follows:
Nal Pro Gin Ser He Asp Trp Arg Asp Tyr Gly Ala Val Asn Glu Val Lys Asn
(SEQ ID ΝO:21) Gly Gly Trp Glu Phe Lys (SEQ ID NO: 22)
Lys Ala Val Asn Gly (SEQ ID NO: 23)
Tyr Trp He Val Arg (SEQ ID NO: 24)
Asn Ser Trp Gly Ser Ser Trp Gly Glu Gly Gly Tyr Val Arg (SEQ ID NO:25)
Thr Ser Leu Asn His Ala He Thr He He Gly Tyr (SEQ ID NO: 26) Leu Ser Glu Gin Pro (Gin) (Glu) Val Leu Asp (Cys) Ala - (SEQ ID NO: 27)
Gly Val Ser Ser Ser Ser Gly Ala Cys Gly He Ala Met Ser Pro Leu - - Thr -
(SEQ ID NO: 28)
Gly Gly Val Phe Ser Gly Pro Ala Gly (SEQ ID NO: 29)
Asn Asn Ala Tyr (SEQ ID NO: 30) Ser Ser Gly Thr Lys Tyr Trp - Val - (SEQ ID NO: 31);
where the bracketed amino acids represent alterntives to the preceding amino acid and a "-" represents an unidentified amino acid. Some of the sequences above differ slightly from those originally disclosed in WO-A-98/38320.
When the peptide sequences from CCX2 were compared to stem bromelain and other proteinase sequences in the DDBJ/EMBL/GenBank database, it was found that the most similar sequence to CCX2 was a stem bromelain-like protease isolated from the pineapple fruit. This proteinase has the database accession number D38534.
However, the only full-length cDNA sequence with which the CCX2 cDNA had a high identity was D 14059.
The present inventors have extracted RNA from a pineapple stem and used a reverse transcriptase polymerase chain reaction (RT-PCR) to isolate and sequence novel cDNA sequences encoding proteins similar to CCX2 and having the same activity.
Two pairs of primers were used in the first instance; one set was made against conserved regions of the mature peptide, whereas the other pair was designed to amplify the full length open reading frame. An additional pair of primers was also designed for use in Example 3 below. All the primers used were derived from the nucleic acid sequence having accession number D 14059 in the
DDBJ/EMBL/GenBank database.
In the present invention there is provided a nucleic acid sequence comprising one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ
ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 or a sequence differing from one of the above by not more than forty point mutations when aligned in the manner shown in Figure 1, or an RNA equivalent of one of these, provided that the sequence does not include the sequences having accession numbers D14059(SEQ ID NO: 17) and D38534 (SEQ ID NO: 19) in the DDBJ/EMBL/GenBank database.
It is preferred that the sequence of the invention differs from SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 or 15 by fewer than 30 point mutations, more preferably fewer than 20 point mutations and most preferably fewer than 10 point mutations from the sequences given above.
The present invention also provides a sequence which is complementary to one of the sequences of the first aspect or its RNA equivalent.
The term "RNA equivalent" when used above indicates that a given RNA molecule has a sequence which is complementary to that of a given DNA molecule (allowing for the fact that in RNA, "U" replaces "T" in the genetic code).
When comparing nucleic acid sequences for the purposes of determining the degree of homology or identity one can use programs such as BESTFIT and GAP (both from the Wisconsin Genetics Computer Group (GCG) software package), BESTFIT, for example, compares two sequences and produces an optimal alignment of the most similar segments. GAP enables sequences to be aligned along their whole length and finds the optimal alignment by inserting spaces in either sequence as appropriate.
Suitably, in the context of the present invention, when discussing identity of nucleic acid sequences, the comparison is made by alignment of the sequences along their whole length.
The sequences listed above encode proteins which are extremely similar to the CCX2 protein disclosed in WO-A-98/38320 and which have the same activity.
Therefore, in a second aspect of the invention, there is provided a protein comprising one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16 or a sequence differing from one of the above by not more than 15 amino acid substitutions, provided that the sequence is not CCX2 as disclosed in WO-A-98/38320 or the protein encoded by the nucleic acid sequence having accession number D14059 or D38534 in the DDBJ/EMBL/GenBank database.
The protein of SEQ ID NO: 2 forms the mature peptide region of a longer protein, SEQ ID NO: 14
Therefore, also included in the scope of the present invention is a protein comprising
SEQ ID NO: 14 or a sequence differing from it by not more than 15 amino acid residues, provided that the sequence is not CCX2 as disclosed in WO-A-98/38320 or the protein encoded by the nucleic acid sequence having accession number D14059 in the DDBJ/EMBL/GenBank database.
It is preferred that the protein sequence of the present invention has not more than 10, and more preferably, not more than five amino acid substitutions compared with the sequences given above.
The proteins of the invention have anti-cancer and immunostimulant activity and, therefore, in a further aspect of the invention, there is provided a protein of the invention for use in human or veterinary medicine, in particular the treatment or prevention of cancer.
When used in medicine, the protein of the invention will preferably be isolated and purified to remove the possibility of side effects arising from the presence of impurities.
There is also provided the use of a protein of the invention in the preparation of an anti-cancer agent.
As a result of this anti-cancer activity, the protein of the invention may be used in a method for the treatment of cancer, the method comprising administering to a patient an effective amount of the isolated and purified protein of the first aspect of the invention.
It was already known that the bromelain mixture has anti-cancer activity and, as discussed in our earlier application WO-A-9500169, this appears to arise from bromelain' s ability to affect intracellular signalling pathways, in particular, pathways which are modulated by MAP kinases. It is therefore possible that this is the mechanism of action of the protein of the present invention. However, the present invention is not dependent upon the correctness or otherwise of this theory.
Ras proteins help relay signals from growth- factor receptors on the surface of cells to transducer molecules to stimulate cell proliferation or differentiation. Oncogenic (or mutant) ras genes produce defective ras proteins that have acquired independence from externally supplied growth factors and, at the same time, may no longer respond to external growth-inhibitory signals. Mutant ras proteins are thus persistently hyperactive and their unbridled catalytic activity has a detrimental effect on the control of cell growth. Oncogenic ras genes therefore promote cancer and tumour formation by disrupting the normal controls on cell proliferation and differentiation. Approximately 30% of human cancers have mutations in a ras gene.
One of the transducer molecules which are activated by ras are the mitogen-activated protein (MAP) kinases (also called extracellular-signal regulated kinases [ERKs]) which transduce growth signals to the nucleus. WO-A-9500169, Figures 2 to 6 show that bromelain can prevent activation of the MAP kinases, ERK-1 and ERK-2. Given that signals transmitted by ras can be blocked via MAP kinase, the bromelain mixture would be expected to block cancer and tumour growth and it is possible that the protein of the present invention also works by this mechanism of action.
An alternative explanation is that the protein of the present invention acts by activating the innate immune system. The immune response has two functional divisions: the innate immune system and the adaptive immune system. The innate immune response is mediated by macrophages, natural killer cells and neutrophils. The adaptive immune response is mediated by B and T cells. When a pathogen invades the body, both the adaptive immune response and the innate immune response are activated. Innate immunity provides the first line of defence against infectious agents and most potential pathogens before they establish infection. During this initial phase of innate immunity, the adaptive immune response is developing. If the first defences are breached, the adaptive immune system should be sufficiently developed to produce a specific reaction to the infectious agent, which normally eradicates this agent. The innate immune system is also critically important in killing tumour cells.
The protein of the present invention has been shown to activate macrophages and natural killer cells (NK), critical mediators of the innate immune system important for controlling tumour growth. The protein has also been shown to increase interferon-γ- mediated nitric oxide (NO) production. Various publications have linked NO production to anti-tumour activity. For example, Hibbs (119, Res. Immunol, 142, 565-569) has shown that when macrophages produce NO, they kill tumour cells in vitro. Thus, increased NO production and activation of innate immunity may be the mechanism by which the protein of the present invention acts against tumours. Again, however, the effectiveness of this protein as an anti-tumour agent is not dependent upon the correctness of this proposition.
The protein of the present invention is useful for treating many different types of cancer including solid cancers such as ovarian, colon, breast or lung cancer and melanoma as well as non-solid tumours and leukaemia.
As mentioned above, CCX2 is able to activate natural killer (NK) cells. NK cells are lymphocytes which can recognise and destroy cells infected with various viral, bacterial or parasitic pathogens. They are also able to kill tumour cells by specifically recognising the expression of virus-induced molecules on tumour cells or other molecules associated with tumours. Therefore, because CCX2 is able to activate NK cells, it will also be of use in the treatment or prevention of virus-induced tumours. Examples of such tumours include hepatocellular carcinoma (which may result from hepatitis B virus); non-Hodgkin's lymphoma, nasopharyngeal carcinoma or Burkitt's lymphoma (resulting from Epstein-Barr virus); Kaposi's sarcoma (resulting from cytomegalovirus in HIV-infected patients); T-cell leukaemia (resulting from human T cell lympho tropic virus); and cervical carcinoma (resulting from human papilloma viruses such as HPV16 and HPV18).
In addition to its use as an anti-tumour agent, the ability of the protein of the invention to activate the innate immune response suggests that it would also be of use in situations where the adaptive immune response, such as B or T cell responses, are not fully functional. This may occur in many secondary immunodeficiencies which may arise because of malnutrition, infection (for example HIV and malaria), tumours (for example lymphoid, myeloma and other), trauma (for example burns, wounds and surgery), medical treatment (for example with drugs such as steriods, cyclosporin and cyclophosphamide), protein loss (such as in diarrhoea and burns), genetic abnormalities (such as those found in combined immunodeficiency patients who lack T and/or B cells), diabetes and old age.
The present inventors have also shown that the protein of the present invention is capable of increasing interferon-mediated NO production. Therefore, the protein of the invention may be used to treat diseases or conditions which respond to increased NO production.
NO has a critical role in host defence against infection. NO and its derivatives have potent anti-microbial activity against many pathogens including fungi, bacteria and viruses. Therefore, the protein may be administered to patients receiving chemotherapy to protect against opportunisic infections. It may also be used to treat pathogenic infections including parasites, such as Babesia, Brugia, Cryptosporidium, Encephalitoxoon, Entamoeba, Leishmania, Naegleria, Ochocerca, Opisthorchis, Plasmodium, Schistosoma, Toxoplasma and Trypanosoma. Bacteria affected by NO inlcude Bacillus, Brucella, Burkholderia, Clostridium, Ehrlichia, Francisella,
Klebsiella, Legionella, Listeria, Micrococcus, Pseudomonas Rickettsia, Salmonella, Staphylococcus, Yersinia, Chlamydia especially C. trachomatis and mycobacteria such as M. avium, M. leprae and M. tuberculosis. NO has activity against fungi such as Aspergillus, Candida, Cryptococcus, Histoplasma, Pneumocystis and Saccharomyces and against viruses, for example, Coxsackievirus, Ectomelia virus,
Encephalomyocarditis virus, Epstein-Barr virus, Herpes simplex virus, Human immunodeficiency virus type 1, Japanese encephalitis virus, mouse hepatitis virus, parvovirus, poliovirus, rabies virus, simian virus 40, vaccinia virus and vesicular stomatitus virus (Fang, 1997, ASM News, 63, 668-673).
The activity of the protein of the present invention in increasing NO production complements its immunostimmulant activity and means that it can be used as an antimicrobial agent against parasites, bacteria, fungi and viruses such as those listed above.
Thus, in further aspects, the invention provides the protein of the present invention for use as an antimicrobial agent and the use of the protein of the present invention in the preparation of an antimicrobial agent. The protein will usually be formulated before administration to patients and so, in a further aspect of the invention there is provided a pharmaceutical or veterinary composition comprising the isolated and purified protein of the first aspect of the invention together with a pharmaceutically or veterinarily acceptable excipient.
The protein may be administered by a variety of routes including enteral, for example oral, nasal, topical, buccal, or anal administration or parenteral administration, for example by the intravenous, subcutaneous, intramuscular or intraperitoneal routes.
In many cases, the oral route may be preferred as this is often the route which patients find most acceptable. The oral route may be particularly useful if many doses of the protein are required as will often be the case in the treatment of cancer.
When oral adminstration is chosen, it may be desirable to formulate the protein in an enteric coated preparation in order to assist its survival through the stomach.
Alternatively, another orally administrable dosage form may be used, for example a syrup, elixir or a hard or soft gelatin capsule, either of which may be enteric coated.
However, if it is intended to administer only a single dose of the protein, it may be more convenient to use a parenteral route.
For parenteral adminstration, the protein may be formulated in distilled water or another pharmaceutically acceptable solvent or suspending agent.
A suitable dose of the protein to be administered to a patient may be determined by the clinician. However, as a guide, a suitable dose may be from about 0.5 to 100 mg per kg of body weight. It is expected that in most cases, the dose will be from about 1 to 50 mg per kg of body weight and preferably from 1 to 20 mg per kg of body weight. For a man having a weight of about 70 kg, a typical dose would therefore be from about 70 to 7000 mg.
The nucleic acid of the invention may be used in recombinant DNA technology and, for that purpose, may be inserted into a vector. The vector may be, for example, a plasmid, cosmid or phage. For the purposes of expression, the nucleic acid in the vector may be under the control of a suitable control sequence and may be operatively linked to a suitable promoter. The promoter may be either constitutive or induceable.
The vector may be used to transform cells, which may be single celled organisms, such as bacteria or yeast cells. Alternatively, however, the nucleic acid may be used in gene therapy, in which case, the transformed cells will be cells of a patient being treated using gene therapy.
The invention will now be further described with reference to the following examples and to the drawings in which:
FIGURE 1 shows a pile up of NF 36/NF 37 RT-PCR product sequences in which the sequence for the PCR products has been aligned with the published sequence of D 14059. An asterisk denotes a nucleotide substitution which does not affect the translation of the reading frame, whereas a dollar ($) denotes a substitution which does alter the translation.
FIGURE 2 shows an alignment of two NF 43/NF 44 RT-PCR product sequences, one NF 76/NF 77 RT-PCR product sequences, D14059 and D38534. The sequences from cDNAs have been aligned and the translation of the open reading frame is also shown.
Peptide ID NO 24 from CCX2 has been aligned with the translation of the reading frame. Where the cDNA sequences differ from D38534 and D14059 it is marked above the sequence. FIGURE 3 shows the predicted amino acid sequence for cDNA sequences pCRScript 569 (SEQ ID NO: 2), pCRScript 571 (SEQ ID NO: 4), 573 (SEQ ID NO: 8) and 574 (SEQ ID NO: 10) shown aligned to the known sequences of stem bromelain-like proteinases D38534 (SEQ ID NO:18) and D38534 (SEQ ID NO: 20). The theoretical translations of the cDNA sequences pCRScript 570, 572 (SEQ ID NO: 6) 575, 576, 578, 579, 580 and 581 (SEQ ID NO: 12) all matched 569 and so are not included in this figure. The theoretical translation of pCRScript 710 is identical to D38534 and so is also not included in this figure. Also aligned to D38534 are 10 of the 11 peptide sequences from the CCX2 proteinase identified in WO-A-98/38320. Peptide SEQ ID NO: 28 is not shown as it lies outside the region covered by primers NF 36 and NF 37. A full stop (.) represents a match to D38534 and a dash (-) indicates that there was no cDNA sequence data available for that section. Where the amino acid sequences differ, the single letter code for the amino acid is shown. Methods Plant Material A pineapple plant, which was a Jamaica Queen cultivar, was generously donated by the Lost Gardens of Heligan, St Austell, UK. The plant was 3 years old and had fruited in the previous year. After being stripped of offsets and leaves, the stem weighed 256 g and measured approximately 20 cm in length and 7 cm in diameter. The stem was immediately cut into 1 cm cubes and snap frozen in liquid nitrogen, and stored in liquid nitrogen until required.
An additional pineapple plant was collected from the Rayong region of Thailand with the invaluable assistance of Mr C. L. Wei of Hong Mao Biochemicals Co. Ltd. The pineapple plant, which was reported to be a Smooth Cayenne cultivar, was approximately eighteen months old and had recently fruited. The leaves and offsets had been removed 24-48 hours prior to processing of the stems. The stems weighed an estimated 400 g and were approximately 30 cm long and 9 cm in diameter. The stem was cut into 1 cm cubes and frozen in ethanol/dry ice. After being shipped to UK on dry ice the sections of stem were stored at -70 °C. RNA extraction
RNA extraction was performed using a RNeasy™ plant mini kit (Qiagen Ltd, Crawley,
UK). 200 mg stem tissue was powdered in liquid nitrogen, and mixed with 950 μl guanidinium isothiocyanate denaturing buffer. The sample was then incubated at 56°C for 3 min and vortexed at 60 second intervals. The lysate was applied to a QIAshredder™ (Qiagen Ltd, Crawley, UK) and the flow-through was cleared with 0.5 volumes of ethanol. The flow-through was then applied to a RNeasy™ mini spin column for isolation of total RNA. Yield and quality of RNA was assessed by measuring the absorbance at 260 nm and 280 nm.
Reverse Transcriptase-Polvmerase Chain Reaction CRT-PCR') In this section, the following abbreviations are used: TAE - tris acetate ethylenediaminetetraacetic acid buffer;
IPTG - isopropyl β-D-thioglactopyranoside.
To determine the DNA sequence for CCX2 the first step is to prepare the complementary DNA (cDNA) from the messenger RNA (mRNA). To do this, an oligo d(T),6 primer is bound to the poly A tail at the 3' end of the mRNA, this enables the enzyme reverse transcriptase to synthesise the first strand of cDNA. The cDNA will cover the entire open reading frame as well as the 5' and 3' untranslated regions at each end. Assuming that CCX2 is similar to the other bromelain-like proteinases, the open reading frame will consist of 3 regions. The first region will encode the prepeptide, or signal peptide, a short sequence of approximately 20 amino acids which is involved in determining the cellular localisation of the protein once it is synthesised. The second region encodes for the propeptide, which consists of almost 100 amino acids that must be removed before the proteinase becomes active. The third region is the section which encodes for the active protein. Ten of the eleven sequences from WO-A-9838320are derived from this region and the first set of PCR primers were designed to amplify this region.
To synthesise the first strand of cDNA, 300 ng of total RNA and 50 pmol oligo d(T)16 primer where mixed with 10 X PCR buffer, dNTP's and 50 U MuLV Reverse Transcriptase (all reagents from Perkin-Elmer, Beaconsfield, UK). The total reaction volume was 20 μl, and reactions were incubated at room temperature for 10 min, followed by 15 min at 42°C, and finally, 5 min at 99°C. The first strand cDNA was stored on ice or at -20°C until required.
Three pairs of primers were designed for amplification of cDNA's encoding proteinases. The first set (NF 36: 5' TGC CTC AAA GTA TTG ATT GG 3' (SEQ ID NO: 32)/NF 37: 5' ATA CGG ACG TAT CCA CCC TC 3' (SEQ ID NO: 33)); see Figure 1) of primers were made to regions where the bromelain proteinases' sequences are conserved and where the amino acid sequence of D 14059 is identical to
CCX2. Because the amino acid sequences in this region were so similar, it was thought that the DNA sequences would also be conserved. Thus NF 36 and NF 37 were based on the D 14059 cDNA sequence, but the primers, in theory, would amplify CCX2 as well as D14059. It was expected that the primers would amplify a short 574 bp fragment of the cDNA corresponding to the mature peptide.
The second set of primers (NF 43: 5' GCA TGG CTT CCA AAG TTC AAC TC 3' (SEQ ID NO: 34)/ NF 44: 5' TTG CTA AAT CAT TTG CTT CTT CCG AC 3' (SEQ ID NO: 35)) were made to less well conserved regions of bromelain proteinases, but when used in the PCR reaction they would amplify the cDNA corresponding to the full length of the open reading frame, which was expected to be 1092 bp.
The third set of primers (NF 76: 5' CCC CCC GCT AGC CGT GAC GAA CCC AGT GAT C 3' (SEQ ID NO: 36) and NF 88: 5' CCC CCC GAG CTC TTA TGA TTG TAG AGT GGG AAA GAG AG 3' (SEQ ID NO: 37))were designed to remove the prepeptide and the C-terminal thirteen residues, which were thought to be removed during post-translational modifications in vivo. Although these primers contained the Nhel and Sad restriction sites, respectively, which enables the PCR product to be inserted in frame into certain expression vectors, the RT-PCR products were blunt end ligated into pCRScript as before.
Each PCR consisted of 5 μl cDNA template, lOOng of each primer, lOx PCR buffer, 2.5 mM dNTPs, AmpliTaq™ DNA Polymerase (all from Perkin-Elmer, Beaconsfield, UK) and water to a total volume of 25 μl. The amplification took place on a thermal cycling block under the following conditions: an initial denaturation of 94°C for 4 min, followed by 30 cycles of 94°C for 30 seconds, annealing temp of 56°C for 30 seconds and extension temperature of 72°C for 30 seconds.
When the PCR resulted in multiple products, they were size fractionated on a 1% agarose/TAE gel. One PCR product was then excised from the gel and purified using a QIAquick gel extraction kit (Qiagen, Crawley, UK). The PCR product was then incubated with 1 unit Pfu DNA polymerase at 72 °C for 30 minutes to remove 5' overhangs and then blunt-end ligated into pCRScript Amp SK (+) cloning vector (Stratagene Ltd, Cambridge, UK). Escherichia coli strain XL-1 transformants were plated onto agar plates containing the antibiotic ampicillin, X-gal and IPTG. E. coli which contain the pCRScript vector are resistant to ampicillin, and so are able to grow on the agar plates. When the PCR product is inserted into the pCRScript vector it disrupts the β-galactosidase gene, an enzyme which processes X-gal into an insoluble blue product. It is therefore possible to select E. coli transformants which have pCRScript containing a PCR product (white colonies) and avoid those which contain pCRScript without a PCR product (blue colonies). The colonies containing the plasmids with a PCR product were grown up in L-broth and 10 μg of the plasmid was purified using a QIAprep™ miniprep (Qiagen Ltd, Crawley, UK). To confirm the size of the PCR product, the plasmid was digested with restriction enzyme EcoRl and Notl (Boehringer-Mannheim, Lewes, UK). The restriction enzymes cleave the plasmid either side of the PCR product, allowing the size of the PCR product to be estimated by gel electrophoresis. Plasmids containing a PCR product of the expected size were then sequenced using an automated sequencer. After the sequencing reaction, each pCRScript construct was given an identity number as indicated in Figure 1.
EXAMPLE 1 - Amplification of cDΝAs encoding the mature peptide
The oligonucleotides ΝF 36/ΝF 37 gave a single band of approximately 600 bp. This is comparable to the expected size of 574 bp, assuming that CCX2 is similar to D14059 and D38534. 24 "white" bacterial colonies that contained the ampicillin resistance marker were selected and the plasmids were analysed by restriction enzyme digest. 17 of the plasmids had a 600 bp PCR product, 6 plasmids had a shorter PCR product of approximate 350 bp, and 1 plasmid contained no detectable PCR product.
12 of the plasmids which contained a 600 bp PCR product and 1 plasmid which contained a 350 bp PCR product were sequenced, the results of which are summarised in Figure 1. For comparison, 7 of the PCR products that had unique sequences are shown aligned with the nucleotide sequence of a stem bromelain-like proteinase which was obtained from fruit (accession number D14059, labelled D14059 nuc in Figure 1
(SEQ ID NO: 17)). These are as follows: pCRScript 569 (SEQ ID NO: 1) pCRScript 570 pCRScript 571 (SEQ ID NO: 3) pCRScript 572 (SEQ ID NO: 5) pCRScript 573 (SEQ ID NO: 7) pCRScript 574 (SEQ ID NO: 9) pCRScript 581 (SEQ ID NO: 11). Beneath the alignment of the nucleotide sequences is the amino acid translation of D14059 (D14059 pro in Figure 1(SEQ ID NO: 18)) aligned with the 11 peptide sequences from WO-A-9838320(SEQ ID NOS: 21 to 31).
Each PCR product was sequenced at least twice, however, for pCRScript 574 and 581, this was not sufficient to reliably sequence the full length of the product, thus the sequence shown in Figure 1 does not represent the full length of the PCR product. All of the sequences shown in Figure 1 differed from each other and from the known sequences in the database.
The differences between sequences are highlighted in Figure 1. An asterisk (*) indicates a substitution which does not effect the translation and a dollar ($) indicates a substitution which does alter the translation of the reading frame. Plasmid 570, which was the only 350 bp PCR product to be sequenced, had a 198 bp deletion of a central section of the sequence which is represented by a series of dots (.).
There were 6 substitutions which altered the theoretical translation of the reading frame, one of which ($6 in Figure 1) is common to all sequences and would cause a glutamine to be substituted for a leucine. Plasmid pCRScript 571 (SEQ ID NO: 3) has an additional mutation ($1 in Figure 1) which would have caused a histidine to be substituted for a tyrosine. Plasmid pCRScript 573 (SEQ ID NO: 7) also has one additional mutation ($5 in Figure 1) which would cause a serine for pro line substitution and pCRScript 574 (SEQ ID NO: 9) has two additional mutations ($2 and $4 in Figure 1) which would cause a glycine for alanine and serine for glycine substitutions respectively. pCRScript 574 also as an ambiguous base, marked by $3 in
Figure 1 , which may be G or A and so could cause aspartic acid to be substituted for glycine. In summary, all of the sequences obtained are extremely similar to D14059, but they all contain a number of unique amino acid substitutions making them different from D 14059. They also differ from other previously published bromelain proteinase sequences, including D38534. Moreover, they also differ from the 11 peptides from CCX2 at various positions.
EXAMPLE 2 - Amplification of cDNAs encoding the full length pre-propeptide RNA extracted from the Jamaica Queen cultivar
The oligonucleotides NF 43/NF 44 resulted in two major PCR products, one of approximately 500 bp and one of approximately 1100 bp, as well as 3 minor PCR products between 600 and 1600 bp. The expected size of the open reading frame for the full length pre-propeptide is 1092 bp. Therefore the 1100 bp PCR product was purified on an agarose gel and ligated into pCRScript. Of the 11 white colonies that were selected and analysed by restriction enzyme digestion, 9 contained inserts of 1100 bp, and of those, two, designated pCR Script 673 and pCR Script 674, were sequenced (Figure 2). Plasmid pCRScript 673 has only been partially sequenced, although over the known sequence it appears to be identical to plasmid pCRScript 674 (SEQ ID NO: 13), with the exception of 1 nucleotide at position 725. This mutation, however, does not affect the translation of the reading frame. Apart from the substitution at position 725, both plasmids match pCRScript 569 in the region which corresponds to the mature peptide. However, in the pro-region, the two sequences have 5 substitutions when compared to D 14059 and they are marked by an asterisk above the sequence. The resultant change to the theoretical translation of the reading frame is shown in bold. Although only two PCR products were sequenced it appears that the sequence obtained, is very similar to D 14059 and the CCX2 peptides, especially in the region corresponding to the mature peptide. There are however, a number of amino acid substitutions.
pCRScript 673 and 674 encode the entire open reading frame and consists of a 351 amino acid peptide, which by homology to other proteinases, contains a 24 amino acid signal peptide, a 98 amino acid pro-peptide and a 229 amino acid peptide v/hich would form an active proteinase. However, it is likely that a portion of the C-terminal would be removed during processing of the mature peptide this would result in a loss of 13 amino acids leaving 216 amino acids, with a predicted molecular mass of 23,490 Da.
EXAMPLE 3 - Amplification of cDNA's encoding the mature peptide from RNA extracted from the Smooth Cayenne cultivar
When the NF 76/NF 77 primers were used in a RT-PCR reaction two bands were seen. The band of the approximately expected size was gel purified and cloned. Of the transformed E. coli which contained recombinant vector that were obtained one, pCRScript 710, was sequenced extensively. It was show to be almost identical to D38354 with the exception of two nucleotide substitutions, firstly a 'T' for 'C substitution and secondly a 'G' for 'A' substitution at the positions marked with the crosses (+) in figure 2. The latter substitution results in a Valine being substituted for an Alanine in the amino acid sequence.
Discussion
In this patent a total of 10 cDNA sequences have been presented which encode 6 different proteinases and were obtained from two different pineapple plant cultivars.
The first cDNA's were obtained from the Jamaica Queen cultivar. Of these one plasmid, pCRScript 570, contained a deletion of 198 bp, this is probably due to alternative splicing of the mRNA, although its is not known what effect such a deletion would have on the protein. The other 4 plasmids (pCRScript 569, 571, 473 and 574) all gave theoretical protein sequences that were all unique, i.e. they had one or more amino acid substitutions that have not been previously reported.
With respect to these sequences it is important to note that none of the amino acid substitutions affect the three amino acids in the catalytic triad, or amino acids that are thought to be involved in conserved disulphide bonds. However, the two cDNA sequences from the Jamaica Queen cultivar that encode the pro-peptide (pCRScript 673 and 674), contain five substitutions when compared to D14059, indicating that there may be less evolutionary pressure to keep such sequences conserved.
Despite the discovery of cDNAs encoding 5 novel proteinases from the Jamaica Queen cultivar, none of the sequences exactly match the peptides that characterise CCX2. These cDNA sequences all have the highest similarity to a bromelain mRNA extracted from the pineapple fruit (accession number D14059), this was to be expected as the primers were designed from the D14059 sequence. Although none of the sequences exactly match D 14059 at either the cDNA level or at the protein sequence level it is interesting to note that at the protein level the sequences are equally similar to both D14059 and D38534. For example the theoretical translation of pCRScript 569 is 99% identical to both D14059 and D38534.
D14059 and D38534 are extremely similar, for example over the region that encodes for the mature peptide, the sequences differ by only four amino acid residues. Moreover, in the region which corresponds to the section between primers NF 36 and NF 37 the amino acid sequence only differs by two amino acids (see positions 27 and 88 as numbered in Figure 3). Neither of these amino acids is covered by the peptide sequences which characterise CCX2. However, peptide sequence (ID NO 28) shows that CCX2 is similar to D38534 at the C-terminal.
In summary, the present inventors have identified six new proteinases in the stem of the pineapple plant. They have high similarity to two bromelain-like proteinases
(D14095 and D38534) found in the fruit of the pineapple plant. All of the sequences from the Jamaica Queen cultivar have a number of nucleotide substitutions that result in one or more unique amino acid substitution. Such changes may indicate different characteristics of the proteinases such as enzyme substrate specifities, or biological activity. In addition, the one sequence obtained from the Smooth Cayenne cultivar differs from a published sequence by two nucleotide substitutions, however, only one results in a change in the amino acid sequence, and this is within the propeptide sequence.
The following table shows which SEQ ID Nos have been allocated to which products.

Claims

1. A nucleic acid sequence comprising one of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 or a sequence differing from one of the above by not more than forty point mutations when aligned in the manner shown in Figure 1, or an RNA equivalent of one of these, provided that the sequence does not include the sequences having accession numbers D14059(SEQ ID NO: 17) and D38534 (SEQ ID NO: 19) in the DDBJ/EMBL/GenBank database.
2. A nucleic acid molecule as claimed in claim 1, which differs from the sequences of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 OR 15 by fewer than 10 point mutations.
3. A nucleic acid molecule which has a sequence complementary to a nucleic acid sequence as claimed in claim 1 or claim 2.
4. A protein comprising one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16 or a sequence differing from one of the above by not more than 15 amino acid substitutions, provided that the sequence is not CCX2 as disclosed in WO-A-98/38320 or the protein encoded by the nucleic acid sequence having accession number D 14059 or D38534 in the DDBJ/EMBL/GenBank database.
5. A protein as claimed in claim 4 which differs from one of SEQ ID NOS : 2, 4,
6. 8, 10, 12, 14 or 16 by not more than 10 amino acid substitutions.
6. A protein as claimed in claim 4 or claim 5 for use in human or veterinary medicine.
7. A protein as claimed in claim 4 or claim 5 or the protein encoded by the nucleic acid sequence having accession number D14059 or D38534 in the DDBJ/EMBL/GenBank database for use in the treatment or prevention of cancer in a human or other mammal.
8. The use of a protein as claimed in claim 4 or claim 5 or the protein encoded by the nucleic acid sequence having accession number D14059 or D38534 in the DDBJ/EMBL/GenBank database in the preparation of an agent for the treatment or prevention of cancer.
9. A protein as claimed in claim 7 or the use as claimed in claim 8, wherein the cancer is a solid cancer such as ovarian, colon, breast or lung cancer or melanoma, a non-solid tumour, leukaemia or a virus-induced tumours such as hepatocellular carcinoma, non-Hodgkin's lymphoma, nasopharyngeal carcinoma, Burkitt's lymphoma, Kaposi's sarcoma, T-cell leukaemia or cervical carcinoma.
10. A protein as claimed in claim 4 or claim 5 or the protein encoded by the nucleic acid sequence having accession number D14059 or D38534 in the DDBJ/EMBL/GenBank database for use as an immunostimmulant.
11. The use of a protein as claimed in claim 4 or claim 5 or the protein encoded by the nucleic acid sequence having accession number D14059 or D38534 in the DDBJ/EMBL/GenBank database in the preparation of an immunostimulant.
12. A protein as claimed in claim 10 or the use as claimed in claim 11, wherein the immunostimulant is for the treatment of secondary immunodeficiencies arising from malnutrition, infection (for example HIV and malaria), tumours (for example lymphoid, myeloma and other), trauma (for example burns, wounds and surgery), medical treatment (for example with drugs such as steriods, cyclosporin and cyclophosphamide), protein loss (such as in diarrhoea and burns), genetic abnormalities (such as those found in combined immunodeficiency patients who lack T and/or B cells), diabetes and old age.
13. A protein as claimed in claim 4 or claim 5 or the protein encoded by the nucleic acid sequence having accession number D14059 or D38534 in the
DDBJ/EMBL/GenBank database for use in the treatment or prevention of diseases or conditions which respond to increased NO production.
14. The use of a protein as claimed in claim 4 or claim 5 or the protein encoded by the nucleic acid sequence having accession number D 14059 or D38534 in the
DDBJ/EMBL/GenBank database in the preparation of an agent for the treatment or prevention of diseases or conditions which respond to increased NO production.
15. A protein as claimed in claim 4 or claim 5 or the protein encoded by the nucleic acid sequence having accession number D14059 or D38534 in the
DDBJ/EMBL/GenBank database for use as an anti-microbial agent.
16. The use of a protein as claimed in claim 4 or claim 5 or the protein encoded by the nucleic acid sequence having accession number D14059 or D38534 in the DDBJ/EMBL/GenBank database in the preparation of an anti-microbial agent.
17. A pharmaceutical or veterinary composition comprising a protein as claimed in in claim 4 or claim 5 together with a pharmaceutically or veterinarily acceptable excipient.
18. A composition as claimed in claim 17 which is formulated for enteral, for example oral, nasal, buccal, topical or anal administration.
19. A composition as claimed in claim 17 which is formulated for parenteral administration, for example by the intravenous, subcutaneous, intramuscular or intraperitoneal routes.
20. A vaccine composition comprising a protein as claimed in claim 4 or claim 5 or the protein encoded by the nucleic acid sequence having accession number D14059 or
D38534 in the DDBJ/EMBL/GenBank database as an adjuvant.
21. A vector comprising a nucleic acid molecule as claimed in any one of claims 1 to 3.
22. A vector as claimed in claim 21 which is a plasmid, cosmid or phage.
23. A cell transformed by a nucleic acid molecule as claimed in any one of claims l to 3.
24. A cell as claimed in claim 23 which is a single celled organism.
25. A cell as claimed in claim 23 which is a cell of a patient to be treated using gene therapy.
EP99940397A 1998-09-02 1999-08-24 Nucleic acids and proteins from pineapple stem Withdrawn EP1078077A1 (en)

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US20020102253A1 (en) 1997-02-25 2002-08-01 Mynott Tracey Lehanne Component of bromelain
JP2001513636A (en) 1997-02-25 2001-09-04 コルテクス（ユーケイ）リミティッド Bromelain ingredients
EP1207204A1 (en) * 2000-11-16 2002-05-22 KWS Saat AG Tissue-specific promoters from sugar beet
IL165334A0 (en) * 2004-11-22 2006-01-15 Mediwound Ltd Debriding composition from bromelain and methods of producing same
EP2036978A1 (en) * 2007-09-14 2009-03-18 URSAPHARM Arzneimittel GmbH & Co. KG Recombinant preparation of selected bromelain fractions
MY190244A (en) * 2009-12-17 2022-04-08 International Islamic Univ Malaysia Method for producing recombinant stem bromelain
EP2821106A1 (en) * 2013-07-05 2015-01-07 Cellquantum GmbH Composition and use thereof for the treatment of tumor indications
EP2821107A1 (en) * 2013-07-05 2015-01-07 Cellquantum AG Composition and use thereof for the treatment of benign and malign diseases
US11382958B2 (en) 2016-01-31 2022-07-12 Mediwound Ltd. Debriding composition for treating wounds

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GB9412711D0 (en) * 1994-06-24 1994-08-17 Cortecs Ltd Medical use of bromelain
JP2001513636A (en) * 1997-02-25 2001-09-04 コルテクス（ユーケイ）リミティッド Bromelain ingredients

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