WO2004055046A2 - Genes adam - Google Patents

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Publication number
WO2004055046A2
WO2004055046A2 PCT/GB2003/005550 GB0305550W WO2004055046A2 WO 2004055046 A2 WO2004055046 A2 WO 2004055046A2 GB 0305550 W GB0305550 W GB 0305550W WO 2004055046 A2 WO2004055046 A2 WO 2004055046A2
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Prior art keywords
variant
seq
adam
polypeptide
polynucleotide
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PCT/GB2003/005550
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English (en)
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WO2004055046A3 (fr
Inventor
Michael Birch
Jayne Louise Brookman
Sandra Elizabeth Lavens
Nuria Rovira Graells
Daniel Scott Tuckwell
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F2G Ltd
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Priority to AU2003292438A priority Critical patent/AU2003292438A1/en
Publication of WO2004055046A2 publication Critical patent/WO2004055046A2/fr
Publication of WO2004055046A3 publication Critical patent/WO2004055046A3/fr

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    • 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)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to ADAM genes, and encoded proteins thereof, including derivatives and analogues thereof, and their uses in diagnosis and therapy.
  • the ADAMs are a family of membrane associated proteases characterised by, and named for, the presence of A Disintegrin and A Metalloproteinase domain (Black and White, 1998, Current Opinion in Cell Biology. 10, 654-659; Evans, 2001 Bioessays 23, 628-639) .
  • the proteins are typically 700-800 amino acids in length and are composed of, from the N-terminus, a pro-peptide region, a reprolysin-type Zn-metalloproteinase domain, a disintegrin domain, a cysteine-rich region, a transmembrane region and a cytoplasmic tail. The removal of the pro-peptide domain is required for activation of the protease.
  • Most of the work on this family of proteases has been carried out in
  • ADAMs which are not membrane-bound (ADAMTS proteins) .
  • ADAMTS proteins membrane-bound proteins.
  • a number of snake venoms contain proteins that are clearly evolutionarily related to the ADAMs, but these lack the C-terminal domains seen in the human ADAMs, particularly the transmembrane region but other domains C-terminal to the disintegrin domain may also be missing.
  • ADAM 17 (TACE) is required for the proteolytic cleavage of the " membrane-associated TNF ⁇ precursor to liberate TNF ⁇ , and is therefore also known as TNF Alpha Converting Enzyme. "ADAM17 is also involved in the cleavage of other cell surface proteins with a variety of biological functions including the growth factor, TGF ⁇ . ADAM 17 is clearly very important for mammalian viability as a knock out of the gene in mice resulted in the majority of pups dying between embryonic day 17.5 and the first day after birth (Peschon et al .
  • the crystal structure of the metalloprotease domain has been resolved, providing the opportunity for the design of inhibitors (Maksos et al . , 1998, Proc. Nat. Acad. Sci. U.S.A. ,95, 3408-3412).
  • the protein substrate of ADAM17, TNF ⁇ is a cytokine which is rapidly increased in levels in response to infection or tissue damage. It has been implicated in rheumatoid arthritis and endotoxic shock and so the protein responsible for its activation, ADAM 17, is a good candidate for the development of therapeutic inhibitors.
  • ADAM 10 has been best characterised in Drosophila melanogaster where it is known as KUZBANIAN (Pan and Rubin, 1997, Cell 90, 271-280) . This is involved in the proteolytic processing of the cell surface protein NOTCH, thereby regulating development in neuronal and other tissues. SUP-17, the Caenorhabditis elegans homologue of ADAM 10 is also involved in NOTCH signalling. The biological function of human ADAM 10 has yet to be defined.
  • ADAM 2 ferrtillin ⁇
  • ADAM 3 cyritestin
  • ADAM12 (meltrin ⁇ ) may also have an adhesive function as its disintegrin domain has been implicated in myoblast fusion.
  • ADAMs are not unique, as there appear to be at least 4 ADAMs, in the genome, including ADAM 10- and ADAM 17- homologues, and an ADAM involved in sperm and epithelial cell fusion, as well as 2 ADAMTS (Daulby et al., (2001) Abstract 558, European Worm meeting) .
  • ADAMs family plays a major role in animal biology, as well as being amenable to manipulation via inhibitors.
  • isolation of fungal ADAMs represents the identification of novel and potentially essential molecules which could be therapeutically manipulated to kill or severely impair the ability of a fungus to grow.
  • ADAM ADAM polypeptide
  • ADAM protein proteins containing a metalloproteinase domain of the adamalysin/reprolysin family and a disintegrin domain. A large number of snake venom metalloproteinases also share these domains, but these are not classified as ADAMs.
  • ADAM gene(s) is defined as a gene encoding an ADAM or ADAM protein
  • ADAM ADAM protein
  • bases nucleotides
  • ADAMs are typically defined and identified at the sequence level. Only a few ADAMs have been defined functionally or structurally (see above) .
  • An ADAM gene can be identified either by a match on BLAST against a known ADAM and/or on the basis of matches to certain sequence patterns at the INTERPRO or SMART protein profile web sites (www.ebi.ac.uk/interpro/scan.html; smart.embl-heidelberg.de).
  • ADAM will match the metalloproteinase ADAM/reprolysin
  • M12B pattern and may also match the disintegrin pattern
  • a polynucleotide comprising an ADAM gene - isolated from an organism independently selected from a group of genera consisting of Aspergillus; Pneumocystis ; ' Encephalitozoon; Cryptococcus ; Trichophyton ; Fusarium; Blumeria ; Leptosphaeria ; Plasmopara ; Pyricularia ; Puccinia and Rhizoctonia .
  • the polynucleotide comprises an ADAM gene isolated from an organism independently selected from a group of genera consisting of Aspergillus; Pneumocystis and Encephali tozoon .
  • the polynucleotide comprises an ADAM gene isolated from an organism independently selected from a group of species consisting of Aspergillus fumigatus ;
  • Aspergillus terreus Aspergillus parasiticus; Aspergillus flavus; Aspergillus niger; Aspergillus nidulans;
  • Trichophyton ruhrum Fusarium solani; Blumeria graminis;
  • Leptosphaeria nod ⁇ rum Plasmopara viticola ; Pyricularia oryzae; Puccinia species; and Rhizoctonia species.
  • the polynucleotide comprises an ADAM gene isolated from an organism independently selected from a group of species consisting of Aspergillus fumigatus; Pneumocystis jiroveci; and Encephali tozoon cuniculi .
  • an ADAM polypeptide isolated from an organism independently selected from a group of genera consisting of Aspergillus; Pneumocystis; Encephalitozoon; Cryptococcus; Tri chophyton; Fusarium; Bl umeria ;
  • Leptosphaeria Leptosphaeria ; Plasmopara ; Pyricularia ; Puccinia and Rhizoctonia .
  • the ADAM polypeptide is isolated from an organism independently selected from a group of genera consisting of Aspergillus; Pneumocystis ; and
  • the ADAM polypeptide is isolated from an organism independently selected from a group of species consisting of Aspergillus fumiga tus; Aspergillus terreus ; Aspergillus parasi ticus; Aspergillus flavus ; Aspergillus niger; Aspergillus nidulans; Pneumocystis j iroveci ; Encephalitozoon cuniculi; Cryptococcus neoformans;
  • Trichophyton interdigitale Trichophyton rubrum; Fusarium solani; Blumeria graminis; Leptosphaeria nodorum; Plasmopara vi ticola ; Pyricularia oryzae ; Puccinia species; and Rhizoctonia species .
  • the ADAM polypeptide is isolated from an organism independently selected from a group of species consisting of Aspergillus fumiga tus; Pneumocystis jiroveci; and Encephalitozoon cuniculi .
  • the isolated polynucleotide comprises an ADAM gene.
  • the isolated polypeptide comprises an ADAM.
  • the polynucleotide may comprise recombinant polynucleotide, and the polypeptide may comprise recombinant polypeptide.
  • the polynucleotide may comprise native, synthetic or recombinant polynucleotide, and the polypeptide may comprise native, synthetic or recombinant polypeptide.
  • the polynucleotide or polypeptide may comprise combinations of native, synthetic or recombinant polynucleotide or polypeptide, respectively.
  • the polynucleotides and polypeptides of the invention may have a sequence which is the same as, or different from, naturally occurring ADAM polynucleotides and polypeptides .
  • references to polynucleotides and polypeptides being “isolated from” a particular organism include polynucleotides and polypeptides which were prepared by means other than obtaining them from the organism, such as synthetically or recombinantly .
  • the polynucleotide and the polypeptide may be isolated from Aspergillus fumiga tus , and more preferably, from A. fumiga tus AF293.
  • the polynucleotide and the polypeptide may be isolated from Encephalitozoon, and more preferably from Encephalitozoon cuniculi .
  • the polynucleotide and the polypeptide may be isolated from Pneumocystis, and more preferably from Pneumocystis jiroveci.
  • ADAMs Prior to the work outlined in this patent, the presence of ADAMs in organisms outside of the animal kingdom had yet to be explored. No ADAM can found in the completed S. cerevisiae genome sequence, although one has been identified in S . pombe . The occurrence and distribution of ADAMs in fungi was therefore still an area of considerable uncertainty.
  • ADAMs genes (arbitrarily named ADM-A and ADM-B) in A. fumiga tus .
  • the inventors have predicted the sequence of the polypeptide, i.e. the ADAM protein, which each of these two genes encodes.
  • the two ADAMs genes show significant differences at the sequence level.
  • the inventors have isolated an "ADAM gene and predicted the sequence of the ADAM protein in Encephalitozoon cuniculi and Pneumocystis jiroveci .
  • ADAMs ' genes and gene products have, to date, been unknown to exist in Aspergillus , Encephalitozoon or Pneumocystis.
  • ADAMs specific regions within the disintegrin domains of mammalian ADAMs have been shown to be important for protein-protein interactions mediated by ADAMs (Evans, 2001; Eto et al . , 2002, J.Biol.Chem. In press ms M200086200) . Examination of the ADM-A and ADM-B disintegrin domains in the light of these data indicated two regions, D and E in Fig.l, which are thereby predicted to be involved in adhesive interactions. Region D contains the putative adhesive sequence E/PCD, although flanking sequence will also be important. Region E is topologically adjacent to region D in disintegrin structures and is therefore also predicted to be involved in interactions. The regions within these boxes marked "+" are 'therefore predicted to be involved in the specificity of the fungal ADAMs .
  • the isolated polypeptide may comprise an amino acid sequence independently selected from at least one of the , following: - (i) a residue shown at position 3 of rows ADM-A/ADM- B/E. cuniculi of box A as shown in Figure 1, or variant thereof; (ii) residues shown at positions 2-4, and/or position 6, and/or position 8, and/or positions 12-14, and/or position 26, of rows ADM-A/ADM-B/.E. cuniculi/ P. jiroveci of box B as shown in Figure 1, or variant thereof; (iii) residues shown at positions 3, and/or position 6, and/or position 8 of rows ADM-A/ADM-B/E. cuniculi/ P. jiroveci of box C as shown in Figure
  • the ADAM polynucleotide is expressed to produce a nascent ADAM polypeptide which is, preferably inactive.
  • the nascent ADAM polypeptide is activated by a proteolytic cleavage, which liberates a propeptide domain producing an activated ADAM polypeptide .
  • A. fumigatus ADM-A the isolated polypeptide comprises a propeptide-proteinase junction comprising ' an amino acid sequence substantially as set out as residues 190-210 of SEQ ID No.2 , or variant thereof.
  • A. fumiga tus ADM-B the isolated polypeptide may comprise a propeptide-proteinase junction comprising an amino acid sequence substantially as set out as residues 263-283 of SEQ ID No. , or variant thereof.
  • E. cuniculi ADAM the isolated polypeptide may comprise a propeptide-proteinase junction comprising an amino acid sequence substantially as set out as residues 148-168 of SEQ ID No. 40, or variant thereof.
  • the propeptide-protease junction is of functional importance.
  • the P. jiroveci EST sequence does not extend to the propeptide.
  • the protease responsible for the cleavage is currently unknown but specificity will reside in the site of cleavage and probably also in spatially adjacent regions in the propeptide and protease domains. The activating protease could therefore be targetted via drugs -based on the ADAM sequences.
  • the cytoplasmic domain is also of functional significance. This is the interface between the extracellular functional domains of the ADAM and the intracellular cell signalling machinery.
  • the cytoplasmic domain of human ADAM17 (TACE) appears to be involved in the regulation of ADAM 17 (Zheng et al . , 2002, J. Biol . Chem. 277, 42463-42470). Thus /the interaction of the cytoplasmic domain with cell sigalling components could also be targetted for therapeutic purposes.
  • the isolated polypeptide may comprise a motif/domain independently selected from at least one of the following: - (i) a zinc binding motif comprising an amino acid sequence substantially as set out as residues of rows ADM-A/ADM-B/i--. cuniculi/ P. jiroveci of a region labelled "1" shown in box B in Figure 1, or variant thereof; and ( ⁇ ) SL protein binding site comprising an amino sequence substantially as set out as residues of rows ADM- / DM-B/-?. cuniculi/ P. jiroveci of a region labelled "2" shown in box D in Figure 1, or variant thereof.
  • the motif or domain may be functional .
  • the variant may be functional .
  • the isolated polypeptide may comprise a propeptide domain comprising an amino acid sequence substantially as set out as residues 1-199 of SEQ ID No.2, or variant thereof.
  • A. fumigatus ADM-B the isolated polypeptide may comprise a propeptide domain comprising an amino acid sequence substantially as set out as residues 1-273 of SEQ ID No.4, or variant thereof.
  • E. cuniculi ADAM the isolated polypeptide may comprise a propeptide domain comprising an amino acid sequence substantially as set out as residues 1-157 of SEQ ID No. 40, or variant thereof.
  • the isolated polypeptide may comprise a motif/domain independently selected from at least one of the following :-
  • a substrate recognition sequence comprising an amino acid sequence substantially as set out as row ADM-A in box A as shown in Figure 1, or variant thereof;
  • a substrate recognition sequence comprising an amino acid sequence substantially as set out as row ADM-A in box B as shown in Figure 1, or variant thereof;
  • a substrate recognition sequence comprising an amino acid sequence substantially as set out as row ADM-A in box C as shown in Figure 1, or . variant thereof;
  • an adhesive region comprising an amino acid sequence substantially as set out as row ADM-A in box D as shown in Figure 1, or variant thereof;
  • an adhesive region comprising an amino acid sequence substantially as set out as row ADM-A in box E as shown in Figure 1, or variant thereof .
  • the isolated polypeptide may comprise a motif/domain independently selected from at least one of the following :-
  • a substrate recognition sequence comprising an amino acid sequence substantially as set out as row ADM-B in box A as shown in Figure 1, or variant thereof;
  • a substrate recognition sequence comprising an amino acid sequence substantially as set out as row ADM-B in box B as shown in Figure 1, or variant thereof;
  • a substrate recognition sequence comprising an amino acid sequence substantially as set out as row ADM-B -in box C as shown in Figure 1, or variant thereof;
  • an adhesive region comprising an amino acid sequence substantially as set out as row ADM-B in box D as shown in Figure 1, or variant thereof;
  • an adhesive region comprising an amino acid ' sequence substantially as set out as row ADM-B in box E as shown in Figure 1, or variant thereof.
  • the isolated polypeptide may comprise a motif/domain independently selected from at least one of the following :-
  • a substrate recognition sequence comprising an amino acid sequence substantially as set out as row E. cuniculi in box A as shown in Figure 1, or variant thereof;
  • a substrate recognition sequence comprising an amino acid sequence substantially as set out as row E. cuniculi in box B as shown in Figure 1, or variant thereof;
  • a substrate recognition sequence comprising an amino acid sequence substantially as set out as row E. cuniculi in box C as shown in Figure 1, or variant thereof;
  • an adhesive region comprising an amino acid sequence substantially as set out as row E. cuniculi in box D as shown in Figure 1, or variant thereof;
  • an adhesive region comprising an amino acid sequence substantially as set out as row E. cuniculi in box E as shown in Figure 1, or variant thereof.
  • the isolated polypeptide may comprise a motif/domain independently selected from at least one of the following :-
  • the isolated polypeptide may comprise a motif/domain independently selected from at least one of the following :-
  • the isolated polypeptide may comprise a motif/domain independently selected from at least one of the following :-
  • a proteinase domain substantially as set out as residues 274-508 of SEQ ID No.4, or variant thereof;
  • a disintegrin domain substantially as set out as residues 509-611 of SEQ ID No.4, or variant thereof; and
  • a cytoplasmic domain substantially as set out as residues 723-785 of SEQ ID No.4, or variant there-of.
  • the isolated polypeptide may comprise a motif/domain independently selected from at least one of the following :-
  • the isolated polypeptide may comprise a motif/domain independently selected from at least one of the following :-
  • the isolated polynucleotide may comprise DNA, preferably genomic DNA.
  • the isolated polynucleotide comprises substantially ' the sequence as shown by bases 606-2536 of SEQ ID No.l, or a complement, or variant thereof.
  • the isolated polynucleotide comprises a first exon as shown by bases 606-666 of SEQ ID No.l, a second exon- as shown by bases 715-1696 of SEQ ID No.l, and a third exon as .shown by bases 1756-2536 of SEQ ID No.l.
  • the isolated polynucleotide comprises substantially the sequence as shown by bases 488-2913 of SEQ ID No.3, or a complement, or variant thereof.
  • the isolated polynucleotide comprises a first exon as shown by bases 488-557 of SEQ ID No.3, and a second exon as shown by bases 626-2913 of SEQ ' ID No.3.
  • the isolated polynucleotide comprises substantially the sequence as shown by bases 1-1662 of SEQ ID No.39, or a complement, or variant thereof.
  • the isolated polynucleotide comprises substantially the sequence as shown by bases 1-515 of SEQ ID No.41, or a complement, or variant thereof.
  • the isolated polynucleotide encodes a fungal ADAM polypeptide which, in a first embodiment, A. fumiga tus ADM-A, preferably, comprises substantially the amino acid sequence as shown as SEQ ID No.2 , or variant thereof, and which, in a second embodiment, A. fumiga tus ADM-B, preferably comprises substantially the amino acid sequence as shown as SEQ ID No. , or variant thereof.
  • A. fumiga tus ADM-A preferably, comprises substantially the amino acid sequence as shown as SEQ ID No.2 , or variant thereof
  • A. fumiga tus ADM-B preferably comprises substantially the amino acid sequence as shown as SEQ ID No. , or variant thereof.
  • E. cuniculi the polynucleotide encodes an ADAM polypeptide which preferably comprises substantially the amino acid sequence as shown as SEQ ID No.40, or variant thereof.
  • P. jiroveci ADAM the polynucleotide encodes an ADAM polypeptide
  • the isolated polynucleotide may comprise RNA, preferably mRNA which is, preferably spliced RNA.
  • A.- fumiga tus ADM-A the isolated polynucleotide comprises substantially the sequence shown as SEQ ID No.31, or a complement, or variant thereof.
  • A. fumiga tus ADM-B the isolated polynucleotide comprises substantially the sequence shown as SEQ ID No.32, or a complement, or variant thereof.
  • E. cuniculi ADAM the isolated polynucleotide comprises substantially the sequence shown as SEQ ID No.33, or a complement, or variant thereof.
  • P. jiroveci ADAM the isolated polynucleotide comprises substantially the sequence shown as SEQ ID No.34, or a complement, or variant thereof.
  • the polypeptide comprises substantially the sequence as shown as SEQ ID No.2, or variant thereof.
  • the polypeptide comprises substantially the sequence as shown as SEQ ID No.4, or variant thereof.
  • the polypeptide comprises substantially the sequence as shown as SEQ ID No.40, or variant thereof.
  • P. jiroveci ADAM the polypeptide comprises substantially the sequence as shown as SEQ ID No.42, or variant thereof.
  • the isolated polypeptide is encoded by exon sequences of the polynucleotide shown in SEQ ID No.l, preferably bases 606- 666, 715-1696 and 1756-2536 shown in SEQ ID No.l.
  • the polypeptide is encoded by exon sequences of the polynucleotide shown in SEQ ID No.3, preferably bases 488- 557 and 626-2913 shown in SEQ ID No .3.
  • the polypeptide is encoded by the sequence shown as bases 1-1662 of SEQ ID No.39.
  • the polypeptide is encoded by the sequence shown as bases 1-515 of SEQ ID No.41.
  • an isolated polynucleotide comprising substantially a nucleotide sequence independently selected from at least one of the sequences referred to in Table 1, or complement thereof, or variant thereof.
  • the isolated polynucleotide comprises an ADAM gene.
  • the isolated polynucleotide comprises substantially a nucleotide sequence independently selected from at least one of the following: - (i) bases 606-2536 of SEQ ID No.l, or complement, or variant thereof;
  • the isolated polynucleotide encodes a gene product, which gene product comprises an amino acid sequence independently selected from at least one of SEQ ID No.2, SEQ ID No.4, SEQ ID No.40 and SEQ ID No.42, or variants thereof.
  • an isolated polypeptide comprising substantially an amino acid sequence independently selected from at least one of SEQ ID No.2, SEQ ID No.4, SEQ ID No. 0 and SEQ ID No.42, or variants thereof.
  • the isolated polypeptide comprises an ADAM.
  • the polypeptide is encoded by a polynucleotide which polynucleotide comprises substantially a sequence independently selected from at least one of the following: - (i) bases 606-2536 of SEQ ID No.l, or complement, or variant thereof;
  • the polynucleotide or polypeptide may be modified prior to use, preferably to produce a derivative or variant thereof.
  • the polynucleotide or polypeptide may be derivatised.
  • the polypeptide may be modified by epitope tagging, addition of fusion partners or purification tags such as glutathione S-transferase or maltose binding protein, addition of green fluorescent protein, covalent attachment of molecules including biotin or fluorescent . tags, incorporation of selenomethionine, inclusion or attachment of radioisotopes or fluorescent/non-fluorescent lanthanide chelates.
  • the polynucleotide may be modified by methylation or attachment of digoxygenin (DIG) or by addition of sequence encoding the above tags, proteins or epitopes .
  • DIG digoxygenin
  • the polynucleotide defined in the first or third aspect, or the polypeptide' defined in the second or fourth aspect, may not be modified or derivatised.
  • drug research purposes comprises use of the polynucleotide or polypeptide as medicament, or in diagnosis, or for the treatment, retarding or prevention of fungal infection.
  • the fungal infection may be in human, animal or plant.
  • Drug research purposes may comprise use of the polynucleotide or polypeptide for the development of a drug.
  • Drug research purposes may comprise the generation of a molecular model of said polynucleotide or said polypeptide.
  • the invention provides a method of screening which may be used to identify modulators of ADMA genes or ADAM polypeptides .
  • a candidate substance is contacted with a polynucleotide or polypeptide of the invention or with a promoter of any of the ADAM genes of the invention whether or not the candidate substance binds or modulates the polynucleotide, polypeptide or promoter is determined.
  • the modulator may increase or decrease expression of the polynucleotide, or may promote (agonise) or inhibit (antagonise) the activity of the polypeptide.
  • a therapeutic modulator (against fungal infection) will generally causes cause decreased expression of an ADAM polypeptide or will inhibit the activity of an ADAM polypeptide.
  • the method may be carried out in vi tro (inside or outside a cell) or in vivo . In one embodiment the method is carried out on a cell, cell culture or cell extract.
  • the cell may or may not be a cell in which the polynucleotide or polypeptide is naturally present.
  • the cell may or may not be a fungal cell, or may or may not be a cell of any of the fungae mentioned herein.
  • Whether or not a candidate substance modulates the activity of the polypeptide may be determined by providing the candidate substance to the ⁇ polypeptide under conditions that permit activity of the polypeptide, and determining whether the candidate substance is able to modulate the activity of the product.
  • the activity which is measured may be any of the activities of the polypeptide of the invention mentioned herein, such as protease activity.
  • the screening method preferably comprises a polypeptide fragment as discussed herein which has a property of an ADAM polypeptide.
  • Promoter activity may be measured in a method comprising: providing a ' test construct . comprising a first polynucleotide sequence with the promoter activity operably linked to a second polynucleotide sequence to be expressed in the form of mRNA; contacting the candidate substance with the test construct under conditions that would permit the second polynucleotide sequence to. be expressed in the form of mRNA in the absence of the substance; and determining whether the substance modulates expression from the construct.
  • a polynucleotide defined in the first or third aspect, or variant thereof, or a polypeptide defined in the second or fourth aspects, or variant thereof for the preparation of a medicament for the treatment of fungal infection.
  • the treatment may comprise .retarding or prevention of the fungal infection.
  • the polynucleotide which is administered causes inhbition of the expression of the ADAM gene of the fungus which has infected the individual by an antisense or RNA interference mechanism.
  • the drug and/or medicament comprises an inhibitor, preferably an ADAM inhibitor.
  • the inhibitor is adapted to inhibit expression and/or activity of the ADAM polynucleotide or a fragment thereof, and/or the function of the ADAM polypeptide or a fragment thereof.
  • the fungal infection comprises an infection by an organism independently selected from a group of genera consisting of Aspergillus; Pneumocystis ; Encephalitozoon; Cryptococcus; Trichophyton; Fusarium; Blumeria ; Leptosphaeria ; Plasmopara ; ⁇ Pyricularia ; Puccinia and Rhizoctonia .
  • the fungal infection comprises an infection by an organism independently selected from a group of species consisting of Aspergillus fumiga tus ; Aspergillus terreus; Aspergillus parasi ticus; Aspergillus flavus ; Aspergillus niger; Aspergillus nidulans; Pneumocystis jiroveci; Encephalitozoon cuniculi; Cryptococcus neoformans ; Trichophyton interdigitale; Trichophyton rubrum; Fusarium solani; Blumeria graminis; Leptosphaeria nodorum; Plasmopara vi ticola ; Pyricularia oryzae; Puccinia species; and Rhizoctonia species .
  • variant and the terms “substantially the amino acid/polynucleotide/polypeptide sequence” are used herein to refer to related sequences. As discussed below such related sequences are typically homologous to (share percentage identity with) a given sequence, for example over the entire length of the sequence or over a portion of a given length. The related sequence may also be a fragment of the sequence or of a homologous sequence. A variant polypeptide may be encoded by a variant polynucleotide.
  • variant and the terms “substantially the amino acid/polynucleotide/polypeptide sequence” we mean that the sequence has at least 30%, preferably 40%, more preferably 50%, and even more preferably, 60% sequence identity with the amino acid/polynucleotide/polypeptide sequences of any one of the sequences referred to.
  • variant and the terms “substantially the amino acid/polynucleotide/peptide sequence” we mean that the sequence has at least 30%, preferably 40%, more preferably 50%, and even more preferably, 60% sequence identity with the amino acid/polynucleotide/peptide sequences of any one of the sequences referred to.
  • An amino acid/polynucleotide/peptide sequence with a greater identity than 65% to any of the sequences referred to is also envisaged.
  • amino acid/polynucleotide/peptide sequence with a greater identity than 75% to any of the sequences referred to is also envisaged.
  • An amino acid/polynucleotide/peptide sequence with a greater identity than 80% to any of the sequences referred to is also envisaged.
  • the amino acid/polynucleotide/peptide sequence has 85% identity with any of the sequences referred to, more preferably 90% identity, even more preferably 92% identity, even more preferably 95% identity, even more preferably 97% identity, even more preferably 98% identity and, most preferably, 99% identity with any of the referred to sequences.
  • a sequence which is "substantially the amino acid/polynucleotide/peptide sequence" may be the same as the relevant sequence.
  • the above mentioned percentage identities may be measured over the entire length of the original sequence or over a region of 15, 20, 50 or 100 amino acids/bases of the original sequence.
  • percentage identity is measured with reference to SEQ ID NO:2.
  • the variant polypeptide has at least 40% identity, such as at least 60% or at least 80% identity with SEQ ID NO: 2 or a portion of SEQ ID NO: 2.
  • the variant sequence has (or comprises sequence which has) at least 40% identity, such as at least 60% or at least 80% identity with amino acids 200 to 526 or with amino acids 334 to 360 of SEQ ID NO:2.
  • Polynucleotide sequences which encode such variant polypeptides are also preferred.
  • a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequences shown in SEQ ID Nos . 1, 3, 31-34, 39, 41, or their complements under stringent conditions .
  • stringent conditions we mean the nucleotide hybridises to filter- bound DNA in 6x sodium chloride/sodium citrate (SSC) at approxmiately 45°C followed by at least one wash in 0.2x SSC/0.1% SDS at approximately 5-65°C.
  • a substantially similar polypeptide may differ by at ' least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID Nos. 2, 4 , 40 or 42. Such differences may each be additions, deletions or substitutions .
  • nucleic acid sequence could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
  • Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
  • suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
  • small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine.
  • Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
  • the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
  • the positively charged (basic) amino acids include lysine, arginine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • Certain organisms, including Candida are known to use non-standard codons compared to those used in the majority of eukaryotes. Any comparisons of polynucleotides and polypeptides from such organisms with the sequences given here should take these differences into account.
  • Align http://www.gwdg.de/ ⁇ dhepper/download/; Hepperle, D., 2001: Multicolor Sequence Alignment Editor. Institute of Freshwater Ecology and Inland Fisheries, 16775 Stechlin, Germany) , although others, such as JalView or Cinema are also suitable.
  • variants also include a fragment of the relevant polynucleotide or polypeptide sequences, including a fragment of the homologous sequences (which have percentage identity to a specified sequence) referred to above.
  • a polynucleotide fragment will typically comprise at least ' 10 bases, such as at least 20, 30, 50, ' 100, 200, 500 or 1000 bases.
  • a polypeptide fragment will typically comprise at least 10 amino acids, such as at least 20, 30, 50, 80, 100, 150, 200, 300, 400 or 500 amino acids.
  • Preferred variant polypeptides may comprise the following domains, and preferred variant polynucleotides may encode the following 1, 2, 3 or 4 of the following domains:
  • the variant comprises, or encodes, at least any of the following combinations :
  • propeptide a propeptide, a protease domain and a disintegrin domain .
  • these domains are defined with reference to Table 1 and/or Figure 1.
  • sequence in the variant polynucleotide which encodes any of the above mentioned domains may be the same as any of the portions of sequence mentioned in Table 1 (of SEQ ID NO' s 1, 3, 31, 32, 33, 34, 39 or 41) or be homologous to such portions .
  • the variant polynucleotide contains sequences which are fragments of the sequences of Table 1 or fragments of homologoues of such sequences .
  • the variant polypeptide may be encoded by any of these variant polynucleotides .
  • the fragments may lack at least 10 amino acids, such as at least 20 or 30 amino acids of the amino acids from either end of the protease or disintegrin domain as defined in Table 1 or Figure 1.
  • the fragments may lack at least 3 amino acids, such as at least 5 or ⁇ 10 amino acids of the amino acids from either end of the propeptide-protease junction as defined in Table 1 or Figure 1.
  • polypeptide sequences included in the term “variant”, and the terms “substantially the amino acid/peptide sequence” may have particular properties and the polynucleotide sequences included by the term “variant”, and the terms “substantially the polynucleotide sequence” may encode polypeptides with such properties .
  • properties may relate to the functionality of the polypeptide.
  • the polypeptide may have 1, 2, 3, 4 or 5 of the following properties:
  • - disintegrin domain activity for example the ability to inhibit or promote cell to cell interactions, or the ability to bind a protein, - ability to bind SH3 domains, or
  • the variant polypeptide is able to complement the activity of the original ADAM polypeptide in a fungal cell, for example the presence of the variant polypeptide in a fungal cell whose native equivalent ADAM gene has been rendered non-functional, may allow the cell to remain alive .
  • a method of detecting the presence of a fungal infection in an individual comprising :-
  • the individual may be a person (human) or animal (such as a mammal or bird) or a plant.
  • the individual may be of any of the species of animal mentioned below in regard to the therapeutic aspects of the invention.
  • the fungal infection may arise from infection with an organism independently selected from a group of genera consisting of Aspergillus; Pneumocystis ; Encephalitozoon;
  • Cryptococcus Trichophyton ; Fusarium; Blumeria ;
  • Leptosphaeria Leptosphaeria ; Plasmopara ; Pyricularia ; Puccinia and Rhizoctonia .
  • the fungal infection may arise from infection with an organism independently selected from a group of species consisting of Aspergillus fumiga tus ; Aspergillus terreus ; Aspergillus parasi ticus; Aspergillus flavus ; Aspergillus niger; Aspergillus nidulans ; Pneumocystis jiroveci; Encephalitozoon cuniculi; Cryptococcus neoformans;
  • Trichophyton interdigitale Trichophyton rubrum; Fusarium solani; Blumeria graminis; Leptosphaeria nodorum; Plasmopara viticola ; Pyricularia oryzae; Puccinia species; and Rhizoctonia species .
  • the sample comprises a biological sample which, preferably, comprises nucleic acid and/or polypeptide.
  • the nucleic acid or polypeptide is purified (at least partially) from the sample before the detection is performed.
  • the sample may comprise: sputum, bronchoalveloar lavage, urine, respiratory specimens, endotracheal aspirates, sterile specimens obtained by an invasive procedure such as vitreous tap, tympanocentesis, brain biopsy or aspiration, nasal or sinus specimens, blood, tissue or autopsy.
  • the sample may comprise expectorated sputum, induced sputum after nebulised saline, bronchoalveolar lavage, open lung biopsy; mouthwashes, or respiratory specimens.
  • the sample may comprise CSF, urine, serum, renal biopsy, respiratory specimens, corneal/conjunctival smear, scraping or biopsy, sinunasal smear-lavage, sputum, bronchoalveolar lavage, liver biopsy, autopsy, or skin biopsy.
  • said detecting of the presence in the said sample of a polynucleotide as defined by the first or third aspect comprises use of at least one oligonucleotide pair adapted to be used for amplification of DNA, preferably genomic, more preferably, fungal genomic DNA.
  • the amplification may be PCR amplification.
  • the PCR amplification employs at least one primer pair comprising a sequence selected from the group consisting of: .
  • Pneumocystis jiroveci (formerly known as Pneumocystis carinii f .sp. hominis) : SEQ ID No.35 5' -tgtttttaggtgcaaaacgaca-3' SEQ ID No.36 5' -cggggacaaatgttatagcaag-3' SEQ ID No.35 + SEQ ID No.36 (483bp)
  • said detecting comprises subjecting the amplified DNA to size analysis, preferably, electrophoresis and, preferably, comparing the results to a positive control and, preferably, a negative control.
  • Said detecting ' may also include sequencing of the amplified DNA to demonstrate the correct sequence.
  • said detecting of the presence in the said sample of a polypeptide as defined by the second or fourth aspect comprises use of a monoclonal or polyclonal antibody directed to part or all of the polypeptide as defined by the second or fourth aspect.
  • a recombinant DNA molecule comprising a polynucleotide as defined in the first or third aspect, or fragment thereof, or variant thereof.
  • the fragment may be a functional fragment of the polynucleotide.
  • the fragment of the polynucleotide may comprise a polynucleotide sequence encoding any region of DNA shown in Table 1, or any combination thereof.
  • the polynucleotide may encode - a motif, region or domain selected from the zinc binding motif, the protein binding site, substrate recognition sequence, the adhesive region, the propeptide domain, the cleavage site, the proteinase domain, the disintegrin domain, the transmembrane domain or the cytoplasmic domain (See Table I) .
  • the recombinant DNA molecule comprises an expression vector.
  • the polynucleotide sequence is operatively linked to an expression control sequence.
  • a suitable control sequence may comprise a promoter, an enhancer etc.
  • a ninth aspect of the present invention there is provided a cell containing a recombinant DNA molecule defined by the eighth aspect.
  • the cell may be transformed or transfected with the recombinant DNA molecule by suitable means.
  • the cell of the eighth aspect produces a recombinant polypeptide.
  • the recombinant polypeptide may comprise the isolated polypeptide defined by the second or fourth aspect, or fragment thereof.
  • the fragment is a functional fragment.
  • the invention also provides an 'organism which is transgenic for the recombinant DNA of the invention (whose cells may be the same as the cells of the invention mentioned herein) .
  • Such an organism is typically a fungus, such as any genera or species of fungus mentioned herein.
  • the organism may be microorganism, such as a bacterium, virus or yeast.
  • the organism may be a plant, animal (including birds and mammals) , such as any of the animals mentioned herein.
  • the organism may be produced by introduction of the polynucleotide of the invention into a cell of the organism, and in the case of a multicellular organism allowing the cell to grow into a whole organism.
  • a cell in which a polynucleotide defined by the first or third aspect, or variant thereof, or a polypeptide defined by the second or fourth aspect, or variant thereof, is non-functional and/or inhibited.
  • the cell may be a mutant cell .
  • the cell is typically a fungal cell, such as of any genera or species of fungus mentioned herein.
  • a preferred means of generating the cell is to modify the polynucleotide according to the first or third aspect, such that the polynucleotide is non-functional.
  • This modification may be to cause a mutation, which disrupts the expression or function of a gene product.
  • Such mutations may be to the nucleic acid sequences that act as 5' or 3' regulatory sequences for the polynucleotide, or may preferably be a mutation introduced into the coding sequence of the polynucleotide.
  • Functional deletion of the polynucleotide may, for example, be by mutation of the polynucleotide in the form of nucleotide substitution, addition or, preferably, nucleotide deletion.
  • the polynucleotide may be made non-functional by:
  • a preferred means of introducing a mutation into a polynucleotide is to utilize molecular biology techniques specifically to target the polynucleotide which is to be mutated. Mutations may be induced using a DNA molecule.
  • a most preferred means of introducing a mutation is to use a DNA molecule that has been specially prepared such that homologous recombination occurs between the target polynucleotide and the DNA molecule. When this is the case, the DNA molecule will ideally contain base sequences complementary to the target polynucleotide to allow the DNA molecule to hybridize to (and subsequently recombine with) the target.
  • the polynucleotide is non-functional and/or inhibited without introducing a mutation into the ADAM gene or its regulatory regions .
  • This may be done by using specific inhibitors .
  • inhibitors include agents that prevent transcription of the polynucleotide, prevent expression or disrupt post-translational modification.
  • the inhibitor may be an agent that increases degradation of the gene product (e.g. a specific proteolytic enzyme) .
  • the inhibitor may be an agent which prevents the polynucleotide product from combining with cytosolic components such as neutralizing antibodies (for instance an anti-ADAM antibody) .
  • the inhibitor may also be an antisense oligonucleotide or any synthetic chemical capable of inhibiting expression of the gene or the stability and/or function of the polypeptide.
  • the inhibitor may an RNA molecule which causes inhibition by RNA interference.
  • the antisense polynucleotide or RNA molecule which causes RNA interference are examples of polynucleotides of the invention.
  • the signalling properties of the ADAM cytoplasmic domains (as shown in Table: I) may be exploited to indicate ADAM function. Mutant cells can be generated which express only the transmembrane and cytoplasmic 5 . domains of ADAMS. This will subvert the signalling processes occurring through the endogenous ADAM molecules, leading to a negative or over-stimulation phenotype . Either will give detailed insights into ADAM function and its role in fungal biology. 0
  • the invention also provides an organism whose cells are the same as the above discussed cell in which a polynucleotide defined by the first or third aspect, or variant thereof, or a polypeptide defined by the second or 5 fourth aspect, or variant thereof, is non-functional and/or inhibited.
  • an antibody exhibiting immunospecificity for a polypeptide of 0 the second or fourth aspect, or fragment or variant thereof.
  • the antibody may be used as a diagnostic reagent.
  • the antibody may be monoclonal or polyclonal.
  • the term "antibody”, 5 includes fragments which bind a polypeptide of the invention. Such fragments include Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragment thereof may be chimeric antibodies, CDR-grafted 0 antibodies or humanised antibodies.
  • a method for treating a fungal infection comprising administering to an individual a polynucleotide defined in the first or third aspect, or variant thereof, or a polypeptide defined in the second or fourth aspect, or variant thereof, each being optionally modified.
  • the polynucleotide may be modified prior to use, and may include derivatisation.
  • the polynucleotide or polypeptide may not be modified or derivatised prior to administration to the individual.
  • the method of treating may comprise antifungal therapy.
  • the individual may be a person (human) , animal (such as a mammal or bird), or a plant.
  • the animal is typically an agricultural animal such as a pig, cow, horse, sheep, goat, camel, chicken, duck, turkey or goose.
  • the animal may be a cat or a dog.
  • polypeptides, polynucleotides, vectors, cells or antibodies of the invention may be present in a substantially isolated form. They may be mixed with carriers or diluents which will not interfere with their intended use and still be regarded as substantially isolated. They may also be in a substantially purified form, in which case they will generally comprise at least 90%, e.g. at least 95%, 98% or 99%, of the proteins, polynucleotides, cells or dry mass of the preparation.
  • any of' he. therapeutic substances e.g. polypeptides, polynucleotides or modulators
  • Any such substance may be administered in a variety of dosage forms. It may be administered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), parenterally, subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The substance may also be administered as suppositories. A physician will be able to determine the required route of administration for each particular patient.
  • the substance is formulated for use with a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical carrier or diluent may be, for example, an isotonic solution.
  • solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g.
  • Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.
  • Liquid dispersions for oral administration may be syrups, emulsions and suspensions.
  • the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • the suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride .
  • Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions .
  • a therapeutically effective non-toxic amount of substance is administered.
  • the dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.
  • a typical daily dose is from about 0.1 to 50 mg per kg, preferably from about O.lmg/kg to lOmg/kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the type ⁇ and severity of the disease and the frequency and route of administration.
  • daily dosage levels are from 5 mg to 2 g.
  • Figure 1 illustrates a multiple sequence alignment of amino ⁇ acid sequences corresponding to protease and disintegrin domains of A. fumiga tus ADM-A, A. fumiga tus ADM-B, S . pombe ADAM, N. crassa ADAM, E . cuniculi ADAM, P. jiroveci ADAM, a sea urchin (su) ADAM, a selection of the closest related mammalian (hu, mo) ADAMs (as judged by BLAST) and snake toxins catrocollaststin ( Crotalus a trox) and HV1 ⁇ Trimeresurus flavoviridis) (closest related as judged by BLAST) ;
  • FIG. 2 illustrates phylogenetic analyses of ADAMs
  • Figure 3 illustrates an electrophoretic gel • on which purified PCR products of A. fumiga tus ADM-A and ADM-B have been run;
  • Figure 4 is a schematic representation of plasmid pPCR- Script Amp SK(+) ;
  • Figure 5 shows- Western blots of samples from bacteria carrying protease or propeptide+protease constructs, probed with anti-V5 antibodies, showing bands of the correct size for ADM-B protease and ADM-B propeptide+protease;
  • Figure 6 shows protease assays to demonstrate the protease function of the ADM-B protease domain against casein, its substrate specificity, and its EDTA dependence.
  • ADM-A protein (SEQ ID No.2) is predicted to have the following regions : -
  • Protease domain amino acid residues 200-425;
  • Disintegrin domain amino acid residues 426-526.
  • Adhesive motif amino acid residues 498-500.
  • ADM-B protein (SEQ ID No.4) is predicted to have • the •following regions :-
  • Protease domain amino acid residues 274-508;
  • Zinc binding consensus amino acid residues 429-440; 3) Disintegrin domain: amino acid residues 509-611; and
  • Adhesive motif amino acid residues 583-585.
  • the P. jiroveci EST identified in this way was also identified after searching P. jiroveci (carinii) at nc.stress-genomics.org.
  • Candida albicans www-sequence . Stanford . edu/group/ca dida/search . html; tblastn, no settings available.
  • Searching database (1) with the ADM-B sequence identified a match to A . fumiga tus ADM-B in Schizosa ccharomyces pombe, YE94_SCHPO.
  • Subsequent searches of the fungal gene sequence databases with YE94_SCHPO also identified putative fungal ADAMs in Neurospora crassa
  • SEQ ID No.41 SEQ ID No.41
  • Trichoderma reesei BM076331.1
  • a predicted gene from Encephalitozoon cuniculi shown as SEQ ID No.39 was annotated as resembling YE94_SCHPO.
  • the E . cuniculi ADAM protein (SEQ ID No.40) is predicted to have the following regions :- 1) Protease domain: amino acid residues 158-355;
  • Zinc binding motif amino acid residues 307-318;
  • Disintegrin domain amino acid residues 356-458;
  • Adhesive motif amino acid residues 430-432; 5) Propeptide domain: 1-157
  • the P. jiroveci ADAM protein (SEQ ID No.42) is predicted to have the following regions :- 1) Protease domain: amino acid residues ⁇ l-95;
  • Disintegrin domain amino acid residues 96->171;
  • Adhesive motif amino acid residues 170-171.
  • a sea urchin ADAM a selection of the closest related mammalian ADAMs (as judged by BLAST) and the snake toxins catrocollaststin ( Crotalus a trox) ' and HV1 ⁇ Trimeresurus flavoviridis) (closest related as judged by BLAST) .
  • the alignment was generated using CLUSTALX (Parameters: Gap opening, 10.0; Gap extension, 0.2; Delay divergent sequences, 30%; Gonnet series matrix) . Manual editing was carried out using the alignment editor ALIGN.
  • Shaded regions in Figure 1 indicate amino acids conserved in at least half of the sequences.
  • the regions of the sequences denoted by “1” relate to. a zinc binding motif, and the regions of the sequences as denoted by “2" relate to an adhesive motif.
  • the boxed regions labelled A, B, and C in ' Figure 1 indicate regions predicted to be involved in substrate recognition based on the structure of ADAM 17- inhibitor co-crystal (Maskos et al . , 1998).
  • the boxed regions D and E indicate regions predicted to be involved • in adhesive interaction ' s by the disintegrin domain (Eto et al . , (2002) J. Biol. Chem. In press (manuscript M200086200) ) .
  • the proteins ADM-A and ADM-B have retained the modular structure of mammalian ADAMs including the transmembrane region, but differed in the N-terminal- propeptide domain as this region did not align well with mammalian proteins.
  • the disintegrin and cysteine-rich domains tended to be more conserved across all alignments.
  • the ADAMs of fillamentous fungi show most similarity to each other (Table 2) and are clearly distinct from other animal sequences.
  • the P. carinii ADAM showed a higher percentage ID to filamentous fungi than to animal sequences.
  • the E. cuniculi ADAM showed lower percentage identities both to other fungal and animal ADAMs, but this is consistent with its early divergence in the fungal lineage.
  • Box B positions 2-4 (NVV) , 6 (R) , 8 (S) , 12-14 (WQV) , and 26 (V) ; and
  • Box C positions 3 (N) , 6 (S/T) and 8 (A).
  • Disintegrin domains of ADAMs mediate cell-cell adhesion.
  • the ADM-B disintegrin contains an ECD sequence as shown in region 2 of Figure 1.
  • ADAM 2 appears to be an adhesive motif for integrins
  • the A. fumiga tus ADM-B may therefore have an adhesive function.
  • protease and disintegrin domains are of interest as therapeutic targets.
  • the protease could be inhibited, while the disintegrin could be the template for the design
  • the linking region between the propeptide and protease domains is also of interest; Since the ADAMs are proteolytically activated by the cleavage of the peptide chain, between the propeptide and the protease, this region could be used as a way of targeting inhibitors 0 of the (unknown) activating protease.
  • the regions outlined below are also of interest; Since the ADAMs are proteolytically activated by the cleavage of the peptide chain, between the propeptide and the protease, this region could be used as a way of targeting inhibitors 0 of the (unknown) activating protease.
  • A. fumiga tus, P. j iroveci and E. 5 cuniculi ADAM proteins expressed on the surface ' of yeast or other fungi would be used to screen for inhibitory compounds. Subsequently (or alternatively) screening would be carried out with recombinant ADAMs, which possibly lack the transmembrane region and cytoplasmic 0 tail, or with individual recombinant propeptide, protease or disintegrin domains, or combinations of these domains. It is anticipated that any reagents directed at the protease or disintegrin region would bind at the regions indicated in Boxes A-E shown in Figure 1 with the indicated residues accounting for fungal- specificity of any reagent. The link between the propeptide and protease domains would be a candidate binding site for the activating protease. Novel allosteric inhibitory sites may also be uncovered by the screening process.
  • genomic sequences of ADM-A and ADM-B were determined as follows .
  • E. coli strain XLIO-Gold ® Kan ultracompetent cells (Genotype: Tet R ⁇ (mcrA) 183 A (mcrCB- hsdSMR-mrr) 113 endAl supE44 thi-1 recAl gyrA96 relAl lac Hte [F ' proAB lacI q Z ⁇ M15 TnlO (Tet R ) Tn5 (Kan R ) Amy]) was used (Stratagene Europe, P.O. Box 12085, 1100 AB Amsterdam, The Netherlands) .
  • A. fumiga tus clinical isolate AF293 (available to the public from the NCPF repository (Bristol, U.K.); the CBS repository (Belgium) or from Dr. David Denning clinical isolate culture collection, Hope Hospital, Salford. U.K.) is the preferred strain according to the present invention.
  • AF293 was isolated in 1993 from the lung biopsy of a patient with invasive aspergillosis and aplastic anaemia. It was donated by Shrewsbury PHLS .
  • the mycelium (fresh or -freeze dried) was ground to a powder using liquid nitrogen in a -20°C cooled mortar.
  • the ground biomass was transferred to 50 ml tubes on ice up to the 10 ml mark.
  • An equal volume of extraction buffer (0.7 M NaCl; 0.1 M Na 2 S0 3 ; 0.1 M Tris-HCl pH 7.5; 0.05 M EDTA; 1% (w/v) SDS; pre-warmed to 65°C) was then added to each tube, mixed thoroughly with a pipette tip and incubated at 65 °C for 20 minutes in a water bath.
  • a volume of chloroform/isoamyl alcohol (24:1) equivalent to the volume of the original biomass was then added to each tube, tubes were mixed thoroughly and incubated on ice for 30 min. Tubes were then centrifuged at 3,500 x g for 30 min and the aqueous phase carefully transferred to fresh 50 ml tubes without disturbing the interface. An equal volume of chloroform/isoamyl alcohol (24:1) was added, the tubes vortexed and incubated on ice for 15 minutes . Tubes were then spun at 3,500 x g for 15 minutes. After this spin, if large amounts of precipitate were still present, the supernatant was removed and the chloroform: isoamyl alcohol step repeated.
  • the supernatant was removed and placed in clean steriie Oak Ridge tubes. An equal volume of isopropanol was added and mixed gently. Tubes were incubated at room temperature for at least 15 minutes. Tubes were then cen-rifuged at 3,030 x g for 10 minutes at 4°C to pellet the DNA. The supernatant was removed and the pellet allowed to air dry for 10-25 minutes . The pellet was suspended in 2 ml sterile water. 1 ml of 7.5 M ammonium acetate was added, mixed and incubated on ice for 1 hour.
  • Tubes were centrifuged at 12,000 x g for 30 min, the supernatants transferred to a fresh tube and 0.54 volumes of isopropanol were added, mixed and incubated at room temperature for at least 15 minutes. Tubes were then was centrifuged at 5,930 x g for 10 min, the supernatant was removed and the pellet washed in 1 ml of 70% ethanol . Tubes were centrifuged at 5,930 x g for 10 min and all the ethanol was removed.
  • the pellet was air dried for 20-30 minutes at room temperature and suspended in 0.5-1.0 ml of TE (10 mM Tris-HCl pH 7.5; ImM EDTA) Finally, the DNA was treated with RNase A (5 ⁇ l of lmg/ml stock) .
  • Primers were designed to the upstream and downstream regions of the A. fumiga tus AF293 ADM-A and ADM-B genes.
  • the ADM-A gene sequence is 1931bp in total.
  • the ADM-A cloning primer pair amplifies 2906bp of gene including upstream and downstream regions .
  • ADMAF1 5'-CTC GAT GCA AGT TGC TGA CA-3' SEQ ID no.5
  • ADMAR1 5'-CGC AAT AGT ACA GAC TGC CT-3' (SEQ ID no.6)
  • the ADM-B gene sequence is 2426bp in total.
  • the ADM-B cloning primer pair amplifies 3390bp of gene including upstream and downstream- regions .
  • ADM-B cloning primer pair (ADM-B cloning primer pair:
  • PCR reactions using either the ADM-A cloning primer pair or the ADM-B cloning primer pair were set up using the Expand Long Template PCR System (Roche Diagnostics Ltd, Bell , Lane, Lewes, East London BN7 1LG, United Kingdom) according to the manufacturers instructions. The following reagent concentrations and volumes were used:
  • Step3 53°C Imin 30sec .
  • FIG. 3A there is shown a band at 2906 bp signifying the amplification of ADM-A (1931bp) and a total of 975 bp of upstream and downstream sequence.
  • FIG. 3B there is shown a band at 3390bp signifying the amplification of ADM-B (2426bp) and a total of 964bp of upstream and downstream sequence.
  • the purified PCR products shown in Figure 3 were cloned into Stratagene's pPCR-Script Amp SK (+) as shown in Figure 4 and then transformed ⁇ into XLIO-Gold ® Kan ultracompetent E. coli cells according to the manufacturers instructions. Vectors are sold predigested with Srf I ⁇ and the blunt ended PCR products are ligated directly into the vector. These cloning vectors work ideally with blunt ended PCR ' products ' as produced when using proofreading Taq. The transformation ' reactions were then plated onto LB agar plates containing ampicillin (100 ⁇ g/ml), 50 ⁇ l X-gal (4%) and 10 ⁇ l IPTG (100 mM) .
  • Restriction digests were carried out using Kpn I to check for the correct orientation of the PCR products within the cloning vector (not shown) using standard techniques known in the art. Clones containing inserts with the correct orientation were freeze-dried and sent away for full- length sequencing.
  • ADMAF1 (SEQ ID No.5, 1) 5'-CTC GAT GCA AGT TGC TGA CA-3'
  • ADMAF3 (SEQ -ID No.9, 283) 5'-GCT GTG CAT ACA CCA TGT CA-3'
  • ADMAF4 (SEQ ID No.10, 702) 5'-CTA ACG TTC TTA GCG CAT TC-3'
  • ADMAF5 (SEQ ID No.11, 1134) 5'-GCC AGT GAA CCG CAG ATG AT-3'
  • ADMAF6 (SEQ ID No.12, 1540) 5' -ACG ATG ACA ATG CAT ACT GG-3'
  • ADMAF7 (SEQ ID No.13, 2205) 5'-CAT GCG ATG TTG AGG AGA TG-3'
  • ADMAR1 (SEQ ID No.6, 2906) 5'-CGC AAT AGT ACA GAC TGC CT-3' ADMAR3 (SEQ ID No.14, 699) 5' -TGC ATA GTG CAT GGC AGA GC-3' ADMAR4 (SEQ ID No.15, 992) 5' -ATG CCG AGC AGA TAC GAC AC-3'
  • ADMAR5 (SEQ ID No.16, 1390) 5'-AAG GTC CGC TCG TAG ACT TC-3'
  • ADMAR ⁇ (SEQ ID No.17, 1842) 5' -GAC AAC GGA CAG CAC TGA CT-3'
  • ADMAR7 (SEQ ID No.18, 2256) 5'-TCC ACG GGA CAT ATG GTC GA-3'
  • ADMBF1 (SEQ ID No.7, 1) 5'-CTG CAT AAT CCA GTC ATT CG-3'
  • ADMBF3 (SEQ ID No.19, 427) 5'-AGG TTA CGT TCC ACA GCC TG-3' ADMBF4 (SEQ ID No.20, 854) 5'-CGA TCC AAC GAC ATG AAC AC-3'
  • ADMBF5 (SEQ ID No.21, 1287) 5'-TGG CCT CAG CAA ACG ACA GT-3'
  • ADMBF ⁇ (SEQ ID ' No.22, 1752) 5'-TAC AGA AGT TAC CGG TGA CG-3'
  • ADMBF7 (SEQ ID No.23, 2226) 5'-CGA TGA TGC CAA CGA CAG CT-3'
  • ADMBF8 (SEQ ID No.24, 2718) 5' -GTG TAT GAT CAA CCG CTG TC-3'
  • ADMBR1 (SEQ ID No .8 , 3390) 5' -AGA AGC TCA GCG ATA CAG C-3'
  • ADMBR3 (SEQ ' ID No.25, 703) 5'-ACC TCG TGA GAC GGT GTG TT-3'
  • ADMBR4 (SEQ ID No.26, 1124) 5'-TGT CGA ATC ATG TCC GAG TC-3'
  • ADMBR5 (SEQ ID No.27, 1575) 5' -AGT CTC AGG ACA GGT CTT GT-3'
  • ADMBR6 (SEQ ID No.28, 2009) 5' -GGT GAG AAC GCA GTG ATA TC-3'
  • ADMBR7 (SEQ ID No.29, 2442) 5' -GCA TTG ATA GTC ACG ACT GG-3'
  • ADMBR8 (SEQ ID No.30, 2909) 5' -GCA TAT CGT GTT GCT GAC TG-3'
  • the experimentally obtained sequence extended beyond the position of the next sequencing primer.
  • The- sequence data ' obtained was compared to sequences in the. Aspergillus fumiga tus genome database (http://www.TIGR.org) .
  • the sequences of ADM-A and ADM-B were identical to those identified by bioinformatic analyses as shown as SEQ ID No.l and SEQ ID- No.3, respectively.
  • the 5', 3' and internal sequence of the ADM-A and ADM-B messages were experimentally, determined by RACE .(Rapid Amplification of cDNA Ends) and cloning and sequencing of cDNA. RACE covered the full extent of the ADM-A message, whereas cDNA sequencing was necessary to generate internal sequence for ADM-B.
  • RACE was carried out using the GeneRacerTM Kit (Invitrogen; cat. No. L1502-01) , essentially as per manufacturers instructions. 1 ⁇ g total RNA prepared as described above was de-phosphorylated in a 10 ⁇ l reaction using 10 units of calf intestinal phosphate (CIP) , 1 ⁇ l 10X CIP buffer and 40U RNaseOutTM (made up to 10 ⁇ l in DEPC water) at 50°C for 1 hour. Samples were then made up to 100 ⁇ l with DEPC water and 100 ⁇ l phenol : chloroform: isoamyl alcohol (25:24:1) were added.
  • CIP calf intestinal phosphate
  • RNaseOutTM made up to 10 ⁇ l in DEPC water
  • RNA was then precipitated by the addition of 2 ⁇ l mussel glycogen (lOmg/ml) , 10 ⁇ l 3M sodium acetate, pH5.2 and 220 ⁇ l 95% ethanol, and the sample was frozen on dry ice for 10 minutes. RNA was pelleted by centrifugation at 14,500 rpm for 20 minutes at 4°C, washed with 70% ethanol, air dried and re- suspended in 8 ⁇ l DEPC water.
  • RNA was de-capped in a 10 ⁇ l reaction with 0.5 U tobacco acid pyrophosphatase (TAP), 1 ⁇ l lOx TAP buffer and 40U RnaseOutTM for 1 hour at 37°C. RNA was extracted with phenol : chloroform and precipitated as above, and then re-suspended in 7 ⁇ l DEPC-treated water.
  • TAP tobacco acid pyrophosphatase
  • RnaseOutTM 40U
  • First-strand cDNA was prepared by the addition of 1 ⁇ l GeneRacerTM Oligo dT .
  • primer- and 1 ⁇ l dNTP mix (lOmM each) to 10 ⁇ l ligated RNA and incubated at 65°C for 5 minutes.
  • the following reagents were added to the 12 ⁇ l ligated RNA and primer mix; 4 ⁇ l 5x first strand buffer, 2 ⁇ l 0.1M
  • PCR reaction was set up using 1 or 2 ⁇ l of the RACE-ready cDNA prepared above, 3 ⁇ l GeneRacerTM 5' primer (10 ⁇ M) , 1 ⁇ l reverse gene- specific primer (SEQ ID No. 51, ADMA-RACE-Rl or SEQ ID No.
  • a second, nested PCR stage was then set up using 1 ⁇ l of the 5' RACE cDNA from the first stage above, 1 ⁇ l nested
  • 5' primer (10 pmol/ ⁇ l, supplied with kit), 1 ⁇ l reverse gene-specific primer (SEQ ID No. 53; ADMA-RACE-Rlnest or SEQ ID No. 54, ADMB-RACE-Rlnest: 10 pmol/ ⁇ l), 1 ⁇ l dNTP solution (lOmM each), 2 ⁇ l 50 mM MgS0, 5 ⁇ l lOxHigh Fidelity PCR buffer, 0.5 ⁇ l Platinum® Taq DNA Polymerase High Fidelity (5U/ ⁇ l) and 38.5 ⁇ l .sterile molecular biology grade water. Cycling parameters are given in Table III below.
  • PCR reaction was set up using 1 ' or 2 ⁇ l of the RACE-ready cDNA prepared above, 3 ⁇ l GeneRacerTM 3' primer (10 ⁇ M) , 1 ⁇ l forward gene-specific primer (SEQ ID No. 55, ADMA-RACE-Fl, or SEQ ID No.
  • a second, nested PCR stage was then set up using 1 ⁇ l of the 3' RACE cDNA from the above first stage, 1 ⁇ l Nested 3' primer (10 pmol/ ⁇ l; supplied with kit), 1 ⁇ l forward gene-specific primer (SEQ ID No. 57, ADMA-RACE-Flnest or SEQ ID No.
  • RNA was then extracted using the Qiagen RNeasy Plant Mini Kit following the protocol for isolation of total RNA from filamentous fungi in the RNeasy Mini Handbook (06/2001, Pages 75-78, http://www.qiagen.com/literature/ handbooks/rna/rnamini/1016272HBRNY_062001WW.pdf) .
  • step 3 RLC was used as the lysis buffer of choice;
  • step 7 the Rneasy column was incubated for 5 min at room temperature after addition of RWl;
  • step 9a was carried out;
  • step 10 30 ⁇ l RNase-free water was added, the samples incubated for 10 min at room temperature, and then centrifuged;
  • step 11 the elution step was repeated to give a total volume of 60 ⁇ l RNA.
  • RNA contamination was removed from. the RNA by the addition of Dnase, using 2 ⁇ l DNase per ⁇ g RNA, in the presence ' of 10X DNase buffer and incubating at 37 °C for 2h.- DNase- treated RNA was cleaned up using the RNeasy Plant Mini Kit following the RNeasy Mini Protocol for RNA Cleanup (RNeasy Mini Handbook 06/2001, pages 79-81) .
  • RNeasy Mini Handbook 06/2001, pages 79-81 To synthesise cDNA from the above RNA the following reaction mixture was prepared: lOOng-l ⁇ g of DNA-free RNA, 3 ⁇ l oligo (dT) (lOOng/ ⁇ l) , and DEPC-treated water to a total volume of 42 ⁇ l.
  • Samples were incubated in a heat block at 65°C for 5 min after which they were allowed to cool slowly to room temperature. Then ' 2 ⁇ l Ultrapure dNTPs, l ⁇ l reverse transcriptase (Stratascript) and 5 ⁇ l 10X reverse transcriptase reaction buffer (Stratascript) were added. Samples were incubated at 42°C for lh, denatured at 90°C for 5 min and then cooled on ice..
  • PCR was carried out using the cDNA above to generate cDNA fragments using the following forward and reverse primer pairs: For ADMA, SEQ ID No. 59, FL-ACDNAF, and SEQ ID No . 60, FL-ACDNAR; For ADMB, SEQ ID No. 61, FL-BCDNAF, and SEQ ID No. 62, FL-BCDNAR. PCR reactions were carried out using- the following reagents and conditions :
  • Reverse primer (5 pmol/ ⁇ l stock) 1 ⁇ l cDNA 2-4 ⁇ l
  • Cycles 2-4 were repeated 40 times in total.
  • the amplicon sizes were " 2131 bp (ADM-A) and 2939 bp (ADM-B) .
  • the PCR products were run on agarose gels, excised and purified using Qiagen' s QIAquick Gel Extraction Kit (Qiagen Ltd, Boundary Court, Gatwick Road, Crawley, Westshire, RH10 9AX, UK) , ligated into pGEM-Teasy and used to transform Select 96 cells. Inserts were sequenced as described above .
  • ADM-A and ADM-B messages have therefore been determined experimentally.
  • the predicted ' sequences previously given in SEQ ID Nos. -31 and 32 were compared to the experimentally " determined sequences and found to be identical.
  • Bases T1297 and A1492 of ADM-B cDNA were C and G respectively, however, it was considered that these were PCR errors.
  • Figure 2A illustrates distance and parsimony analysis carried out on an alignment of protease and disintegrin domains as shown in Figure 1
  • Figure 2B illustrates distance and parsimony analysis carried out on a truncated alignment including the P. jiroveci sequence shown in Figure 1.
  • Distance trees are shown with nodes supported by bootstrapping values >50% marked by black dots.
  • the values given for the nodes are the bootstrapping percentages (A; first value, distance; second value, parsimony. B; distance value) .
  • the bar line corresponds to the -expected number of substitutions per site.
  • the fungal ADAMs formed a distinct clade and did not group closely with any one member or set of the mammalian ADAMs . Thus, the fungal ADAMs are not predicted to share close functional similarity with the mammalian ADAMs.
  • the duplication that gave rise to ADM-A and ADM-B appears not to have occurred early in fungal evolution. Identification of a single -clade of fungal ADAMs raises the -possibility of developing an anti-ADAM therapeutic reagent which will target multiple fungi, rather than A. fumiga tus alone. Furthermore, the separation of fungal and animal ADAMs indicates that this approach could be employed with the minimum risk to the patient.
  • the ADAM phylogeny was compared with the phylogenetic trees inferred for fungi (http: //megasun.bch.umontreal . ca/People/lang/FMGP/phylogen y . html; Liu et al . , 1999, Mol . Biol. Evol . 16, 1799-1808; Keeling et al., 2000, Mol. Biol. Evol. 17, 23-31). These trees showed that C. albicans and S . cerevisiae cluster together in the order Saccharomycetales . Thus the two fungi lacking an ADAM group together. The other ADAM- containing fungi (A. fumiga tus, E . cuniculi , N.
  • ADAMs are widely dispersed throughout the kingdom Fungi. This, coupled with the presence of ADAMs in animals (the nearest kingdom to fungi) allows us to propose that ADAMs were present in the earliest fungi and that the lack of ADAMs in a particular species, genus or higher group (including S . cerevisiae and C. albicans) is due to a specific event.
  • the ADAM sequences described in the invention may be exploited to diagnose fungal infections .
  • Samples from patients or plants potentially carrying an infection with A . fumiga tus, P. carinii , E. cuniculi , or other organisms are processed to extract DNA using the DNAeasy Tissue kit, QIAamp DNA Blood Minikit or DNeasy Plant Minikit (Quiagen, Crawley, UK) , although other DNA preparation methods are available and suitable.
  • PCR reactions are set up as follows :
  • Pneumocystis jiroveci SEQ ID No.35 + SEQ ID No.36 (483bp)
  • Appropriate controls include; (i) template DNA but no primers; primers but no template (negative controls); (ii) cDNA encoding fungal ADAM or DNA from cultured fungi instead of patient/sample DNA (positive control) .
  • PCR reactions are run as follows :
  • PCR products are examined on agarose gels .
  • the production of a band of the correct molecular weight is diagnostic of the presence of the particular fungus. It may be additionally necessary to carry out diagnostic restriction digests of the PCR products. If necessary, PCR products are subcloned into a vector, such as pGEM-Teasy, and sequenced to verify that the PCR products are from the appropriate fungus .
  • the presence of an infection with A. fumiga tus, P. carinii , E. cuniculi , or other organisms may be detected by means of antibodies raised against the fungal ADAMs .
  • One suitable means is the use of a capture ELISA.
  • microtitre plates are coated with a monoclonal antibody raised against the fungal ADAM. Then the plates are incubated with diluted patient samples, or appropriate protein extracts of samples (particularly if the samples are biopsies or plants) . Plates are then incubated with a polyclonal antibody (again against the ADAM) . Finally, binding of the second antibody is detected by means of an enzyme-coupled or fluorescently-labelled antibody directed against the polyclonal.
  • two monoclonal or polyclonal antibodies or various combinations may be used.
  • ADAM fragments are expressed to enable detailed study of ADAM function and as the starting point for the development of a high-throughput screen for inhibitory compounds .
  • RNA was prepared from A. fumiga tus cultures and used as the template for the production of ADAM cDNA:
  • RNA was then extracted using • 5 the Qiagen RNeasy Plant Mini Kit following the protocol for isolation of total RNA from filamentous fungi in the RNeasy. Mini Handbook (06/ ' 2001, Pages 75-78, http: //www. qiagen. com/literature/handbooks/rna/rnamini/101 6272HBRNY_06200lWW.pdf) . The following modifications were
  • step 10 used: At step 3, RLC was used as the lysis buffer of choice; At step 7, the Rneasy column -was incubated for 5 min at room temperature after addition of RW1; The optional step 9a ' was carried out; At step 10, 30 ⁇ l RNase- free water was added, the samples incubated for 10 min at
  • DNA contamination was removed from the RNA by the addition of Dnase, using 2 ⁇ l DNase per ⁇ g RNA, in the presence of 20 10X DNase buffer and incubating at 37 °C for 2h.
  • DNase- ' treated RNA was cleaned up using the RNeasy Plant Mini Kit following the RNeasy Mini Protocol for RNA Cleanup (RNeasy Mini Handbook 06/2001, pages 79-81) .
  • PCR is carried out using the cDNA above to generate cDNA fragments corresponding to the following domain combinations (although other combinations can be envisaged) .
  • the appropriate primer pairs are given here, to correspond with A+B pairs in the method below.
  • Primers with SEQ .ID Nos. 43, 44, 47 and 48 included the sequence CACCATG at the 5' end to enable cloning into a pETlOO/D- TOPO vector (Invitrogen) .
  • the figures in brackets after each primer pair corresponding to the first and last bases of the expected PCR product as read from the cDNA sequences for ADM-A and ADM-B given as SEQ ID Nos.31 and 32 respectively.
  • Propeptide protease and disintegrin domains - ADM-A, SEQ ID No.43 + SEQ ID No.46 (64-1581) ADM-B, SEQ ID No.47 + SEQ ID No.50 (73-1836)
  • Propeptide and protease domain - ADM-A, SEQ ID No.43 + SEQ ID No.45 (64-1278) ADM-B, SEQ ID No.47 + SEQ ID No.49 (73-1527)
  • ADM-A SEQ ID No.44 + SEQ ID No.45 (592-1278)
  • ADM-B SEQ ID No.48 + SEQ ID No.49 (817-1527)
  • PCR products are purified using Qiagen' s QIAquick PCR Purification Kit (Qiagen Ltd, . Boundary Court, Gatwick Road, Crawley, West Canal, RH10 9AX, UK) according to the manufacturers instructions.
  • the purified PCR products are examined on agarose gels. cDNA fragments are then cloned in to the pETlOO/D-TOPO vector (Invitrogen) .
  • the plasmids are then transformed in to TOP10 chemically competent E-coli cells and plated on to a prewarmed ampicillin (+) selection plate. After an overnight incubation at 37° C, ampicillin resistant colonies are selected and grown up in ampicillin containing LB medium. Plasmid DNA is isolated using the Plasmid Mini Kit (qiagen) . Confirmation of the presence and correct orientation of the inserts is determined by restriction analysis and sequencing of the construct.
  • Purified plasmid DNA which has been confirmed to be of the correct sequence and orientation, is transformed into chemically competent BL21 Star (DE3) One Shot E. coli cells and grown overnight at 37° C. Protein expression is then induced by the addition of isopropylthio- ⁇ - galactosidase (IPTG) and detected by SDS PAGE and western blotting ⁇ using an anti-His antibody. Purification of the recombinant protein is then be performed by metal affinity chromatography.
  • Alternative expression systems can be used for expression in bacteria, such as the glutathione S-transferase or mannose-binding fusion-protein system.
  • cDNA constructs corresponding to the ADM-B protease domain and to the ADM-B propeptide domain plus protease domain were generated and the encoded recombinant proteins expressed.
  • PCR reactions were carried out using the following reaction mixture and conditions . All Reagents were present in the KOD kit (Novagen) : Reaction mix: 2.5 ⁇ l lOx PCR Buffer 5 ⁇ ls dNTPs (2mM) 2 ⁇ l MgS0 4 (25mM)
  • primers A and B were SEQ ID Nos. 48 and 49;
  • primers A arid B were SEQ ID Nos. 47 and 49.
  • Step6 10°C Hold 40 cycles of steps 2-4 were carried out and the PCR products were purified using Qiagen' s QIAquick PCR Purification Kit (Qiagen Ltd, Boundary Court, Gatwick Road, Crawley, Westshire, RH10 9AX, UK) according to the manufacturers instructions .
  • the purified PCR products were examined on agarose gels.
  • cDNA constructs encoding the corresonding fragments of ADMA can be produced using the following primer pairs: Propeptide+protease: SEQ ID No. 63 and SEQ ID No. 65; Protease: SEQ ID No. 64 and SEQ ID No. 65.
  • cDNA fragments were cloned in to the pETlOl/D-TOPO vector (Invitrogen) .
  • the plasmids were then transformed in to TOP10 chemically competent E-coli cells and plated on to a prewar ed ampicillin (+) selection plate. After an overnight incubation at 37° C, ampicillin resistant colonies were selected and grown up in ampicillin containing LB medium. Plasmid DNA was isolated using the Plasmid Mini Kit (Qiagen) . Confirmation of the presence and correct orientation of the inserts was determined by restriction analysis and sequencing of the construct.
  • Figure 5 shows Western blots of recombinant ADMB domains.
  • Samples from bacteria carrying the protease or propeptide+protease constructs were run on SDS page gels, transferred to nitrocellulose and probed with anti-V5 antibody.
  • ⁇ The detection of a band of the correct size of approximately 22.9 kDa for ADM-B protease (A), and 5 approximately 51.8 kDa for ADM-B propeptide+protease (B) , indicated the successful expression of these constructs.
  • Clones identified .as producing recombinant protein of the correct molecular weight were then used for bulk
  • Bacteria were harvested by centrifugation at 14,000 rpm for 10 minutes and the pellets lysed in lysis buffer (10 ml Bugbuster (Novagen), 10 ⁇ l Benzonase (Novagen), 0.4 ⁇ l 20 lysozyme (Novagen) and 100 ⁇ l Imadazole (IM) for 20 minutes at room temperature.
  • lysis buffer 10 ml Bugbuster (Novagen), 10 ⁇ l Benzonase (Novagen), 0.4 ⁇ l 20 lysozyme (Novagen) and 100 ⁇ l Imadazole (IM) for 20 minutes at room temperature.
  • Supernatant was obtained by centrifugation at ' 1,000 rpm for 20 minute, .4° C, and passed down a nickel column (Novagen) equilibrated in 50 mM sodium phosphate (pH 8.0), 500 mM MaCl, 20 mM ' 25 imidazole, 0.1% Tween 20, and the recombinant protein eluted with 50 mM sodium phosphate pH 8.0, 500 mM NaCl, 250 mM imidazole, 0.1% Tween 20.
  • ADAM cytoplasmic domains Use of the ADAM cytoplasmic domains to generate ADAM 30 mutant strains.
  • the cytoplasmic domains of the fungal ADAMs can be exploited to generate fungal cells in which ADAM function is perturbed, without the need for mutating the ADAM gene itself; this will give insights into ADAM function.
  • Over-expression of constructs encoding only the transmembrane and cytoplasmic domains of the ADAMs in A. fumiga tus or other fungi would present the cellular signalling machinery with excess target and thus greatly diminish any signalling to or from the ADAMs. This can be accomplished as follows :
  • PCR is used to generate cDNA fragments encoding the transmembrane region and cytoplasmic domain, .with the addition of an N-terminal methionine start and a leader sequence, e.g. from the appropriate ADAM itself.
  • the fragments are then cloned into an expression vector suitable for transformation into A. fumigatus or the chosen host; e.g. including a fungal antibiotic resistence gene such as hygromycin or an auxotrophic marker such as PyrG.
  • Other properties of the vector should include an inducible promoter and an epitope tag.
  • the resulting constructs are transfected into A. fumiga tus, and the effects of induction observed. This involves examining the morphology and growth of the cells in induced and uninduced conditions . It is also important to determined whether the. construct is being expressed, exploiting the epitope tag, by western blotting or immunohistochemistry . 7. Assays for protease function using recombinant ADAMs
  • Functional assays for recombinant ADAM protease domains enable both the identification of potential in vitro and in vivo substrates and screening for potential inhibitors.
  • a relatively non-specific substrate is employed, such as azocasein.
  • proteolytic cleavage of the substrate liberates soluble coloured peptides .
  • Stopping the reaction with trichloracetic . acid precipitates enzyme and undigested • substrate.
  • Centrifugation of the digestion mixture leaves reaction products in solution, enabling these .to be read spectrophotometrically.
  • substrates such as FITC-labelled casein or more complex derivatives are used and their cleavage' measured using a fluorescence plate reader or techniques such as FRET.
  • TACE and matrix metalloproteinase substrates such as OmniMMP; (7-methylcoumarin-4-yl) acetyl- Pro-Leu-Ala-Gln-Ala-Val- (N-3- (2, 4-dinitrophenyl) -L- ⁇ , ⁇ - diaminopropionyl) -Arg-Ser-Ser-Ser-Arg-NH 2 ; and (2,4- dinitrophenyl) -Pro- ⁇ -cyclohexyl-Ala-Gly-Cys (N-Me- anthranilic acid) -His-Ala-Lys (N-Me-2-aminobenzoyl) -NH 2 ;
  • ADMB protease domain described in Example 5.3 was assayed for protease activity. Assays were carried out in 96-well microtitre plates.
  • Antibodies against the fungal ADAMs will be of considerable use as diagnostic reagents (see example 4- above) .
  • diagnostic reagents see example 4- above.
  • As an immunogen recombinant. domains are used (as described in example 5) .
  • synthetic polypeptides encoding regions either unique to the individual ADAMs or likely to provide cross-reactivity within a selected species or genus range are used. Peptides may need to be conjugated to carrier proteins before immunization.
  • Preimmune sera from animals to be immunised are screened against the immunogen to ensure that there is no endogenous cross reactivity. Animals (sheep, rabbits or mice) are then immunised.
  • the resulting sera can be affinity purified using the immunogen cross-linked to a chromatography matrix.
  • purification of the antibody fraction from the serum e.g. using protein G or protein A cross-linked to a matrix, may be sufficient.
  • Monoclonal antibody production proceeds by methods familiar to those skilled in the art.
  • the specificities of the resulting polyclonal and/or monoclonal antibodies are checked by ELISA and/or western blotting using the immunogen, related constructs or whole cell lysates and extracts as targets.
  • Negative controls such as different ADAMs, different constructs or different species are also employed to test specificity and/or to determine the range of species and/or genus cross- reactivity.
  • a BAG (bacterial artificial chromosome) clone library containing the ⁇ . fumiga tus genome, partially digested with BamHT and inserted into the vector pBACe3.6 was purchased from the Sanger Centre, Cambridge, UK.
  • the BAC clone containing the gene to be inactivated was identified by bioinformatics (BLAST searching of Sanger BAC and related databases) and the glycerol stock of the clone grown up in 50 ml LB, 20 ⁇ g/ l chloramphenicol at 37 °C overnight. The overnight culture was centrifuged at 4,500 rpm for 15 min.
  • the bacterial pellet was resuspended in 4 ml of Buffer PI (Qiagen plasmid miniprep kit) , then 4 ml of buffer P2 (Qiagen plasmid miniprep kit, lysis buffer) was added and mixed gently by inverting 3-6 times.
  • Buffer PI Qiagen plasmid miniprep kit
  • buffer P2 Qiagen plasmid miniprep kit, lysis buffer
  • - Proteins and genomic DNA were precipitated by adding 4 ml of buffer P3 (Qiagen plasmid miniprep kit, neutralizing buffer) and incubated on ice for 10 minutes.
  • the supernatant was transferred into a 50 ml falcon tube, an equal volume of phenol/chlorophorm (1:1) mixture was added, and the mixture centrifuged for 15 min at 4500 rpm. The supernatant was then transferred into an oakridge tube and 0.7 volumes isopropanol were added.' After mixing, the tube was centrifuged at 10,000 rpm (Beckman centrifuge, rotor JA-17) for 30 min at 4°C. The resulting pellet was washed with 2 ml 70% ethanol at the same speed.. The resulting BAC DNA was resuspended in 100 ⁇ l buffer EB. The transposition reaction was carried out as follows.
  • coli cells (Invitrogen) were then transformed with the transposed BAC, the cells plated onto LB agar, 25 ⁇ g/ml kanamycin, 20 ⁇ g/ml chloramphenicol, and plates incubated overnight at 37°C.
  • BAC DNA was then purified using the Millipore montage 96 BAC KIT using a MWG ROBOSEQ 4200 robot. BACs containing the transposon inserted into the gene of interest were identified by PCRs using primer pairs that spanning the gene of interest and pairs that extended from the transposon into the BAC.
  • the BAC was then linearised using a restriction enzyme determined to cut in the vector backbone but not the BAC
  • A. fumiga tus (haploid) protoplasts were prepared using 5% Glucanex (Novo Nordisk A/S) solution (in 0.6 M KCl ) and shaking for 2 h at 80 rpm in 30 ° C .
  • the protoplasts were washed with 0.6 M KCl and then ' with STC (Sorbitol, Tris, CaC12) .
  • the washed protoplasts were diluted in STC to 10 5 /ml and 100 ⁇ l transferred into 14 ml falcon tubes . 7 ⁇ l of linearised BAC were added to the tube and whole mixture incubated on ice for 20 min.
  • Transformation was carried out by adding 200 ⁇ l of PEG 8000 solution (60%w/v, pH 7.5) dropwise over 2 min and then adding 800 ⁇ l PEG. The mixture was left at room temperature for 20 min. Transformed protoplasts were washed with STC, resuspended in 1 ml STC, spread onto CM-sorbitol- Zeocin (250 ⁇ g/ml) plates and incubated at 37 ° C.
  • zeocin resistant colonies were picked and checked for presence of the knocked-out gene by PCR using primers which specifically amplify the whole gene of interest. Usually 10-20 transformants were checked.
  • the fungae produced in Example 9 may be tested as follows.
  • the ectopic integration of the BAC gives two bands by PCR, one for the endogenous gene and one for the BAC/transposon construct, which has a higher molecular weight.
  • Assays are carried out in 96-well microtitre plates. The following amounts are added per well: 125 ul of Tris-HCl pH7.5 (lOOmM), 62.5 ul Casein (2mg/ml) , 0.1-1000 ug recombinant or control protease, and a candidate modulator solution; well volumes are made up to 250 ul with nuclease-free water. Plates are incubated at 37° C for 30 min after which 100 ul samples are transferred to wells of a fresh plate and the following added per well: 50 ul nuclease free water, 100 ul of 1:2 Coomassie solution (0.05% w/v Coomassie G250 in 23.5% ethanol/42.5% phosphoric acid) . - Plates are incubated for 10 mins at RT and absorbarice are then read at 595 nm.
  • protease domains can be identified using peptides and synthetic molecules. These can be labelled, e.g. using fluorescent tags, and binding detected using a fluorescence plate reader.

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Abstract

Cette invention se rapporte à un polynucléotide comprenant un gène ADAM isolé à partir d'un organisme sélectionné dans le groupe des genres constitués par Aspergillus; Pneumocystis; Encephalitozoon; Cryptococcus; Trichophyton; Fusarium; Blumeria; Leptosphaeria; Plasmopara; Pyricularia; Puccinia et Rhizoctonia. Cette invention concerne en outre un polypeptide ADAM isolé à partir d'un organisme sélectionné dans le groupe des genres constitués par Aspergillus; Pneumocystis; Encephalitozoon; Cryptococcus; Trichophyton; Fusarium; Blumeria; Leptosphaeria; Plasmopara; Pyricularia; Puccinia et Rhizoctonia
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