WO2000044780A1 - Polypeptide capable de se lier au mannose et utilisations - Google Patents

Polypeptide capable de se lier au mannose et utilisations Download PDF

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Publication number
WO2000044780A1
WO2000044780A1 PCT/AU2000/000039 AU0000039W WO0044780A1 WO 2000044780 A1 WO2000044780 A1 WO 2000044780A1 AU 0000039 W AU0000039 W AU 0000039W WO 0044780 A1 WO0044780 A1 WO 0044780A1
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Prior art keywords
polypeptide
cells
plant
mannose
sequence
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PCT/AU2000/000039
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English (en)
Inventor
Therese Rosa Clark
Robyn Myra Minchinton
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Australian Red Cross Blood Service
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Priority to AU26466/00A priority Critical patent/AU2646600A/en
Publication of WO2000044780A1 publication Critical patent/WO2000044780A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C07K14/42—Lectins, e.g. concanavalin, phytohaemagglutinin
    • 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 generally to a novel polypeptide and to derivatives, homologues, analogues, mimetics and functional equivalents thereof capable of interaction with surface glycoproteins on polymorphonuclear cells. More particularly, the present invention provides a polypeptide capable of binding mannose and interacting with glycoproteins on neutrophils or neutrophil precursor cells.
  • the polypeptide of the present invention preferably exhibits lectin-like properties and in a most preferred embodiment, corresponds to a plant lectin.
  • the polypeptide of the present invention is useful inter alia in targeting neutrophil glycoproteins in a range of therapeutic and diagnostic applications.
  • polypeptide of the present invention and its derivatives, homologues, analogues, mimetics and functional equivalents are useful in assessing the function, make-up and status of the immune system such as the functional capacity of polymorphonuclear cells. They are also useful in "typing" polymorphonuclear cells.
  • polypeptide of the present invention and/or its derivatives, homologues, analogues, mimetics and functional equivalents thereof are useful in the generation of transgenic plants which exhibit enhanced resistance to micro-organisms, fungi viruses and insects.
  • Plants have provided a useful environment in which to search for natural products. However, despite the identification of a range of plant-derived molecules, there is still a need to identify molecules which interact with cellular receptors and ligands. The availability of such molecules would assist in the understanding of the role certain cells have in diseases and other physiological processes.
  • neutrophils are required for cellular functions such as the passage of neutrophils along blood vessel endothelium, chemotaxis of neutrophils across vessel endothelium into infected and inflamed body tissues and bacterial attachment to neutrophil surfaces (Berman & Muller, 1995; Hogg & Berlin, 1995; De Haas et al, 1995). Individually or in cooperation, neutrophil glycoproteins also mediate signalling from the cell's exterior to its cytosol to initiate generation of peroxides for bacterial killing (Petty & Todd, 1996; Zhou et al, 1993). These basic neutrophil functions constitute some of the primary defences against bacterial infection.
  • neutrophils also carry the blood group antigens specific to this human cell type (Clay & Stoncek, 1994, de Haas et al, 1995; Stoncek et al, 1994).
  • Neutrophil blood groups are clinically important and have been implicated in the pathogenesis of severe, immune neutropenias with associated local and generalised bacterial infections (Stroncek et al, 1994).
  • Life threatening transfusion reactions in which lung function is compromised have also been linked with interactions between neutrophil antigens and their corresponding antibodies (Bux et al, 1996; Popovsky et al 1992).
  • the inventors sought to identify plant products capable of interaction with surface molecules on polymo ⁇ honuclear cells.
  • the inventors undertook a screening program of plant species and isolated, purified and characterised a novel lectin and molecule. It was determined that the seeds of Hernandia moerenhoutiana subspecies samoensii contain a novel mannose binding lectin molecule capable of activating human neutrophils in an agglutination-aggregation test. The availability of this novel lectin permits a range of agents and assays useful in the study of neutrophil structure and function.
  • the lectin molecule of the present invention and agonists and antagonists thereof are also useful in a range of therapeutic and diagnostic applications.
  • one aspect of the present invention is directed to a polypeptide or derivative, homologue, analogue, mimetic or functional equivalent thereof wherein said polypeptide is isolatable from a species of the plant Hernandia or a physiological, biochemical or genetic relative thereof and which polypeptide is capable of binding to and/or interaction with mannose or a sugar chemically related to mannose.
  • polypeptide or derivative, homologue, analogue, mimetic or functional equivalent thereof wherein said polypeptide is isolatable from a species of the plant Hernandia or a physiological, biochemical or genetic relative thereof and which polypeptide is capable of activating polymo ⁇ honuclear cells from an animal or avian species.
  • the present invention contemplates a polypeptide or a derivative, homologue, analogue, mimetic or functional equivalent thereof which polypeptide comprises an amino acid sequence substantially as set forth in ⁇ 400>2 or an amino acid sequence with at least about 50% similarity to the amino acid sequence set forth in ⁇ 400>2.
  • polypeptide or derivative, homologue, analogue, mimetic or functional equivalent thereof where said polypeptide is isolatable from a species of the plant Hernandia or a physiological, biochemical or genetic relative thereof and which polypeptide is capable of interaction with mannose or a sugar chemically related to mannose and is capable of activating polymo ⁇ honuclear cells from an animal or avian species and which polypeptide comprises an amino acid sequence substantially as set forth in ⁇ 400>2 or an amino acid sequence with at least about 50% similarity to the amino acid sequence set forth in ⁇ 400>2.
  • polypeptide of the present invention or its derivative, homologue, analogue, mimetic or functional equivalent thereof is preferably in isolated or purified form. This means that the molecule has undergone at least one purification step away from other molecules.
  • the polypeptide of the present invention is in multimeric form such as a dimer.
  • the present invention extends to other forms of the molecule such as in monomeric form.
  • polymorphonuclear cell refers to a type of phagocytic white blood cell characterised by a multipartite nucleus and which ingests invading organisms and includes cells such as neutrophils and granulocytes.
  • reference herein to a polymo ⁇ honuclear cell means reference to a neutrophil.
  • similarity includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, "similarity” includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity.
  • references to describe sequence relationships between two or more polynucleotides or polype tides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length.
  • two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a comparison window refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence.
  • the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • sequence similarity and “sequence identity” as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by- nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g.
  • sequence identity will be understood to mean the "match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
  • activating includes, for example, inducing migration and/or agglutination of polymo ⁇ honuclear cells in vitro and can be measured using a granulocyte agglutination tests (GAT).
  • GAT granulocyte agglutination tests
  • the polypeptide binds to the neutrophil membrane glycoprotein CD9 (Aager et al. , 1997) or its homologue.
  • the CD9 molecule is involved in cell adhesion and migration, platelet activation and may also be important in ion transport (Aager et al. , 1997; Wright & Tomlinson, 1994).
  • another aspect of the present invention is directed to a polypeptide or derivative, homologue, analogue, mimetic or functional equivalent thereof where said polypeptide is isolatable from a species of the plant Hernandia or a physiological, biochemical or genetic relative thereof and which polypeptide is capable of interaction with mannose or a sugar chemically related to mannose and which polypeptide is capable of interaction with glycoprotein CD9 or its immunological homologue.
  • polypeptide or derivative, homologue, analogue, mimetic or functional equivalent thereof where said polypeptide is isolatable from a species of the plant Hernandia or a physiological, biochemical or genetic relative thereof and which polypeptide is capable of activating neutrophil cells from an animal or avian species and which polypeptide is capable of interaction with glycoprotein CD9 or its immunological homologue.
  • the present invention contemplates a polypeptide or a derivative, homologue, analogue, mimetic or functional equivalent thereof which polypeptide comprises an amino acid sequence substantially as set forth in ⁇ 400>2 or an amino acid sequence with at least about 50% similarity to the amino acid sequence set forth in ⁇ 400>2 and which polypeptide is capable of interaction with glycoprotein CD9 or its immunological homologue.
  • polypeptide or derivative, homologue, analogue, mimetic or functional equivalent thereof where said polypeptide is isolatable from a species of the plant Hernandia or a physiological, biochemical or genetic relative thereof and which polypeptide is capable of interaction with mannose or a sugar chemically related to mannose and is capable of activating neutrophil cells from an animal or avian species and comprises an amino acid sequence substantially as set forth in ⁇ 400 > 2 or an amino acid sequence with at least about 50% similarity to the amino acid sequence set forth in ⁇ 400>2 and which polypeptide is capable of interaction with glycoprotein CD9 or its immunological homologue.
  • CD9 glycoprotein is considered to belong to a super family of proteins which span the neutrophil cellular membrane.
  • CD9 has been characterised by its ability to bind murine monoclonal antibody (Aager et al., 1997).
  • derivative as used herein is meant any single or multiple amino acid substitutions, deletions and/or additions relative to the naturally-occurring polypeptide for Hernandia species such as exemplified in ⁇ 400>2.
  • derivative includes parts, fragments, portions, homologues and further extends to functional equivalents and homologues as well as monomeric and heteromultimeric forms of the molecule.
  • Homologues include functionally, structurally or sterochemically similar polypeptides from other related plant species.
  • Analogues and mimetics include molecules which contain non-naturally occurring amino acids as well as molecules which do not contain amino acids but nevertheless behave functionally the same as the polypeptide.
  • Natural product screening is one useful strategy for identifying analogues and mimetics. Natural product screening involves screening environments such as bacteria, plants, riverbeds, seabeds, aquatic environments, coral and antarctic or arctic environments for naturally occurring molecules which mimic, agonise or antagonise the subject polypeptide of the present invention.
  • Analogues of the subject peptides contemplated herein include modifications to side chains, inco ⁇ oration of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the peptide molecule or their analogues.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5- phosphate followed by reduction with NaBH 4 .
  • modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS
  • the guamdine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4- chloromercuriphenylsulphomc acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of inco ⁇ orating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5- phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D- isomers of amino acids.
  • a list of unnatural amino acid contemplated herein is shown in Table 1.
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • peptides can be conformationally constrained by, for example, inco ⁇ oration of C ⁇ and ⁇ -methylamino acids, introduction of double bonds between C ⁇ and C p atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • the present invention further contemplates functional equivalents of the subject polypeptides.
  • Functional equivalents may not necessarily be derived from the polypeptides themselves but may share certain conformational similarities.
  • functional equivalents may be specifically designed to mimic certain physiochemical properties of the polypeptides.
  • Functional equivalents may be chemically synthesised or may be detected following, for example, natural product screening.
  • polypeptide of the present invention should be read as including reference to all forms of the polypeptide including, by way of example, isoforms, monomer ic, dimeric and multimeric forms and peptide fragments of the polypeptide.
  • the present invention is exemplified with respect to a novel lectin polypeptide from seeds of Hernandia moerenhoutiana subspecies samoensii.
  • the present invention extends to any similar or equivalent polypeptides isolated from any other part of the plant or any other related plant species.
  • the term "related" in this context means a plant related at the genetic, immunological and/or biochemical level to H. moerenhoutiana subspecies samoensii.
  • the polypeptide of the present invention may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the polypeptide such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • the polypeptide is unglycosylated.
  • the polypeptide of the present invention may be produced by chemical synthetic techniques or may be produced by recombinant DNA technology.
  • the polypeptide may also be a fragment of the lectin molecule.
  • the fragments may be naturally occurring fragments or generated by the action of proteases, peptidases, amidases, lysins or other enzymes as well as by sonic disruption, heat, chemical disruption and/or shearing.
  • the preferred amino acid sequence of the polypeptide of the present invention is substantially as set forth in ⁇ 400>2.
  • polypeptide of the present invention may be accomplished by any suitable means such as by chromatographic separation, for example using affinity chromatography. Many other techniques are available including HPLC and PAGE.
  • the polypeptide can be partially sequenced and/or fragments produced and used directly as a source of polypeptides or as a template for polypeptide synthesis.
  • partial N-terminal, C-terminal or internal amino acid sequence can be obtained in order to deduce a nucleotide sequence of oligonucleotide primers such as for hybridisation to cDNA libraries or for use in PCR.
  • Polypeptides may be synthesized by solid phase synthesis using F-moc chemistry as described by Ca ⁇ ino et al., (1991).
  • the polypeptide and fragments thereof may also be synthesized by alternative chemistries including, but not limited to, t-Boc chemistry as described in Stewart et al., (1985) or by either classical methods of liquid phase polypeptide synthesis.
  • the polypeptides of the present invention are useful in evaluating the makeup and nature of immune cells such as the activity of polymo ⁇ honuclear cells and in particular neutrophils by, for example, analysing migration, agglutination and/or aggregation in a subject such as a mammal (e.g. a human).
  • a mammal e.g. a human
  • non-human animals are also contemplated such as primates, livestock animals (e.g. sheep, cows, horses, donkeys, pigs), companion animals (e.g. dogs, cats), laboratory test animals (e.g. mice, rats, guinea pigs, rabbits, hamsters), captive wild animal (e.g. deer, foxes), caged birds (e.g.
  • the subject is a mammal. Most preferably, the subject is a human.
  • the subject polypeptides are also useful in typing such as blood group typing of polymo ⁇ honuclear cells.
  • polypeptide of the present invention is produced by recombinant means.
  • Another aspect of the present invention is directed to genetic sequences encoding the polypeptide herein described.
  • the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or a sequence complementary to sequence or sequences encoding a polypeptide or derivative, homologue, analogue, mimetic or functional equivalent thereof wherein said polypeptide is isolatable from a species of the plant Hernandia or a physiological, biochemical or genetic relative thereof and which polypeptide is capable of interaction with mannose or a sugar chemically related to mannose.
  • an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or a sequence complementary to sequence or sequences encoding a polypeptide or derivative, homologue, analogue, mimetic or functional equivalent thereof where said polypeptide is isolatable from a species of the plant Hernandia or a physiological, biochemical or genetic relative thereof and which polypeptide is capable of activating polymo ⁇ honuclear cells from an animal or avian species.
  • the present invention contemplates an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or a sequence complementary to sequence or sequences encoding a polypeptide or a derivative, homologue, analogue, mimetic or functional equivalent thereof which polypeptide comprises an amino acid sequence substantially as set forth in ⁇ 400 > 2 or an amino acid sequence with at least about 50% similarity to the amino acid sequence set forth in ⁇ 400> 2.
  • Another aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or a sequence complementary to sequence or sequences encoding a polypeptide or derivative, homologue, analogue, mimetic or functional equivalent thereof where said polypeptide is isolatable from a species of the plant Hernandia or a physiological, biochemical or genetic relative thereof and which polypeptide is capable of interaction with mannose or a sugar chemically related to mannose and is capable of activating polymo ⁇ honuclear cells from an animal or avian species and which polypeptide comprises an amino acid sequence substantially as set forth in ⁇ 400> 2 or an amino acid sequence with at least about 50% similarity to the amino acid sequence set forth in ⁇ 400 > 2.
  • the present invention provides a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in ⁇ 400> 1 or a complementary form thereof or a nucleotide having at least 60% similarity to ⁇ 400> 1 or a nucleotide sequence capable of hybridising to ⁇ 400> 1 under low stringency conditions.
  • the percentage similarity to amino acid or nucleotide sequences is at least about 70%, more preferably at least about 80% and still more preferably at least about 90-95% or above relative to ⁇ 400>2 or ⁇ 400> 1, respectively.
  • Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1M to at least about 2M salt for hybridization, and at least about 1M to at least about 2M salt for washing conditions.
  • low stringency is conducted at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions.
  • Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridization, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31 % v/v to at least about 50% v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridization, and at least about 0.01M to at least about 0.15M salt for washing conditions.
  • T m of a duplex DNA decreases by 1°C with every increase of 1 % in the number of mismatch base pairs (Bonner and Laskey, 1974).
  • Formamide is optional in these hybridization conditions.
  • particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1 % w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1 % w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1 % w/v SDS at a temperature of at least 65 °C.
  • the nucleic acid includes the naturally-occurring nucleotide sequence encoding the polypeptide of the present invention or may contain single or multiple nucleotide substitutions, deletions and/or additions to the naturally-occurring sequence.
  • the nucleic acid molecule of the present invention or its complementary form may also encode a "part" of the subject polypeptide, whether active or inactive, and such a nucleic acid molecule may be useful as an oligonucleotide probe or primer for polymerase chain reactions amongst other uses.
  • the nucleic acid molecule may be single or double stranded, linear or a covalenty closed circle.
  • the nucleic acid molecule may be alone or in association, combination or otherwise part of a vector such as an expression or purification vector.
  • Still another aspect of the present invention is directed to antibodies to the subject polypeptide or its derivatives, homologues, analogues, mimetics and functional equivalents thereof.
  • a derivative comprises a small peptide
  • such a molecule may first need to be associated with a carrier molecule in order to induce antibody production.
  • the antibodies of the present invention are particularly useful as therapeutic or diagnostic agents.
  • specific antibodies can be used to screen for polypeptides using immunoassays or used as antagonists to inhibit polypeptide activity under certain circumstances.
  • Techniques for such immunoassays are well known in the art and include, for example, sandwich assays and ELISA. Knowledge of polypeptide levels may be important for monitoring certain therapeutic protocols.
  • Antibodies to the polypeptides (or their derivatives, homologues, analogues, mimetics or chemical equivalents thereof) of the present invention may be monoclonal or polyclonal. Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody” is considered herein to include fragments and hybrids of antibodies.
  • specific antibodies can be used to screen for the subject polypeptide of the present invention.
  • the latter would be important, for example, as a means for screening for levels of polypeptides in a cell extract or other biological fluid or purifying polypeptides made by recombinant means from culture supernatant fluid.
  • second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody.
  • An antibody as contemplated herein includes any antibody specific to any region of polypeptide.
  • Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays.
  • the methods of obtaining both types of sera are well known in the art.
  • Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of polypeptide, or antigenic parts thereof, collecting serum from the animal and isolating specific sera by any of the known immunoadsorbent techniques.
  • antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
  • the use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.
  • Another aspect of the present invention contemplates a method for detecting a subject polypeptide in a biological sample from a plant extract or culture supernatant fluid or other source said method comprising contacting said biological sample with an antibody specific for said polypeptide or its derivative, homologue, analogue, mimetic or chemical equivalent thereof for a time and under conditions sufficient for a complex to form with said antibody, and then detecting said complex.
  • the presence of the sugar polypeptide may be accomplished in a number of ways such as by Western blotting and ELISA procedures.
  • a wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These, of course, include both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.
  • Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody.
  • the sample is one which might contain an analgesic peptide including cell extract, culture supernatant tissue biopsy, serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid.
  • the sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.
  • a first antibody having specificity for the polypeptide or antigenic parts thereof is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample.
  • an aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g. from room temperature to about 37°C) to allow binding of any subunit present in the antibody.
  • the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten.
  • the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.
  • An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody.
  • a second labelled antibody specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, luciferase glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase.
  • the enzyme-labelled antibody is added to the first antibody-peptide complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen- antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.
  • Reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest.
  • Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • polypeptide molecules of the present invention may themselves be labelled with a reporter molecule and used in place of antibodies.
  • tagged polypeptides are useful for detecting antigens and receptors on polymo ⁇ honuclear cells.
  • the present invention extends, therefore, to the ragged polypeptides.
  • another aspect of the present invention provides a method for evaluating immune cells including detecting antigens and receptors on polymo ⁇ honuclear cells said method comprising contacting a biological sample containing immune cells with polypeptides or derivatives, homologues, analogues, mimetics or functional equivalents thereof immobilized to a solid support in order for immune cells to bind to said polypeptides via mannose or a sugar chemically related to mannose and then detecting bound immune cells.
  • bound immune cells are detected by a labelled antibody directed to an antigen compound of the cells.
  • labelled lectins may be used to define the cells.
  • polypeptides of the present invention have a range of uses such as in the diagnosis and treatment of disorders associated with polymo ⁇ honuclear cells and in particular neutrophils or aberrations in such cells.
  • another aspect of the present invention contemplates a method of activating polymo ⁇ honuclear cells in an animal, such as a human, said method comprising contacting said cells with an effective amount of a polypeptide as herein before defined for a time and under conditions sufficient to effect polymo ⁇ honuclear cell activation.
  • the present invention contemplates a method of activating neutrophils said method of comprising contacting said neutrophils with an effective amount of a polypeptide as hereinbefore defined for a time under conditions sufficient to effect neutrophil activation.
  • a metliod of determimng the activity of a neutrophil said method comprising contacting said neutrophils with an effective amount of polypeptide as hereinbefore defined for a time under conditions sufficient to effect neutrophil activation and assessing the extent of activation.
  • the present invention contemplates a method of identifying an individual with improved neutrophil cell function said method comprising contacting a population of neutrophils with an effective amount of a polypeptide as hereinbefore defined and detecting the extend of activation.
  • the present invention provides a method for down-regulating neutrophil activity.
  • the polypeptides of the present invention may be used to bind but not to actuate to neutrophils.
  • the blocking phenomenon may be useful, for example, in the treatment of rheumatoid arthritis where it would be desirable to reduce neutrophils activation and/or migration of neutrophils into tissue.
  • polypeptides of the present invention are also useful in "typing" polymo ⁇ honuclear cells such as neutrophils and in determining the nature, functional capacity and status of the immune system through the neutrophil component of the immune cells population.
  • the polypeptides of the present invention can also be used to generate transgenic plants.
  • Reference to "transgenic plants” includes parts of transgenic plants as well as transgenic plant cells. Parts of plants includes flowers, stems, leaves, roots and reproductive portions, such as pollen and seeds. Extracts from the transgenic plants have a range of uses including providing an agent useful for protection against insect attack or bacterial, viral or fungal infection. Expression of the polypeptides as herein described in plants and plant cells also reduces the incidence of infestation by insects.
  • Reference to "insects” includes all stages in the lifecycle of an insect including larval, pupatal, hatchling, neonatal and instar stages. The insects may also be boring, sucking or chewing insects. However, any insect is contemplated by the present invention.
  • the carbohydrate specificity of the polypeptides of the present invention can also be used to target enzymes and allow for components of the infecting organism.
  • one aspect of the present invention contemplates a method of generating a transgenic plant with enhanced resistance to a micro-organism, fungus, virus or insect, said method comprising introducing into cells of a plant a genetic construct as hereinbefore defined, selecting for cells carrying said genetic construct and then regenerating a plant therefrom.
  • the means for introducing the genetic constructs may be by, for example, Agrobacterium-mediated transformation, electroporation, protoplast fusion or micro-particle bombardment amongst other means.
  • the resulting trangenic plants are genetically modified and produce, constitutively or following induction, a polypeptide as hereinbefore defined.
  • genetically modified is used in its broadest sense to include introducing exogenous genetic material such as in the generation of a transgenic plant.
  • the term also encompasses introducing antisense molecules, ribozymes and sense molecules such as for use in co-suppression.
  • a “mutation” includes the introduction of a single or multiple nucleotide substitution, addition and/or deletion.
  • the genetic material to be introduced may be a gene or may correspond to a gene or may be a gene fragment, segment, portion and/or a gene hybrid or fusion or a combination of genes.
  • the genes may be in monocistronic form or in multicistronic form.
  • the gene encoding the subject polypeptide may be use alone or in combination with two or more other genes such as genes encoding peptides, polypeptides, or genetic sequences which are beneficial for the action of the lectin polypeptide or which otherwise confer resistance of the plant to a particular infestation.
  • the multi-gene construct may be in monocistronic or polycistronic form.
  • useful other genes include genes encoding a proteinase inhibitor or precursor thereof.
  • An example of a proteinase inhibitor precursor is a serine proteinase inhibitor precursor described in International Patent Application No. PCT/AU93/00659 (International Publication No. WO 94/13810).
  • gene is used in its broadest sense and includes cDNA corresponding to the exons of a gene. Accordingly, reference herein to a “gene” is to be taken to include :-
  • a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); or
  • gene is also used to describe synthetic or fusion molecules encoding all or part of a functional product.
  • the genetic material is generally in the form of a genetic construct and comprises a gene or nucleic acid molecule to be introduced into a plant cell, operably linked to a promoter and optionally various regulatory sequences.
  • the genetic material of the present invention may comprise a sequence of nucleotides or be complementary to a sequence of nucleotides which comprise one or more of the following: a promoter sequence, a 5' non-coding region, a cw-regulatory region such as a functional binding site for transcriptional regulatory protein or translational regulatory protein, an upstream activator sequence, an enhancer element, a silencer element, a TATA box motif, a CCA AT box motif, or an upstream open reading frame, transcriptional start site, translational start site, and/or nucleotide sequence which encodes a leader sequence.
  • 5' non-coding region is used herein in its broadest context to include all nucleotide sequences which are derived from the upstream region of an expressible gene, other than those sequences which encode amino acid residues which comprise the polypeptide product of said gene, wherein the 5' non-coding region confers or activates or otherwise x facilitates, at least in part, expression of the gene.
  • a “promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e.
  • upstream activating sequences which alter gene expression in response to developmental and/or environmental stimuli, or in a tissue-specific or cell-type-specific manner.
  • a promoter is usually, but not necessarily, positioned upstream or 5', of a structural gene, the expression of which it regulates.
  • the regulatory elements comprising a promoter are usually positioned within 2kb of the start site of transcription of the gene.
  • promoter is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of a structural gene or other nucleic acid molecule, in a plant cell.
  • Preferred promoters according to the invention may contain additional copies of one or more specific regulatory elements to further enhance expression in a cell, and/or to alter the timing of expression of a structural gene to which it is operably connected.
  • operably connected means placing a structural gene under the regulatory control of a promoter which then controls expression of the gene. Promoters and the like are generally positioned 5' (upstream) to the genes which they control.
  • Figure 1 is a photographic representation of (a) SDS-PAGE and (b) native PAGE of the fractions eluted from mannose agarose.
  • Lane 1 shows the crude material: Lanes 2 and 3 show non-binding material washed off the column; Lanes 4-7 show lectin released upon addition of 0.3 M and 0.5 M mannose.
  • Figure 2 is a graphical representation of mass spectrometry of Hernandia lectin. Top: - reverse HPLC chromatogam; middle:- mass spectrum of predominant isoform at centre of peak (sum of two scans); bottom: -molecular mass (reconstructed).
  • Figure 3 is a representation of the results of N-Terminal Sequencing of Deglycosylated Hernandia bands H4, H6, H7 and H8 run on Native PAGE.
  • Figure 4 is a representation of the cDNA and deduced amino acid sequences for Hernandia lectin derived from mRNA. Below this is the sequence for Galanthus nivalis which shares 65% sequence homology with Hernandia lectin. (*) Denotes those residues responsible for mannose binding in Galanthus nivalis.
  • ( t ) denotes the site of post translation cleavage in Galanthus nivalis.
  • ( 1 ) denotes the putative cleavage site in the Hernandia lectin derived using mass spectrometry data.
  • Figure 5 is a photographic representation of a Western immunoblot of solubilised neutrophils run on SDS-PAGE under reducing (Lane 1) and Non-Reducing Conditions (Lane 2) probed with Hernandia lectin.
  • Figure 6A is a graphical representation showing mortality of Helicoverpa punctigera larvae fed on lectin and control diets. -•- control - ⁇ - + Lectin: Trial 1 -*- + Lectin: Trial 2
  • Figure 6B is a graphical representation showing growth of larvae fed on lectin and control diets.
  • Lectin Trial 2
  • Freshly harvested seeds were allowed to air dry for 3-4 days before further manipulation. Seeds were re-hydrated overnight in phosphate buffered saline (PBS pH 7.4) and blended to a pulp. Proteins were extracted by standing the buffered pulp overnight at 4°C. An initial 40% w/v ammonium sulphate precipitation of the solubilised material removed unwanted proteins. A second 80% ammonium sulphate cut of the supernatant precipitated the proteins of interest. The precipitate was solubilised in PBS and dialysed in several changes of PBS. Protein concentration was assayed using the Bio-RAD protein assay kit (BioRAD Laboratories, Hercules, CA) with IgG as the standard protein.
  • Bio-RAD protein assay kit BioRAD Laboratories, Hercules, CA
  • the crude extract was further purified using affinity chromatography.
  • the sugar specificity of Hernandia lectin was determined for a range of pure sugars by blocking the binding of the crude lectin to neutrophils in a granulocyte immunofluorescence test (see later).
  • Four milligram of crude Hernandia extract was applied to a 5 mL mannose-bound agarose column (Sigma, St Louis, MO) and 1.8 mg of protein was released with the addition of first, 0.3 M then 0.5 M Mannose (Sigma, St. Louis, MO). This represented a yield of 0.1 % of the total crude protein extracted from the Hernandia seeds.
  • An alternative method for lectin removal from Hernandia seeds involves a chloroform extraction.
  • the Hernandia seeds were soaked in water to soften the outer fibrous layer. This layer was removed by scraping with a scalpel. The remaining kernel was pulped in a blender. Approximately 41.3 g of seed flesh was pulped and soaked overnight in IL of PBS/ 0.1 % NaN 3 . The resultant slurry was centrifuged at 9550 ⁇ m at 4°C to remove the granular material. Approximately 700 mL of dark brown liquor was retrieved including a fatty surface layer. Fats and organic plant products were removed by adding 100 ml of chloroform to the protein solution.
  • the mixture was shaken thoroughly and placed at 4°C overnight to produce a hard white layer in the bottom of the vessel. Approximately 670 ml of clarified protein solution was easily removed and stored in frozen aliquots. Prior to affinity purification, the protein solution was centrifuged at 11,000 g and filtered (0.2 ⁇ M). At this stage, the protein concentration was 6.6 mg/mL. The mannose affinity purified lectin constitutes 6% of the total protein extract.
  • a commercial glycosylation detection kit (BioRAD Laboratories, Hercules, CA), relying on electroblot of SDS-PAGE separated proteins, was used to detect the presence of carbohydrates and sialic acid on the Hernandia proteins.
  • the kit was used according to manufacturer's instructions.
  • the crude extract, mannose affinity purified lectin and deglycosylated, affinity purified lectin were tested for biological activity by titrating solutions of known protein concentration in the granulocyte agglutination test (GAT) and granulocyte immunofluoresence test (GIFT) (McCulloch et al , 1988).
  • Biological activity of this lectin is represented by its ability to initiate active migration and agglutination of neutrophils in vitro and to bind directly to neutrophils in the immunofluorescence test, quantitated by flow cytometry.
  • Activity is expressed as agglutination score (0-3 +) in the GAT or in units of mean channel fluorescence (MCF) in the GIFT.
  • the protein concentration at which binding or agglutination ceases is significant.
  • 250 ⁇ L Hernandia lectin at 2 mg/mL was dialysed against pH 7.8. NaHCO 3 before exposure to 40 ⁇ l freshly prepared 2 mg/mL N-succinimido biotin (Sigma, St Louis, MO) at room temperature in darkness for 2 hours (Sigma, St. Louis, MO). Excess biotin was removed by dialysis against PBS. Binding was visualised with fluorescein labelled strep tavidin (Dakopatts, Ca ⁇ interia, CA).
  • the sugar blocking assay was based on the GIFT using the flow cytometer to quantitate the percentage decrease in binding (measured as MCF) of biotinylated Hernandia lectin to neutrophils caused by different concentrations and types of chemically pure monosaccharides (Sigma, St. Louis, MO).
  • MCF percentage decrease in binding
  • a "no-block" baseline MCF was established for Hernandia lectin using a "sham” blocking step PBS. Blocking tests were performed using 1 mg/mL Hernandia lectin incubated for 30 minutes at room temperature with 0.1 M, 0.25 M and 0.5 M solutions of sugars in PBS.
  • Sugars included were:- glucose, mannose, lactose, fucose, galactose, maltose, N-acetyl galactosamine and N-acetyl glucosamine. Reduction of the MCF greater than 50% was considered significant blocking.
  • the affinity purified Hernandia lectins were analysed by liquid chromatography/mass spectrometry (LC/MS) to determine the number of protein isoforms that were present and their respective molecular weights.
  • LC/MS liquid chromatography/mass spectrometry
  • a solution of the lectin in 20 mM phosphate buffer (10 ⁇ L at a protein concentration of about 0.5 mg/mL) was applied to a reverse phase MPLC column (Vydac C4, 300 ⁇ pore size, 5 ⁇ m particle size, (2.1 x 150mm) and was eluted at a flow rate of 130 ⁇ L/min with H 2 0 + 0.05 % v/v TFA for 2 min, followed by a linear gradient to 60% v/v acetonitrile: 40% v/v H 2 0 + 0.05% v/v TFA over a further 30 min.
  • the eluent flowed directly into a PE-SCIEX API-3 turbo assisted electrospray mass spectrometer and data were acquired from 900-1200 Da at intervals of 0.2 Da and a dwell time of 0.5 ms.
  • RNA samples from Hernandia moerenhoutiana were collected at various stages of development and immediately flash frozen in liquid nitrogen (Walling et al. , 1986). The frozen tissue was ground with mortar and pestle and weight aliquots of approximately 0.140 g were applied to the QLAGEN RNeasy plant mini kit (QIAGEN Pty. Ltd., Clifton Hill, Vic, 3068) for total RNA extraction. RNA product was eluted in 2 x 30 ⁇ L RNase free at H 2 0 at a concentration of 0.6 mg/ml. RNA samples were analysed for purity and integrity by 2% w/v NuSieve agarose gel and visualised using ethidium bromide.
  • First strand copy DNA was made using SuperscriptTM (II) RNase H Reverse Transcriptase with 5 ⁇ g of purified RNA according to manufacturers instructions.
  • 3 'RACE technology was used to provide an adapter sequence for the unknown 3' end of 5 cDNA : 5' CGCCTAGTT(TTT) 5 T 3' ( ⁇ 400> 8).
  • N-terminal amino acid analysis provided the 5 'end cDNA degenerate primers, HOI 5' GGIGAGCAGATGIIICCIGGICA 3' ( ⁇ 400 >4) , HO2: 5' GGIGAGCAAATGIIICCIGGICA 3' ( ⁇ 400 > 5), HO3 5' GGIGAACAGATGi ⁇ CCIGGICA 3' ( ⁇ 400>6), HO45' GGIGAACAAATGIIICCIGGICA 3' ( ⁇ 400 > 7).
  • Primers and oligo d(T) were supplied by Pacific Oligos.
  • Buffer II 500 mM KC1, lOOmM Tris pH 8.3, 6 ⁇ L MgCI 2 (25mM), (Roche), 2 ⁇ L dNTP (10 mM), 1 ⁇ L 5' primer (10 ⁇ M) and 1 ⁇ L 3' RACE adapter primer (10 ⁇ M), 2 ⁇ L cDNA (approximately 1 ⁇ g), 77 ⁇ L water and 1 ⁇ L Taq (added after the reaction had reached
  • the PCR was carried out using a Perkin Elmer 2400 with touchdown thermal cycling. The protocol started with 5 min at 94 °C during which time the Taq was added, followed by 40 cycles in total where denaturation of DNA was achieved by 15s. at 94 °C followed by a decreasing annealing temperature from 65 °C to 50 °C for 30 seconds over 30 cycles with 30s extension time of PCR product at 72°C. the last 10 cycles had a constant
  • Resultant purified products A, B and C were subjected to PCR amplification using the same reaction mix as above but replacing cDNA with 1 ⁇ L of PCR product: A, B at 1/lOdilution
  • the heating protocol was followed for Perkin Elmer 2400, i.e. 5s. at 50°C 10s at 94°C, 5s. at 50°C, 4 min at 60°C for 25 cycles.
  • the resultant purified products were directly sequenced using the forward primer HO2 and labelled with Big Dye terminators according to manufacturers instructions. Sequencing was performed using an ABI Prism sequencer.
  • biotinylated Hernandia lectin was used at 2 ⁇ g/mL to probe the blotted neutrophil proteins. Bound lectin was visualised with peroxidase labelled streptavidin (BioRAD Hercules, CA).
  • Figure la shows the SDS-PAGE of the original crude extract and the fractions eluted from the mannose affinity column.
  • Two significant proteins of approximate molecular weight 14.5 and 16.4 kDa were released by the addition of mannose to the affinity column. Together these proteins constituted more than half of the total protein present in the crude extract.
  • Figure lb (Lane 1) provides a comparison of major species present in the crude extract (no affinity purification) run under the same electrophoretic conditions.
  • Figure 4 illustrates the cDNA sequence and deduced amino acid sequence for Hernandia moerenhoutiana together with the sequence of a homologous lectin protein from Galanthus nivalis.
  • MCF glycosylated affinity purified lectin
  • the GAT assay is only semi-quantitative but demonstrates stimulation and activation of neutrophils as well as binding.
  • One mg/mL solutions of glycosylated and deglycosylated affinity purified lectin and crude protein extract all caused strong neutrophil agglutination (score 3+).
  • Table 2 highlights the sensitivity of Hernandia protein inhibition by 0.5 M mannose solution.
  • Hcoverpa punctigera larvae were raised on an artificial diet based on lima beans (Table 3). Lima beans were ground to a fine powder in a coffee bean grinder then combined with the wheatgerm, oils and 15 ml of water. The remaining water and agar were heated to 100°C to dissolve the agar, and added to the preparation. The mixture was cooled to 50 °C and the remaining ingredients were added and mixed well. The blended diet was poured into trays and after setting was used immediately or stored at -20 °C for no longer than two weeks. The test diet was supplemented with the lectin (0.3 % w/v) replacing casein (0.3 % w/v) in the control diet.
  • the amount of lectin (0.3 % w/v) used in this Example is comparable to the amount described by Fitches et al. (1997) in their test of the effect of snowdrop lectin on the development and growth of larvae of the tomato moth.

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Abstract

L'invention concerne de manière générale un nouveau polypeptide et des dérivés, des homologues, des analogues, des mimétiques et des équivalents fonctionnels de ce polypeptide, capables d'interagir avec les glycoprotéines de surface des cellules polynucléaires. L'invention concerne plus particulièrement un nouveau polypeptide capable de se lier au mannose et d'interagir avec les glycoprotéines présents à la surface des neutrophiles ou des précurseurs de neutrophiles. Ce polypeptide présente de préférence des propriétés de type lectine et correspond, dans sa forme préférée, à une lectine végétale. Il permet notamment de cibler les glycoprotéines de neutrophiles dans un éventail d'applications thérapeutiques et diagnostiques. Ce polypeptide et ses dérivés, homologues, analogues, mimétiques et équivalents fonctionnels sont particulièrement utiles pour l'évaluation de la fonction, de la constitution et de l'état du système immunitaire, par exemple l'évaluation de la capacité fonctionnelle des cellules polynucléaires. Ils permettent en outre de déterminer à quel le type appartiennent les cellules polynucléaires. Dans des formes différentes, ce polypeptide et/ou ses dérivés, homologues, analogues, mimétiques et équivalents fonctionnels servent à la production de plantes transgéniques présentant une résistance améliorée aux micro-organismes, virus, champignons et insectes.
PCT/AU2000/000039 1999-01-29 2000-01-28 Polypeptide capable de se lier au mannose et utilisations WO2000044780A1 (fr)

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Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK, "Hernandia moerenhoutiana subsp. samoensis seed lectin mRNA" *
KOIKE T. ET AL.: "The complete amino acid sequence of a mannose-binding lectin from "Kidachi Aloe" (Aloe arborescens Miller var. natalensis Berger)", BIOCHEMICAL BIOPHYSICAL RESEARCH COMMUNICATION,, vol. 214, no. 1, 1995, pages 163 - 170 *
VAN DAMME E.J. ET AL.: "Cloning and characterization of the lectin cDNA clones from onion, shallot and leek", PLANT MOLECULAR BIOLOGY,, vol. 23, 1993, pages 365 - 376, XP008163421, DOI: doi:10.1007/BF00029011 *
VAN DAMME E.J. ET AL.: "Molecular cloning of mannose-binding lectins from Clivia miniata", PLANT MOLECULAR BIOLOGY,, vol. 24, 1994, pages 825 - 830 *
VAN DAMME E.J.: "Molecular cloning of two different mannose-binding lectins from tulip bulbs", EUROPEAN JOURNAL OF BIOCHEMISTRY,, vol. 236, 1996, pages 419 - 427 *
WRIGHT C.S. ET AL.: "uTe 2.0 A structure of a cross-linked complex between snowdrop lectin and a branched mannopentaose: evidence for two unique binding modes", STRUCTURE,, vol. 4, 1996, pages 1339 - 1352, XP004671702, DOI: doi:10.1016/S0969-2126(96)00141-4 *

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