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EP0705435A1 - Nouvelle famille de proteinases de pollen, procedes pour leur utilisation et compositions derivees - Google Patents

Nouvelle famille de proteinases de pollen, procedes pour leur utilisation et compositions derivees

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Publication number
EP0705435A1
EP0705435A1 EP94921321A EP94921321A EP0705435A1 EP 0705435 A1 EP0705435 A1 EP 0705435A1 EP 94921321 A EP94921321 A EP 94921321A EP 94921321 A EP94921321 A EP 94921321A EP 0705435 A1 EP0705435 A1 EP 0705435A1
Authority
EP
European Patent Office
Prior art keywords
leu
ser
asp
glu
val
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94921321A
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German (de)
English (en)
Other versions
EP0705435A4 (fr
Inventor
James Travis
Philip J. Barr
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University of Georgia Research Foundation Inc UGARF
LXR Biotechnology Inc
Original Assignee
University of Georgia Research Foundation Inc UGARF
LXR Biotechnology Inc
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Application filed by University of Georgia Research Foundation Inc UGARF, LXR Biotechnology Inc filed Critical University of Georgia Research Foundation Inc UGARF
Publication of EP0705435A1 publication Critical patent/EP0705435A1/fr
Publication of EP0705435A4 publication Critical patent/EP0705435A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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

Definitions

  • This invention relates to the field f pollen allergies, pollen proteinases, composit ons comprising the proteinases and methods of use thereof.
  • pollens are allergenic.
  • An immunological response to pollens occurs once the immune system has been exposed to the proteins associated with pollen.
  • the physiological responses to allergens are well defined, the precise mechanisms by which these responses are effected are unknown.
  • LolpI A family of allergenic proteins, LolpI, LolpII and LolpIII obtained from rye grass pollen has been characterized. Perez et al. (1990) J. Biol. Chem. , 2_£5.16210-16215; and Griffith et al. (1991) FEBS. 279;210-215.
  • the proteins were obtained from Lolium perenne (rye grass) and it appears that LolpI is the major allergen responsible for rye grass allergy. Mature, glycosylated LolpI has a molecular weight (MW) of about 35,000 Daltons and has been shown to be the major IgE binding protein. These proteins are found in the cytosol of pollen. Their function is unknown.
  • Der pi Proteins from other common allergens have also been isolated. In dust mites, a protein termed Der pi has been identified as reacting with anti-mite IgE antibodies in up to 80% of allergic sera. Der pi has been cloned and sequenced. Chira et al. (1988) J. Exp. Med.. 167:175-182. Sequence analysis showed that Der pi is homologous to cysteine proteinases and in fact has cysteine proteinase activity.
  • Aspergillus fumigatus responsible for allergic bronchopulmonary aspergillosis, contains an allergen that has been proposed to be a proteinase capable of inducing human epithelial detachment.
  • A. fumigatus grows within sputum plugs adjacent to the airway wall for prolonged periods. Since the proteinase is released only by the hyphal tip of the fungus, it has been theorized that the proteinase may be responsible for the resulting chronic lung damage that occurs upon colonization of the airways. Robinson et al. (1990).
  • the protein or proteins responsible for the serine protease activity are from 20-35 kD in molecular weight. It has not yet been determined whether proteinases are involved in allergic responses to pollen. For instance, the Lolp proteins discussed above are highly allergenic but do not have proteolytic activity. Identification of proteinases involved in immune response would be particularly useful in testing whether an individual is allergic to pollen, identifying agents that could ameliorate allergic responses, developing treatments involving such agents, and in monitoring the response of a patient to such treatments or therapies. The present invention meets these and other needs. _._, paragraph
  • the proteins are derived from allergenic pollens and exhibit serine proteinase-like activity.
  • the proteins have a molecular weight of about 85-95 kD as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis; and a molecular weight of about 67 kD as determined by Fast Protein Liquid Chromatography. The apparent molecular weight is dependent on the glycosylation state of the molecule.
  • the proteins are resistant to inhibition by o.-2-macroglobulin ( ⁇ -2-M) , ⁇ -1-proteinase inhibitor ( ⁇ -l-PI) , and trypsin inhibitors and are sensitive to.
  • PMSF phenyl methane sulfonyl fluoride
  • DFP difluorophenol
  • TLCK - benzamidine
  • Figure 1 is a comparison of various serine proteinases.
  • Figure 2 depicts the subclones which were combined to obtain the full length gene encoding the mesquite pollen proteinase.
  • Figure 3 depicts the pBluescript construct containing the full length gene encoding the mesquite pollen proteinase.
  • Figure 4 depicts the pYT construct containing the full length gene encoding the mesquite pollen proteinase.
  • Figure 5 depicts the proteinase activity of the recombinant pollen proteinase expressed in yeast.
  • the proteinases exhibit serine proteinase-like activity, displaying a substrate specificity for Arg-Arg and Arg-lie. The proteinase activity is correlated with the allergenicity of these pollens.
  • the molecular weight (MW) of the proteinases When isolated from pollen, the molecular weight (MW) of the proteinases is about 70-90 kD. As determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) the MW is about 85-95 kD and as determined by Fast Protein Liquid Chromatography (FPLC) the MW is about 67 kD.
  • the proteinases are resistant to inhibition by ⁇ -2-M, ⁇ -l-PI, and trypsin inhibitors and sensitive to inhibition by PMSF, DFP, benzamidine, antipain, leupeptin and TLCK.
  • the mesquite DNA encoding the pollen proteinase has now been cloned and sequenced. The invention thus encompasses the recombinant gene and protein encoded thereby.
  • the proteinase has homology to various serine proteinases. Several serine proteinase families are depicted in Figure 1.
  • allergenic pollens have significantly higher proteinase activity than non-allergenic pollens.
  • the degree of proteinase activity is thus correlated with allergenicity, although it is possible that proteinase activity may not be the sole mechanism effecting allergenicity and other nonallergic responses.
  • Inactivation of the trypsin-like activity (cleavage after arginine ubstrates) by DFP or PMSF causes total loss of proteinase activity in the crude pollen extracts, indicating that this is the primary proteinase in the pollen sample.
  • the proteinases in purified form are useful, for example, in formulating both diagnostic and therapeutic regimens for allergy sufferers, in formulating diagnostics for testing individuals for allergic responses, in monitoring the effectiveness of allergy treatments and therapies, in vaccines capable of treating an allergy, and in methods for identifying agents that modulate the effect of the proteinases on cells involved in a. rgic responses.
  • Antibodies raised against the purified proteinases are useful for a number of purposes, including immunodiagnostics.
  • the present invention also embraces the cloning and recombinant expression of polynucleotides encoding the proteinases and related genes made possible by the newly determined amino acid sequence of the purified polypeptide.
  • compositions and methods of the present invention are widely applicable to other allergenic pollens as well.
  • other highly allergenic pollens such as Japanese cedar are now thought to produce homologous peptides and thus are encompassed by the embodiments discussed herein.
  • the proteinases have proteolytic activity, it is possible their biological effects may not be directly related to this activity.
  • the initial non- immunological response described above may be due to cell binding, for instance, or proteolytic activity on other proteins which renders these newly digested proteins irritating.
  • non-immunologic responses may be directly caused by the proteolytic activity whereas immune responses may be due to non-proteolytically active epitopes.
  • the absence of widespread substrates indicates that the role of these proteinases in generating a physiological response may be due to a non- proteolytic event.
  • Such events include but are not limited to modulating cell surface events so as to modulate receptor signalling.
  • the proteinases include variants or fragments of full length proteinases as isolated from pollen or produced by chemical synthesis or recombinant means. Ordinarily, such proteins will be at least about 50% homologous to the native (wild-type) proteinase, preferably in excess of about 90%, and, more preferably, at least about 95% homologous. Also included are proteins encoded by polynucleotides which hybridize under stringent conditions to proteinase-encoding polynucleotides and closely related polypeptides retrieved by antisera raised against a proteinase.
  • the length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 to 24 residues, typically at least about 28 residues, and preferably more than about 35 residues.
  • the term “substantial homology” or “substantial identity” indicates that the polypeptide or protein in question exhibits at least about 30% homology with a naturally occurring protein or a portion thereof, usually at least about 70% homology, and preferably at least about 95% homology.
  • sequence analysis software See, e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • a polypeptide "fragment, " "portion, “ or “segment” is a stretch of amino acid residues of at least about 5 amino acids, often at least about 7 amino acids, typically at least about 9 to 13 amino acids, and, in various embodiments, at least about 17 or more amino acids.
  • substantially pure protein typically comprises about 60 to 90% W/W of a protein sample, more usually about 95%, and preferably over about 99%. Protein purity or homogeneity may be indicated by a number of means known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a substantially single polypeptide band upon staining the gel. For certain purposes higher resolution may be provided by using HPLC or other means known in the art.
  • the proteinases are substantially free of naturally associated components when separated from the native contaminants which accompany them in their natural state.
  • a proteinase that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.
  • Techniques for synthesis of polypeptides are described, for example, in Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2156.
  • Recombinant proteinases can be obtained by culturing host cells transformed with the expression systems described below under conditions suitable to attain expression of the proteinase-encoding sequence.
  • a chemically synthesized proteinase is considered an "isolated" polypeptide, as is a proteinase produced as an expression product of an isolated proteinase-encoding polynucleotide which is part of an expression vector (i.e., a "recombinant proteinase"), even if expressed in a homologous cell type.
  • Example 1 describes the purification of a trypsin-like proteinase from mesquite, its natural source.
  • Various methods for the isolation of the proteinases from other biological material, such as from cells transformed with recombinant polynucleotides encoding such proteins may be accomplished by methods known in the art.
  • Various methods of protein purification are known in the art, including those described, e.g., in Guide to Protein Purification, ed. Academic Press, Inc.: San Diego, 1990) and Scopes, Protein Purification: Principles and Practice (Springer-Veriag: New York, 1982) .
  • the partial amino acid sequence of the mesquite proteinase was obtained as described in Example 1, with enzymes such as trypsin, clostripain, or Staphylococcus proteinase or with chemical agents such as cyanogen bromide (CnBr) , O-iodosobenzoate, hydroxylamine or 2- nitro-5-thiocyanobenzoate.
  • the peptide fragments thus obtained were separated for instance by reverse-phase high performance liquid chromatography (HPLC) and analyzed by gas-phase sequencing. Other means for generating peptide fragments and obtaining the amino acid sequence of such fragments or an unfragmented protein are known in the art.
  • the method used herein is described by Methods of Enzymology XI and subsequent volumes.
  • the partial internal amino acid residue sequences for fragments of the mesquite proteinase were then used to obtain the DNA sequence encoding the proteinase as described in Example 1.
  • amino acid residues as used herein are as follows: Ala (A), alanine; Val (V), valine; Leu (L) , leucine; lie (I), isoleucine; Pro (P) , proline; Phe (F) , phenylalanine; Trp (W) , tryptophan; Met (M) , methionine; Gly (G) , glycine; Ser (S) , serine; Thr (T) , threonine; Cys (C) , cysteine; Tyr (Y) , tyrosine; Asn (N) , asparagine; Gin (Q) , glutamine; Asp (D) , aspartic acid; Glu (E) , glutamic acid; Lys (K) , lysine; Arg (R) , arginine; and His (H) , histidine.
  • the proteinases include modified forms of the basic polypeptide sequences thereof but which are substantially homologous to that primary sequence and which possess a biological activity characteristic of a proteinase (e.g., serine proteinase-like activity, allergenicity, or immunologic activity, i.e., possession of one or more antigenic determinants recognized by an antibody specific for a proteinase) .
  • a biological activity characteristic of a proteinase e.g., serine proteinase-like activity, allergenicity, or immunologic activity, i.e., possession of one or more antigenic determinants recognized by an antibody specific for a proteinase
  • Such .in vivo or in vitro chemical and biochemical modifications include, e.g., unusual amino acids, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labelling, e.g., with radionuclides and other modifications.
  • radioactive isotopes such as 32 P
  • ligands which bind to labeled antiligands e.g., antibodies
  • fluorophores e.g., fluorophores
  • chemiluminescent agents e.g., enzymes
  • antiligands which can serve as specific binding pair members for a labeled ligand.
  • the choice of label depends on the sensitivity required, ease of conjugation with the proteinase, stability requirements, and available instrumentation. Methods of labeling polypeptides are described, e.g., in Molecular Cloning: A Laboratory Manual. 2nd ed. , Vols. 1-3, ed. Sambrook et al.
  • the present invention provides for fragments or segments of the polypeptides.
  • Such fragments or segments will ordinarily be at least about 5 to 7 contiguous amino acid residues, typically at least about 9 to 13 contiguous amino acids, and most preferably at least about 20 to 30 or more cont; such amino acid residue.
  • the fragments may retain proteinase activity or a proteinase specific epitope.
  • Tandemly repeated proteinase fr ments may also be useful, e.g., as immunogens or as highly efficient competitors for specific binding. Production of antibodies specific for proteinases or fragments thereof is described below. Methods of recombinantly or synthetically producing tandemly repeated segments are within the skill of one in the art. Methods of producing antibodies, both polycr ⁇ nal and monoclonal, are within the skill of one in the art.
  • the present invention also provide:- for fusion polypeptides comprising proteinases or fragments thereof.
  • Homologous polypeptides may be fusions between two or more proteinase sequences or between the sequences of a proteinase and a related protein.
  • heterologous fusions may be constructed which would exhibit a combination of properties or activities of the proteins from which they are derived. Fusion partners include, but are not limited to, immunoglobulins, ubiquitin bacterial 3-galactosidase, trpE, protein A, 3-lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha mating factor. Godowski et al. (1988) Science. 241:812- 816.
  • Fusion proteins will typically be made by recombinant methods, but may be chemically synthesized.
  • polynucleotides which encode the proteinase fragments, homologs or variant thereof, including, e.g., protein fusions or deletions, as well as expression systems are provided. Expression systems are defined as polynucleotides which, when transformed into an appropriate host cell, can express a proteinase.
  • the polynucleotides possess a nucleotide sequence which is substantially similar to a natural proteinase-encoding polynucleotide or a fragment thereof.
  • the polynucleotides include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or contain non-natural or derivatized nucleotide bases.
  • Recombinant polynucleotides comprising sequences otherwise not naturally occurring are also provided by this invention, as are alterations of a wild type proteinase sequence, including but not limited to deletion, insertion, substitution of one or more nucleotides or by fusion to other polynucleotide sequences.
  • suitable cDNA (particularly pollen cDNA libraries) or genomic libraries may be screened as natural sources of the polynucleotides of the present invention, or such polynucleotides may be provided by methods including but not limited to amplification of sequences resident in mRNA, cDNA or genomic DNA, e.g., by amplification methods such as the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • amino acid residue sequences of various peptide fragments of a mesquite proteinase have been obtained as described in the Examples Section.
  • oligonucleotides were chemically synthesized to represent most or all possible DNA sequences capable of encoding such a peptide fragment or one of a small number of sequences having a high likelihood of hybridizing to a native proteinase-encoding sequence encoding such a peptide fragment.
  • the oligonucleotide or pool of oligonucleotides was then used to probe a cDNA or genomic library which contains a proteinase-encoding DNA sequence in order to identify the sequence, which was then isolated and purified.
  • the polynucleotide sequence obtained either by probing a library or an amplification method using such degenerate oligonucleotides was then itself used as a probe or primer in order to obtain full length cDNA or genomic sequences of the proteinase.
  • oligonucleotides are also useful as primers for directly cloning all or a portion of the gene or genes encoding homologous proteinases in a variety of plants by selectively amplifying the proteinase gene present in a genomic DNA or mRNA, or in a cDNA or genomic library by polynucleotide amplification methods known in the art (e.g., the polymerase chain reaction, or PCR). Cloning by PCR or screening libraries using such degenerate oligonucleotides is facilitated where oligonucleotides are synthesized which correspond to two or more peptide sequences are available, as is true in the present case.
  • probes or primers are fashioned which contain these homologous regions. Such probes and primers are useful in directly cloning or screening libraries derived from a variety of plants in order to obtain other, related genes.
  • the polynucleotides will usually comprise sequences corresponding to at least about 12 to 15 nucleotides (or base pairs) , more usually at least about 21 nucleotides, and most preferably at least about 35 nucleotides, the length depending on the desired use.
  • One or more introns may also be present.
  • a polynucleotide or fragment thereof is “substantially homologous" (or “substantially similar") to another polynucleotide if, when optimally aligned (with appropriate nucleotide insertions or deletions) with another polynucleotide, there is nucleotide sequence identity approximately 60% of the nucleotide bases, usually approximately 70%, more usually about 80%, preferably about 90%, and more preferably about 95 to 98% of the nucleotide bases. Alternatively, substantial homology (or similarity) exists when a polynucleotide or fragment thereof will hybridize to another under polynucleotide under selective hybridization conditions.
  • Selectivity of hybridization exists under hybridization conditions which allow one to distinguish the target polynucleotide of interest from other polynucleotides.
  • selective hybridization will occur when there is approximately 55% similarity over a stretch of about 14 nucleotides, preferably approximately 65%, more preferably approximately 75%, and most preferably approximately 90%. See Kanehisa (1984) Nucl. Acids Res.. 12.:203-213.
  • the length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of about 17 to 20 nucleotides, and preferably about 36 or more nucleotides.
  • hybridization of polynucleotides is affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing polynucleotides, as will be readily appreciated by those skilled in the art.
  • Stringent temperature conditions will generally include temperatures in excess of 30°C, typically in excess of 37°C, and preferably in excess of 45°C.
  • Stringent salt conditions vr.ll ordinarily be less than 1 M, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter.
  • an “isolated” or “substantially pure” polynucleotide is a polynucleotide which is substantially separated from other polynucleotide sequences which naturally accompany a native proteinase sequence.
  • the term embraces a polynucleotide sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a polynucleotide is said to "encode” a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide of a fragment thereof.
  • the anti-sense strand of such a polynucleotide is also said to encode the sequence.
  • a polynucleotide sequence is operably linked when it is placed into a functional relationship with another polynucleotide sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
  • operably linked means that the sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • certain genetic elements such as enhancers, may be operably linked even at a distance, i.e., even if not contiguous.
  • polynucleotide refers to a polynucleotide which is made by the combination of two otherwise separated segments of sequence accomplished by the artificial manipulation of isolated segments of polynucleotides by genetic engineering techniques or by chemical synthesis. In so doing one may join together polynucleotide segments of desired functions to generate a desired combination of functions.
  • Polynucleotide probes include an isolated polynucleotide attached to a label or reporter molecule and may be used to identify and isolate other proteinase sequences. Probes comprising synthetic oligonucleotides or other polynucleotides may be derived from naturally occurring or recombinant single or double stranded nucleic acids or be chemically synthesized. Polynucleotide probes may be labelled by any of the methods known in the art, e.g., random hexamer labeling, nick translation, or the Klenow fill-in reaction.
  • polynucleotides may be produced by replication in a suitable host cell.
  • Natural or synthetic DNA fragments coding for a proteinase or a fragment thereof will be incorporated into recombinant polynucleotide constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell.
  • the construct will be suitable for replication in a unicellular host, such as yeast or bacteria, but a multicellular eukaryotic host may also be appropriate, with or without integration within the genome of the host cells.
  • prokaryotic hosts include strains of Escherichia coli , although other prokaryotes, such as Bacillus subtilis or Pseudo.rno.nas may also be usec Mammalian or other eukaryotic host cells include ⁇ e yeast, filamentous fungi, plant, insect, amphibian or avian species. Such factors as ease of manipulation, ability to appropriately glycosylate expressed proteins, degree and control of protein expression, ease of purification of expressed proteins away from cellular contaminants or other factors may determine the choice of the host cell.
  • the polynucleotides may also be produced by chemical synthesis, e. ⁇ ., by the phosphoramidite method described by Beaucage nd Carruthers (1981) Tetra.
  • DNA constructs prepared for introduction into a prokaryotic or eukaryotic host will typically comprise a replication system recognized by the host, including the intended DNA fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment.
  • Expression systems may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences.
  • Signal peptides may also be included where appropriate from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.
  • an appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al. (1989); Ausubel et al. (1987); and Metzger et al. (1988) Nature, 134:31-36. Many useful vectors for expression in bacteria, yeast, mammalian, insect, plant or other cells are well known in the art and may be obtained from such vendors as Stratagene, New England Biolabs, Promega Biotech, and others. In addition, the construct may be joined to an amplifiable gene (e.g., DHFR) so that multiple copies of the gene may be made.
  • amplifiable gene e.g., DHFR
  • Enhancers and Eukaryotic Gene Expression may replicate autonomously, they may less preferably replicate by being inserted into the genome of the host cell.
  • Expression and cloning vectors will likely contain a selectable marker, that is, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector. Although such a marker gene may be carried on another polynucleotide sequence co-introduced into the host cell, it is most often contained on the cloning vector. Only those host cells into which the marker gene has been introduced will survive and/or grow under selective conditions. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxic substances, e.g. ampicillin, neomycin, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media. The choice of the proper selectable marker will depend on the host cell; appropriate markers for different hosts are known in the art.
  • the vectors containing the polynucleotides of interest can be introduced (transformed, transfected) into the host cell by any of a number of appropriate means, including electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is an infectious agent, such as a retroviral genome) .
  • electroporation employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances
  • microprojectile bombardment a microprojectile bombardment
  • lipofection where the vector is an infectious agent, such as a retroviral genome
  • polynucleotides and polypeptides of the present invention may be prepared by transforming suitable prokaryotic or eukaryotic host cells with proteinase-encoding polynucleotides of the present invention in compatible vectors or other expression vehicles and culturing such transformed host cells under conditions suitable to attain expression of the proteinase-encoding gene. The proteinase may then be recovered from the host cell and purified.
  • polyclonal and/or monoclonal antibodies capable of specifically binding to a proteinase or fragments thereof are provided.
  • the term antibody is used to refer both to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities.
  • Monoclonal or polyclonal antibodies specifically reacting with the proteinases may be made by methods known in the art. See, e.g.. Harlow and Lane (1988) Antibodies: A Laboratory Manual. CSH Laboratories; Goding (1986)
  • Monoclonal Antibodies Principles and Practice. 2d ed, Academic Press, New York; and Ausubel et al. (1987) .
  • recombinant immunoglobulins may be produced by methods known in the art, including but not limited to the methods described in U.S. Patent No. 4,816,567.
  • Antibodies specific for proteinases may be useful, for example, as probes for screening cDNA expression libraries or for detecting the presence of a proteinase in a test sample.
  • the polypeptides and antibodies will be labeled by joining, either covalently or noncovalently, a substance which provides a detectable signal.
  • Suitable labels include but are not limited to radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. United States Patents describing the use of such labels include but are not limited to Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
  • Antibodies specific for proteinases and capable of inhibiting proteinase activity may be useful in treating animals including man suffering from the effects of pollen proteinases. Such antibodies can be obtained by the methods described above and subsequently screening the pollen proteinase specific antibodies for their ability to inhibit proteinase activity.
  • compositions and vaccine preparations comprising substantially purified proteinase(s) derived from a pollen(s) and a suitable carrier therefor are provided.
  • Such vaccines are useful, for example, in immunizing an animal, including humans, against an allergic response to pollens.
  • the vaccine preparations comprise an immunogenic amount of at least one proteinase or immunogenic fragments or subunits thereof.
  • Such vaccines may comprise one or more proteinases, or a proteinase in combination with another protein or other immunogen.
  • immunogenic amount is meant an amount capable of eliciting the production of antibodies directed against the proteinase in an individual to which the vaccine has been administered.
  • Immunogenic carriers may be used to enhance the immunogenicity of the proteinases.
  • Such carriers include, but are not limited to, proteins and polysaccharides, liposomes, and bacterial cells and membranes.
  • Protein carriers may be joined to the proteinases to form fusion proteins by recombinant or synthetic means or by chemical coupling. Useful carriers and means of coupling such carriers to polypeptide antigens are known in the art.
  • the vaccines may be formulated by any of the means known in the art. Such vaccines are typically prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also, for example, be emulsified, or the protein encapsulated in liposomes.
  • the active immunogenic ingredients are often mixed with excipients or carriers which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • concentration of the immunogenic polypeptide in injectable formulations will usually be in the range of 0.2 to 5 mg/ml.
  • the vaccines may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • adjuvants which may be effective include, but are not limited to: aluminum hydroxide; N- acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) ; N- acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP) ; N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2- (1' -2' -dipalmitoyl-sn-glycero- 3-hydroxyphosphoryloxy) -ethylamine (CGP 19835A, referred to as MTP-PE) ; and RIBI, which contains three components extracted from thr-MDP
  • the effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against the immunogen resulting from administration of the immunogen in vaccines which are also comprised of the various adjuvants. Such additional formulations and modes of administration as are known in the art may also be used.
  • the proteinases and fragments thereof may be formulated into vaccines as neutral or salt forms.
  • Pharmaceutically acceptable salts include but are not limited to the acid addition salts (formed with free amino groups of the peptide) which are formed with inorganic acids, e.g., hydrochloric acid or phosphoric acids; and organic acids, e.g., acetic, oxalic, tartaric, or maleic acid.
  • Salts formed with the free carboxyl groups may also be derived from inorganic bases, e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases, e.g., isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, and procaine.
  • inorganic bases e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides
  • organic bases e.g., isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, and procaine.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.
  • the quantity tn be administered which is generally in the range of - out 100 to 1,000 ⁇ g of protein per dose, more generally in the range of about 5 to 500 ⁇ g of protein per dose, depends on the subject to be treated, the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of the active ingredient required to be administered may depend on the judgment of the physician and may be peculiar to each individual, but such a determination is within the skill of such a physician.
  • the vaccine may be given in a single dose or multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of vaccination may include 1 to 10 or more separate doses, followed by other doses administered at subsequent time intervals as required to maintain and or reinforce the immune response, e.g., at l to 4 months for a second dose, and if needed, a subsequent dose(s) after several months.
  • a method of monitoring the exposure of an animal to a proteinase is provided. Such monitoring methods are useful, for example, in determining whether pollen spores from which the proteinases of the present invention are derived are responsible for an allergic condition, or for monitoring the progress of a therapy designed to lessen the symptoms of such an allergic condition.
  • a biological sample obtained from the animal e.g., blood, saliva, tissue
  • a proteinase or portions thereof under conditions suitable for antibody-antigen interactions.
  • the detection of the formation of such interactions is indicative of prior exposure of the animal and the subsequent development of an immune response to the proteinase.
  • tests include but are not limited to radioallergosorbent tests (RAST) and enzyme- linked immunosorbent assays (ELISA) .
  • the subject may be exposed to a proteinase and the subsequent reaction monitored.
  • Such exposure may be cutaneously (e.g., by application to the skin via pricking or scratching), intracutaneously (e.g., via intracutaneous injection) or introduced in the form of an aerosol (generally an aqueous aerosol) into the nasal or bronchial passages (nasoprovocation or bronchoprovocation, respectively) , using methods well known in the art.
  • Typical reactions e.g., a wheal and erythema in skin testing, or asthma or rhinitis in naso- or bronchoprovocation, indicate an immunological response to the proteinase. See, e.g., Basic and Clinical Immunology. 6th ed., Stites et al., eds., (Appleton & Lange, 1987) , pp. 436-438, for a general description.
  • the proteinases may also be used in methods of identifying agents that modulate proteinase activity, whether by acting on the proteinase itself or preventing the interaction of a proteinase with a cell of an allergic individual.
  • One such method comprises the steps of incubating a proteinase with a putative therapeutic agent; determining the activity of the proteinase incubated with the agent; and comparing the activity obtained with the activity of a control sample of proteinase that has not been incubated with the agent.
  • immunotherapeutic methods in which the proteinase is repeatedly injected in increasing dosage over a prolonged period of time in order to reduce the symptoms of allergic rhinitis in patients with pollen allergies are provided. See, e.g., Stites et al. (1987), p. 442, for a general description.
  • methods of treating or ameliorating the affects of pollen proteinases on animals including humans include administering to the animal an effective amount of a physiologically acceptable pollen proteinase inhibitor.
  • Known proteinase inhibitors are generally not physiologically acceptable, acceptable inhibitors will include agents that inhibit the pollen proteinases but do not affect, or affect only marginally, the activity of endogenous proteinases.
  • inhibitors can be obtained from a variety of sources includir ⁇ , but not limited to, inhibitory antibodies and small mc ecules.
  • the inhibitors can be administered by a variety of methods including, but not limited to, topically, via aerosol to the nasal passages or lungs, gastrointestinally, subdermally, intravenously and intraperitoneally.
  • the inhibitors can be administered as needed, particularly when applied topically or via aerosol. These methods of administration are known in the art and will not be described in detail herein.
  • proteinases from this novel family have also been purified from ragweed, almond and typha.
  • the supernatant was brought to 30% saturation with solid ammonium sulfate, allowed to sit for 10 min, and then centrifuged at 48,200 x g for 20 min. The supernatant was then brought to 60% saturation with solid ammonium sulfate, allowed to sit for 10 min, and centrifuged as above.
  • the precipitate was dissolved in 100 ml of 0.02 M Bis-Tris, pH 6.5, containing 5 mM CaCl 2 , and dialyzed with two changes of the same buffer for four hours.
  • the dialyzed protein solution was brought to pH 4.5 with 0.4 M sodium acetate buffer, pH 3.8, and centrifuged at 48,200 x g for 15 min.
  • the supernatant was brought to pH 6.5 with 1.0 M Tris-HCl, pH 8.0 and dialyzed against 0.02 M Bis-Tris, pH 6.5, containing 5 mM CaCl ⁇ for 24 hr with two changes of buffer.
  • the dialysate was put onto a Cibacron Blue Sepharose column (3.0 x 27.0 cm (190 ml volume)) equilibrated with 0.02 M Bis-Tris buffer, pH 6.5, containing 5 mM CaCl2 and the column was washed with the same buffer. Collection was started immediately because the proteinase elutes in the void volume. Assays for proteinase and amidase activity were performed, as described in Example 5, on individual fractions and the peak of activity retained. The active fractions were pool.d and applied to a DEAE-Sephacel column (3.0 x 18.0 cm (127 ml vol.)) and washed until the A 28 0 nm was less than 0.050.
  • Sepharose column were pooled, concentrated on an Amicon filter to approximately 7.0 ml and applied to a Sephadex G-150 column (2.2 x 90.0 cm (342 ml column volume)), equilibrated with 0.02 M Bis-Tris, pH 6.5, 5 mM CaCl 2 , 0.2 M NaCl. The protein was eluted with the same buffer. Active fractions were pooled, concentrated to 5-10 ml, dialyzed against 0.02 M Bis-Tris, pH 7.0, 5 mM CaCl 2 and applied to a Mono-Q-ion-exchange column in association with a Fast Protein Liquid Chromatography (FPLC) system.
  • FPLC Fast Protein Liquid Chromatography
  • the proteinase purified as described in Example 1 has a varying apparent MW, depending on the mechanism used for its measurement.
  • SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • the MW was also determined by gel filtration using the FPLC system.
  • the MW appeared to be near 67 kD, since the peak of mesquite proteinase eluted in the same position as bovine serum albumin.
  • the hydrodynamics of the protein are such as to give aberrant MW values, depending on the method used for this measurement.
  • the proteinases have a blocked amino terminus, since no amino acid residues could be detected after ten cycles using a protein sequencer. Protein sequencing was thus performed by first cleaving the proteinases into small fragments and sequencing the fragments.
  • TPCK- treated bovine trypsin (at a ratio of 1:50, trypsin to proteinase) together with 5 mM CaCl was added to the dialyzed proteinase, and the mixture was incubated for 6 hr, then lyophilized.
  • the lyophilized sample was brought up to 1.0 ml with HPLC-grade water and run on a Waters Associates HPLC using a Micro Pak SP column. The peptide peaks were collected and lyophilized for sequencing.
  • cyanogen bromide (CNBr) digestion 200 ⁇ g (2.2 nmol) mesquite pollen proteinase was incubated in 700 ⁇ l containing 300 ⁇ g of CNBr (1:100 molar ratio of number of methionine resides in proteinase to CNBr) and 70% formic acid at 20°C for 20 hrs. in the dark flushing with nitrogen.
  • the sample was diluted ten- fold with HPLC-grade water and lyophilized.
  • the lyophilized sample was solubilized in sample buffer, run on SDS-PAGE followed by electroelution from a PVDP membrane which was stained for protein and the bands cut out for sequencing. Sequencing was performed using the Applied
  • IKEDDTTAPLR tryptic digest
  • DEILRPDXAXLXRLGTEQX cyanogen bromide digest
  • the cDNA library was used to generate a probe by amplifying a sequence with PCR using degenerate primers based on the known amino acid sequences of tryptic peptide sequences of mesquite pollen proteinase.
  • the primers were made on ABI Model No. 394 DNA/RNA synthesizer by phosphoramidite chemistry in accordance with the manufacturer's instructions.
  • the following nucleotide, MK-39-50, a 256-fold degenerate primer, corresponds to the coding strand. - 3.1 -
  • nucleotide MK-42-44, a 32-fold degenerate primer corresponds to the non-coding strand.
  • Both primers include inosines at wobble positions encoded by 4 nucleotides as well as EcoRl restriction endonuclease sites to facilitate cloning of PCR fragments.
  • Hot start PCR was employed to reduce non ⁇ specific annealing as follows. 100 pmol each primer plus dNTPs at a final concentration of 200 ⁇ M, 2.5 mM MgCl 2 and IX PCR Buffer were heated to 80°C with one Ampliwax bead. The reaction was cooled to 25°C and the remaining 5 ⁇ l first strand cDNA template, 2 units Taql Polymerase and water to 100 ⁇ l were added. The reaction was cycled 10 times at 94°C, 1 min then 32°, 10 min followed by 25 cycles at 94°C, 1 min; 55°C, 1 min; 72°C, 1 min with a 7 min 72°C final polish.
  • a PCR product of 300 bp was identified by southern blot analysis with the following probe, HG-3-23, a 12-fold degenerate non-coding strand oligomer based on a tryptic peptide
  • This 300 bp PCR fragment was cloned into the EcoRI site of pCR-script (Stratagene) using the EcoRI sites generated by the PCR primers, according to the manufacturer's instructions.
  • This clone, designated A6, was identified by dot and southern blot with probe HG-3- 23. Sequence analysis showed this clone to contain the 3 regions used as primers and probe.
  • the mesquite pollen library was screened with the 300 bp EcoRI fragment from clone A6 to identify a partial cDNA clone. A 2.1 Kb cDNA was identified.
  • Figure 2 shows the restriction endonuclease map of this clone, designated MPP1-1 (1-1) .
  • MPP8-3 8-3) .
  • Figure 2 shows the restriction endonuclease map of MPP8-3. This clone appears to have been internally primed in an A rich region and, as shown in Figure 2, extends through the putative initiator methionine at the 5' end and overlaps MPPl-1 at a unique EcoRI site close to the 3' end.
  • GAG G-5A CTT AAA GTT TTG ACA CCA CGC ATG GAG GGT ATT GAT ACA Glu Gly Leu Lys Val Leu Thr Pro Arg Met Glu Gly He Asp Thr>
  • the MPP cDNA encodes one open reading frame of 772 amino acid residues.
  • the predicted molecular weight of the full length protein is 88 kD with a predicted pi of 5.3.
  • pYT is a 15.2 Kb vector containing the yeast 2 ⁇ m sequence for autonomous replication, URA3 and LEU2d genes for selection in yeast, and the pBR322 sequences containing both the E. coli origin of replication and the ampicillin resistance gene.
  • pYT also contains the a-factor terminator required for translation termination downstream from BamHI and Sail unique cloning sites.
  • ADH2 glucose regulatable alcohol dehydrogenase II
  • the ADH2 promoter was cloned by PCR and ligated into pBluescript Notl/Xhol restriction endonuclease sites. Also included in the PCR fragment is the cloning site Xbal at the 3' end of the promoter, for in-frame insertion of the gene to be expressed.
  • the entire cassette can be excised at the BamHI site 5' of the ADH2 promoter and the Xhol site at the 3' end of MPP and cloned into pYT at the BamHI/Sall sites to obtain plasmid MPPpYT.
  • the pBluescript construct is shown in Figure 3, and the PYT construct is shown in Figure 4.
  • the MPPpYT plasmid was used to transform the yeast strain designated LXR1 ( [cir°] , MATa,leu2,trpl, ura3-52, prbl-1122,pep4-3, prcl-407) , an endogenous plasmid cured strain of BJ2168 (on deposit at the Berkeley Type Culture Collection) by the spheroplast method using uracil selection as described by Barr et al. (1987) Biotech. 5:486. Transformants were maintained o leucine minus plates. Transformants were grown in YEPD media for 48-72 hours at which point the glucose is depleted and the ADH2 promoter is derepressed to express the recombinant MPP.
  • the inhibitor profile of the protein purified as described in Example 1 was determined as follows.
  • a control consisting of an incubation mixture of pollen extract, buffer (0.1 M sodium phosphate, pH 7.4, 0.15 M NaCl), and Bz-L-Arg-pNA (substrate) in a total volume of 1.0 ml. The mixture was incubated for 10 min at room temperature. 50 ⁇ l glacial acetic acid was added to stop the reaction, after which readings were made a. ⁇ 405 nm to determine the appearance of yellow pigmentation indicating proteolytic cleavage. Release of P-nitroaniline can be quantified to indicate the extent of hydrolysis.
  • TPCK 1.0 mM
  • E-64 0.2 mM
  • P-aminobenzamidine 10 mM
  • aprotonin 0.5 mg/ml
  • ovomucoid 0.5 mg/ml
  • SBTI 0.5 mg/ml
  • BTI 0.5 mg/ml
  • 2-1-PI 1.2 nmol
  • the assay consisted of incubation mixtures of 0.4 ml of pollen extract, 0.1 ml of buffer (0.1 M sodium phosphate, pH 7.4, 0.15 M NaCl) and 0.25 ml 3% azocasein. The mixture was incubated for either 24 or 48 hrs at 37°C, at which time 0.75 ml 10% trichloroacetic acid (TCA) was added. Inhibitor (5 mM, 0.4 ml) was added to the basic incubation mixture for 5 to 10 min prior to addition of the azocasein solution. Controls of azocasein alone and extracts alone were also performed. When proteolytic cleavage occurs, indicating enzyme activity, soluble yellow color remains in the supernatant and casein and uncleaved azocasein remain in the precipitates.
  • buffer 0.1 M sodium phosphate, pH 7.4, 0.15 M NaCl
  • Inhibitor 5 mM, 0.4 ml
  • Table 1 depicts the inhibition spectra of trypsin- like activity in selected pollens and Table 2 depicts the inhibition spectra of caseinolytic activity in pollen.
  • the proteinases are not affected by any of the common proteinase inhibitors. These include ⁇ -l-PI, soybean trypsin inhibitor, lima bean trypsin inhibitor, tomato trypsin inhibitors I, II and PCI, potato trypsin inhibitor PCT1 and tobacco inhibitor Til, tosyl-L- phenylalanine chloromethyl ketone (TPCK) , E-64 (trans- EpoxySuccinyl-leucylamido- (4-guanido) butaine) PMSF, ethylenediaminetetraacetic acid EDTA, benzamidine, p- aminobenzamidine and the Kunitz basic pancreatic trypsin inhibitor. The tomato, potato and tobacco inhibitors were tested later on the purified proteinases and found to be inactive.
  • TPCK tosyl-L- phenylalanine chloromethyl ketone
  • E-64 trans- EpoxySuccinyl-leucylamido- (4-guanido)
  • Fan Palm Wolfberry Japanese cedar* The crude preparations were made as follows: 1.0 g pollen were mixed with 12.0 ml 0.1 M Tris-HCl, pH 8.0; and 0.15 M NaCl, in the presence of 3.0 g 0.5 mm glass beads. The sample was vortexed for 10 min, followed by centrifugation for 20 min at 48,000 x g.
  • the crude fractions were then tested for activity in the following systems: azocasein degradation, hide powder, elastin esterase, cathepsin G esterase and trypsin-like activity.
  • the azocasein degradation assay was performed as described in Example 5 to measure the inhibition of caseinolytic activity described. The results obtained are presented in Table 4. In Table 4 the plants labeled with the asterisks are weakly or non-allergenic.
  • the hide powder assay was performed as follows: 10 mg of blue hide powder was mixed with 0.8 ml buffer (1.0 M Tris-HCl, pH 8.0, 0.15 M NaCl, 1% Brij) and 0.4 ml of pollen extract. Controls of hide powder or pollen extracts alone were also used. Incubation was for 2.5, 5.0, and 24 hrs at 37°C with the sample being continuously shaken. At the end of a given time period the material was allowed to settle and the color development in the supernatant was read at 595 nm. The results obtained are presented in Table 5. In Table 5 the plants marked with the asterisks are weakly or non- allergenic.
  • the blue hide powder is insoluble but, if the enzyme has activity, there will be proteolytic release of peptides bound to the dye, i.e., soluble peptide fragments containing the dye will be quantita ⁇ tively measurable as an indication of the effectiveness of the proteinase being tested.
  • the elastin esterase assay was performed as follows: Zero to 0.5 ml of pollen extract or purified enzyme was incubated in 0.1 M sodium phosphate buffer, pH 7.4, 0.15 M NaCl and 0.04 ml of Suc-Ala-Ala-Ala-pNA (5 mM) (Sigma) for 20 min. The absorbance at 405 nm was then read as a measure of substrate digestion as described above. The results obtained are presented in Table 6. In Table 6 the plants marked with the asterisks are weakly or non-allergenic.
  • the trypsin-like and plasma kallikrein activity assay was performed as follows: Zero to 0.5 ml of pollen extract or purified enzyme was incubated in 0.1 M sodium phosphate buffer, pH 7.4, 0.15 M NaCl and 40 ⁇ l of Pro- Phe-Arg-pNA (5 mM) (Sigma) for 10 min. The absorbance at 405 nm was then read as a measure of substrate digestion. The results obtained are presented in Table 8. In Table 8 the plants marked with the asterisks are weakly or non- allergenic
  • the mesquite pollen proteinase purified from pollen was tested to determine its amino acid residue specificity in order to more fully characterize its activity.
  • the protocol to measure the amino acid residue specificity used peptides of known sequence and was as follows. 1.0 ml of 25 mM ammonium bicarbonate buffer, pH 7.8; 5 mM calcium chloride; and 0.125% sodium azide; was added to 250 ⁇ g of either insulin; mellitin; or dynorphin fragment 1-13 and mixed well. 80 ⁇ l was removed at time zero while 80 ⁇ l was also incubated overnight at 37°C.
  • the protocol for activation of prothrombin was as follows: 0.74, 1.48 or 2.22 ⁇ g of mesquite proteinase purified as described in Example 1 was incubated with 10 or 20 ⁇ g of prothrombin (Calbiochem) in 0.1 M Tris-Hcl, pH 8.0; 0.15 M NaCl; and 5 mM calcium chloride in a final volume cf 0.2 ml for up to 90 min. Then, 0.8 ml of the same buffer and H-D-Phe-Plp-Arg-pNA (Kabi Pharmaceuticals) , a substrate for thrombin, were added. After 10 min incubation, the reaction was stopped with the addition of acetic acid and the absorbance measured at 405 nm. Since the mesquite proteinase also cleaves this substrate, although much more slowly, controls without prothrombin and also controls without proteinase were run and the rates of hydrolysis subtracted from that found for the mixture of proteinase and prothrombin.
  • Active proteinase fractions were found to hydrolyze both synthetic substrates and proteins specifically after arginine residues and, to a much lesser extent, lysine residues. Proteins tested, including mellitin and the oxidized insulin B chain, were not hydrolyzed by the enzyme. Nor was there any activation of trypsinogen which requires cleavage at a Lys-lie bond. However, the small peptide fragment of dynorphin (1-13) was cleaved between an Arg-Arg residue and an Arg-lie residue.
  • PK is activated to kallikrein which in turn activates kininogen to bradykinin, a vasodilator.
  • Bradykinin is responsible for some allergic reaction symptoms.
  • the proteinases activate PK in Hageman factor-deficient plasma.
  • the enzyme may be indirectly responsible for the generation of the vasoagent bradykinin.
  • the assay was performed by adding 60 ⁇ l of a 1:25 dilution of the crude pollen extracts prepared as described in Example 5 to 60 ⁇ l of PK (a gift from Dr. Bruce Zoran, Scripps Research Institute, LaJolla, California) incubated for 30 min at 37°C in a buffer containing 0.1 M NaP0 4 buffer, pH 7.4; 0.15 M NaCl. The samples were then split into two equal aliquots and one half was treated with 0.3 ⁇ g SBTI (Sigma) and the other half with buffer.
  • PK a gift from Dr. Bruce Zoran, Scripps Research Institute, LaJolla, California
  • Guinea pigs were anaesthetized using 80 mg/kg of ketamine, after which they were injected with Evans Blue Dye (30 mg/kg) in a metatarsal vein. Proteinase solutions were first subject to a 30 min incubation with human plasma. The preincubated proteinase solutions were then injected intradermally into the shaved back of the guinea pig. After 15 min the guinea pigs were euthanized by exsanguination using a carotid artery cut-down procedure while still under the Metofane (methoxyflurane) anaesthesia.
  • Metofane methoxyflurane
  • Yeast control and MPP expressing cultures were centrifuged and the cell pellets lysed in TE + 0.1% Triton X-100 by vortexing with 0.5 mM glass beads.
  • the clarified lysates were assayed for proteolytic activity against 5 mM pyro-GluGlyArgpNA (pNA) in 5 mM K 2 HP0 4 , 15 mM NaCl, pH 8 according to the method described in Example 6.
  • Samples were measured in a spectrophotometer at 405 nm in a kinetic assay to measure the rate of pNA conversion or simply incubated at 37° and a single reading taken.
  • the rMPP cultures grown at various pH levels showed 5-8.5 times more activity than the yeast control. The results obtained are shown in Figure 5.

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Abstract

L'invention concerne l'identification d'une nouvelle famille de protéines dérivées de pollens allergènes et ayant une activité du type sérine-protéinase. La protéine a un poids moléculaire d'environ 85-95 kD quand il est déterminé par électrophorèse sur gel polyacrylamide en présence de dodécyle sulfate de sodium, et un poids moléculaire d'environ 67 kD quand il est déterminé par une chromatographie en phase liquide rapide d'une protéine. Les protéines sont résistantes à l'inhibition par l'α-2-macroglobuline, à l'inhibiteur de l'α-1-protéinase, aux inhibiteurs de trypsine et sont sensibles à l'inhibition par le fluorure de phényle méthane sulfonyle, le difluorophénol, la benzamidine, un antalgique, la leupeptine et la cétone de tosyle-L-lysine chlorométhylée.
EP94921321A 1993-06-18 1994-06-17 Nouvelle famille de proteinases de pollen, procedes pour leur utilisation et compositions derivees Withdrawn EP0705435A4 (fr)

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AU2001253739A1 (en) * 2000-04-20 2001-11-07 Salman Baig Method for the identification of active site protease inactivators
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Title
BIOLOGICAL ABSTRACTS, vol. BA92, Philadelphia, PA, US; abstract no. 36277, THAKUR I S: "PURIFICATION AND CHARACTERIZATION OF THE GLYCOPROTEIN ALLERGEN FROM PROSOPIS -JULIFLORA POLLEN." XP002042005 & BIOCHEM INT 23 (3). 1991. 449-460. CODEN: BIINDF ISSN: 0158-5231, *
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