EP0787184A1 - Recombinant alternaria alternata allergens - Google Patents

Recombinant alternaria alternata allergens

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Publication number
EP0787184A1
EP0787184A1 EP95935287A EP95935287A EP0787184A1 EP 0787184 A1 EP0787184 A1 EP 0787184A1 EP 95935287 A EP95935287 A EP 95935287A EP 95935287 A EP95935287 A EP 95935287A EP 0787184 A1 EP0787184 A1 EP 0787184A1
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Prior art keywords
alt
ala
recombinant
lys
allergens
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German (de)
French (fr)
Inventor
Gernot Achatz
Andrea Unger
Erich Lechenauer
Hannes Oberkofler
Birgit Simon
Christoph Ebner
Dietrich Kraft
Hans-Jörg PRILLINGER
Michael Breitenbach
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Biomay AG
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Biomay AG
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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
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to recombinant DNA molecules which code for polypeptides which have the antigenicity of the allergens Alt a 45 and Alt a 12 or for peptides which have at least one epitope of these allergens.
  • the invention relates to the complete cDNA sequences Alt a
  • recombinant allergens or immunogenic partial peptides can also be used to induce immune tolerance or anergy of T lymphocytes in vivo or in vitro.
  • Immunological mechanisms that have been established to ward off antigens from the environment in the course of evolution can normally distinguish between self and non-self. However, as is often the case with complex control mechanisms, the immune system is also subject to a certain frequency of errors and in the event of failure an attack on its own tissue occurs.
  • a basic characteristic of allergies is that an applied substance induces increased sensitivity or hypersensitivity instead of protection. Some of these substances are toxins, others harmless proteins.
  • Type I-IV Type I-III are mediated by antibodies, while type IV is mediated by T lymphocytes.
  • Type I hypersensitivity is also referred to as immediate type hypersensitivity because its effect occurs within hours of contact with the antigen.
  • antigen enters the body, is absorbed and processed by antigen-presenting cells (APC).
  • APC antigen-presenting cells
  • the processed antigen is then presented together with MHC II to the T helper cells (Th) on the surface of the APC.
  • the Th produce Lvmphokine which help the B-lymphocytes to differentiate into antibody-producing plasma cells.
  • the B-lymphocytes recognize the allergen over 'their Surface receptors and secrete IgE. They bind to receptors from mast cells and basophils. However, the initial binding has no obvious effect on the cells.
  • the multivalent allergen binds to IgE and via other epitopes to other IgE molecules, so that bridging (cross-linking) between the IgE molecules occurs.
  • the aggregation immobilizes the receptor and induces a signal transduction chain, which ultimately leads to the degranulation of the mast cells. Degranulation leads to the production of prostagiandins and leukotrienes.
  • the substances released are mainly histamine and heparin. Histamine stimulates smooth muscle cells, vascular endothelial cells and nerve endings. Heparin has an inhibitory effect on platelets.
  • the allergic response is influenced by the nervous system both in the acute and in the late phase.
  • the neurotransmitters interact with the corresponding receptors on the effector cells and activate them either via cAMP or via cGMP.
  • the target cells of the neurotransmitters are in turn the mast cells, the smooth muscles and the epithelial and secretory cells. Not all people who are exposed to the allergen develop an allergy.
  • allergen avoidance Since allergen avoidance can hardly be achieved one hundred percent, there are further possibilities in the use of antihistamines.
  • immunotherapy is also being tried today. This induces an inability to respond to specific allergens. For this purpose, the patient is repeatedly immunized with an allergen mixture. You start with low doses and slowly increase until the patient stops responding. Success is achieved with immunotherapy for hay fever, insect bites and allergic asthma. Treatment appears to induce some form of partial immunological tolerance. The patient's desensitization is never absolute.
  • IgE-related allergies such as allergies to fungal spores
  • hyposensitization Bosensitization
  • This therapy consists of the supply of allergen extracts in the form of injections or oral application in aqueous form as drops in increasing doses until a maintenance dose is reached over several years.
  • the result of this therapy is tolerance towards the allergens used, which is reflected in a decrease in the symptoms of the disease (Birkner et al. 1990).
  • the problem with this type of treatment lies in the large number of side effects that it causes.
  • Hyposensitization therapy has seen cases of anaphylactic shock during treatment.
  • the problem here is the difficulty in standardizing the fungal protein isolates. When using allergen-derived, but not anaphylactic peptides, could higher doses can be administered without risk and thus a significant improvement in hyposensitization can be achieved.
  • Alternaria alternata can be found practically everywhere in nature. Popular locations and habitats of the mushroom are different soil types, grain silos, rotten wood, but also living plants, composting places and bird nests. If black spots are found on tomatoes, they are also likely to come from Alternaria. But Alternaria alternata is not only found in the wild. The fungus is very often found in damp interiors and on window frames. Warm temperatures and high humidity generally favor the growth of the fungus.
  • Alt a 45 is a major allergen and Alt a 11 a minor allergen.
  • RNA was obtained from self-grown mushroom material using the acid guanidium phenol extraction method.
  • poly (A) plus enrichment was carried out with
  • Oligo (dT) cellulose from Boehringer.
  • the cDNA synthesis (1st and 2nd strand) was carried out as in the manual of the Lambda ZAP system from Stratagene
  • the cDNA was then (3 'side) with EcoRI and (5' side) with Provided Xbal linkers, ligated into pre-digested Lambda ZAP arms and packaged.
  • the primary bank titer was 900,000 clones.
  • the expression bank was scanned by incubating the "lifted" phage plaques with a sera mixture of 2 patients, who were known by western blotting to cover the spectrum of the detected antigens. The detection was again carried out using anti-human IgE RAST antibody from Pharmacia. After secondary and tertiary screening, 150 positive clones remained. The 2 clones described were sequenced according to the Sanger method (Sanger 1977).
  • Alt a 45 and Alt a 12 cDNAs as ⁇ -galactosidase fusion protein
  • the respective recombinant plasmids were transformed into the E.coli strain XLl-Blue and induced with IPTG (isopropyl- ⁇ -D-thiogalactopyranoside).
  • the E. coli total protein extract was then electrophoresed and blotted onto nitrocellulose.
  • the fusion protein was detected by means of serum IgE from fungal allergy sufferers and an iodine-labeled rabbit anti-human IgE antibody (Pharmacia, Uppsala Sweden).
  • the ⁇ -galactosidase portion of the fusion protein is 36 amino acids, which is equivalent to a molecular weight of 3800 daltons. Taking this "enlargement" into account, it can be seen precisely that the recombinant fusion proteins Alt a 45 and Alt a 12 also show IgE binding.
  • the derived amino acid sequence of the allergens provides the prerequisite for the prediction of B and T cell epitopes using suitable computer programs.
  • T and B cell epitopes can be defined that have the ability, e.g. To stimulate T lymphocytes and stimulate proliferation, but also to put the cells (at a precisely defined dose) into a state of tolerance or non-reactivity (anergy) (Rothbard et al. 1991).
  • the specific epitopes are given in the description of the recombinant protein in separate figures.
  • the search for B cell epitopes was carried out using the GCG program (Genetics Computer Group).
  • the determination is based on a weighing of the parameters hydrophilicity (Kyte-Doolittle), secondary structure (Chou-Fasman), surface localization (Robson-Garnier) and flexibility, whereby the antigenicity of partial peptides is calculated.
  • the principle of the T cell epitope prediction was in principle based on the
  • the principle consists in the search for amphipathic helices according to the primary sequence of the protein to be determined, flanked by hydrophilic areas.
  • the calculated score must be greater than 10 for relevant T cell epitopes.
  • No consensus can be defined for MHC II-associated peptides, neither in terms of the sequence nor the length of the peptide, as associated with HLA-A2 (human leucocyte antigen) (MHC I).
  • MHC II-associated peptides In the case of HLA-A2-associated peptides, the length of the peptide is 10 amino acids, the second amino acid being a tyrosine and the last amino acid being a leucine (Rammenee et al. 1993).
  • the calculated epitopes are listed separately in the description of the individual allergenic sequences.
  • the DNA molecules according to the invention therefore have a nucleic acid sequence which coincidentally matches the subsequent sequences (1-6) or with partial regions of these sequences, or nucleic acid sequences which hybridize with the nucleic acid sequences mentioned under stringent conditions.
  • the degree of stringency is defined by 0.1 x SSC.
  • the degree of homology is said to be more than 60%.
  • Alt a 45 Sequence 1 shows the complete cDNA sequence of Alt a 45 starting with the start ATG.
  • the length of the cDNA is 1302 bp, which corresponds to a calculated molecular weight of 45904 daltons.
  • GCT ⁇ C GQ ATC CAG CGC ACC GAG AAG AAC GAG AAG TTC CCT ACG AAC CAG GAG GCC AAG ala phe ala ile gln arg thr glu lys asn glu lys phe pro thr asn gln glu ala lys
  • Alt a 45 is a protein disulphide isomerase (PDI).
  • PDI protein disulphide isomerase
  • a "multiple alignment" with PDI sequences from several organisms reflected the high homology of Alt a 45 to PDI sequences.
  • the consensus found shows identities of amino acids across all organisms.
  • the central motif of homology is given by the sequence "EFYAPWCGHCK”.
  • the function of protein disulfide isomerase (PDI) is to support the folding process of 30 proteins. The exact mechanism is not yet known.
  • the active site of PDI is similar to that of Thioredoxm.
  • the high consensus between the homologous proteins is found from bacteria to plants and further to higher mammals.
  • the PDI protein is found in the lumen of the endoplasmic reticulum.
  • T cell epitopes are calculated from the amino acid positions of the midpoints, which are flanked N-terminally by a lysine (K), C-terminally by a proline (P). Potential T cell epitopes are only present if the "score index" is greater than 10.
  • Sequence 4 shows the complete cDNA sequence of Alt a 12 and the amino acid sequence derived from it.
  • the open reading frame comprises 333bp or 111 amino acids.
  • the calculated molecular weight is 11,728 daltons and thus corresponds to the llkD-sized antigenic protein, which is recognized by 8% of the patients in western emblot.
  • TYPE OF MOLECULE cDNA to mRNA / protein
  • HYPOTHETICAL no
  • ANTISENSE no
  • TYPE OF FRAGMENT total sequence
  • ORIGINAL ORIGIN (A) ORGANISM: Alternaria alternata (C) DEVELOPMENT STAGE: Spores and vegetative hyphae
  • GAC ATC ACT GCC GAC AAG CTT CAA TCC CTC ATC AAG GCC GCA AAG ATC GAG GAG GTC GAG asp i le thr al a asp lys leu gln ser leu i le lys ala ala lys i le glu glu val glu
  • the human autoantibodies cross-react with similar proteins, which means that epitopes are recognized that have been highly conserved in evolution.
  • the basis of the immunological cross-reactivity is the 17 amino acid residue carboxy-terminal region KEESEESD (D / E) DMGFGLFD. Whether one in childhood and 0
  • Anti-P autoantibody production requires P1 / P2 as immunogens but is not driven by exogenous self-antigen in mrl mice.

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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

The invention relates to the complete cDNA sequences of the Alternaria alternata allergenes Alt a 45 and Alt a 12. It has been possible in the course of molecular biological analysis of the allergens to identify Alt a 45 as a protein-disulphide isomerase. This protein is a 45 kD large protein to which 47 % of patients react and is thus an important primary allergen. Alt a 12 (to which 8 % of patients react) was identified as a ribosomal protein which is of interest because auto-antibodies for ribosomal proteins are found in patients suffering from lupus. It remains to be seen whether there is any connection between allergy to mould fungus and autoimmune diseases. Using this recombinant sequence and computer analysis, it has been possible to identify highly potent B- and T-cell epitopes and to use the peptides derived from the recombinant proteins for diagnosis of an allergy to Alternaria alternata mould fungus. The peptides has also proved suitable for both in vitro and in vivo allergen-specific stimulation of T-cells (proliferation, interleukin production), and for blocking the T-cells resulting in a tolerance of the allergen-specific T-lymphocytes.

Description

Rekombinante Alternaria Alternata Allergene Recombinant Alternaria Alternata allergens
Die Erfindung betrifft rekombinante DNA Moleküle, die für Polypeptide kodieren, die die Antigenität der Allergene Alt a 45 und Alt a 12 besitzen oder für Peptide, die mindestens ein Epitop dieser Allergene aufweisen. Insbesondere betrifft die Erfindung die vollständigen cDNA Sequenzen Alt aThe invention relates to recombinant DNA molecules which code for polypeptides which have the antigenicity of the allergens Alt a 45 and Alt a 12 or for peptides which have at least one epitope of these allergens. In particular, the invention relates to the complete cDNA sequences Alt a
45, und Alt a 12 von Alternaria alternata, sowie die von diesen Primärsequenzen abgeleiteten Peptidsequenzen, die bei Pilzallergikern zu einer pathologischen Immunantwort mit einem Überschießen von IgE-Antikörpern führt. Rekombinante Allergene, bzw. immunogen wirkende Teilpeptide können neben einer verbesserten Diagnostik auch zu einer in vivo oder in vitro Induktion einer Immuntoleranz bzw. Anergie von T-Lymphozyten Verwendung finden.45, and Alt a 12 from Alternaria alternata, and the peptide sequences derived from these primary sequences, which leads to a pathological immune response with an overshoot of IgE antibodies in allergy sufferers from fungi. In addition to improved diagnostics, recombinant allergens or immunogenic partial peptides can also be used to induce immune tolerance or anergy of T lymphocytes in vivo or in vitro.
Immunologische Mechanismen die zur Abwehr von Antigenen aus der Umwelt im Laufe der Evolution etabliert wurden, können normalerweise zwischen Selbst und nicht-Selbst unterscheiden. Wie es jedoch bei komplexen Kontrollmechanismen oft der Fall ist, unterliegt auch das Immunsystem einer gewissen Fehlerhäufigkeit und bei Versagen erfolgt eine Angriff auf eigenes Gewebe. Es gibt 4 prinzipielle Situationen, in denen der Körper von seinem eigenen Immunsystem angegriffen wird. Die Situationen unterscheiden sich in der Herkunft der Antigene, die den Angriff auslösen und in dessen Mechanismus und deren Manifestation. Die Antwort kann durch Umweltantigene, durch ein infektiöses Agens (aber auch durch harmlose Substanzen), durch Gewebsantigene die von einer anderen Person stammen und durch Antigene des Individuums selbst ausgelöst werden. Die Reaktion bezeichnet man als "allergisch". Ein prinzipielles Merkmal bei Allergien ist, daß eine applizierte Substanz statt einer Protektion eine erhöhte Sensibilität oder Hypersensibilität induziert. Einige dieser Substanzen sind Toxine, andere harmlose Proteine.Immunological mechanisms that have been established to ward off antigens from the environment in the course of evolution can normally distinguish between self and non-self. However, as is often the case with complex control mechanisms, the immune system is also subject to a certain frequency of errors and in the event of failure an attack on its own tissue occurs. There are 4 basic situations in which the body is attacked by its own immune system. The situations differ in the origin of the antigens that trigger the attack and in its mechanism and its manifestation. The answer can be triggered by environmental antigens, by an infectious agent (but also by harmless substances), by tissue antigens that come from another person and by antigens of the individual himself. The reaction is called "allergic". A basic characteristic of allergies is that an applied substance induces increased sensitivity or hypersensitivity instead of protection. Some of these substances are toxins, others harmless proteins.
Heute unterscheidet man 4 Typen der Hypersensibilität: Typ I-IV. Typ I-III werden durch Antikörper vermittelt, wahrend Typ IV durch T-Lymphozyten vermittelt wird. Die Typ I Hypersensibilität wird auch Hypersensibilität vom Soforttyp bezeichnet, weil ihre Wirkung innerhalb von Stunden nach Antigenkontakt auftritt. In der initialen Sensibilisierungsphase dieses Mechanismus gelangt Antigen (AUergen) in den Körper, wird von Antigen präsentierenden Zellen (APC) aufgenommen und prozessiert. Das prozessierte Antigen wird anschließend zusammen mit MHC II an der Oberfläche der APC den T-Helferzellen (Th) präsentiert. Die Th produzieren Lvmphokine die den B-Lymphozyten helfen zu Antikörper-produzierenden Plasmazellen zu differenzieren. Die B-Lymphozyten erkennen das Allergen über 'ihre Oberflächenrezeptoren und sezemieren IgE. Sie binden an Rezeptoren von Mastzellen und Basophilen. Die initiale Bindung hat jedoch keinen offensichtlichen Effekt auf die Zellen.Today there are 4 types of hypersensitivity: Type I-IV. Type I-III are mediated by antibodies, while type IV is mediated by T lymphocytes. Type I hypersensitivity is also referred to as immediate type hypersensitivity because its effect occurs within hours of contact with the antigen. In the initial sensitization phase of this mechanism, antigen (AUergen) enters the body, is absorbed and processed by antigen-presenting cells (APC). The processed antigen is then presented together with MHC II to the T helper cells (Th) on the surface of the APC. The Th produce Lvmphokine which help the B-lymphocytes to differentiate into antibody-producing plasma cells. The B-lymphocytes recognize the allergen over 'their Surface receptors and secrete IgE. They bind to receptors from mast cells and basophils. However, the initial binding has no obvious effect on the cells.
Wenn jedoch das Allergen erneut in den Körper kommt beginnen die Schwierigkeiten. Das multivalente Allergen bindet an IgE und über andere Epitope an weitere IgE-Moleküle, so daß es zu einer Brückenbildung (Kreuzvemetzung) zwischen den IgE-Molekülen kommt. Die Aggregation immobilisiert den Rezeptor und induziert eine Signaltransduktionskette, die schließlich zur Degranulation der Mastzellen führt. Die Degranulation führt zur Produktion von Prostagiandinen und Leukotrienen. Die freigesetzten Substanzen sind vorallem Histamin und Heparin. Histamin stimuliert glatte Muskelzellen, Gefäßendothelzellen und Nervenendigungen. Heparin übt eine inhibitorische Wirkung auf Thrombozyten aus. Die allergische Antwort wird sowohl in der Akut- als auch in der Spätphase vom Nervensystem beeinflußt. Die Neurotransmitter interagieren mit den korrespondierenden Rezeptoren auf den Effektorzellen und aktivieren diese entweder über cAMP oder über cGMP. Die Zielzellen der Neurotransmitter sind wiederum die Mastzellen, die glatte Muskulatur und die epithelialen und sekretorischen Zellen. Nicht alle Personen die dem Allergen ausgesetzt sind entwickeln eine Allergie.However, when the allergen comes back into the body, the difficulties begin. The multivalent allergen binds to IgE and via other epitopes to other IgE molecules, so that bridging (cross-linking) between the IgE molecules occurs. The aggregation immobilizes the receptor and induces a signal transduction chain, which ultimately leads to the degranulation of the mast cells. Degranulation leads to the production of prostagiandins and leukotrienes. The substances released are mainly histamine and heparin. Histamine stimulates smooth muscle cells, vascular endothelial cells and nerve endings. Heparin has an inhibitory effect on platelets. The allergic response is influenced by the nervous system both in the acute and in the late phase. The neurotransmitters interact with the corresponding receptors on the effector cells and activate them either via cAMP or via cGMP. The target cells of the neurotransmitters are in turn the mast cells, the smooth muscles and the epithelial and secretory cells. Not all people who are exposed to the allergen develop an allergy.
Wieso? Man denkt, daß die primäre Ursache des allergischen Zustandes ein Defekt im Immunsystem ist.How so? It is thought that the primary cause of the allergic condition is a defect in the immune system.
Die neo- und postnatalen Serum-Immunglobulin-Spiegel, besonders I A, sind sehr niedrig. Allergiker haben oft weniger T-Lymphozyten (CD8+). Viele Allergiker hatten bei der Geburt einen erhöhten IgE-Spiegel. Ihre Basophilen degranulieren leichter. Manche haben auch einen Defekt in den Suppressor-T-Zellen. Der IgE-Spiegel steigt wenn Ts abnimmt.The neo- and postnatal serum immunoglobulin levels, especially I A, are very low. Allergy sufferers often have fewer T lymphocytes (CD8 +). Many allergy sufferers had an elevated IgE level at birth. Your basophils degranulate more easily. Some also have a defect in the suppressor T cells. The IgE level increases as Ts decreases.
Ein starkes Argument, gegen einen immunologischen Defekt liegt darin, daß der asthmatische Zustand auch oft ohne Beteiligung des Allergens ausgelöst werden kann. Die Befürworter argumentieren, daß die primäre Ursache der Allergien eher physiologisch als immunologisch ist. Vielleicht sind die Nervenendigungen in der glatten Muskulatur den sekretorischen Drüsen und den Blutgefäßen der Zielorgane bei allergische Patienten genetisch hyperaktiv.A strong argument against an immunological defect is that the asthmatic condition can often be triggered without the allergen being involved. Proponents argue that the primary cause of allergies is physiological rather than immunological. Perhaps the nerve endings in the smooth muscles of the secretory glands and blood vessels of the target organs are genetically hyperactive in allergic patients.
Sie wird durch charakteristische Veränderungen nach einer Zweitexposition mit dem gleichen Antigen ausgelöst. Die für eine Sensibilisierung erforderliche Antigenmenge schwankt erheblich. Eine zu geringe Dosis bewirkt keine Antwort und eine zu große Dosis kann in einer Protektion statt in einer Sensibilisierung enden. Ein desensibilisierter Zustand kann erzeugt werden wenn man einen Schock-Zustand 96/14407It is triggered by characteristic changes after a second exposure to the same antigen. The amount of antigen required for sensitization fluctuates considerably. A dose that is too low will not respond and a dose that is too large may result in a protection rather than a sensitization. A desensitized state can be created when you have a shocked state 96/14407
3 überstanden hat. Der Spiegel von reaktivem IgE kehrt aber nach wenigen Wochen wieder zurück.3 survived. The level of reactive IgE returns after a few weeks.
Die beste Behandlung ist die Allergen Vermeidung. Da die Allergenvermeidung ja kaum hundertprozentig zu erreichen ist, hegen weitere Möglichkeiten im Einsatz von Aπtihistaminika. Probiert wird heute auch eine sogenannte Immuntherapie. Dabei wird eine Unfähigkeit induziert, auf spezifische Allergene zu antworten. Zu diesem Zweck wird der Patient wiederholt mit einer Allergenmixtur immunisiert. Man beginnt mit niedrigen Dosen und erhöht langsam bis der Patient nicht mehr darauf reagiert. Erfolge erziehlt man mit der Immuntherapie bei Heuschnupfen, Insektenstichen und allergischem Asthma. Die Behandlung scheint eine Form der partiellen immunologischen Toleranz zu induzieren. Die Desensibilisierung des Patienten ist aber niemals absolut.The best treatment is allergen avoidance. Since allergen avoidance can hardly be achieved one hundred percent, there are further possibilities in the use of antihistamines. So-called immunotherapy is also being tried today. This induces an inability to respond to specific allergens. For this purpose, the patient is repeatedly immunized with an allergen mixture. You start with low doses and slowly increase until the patient stops responding. Success is achieved with immunotherapy for hay fever, insect bites and allergic asthma. Treatment appears to induce some form of partial immunological tolerance. The patient's desensitization is never absolute.
Bis zum heutigen Zeitpunkt ist jedoch weder die Diagnose noch die Therapie von allergischen Erkrankungen zufriedenstellend. Die molekulare Charakterisierung der Hauptallergene von Alternaria mittels cDNA-Klonierung, Sequenzierung, Sequenzvergleich des allergenen Proteins mit Proteindatenbanken, sowie die Produktion von rekombinanten Allergenen wird mehr Aufschluß über die in vivo Funktion der Proteine geben, die die falschen Immunreaktionen auslösen. Diese Informationen sind aus folgenden Gründen interessant:To date, however, neither the diagnosis nor the therapy of allergic diseases has been satisfactory. The molecular characterization of the main allergens of Alternaria using cDNA cloning, sequencing, sequence comparison of the allergenic protein with protein databases, and the production of recombinant allergens will provide more information about the in vivo function of the proteins that trigger the wrong immune reactions. This information is interesting for the following reasons:
1) Hochreine rekombinante Allergene können für eine sorgfältigere Diagnose, besser als es heute mit Rohextrakten möglich ist, herangezogen werden.1) Highly pure recombinant allergens can be used for a more careful diagnosis, better than is currently possible with crude extracts.
2) Die Sequenz der Allergene wird dabei helfen, tolerogene Peptide zu definieren und eventuell auch den "IgE-Class-switch", der bei der Immunisierung mit dem Allergen passiert, verstehen zu lernen.2) The sequence of the allergens will help to define tolerogenic peptides and possibly also to understand the "IgE class switch" that occurs during the immunization with the allergen.
Seit Jahrzehnten werden IgE bedingte Allergien, so z.B. auch Allergien gegen Pilzsporen, durch Hyposensibilisierung therapiert (Bousquet et al. 1991). Diese Therapie besteht in der Zufuhr von Allergenextrakten in Form von Injektionen oder peroraler Applikation in wässriger Form als Tropfen in steigender Dosierung bis eine Erhaltungsdosis über mehrere Jahre erreicht ist. Resultat dieser Therapie ist das Erreichen einer Toleranz gegenüber den eingesetzten Allergenen, was sich in einer Abnahme der Krankheitssymptome äußert (Birkner et al. 1990). Das Problem bei dieser Art der Behandlung liegt in der Vielzahl der dadurch auftretenden Nebenwirkungen. Bei der Hyposensibilisierungstherapie sind Fälle von anaphylaktischem Schock während der Behandlung aufgetreten. Das Problem hiebei liegt in der schweren Standardisierbarkeit der Pilzprotein-Isolate. Bei einem Einsatz von Allergen-abgeleiteten, aber nicht anaphylaktisch wirkenden Peptiden, könnten risikolos höhere Dosen verabreicht werden und damit eine wesentliche Verbesserung der Hyposensibilisierung erreicht werden.For decades, IgE-related allergies, such as allergies to fungal spores, have been treated by hyposensitization (Bousquet et al. 1991). This therapy consists of the supply of allergen extracts in the form of injections or oral application in aqueous form as drops in increasing doses until a maintenance dose is reached over several years. The result of this therapy is tolerance towards the allergens used, which is reflected in a decrease in the symptoms of the disease (Birkner et al. 1990). The problem with this type of treatment lies in the large number of side effects that it causes. Hyposensitization therapy has seen cases of anaphylactic shock during treatment. The problem here is the difficulty in standardizing the fungal protein isolates. When using allergen-derived, but not anaphylactic peptides, could higher doses can be administered without risk and thus a significant improvement in hyposensitization can be achieved.
Alternaria alternata ist praktisch überall in der Natur anzutreffen. Beliebte Standorte, bzw. Lebensräume des Pilzes sind verschiedene Bodentypen, Getreidesilos, verottetes Holz, aber auch lebende Pflanzen, Kompostplätze und Vogelnester. Findet 5 man auf Tomaten schwarze Flecken, so rühren auch sie mit großer Wahrscheinlichkeit von Alternaria her. Aber nicht nur in freier Natur ist Alternaria alternata anzutreffen. Sehr häufig findet man den Pilz in feuchten Innenräumen und an Fensterrahmen. Warme Temperaturen und hohe Luftfeuchtigkeit begünstigen das Wachstum des Pilzes im allgemeinen.Alternaria alternata can be found practically everywhere in nature. Popular locations and habitats of the mushroom are different soil types, grain silos, rotten wood, but also living plants, composting places and bird nests. If black spots are found on tomatoes, they are also likely to come from Alternaria. But Alternaria alternata is not only found in the wild. The fungus is very often found in damp interiors and on window frames. Warm temperatures and high humidity generally favor the growth of the fungus.
10 a) Beschreibung der allergenen Proteine von Alternaria alternata mittels Western-Blotting10 a) Description of the allergenic proteins from Alternaria alternata using Western blotting
Für die Klonierung der vorliegenden Allergene von Alternaria alternata standen 128 Patientensera zur Verfügung. Um die Reaktivität der Patienten mit Pilzproteinextrakt zu testen wurde Alternaria alternata (Sammlung Prof. Windisch Berlin Nummer:128 patient sera were available for cloning the existing allergens from Alternaria alternata. In order to test the reactivity of the patients with fungal protein extract, Alternaria alternata (Prof. Windisch Berlin number:
15 08-0203) auf festem Medium (2% Glukose, 2% Pepton, 1 % Hefeextrakt) gezüchtet. Für die Proteinextraktion wurde die Pilzmatte nach 3 Tagen Wachstum bei 28°C abgezogen und mit flüssigem Stickstoff aufgebrochen. Die Auftrennung der extrahierten Proteine erfolgte auf einem denaturierenden Polyacrylamidgel, das anschließend geblottet, mit Patientenserum inkubiert und mit ,2SI-markiertem anti human IgE detektiert wurde. In Prozentzahlen ausgedrückt reagierten die Patienten 0 auf die allergenen Proteine wie folgt:15 08-0203) on solid medium (2% glucose, 2% peptone, 1% yeast extract). For the protein extraction, the mushroom mat was removed after 3 days of growth at 28 ° C. and broken up with liquid nitrogen. The extracted proteins were separated on a denaturing polyacrylamide gel, which was then blotted, incubated with patient serum and detected with 2S I-labeled anti-human IgE. Expressed in percentages, patients 0 reacted to the allergenic proteins as follows:
Alt a 45 47%Old a 45 47%
Alt a 12 8%Old a 12 8%
Wie aus diesen Zahlen ersichtlich ist, handelt es sich bei Alt a 45 um ein Haupt-, und bei Alt a 11 um ein Nebenallergen.As can be seen from these figures, Alt a 45 is a major allergen and Alt a 11 a minor allergen.
25 b)Konstruktion der cDNA Expressionsbank25 b) Construction of the cDNA expression bank
Gesamt RNA wurde nach der sauren Guanidium-Phenol-Extraktionsmethode aus selbst gezüchtetem Pilzmaterial gewonnen. poly(A)plus Anreicherung erfolgte mitTotal RNA was obtained from self-grown mushroom material using the acid guanidium phenol extraction method. poly (A) plus enrichment was carried out with
Oligo(dT) Cellulose der Firma Boehringer. Die cDNA Synthese ( 1. und 2. Strang) wurde durchgeführt, wie im Manual des Lambda ZAP-Systems der Firma StratageneOligo (dT) cellulose from Boehringer. The cDNA synthesis (1st and 2nd strand) was carried out as in the manual of the Lambda ZAP system from Stratagene
3Q beschrieben. Die cDNA wurde anschließend (3'- seitig) mit EcoRI und (5'-seitig) mit Xbal Linkern versehen, in vorverdaute Lambda-ZAP-Arme ligiert und verpackt. Der Titer der Primärbank betrug 900000 Klone.3Q. The cDNA was then (3 'side) with EcoRI and (5' side) with Provided Xbal linkers, ligated into pre-digested Lambda ZAP arms and packaged. The primary bank titer was 900,000 clones.
c) Screening der cDNA Genbank mit Patientensera, in vivo Excision, Sequenzierungc) Screening of the cDNA library with patient sera, in vivo excision, sequencing
Das Screεnen der Expressionsbank erfolgte mittels Inkubation der "gelifteten" Phagenplaques mit einem Seragemisch aus 2 Patienten, von denen man durch das Westernblotting wußte, daß sie das Spektrum der detektierten Antigene abdecken. Die Detektion erfolgte wieder mit anti human IgE RAST Antikörper der Firma Pharmacia. Nach Sekundär- und Tertiärscreening blieben 150 positive Klone übrig. Die beschriebenen 2 Klone wurden nach der Sangermethode (Sanger 1977) sequenziert.The expression bank was scanned by incubating the "lifted" phage plaques with a sera mixture of 2 patients, who were known by western blotting to cover the spectrum of the detected antigens. The detection was again carried out using anti-human IgE RAST antibody from Pharmacia. After secondary and tertiary screening, 150 positive clones remained. The 2 clones described were sequenced according to the Sanger method (Sanger 1977).
d) Expression der Alt a 45 und Alt a 12 cDNAs als ß-Galaktosidasefusionsproteind) Expression of the Alt a 45 and Alt a 12 cDNAs as β-galactosidase fusion protein
Die jeweiligen rekombinanten Plasmide wurden in den E.coli Stamm XLl-Blue transformiert und mit IPTG (isopropyl-ß-D-thiogalactopyranosid) induziert. Der E.coli Gesamtproteinextrakt wurde anschließend elektrophoretisch aufgetrennt und auf Nitrozellulose geblottet. Das Fusionsprotein wurde mittels Serum IgE von Pilzallergikern und einem jodmarkiertem Kaninchen anti human IgE Antikörper (Pharmacia, Uppsala Schweden) detektiert.The respective recombinant plasmids were transformed into the E.coli strain XLl-Blue and induced with IPTG (isopropyl-β-D-thiogalactopyranoside). The E. coli total protein extract was then electrophoresed and blotted onto nitrocellulose. The fusion protein was detected by means of serum IgE from fungal allergy sufferers and an iodine-labeled rabbit anti-human IgE antibody (Pharmacia, Uppsala Sweden).
Der ß-Galaktosidaseanteil des Fusionsproteins beträgt 36 Aminosäuren, was einem Molekulargewicht von 3800 Dalton gleichkommt. Unter Berücksichtigung dieser "Vergrößerung" zeigt sich genau, daß die rekombinanten Fusionsproteine Alt a 45 und Alt a 12 ebenfalls IgE-Bindung zeigen.The β-galactosidase portion of the fusion protein is 36 amino acids, which is equivalent to a molecular weight of 3800 daltons. Taking this "enlargement" into account, it can be seen precisely that the recombinant fusion proteins Alt a 45 and Alt a 12 also show IgE binding.
e) Bestimmung von B- und T-Zell Epitopen bei den rekombinanten Allergenene) Determination of B and T cell epitopes in the recombinant allergens
Die abgeleitete Aminosäuresequenz der Allergene bietet die Voraussetzung für die Vorhersage von B- und T-Zellepitopen mittels geeigneter Computeφrogramme. Mit diesen Untersuchungen können spezifische T- und B-Zell-Epitope definiert werden, die die Fähigkeit besitzen, zB. T-Lymphozyten zu stimulieren und zur Proliferation anzuregen, aber die Zellen (bei genau definieπer Dosis) auch in einen Zustand der Toleranz bzw. Nicht-Reaktivität (Anergie) zu versetzen (Rothbard et al. 1991). Die bestimmten Epitope werden jeweils bei der Beschreibung des rekombinanten Proteins in eigenen Figuren angeführt. Die Suche nach B-Zellepitopen wurde mit Hilfe des GCG-Programmes (Genetics Computer Group) durchgeführt. Die Bestimmung beruht auf einer Abwägung der Parameter Hydrophilität (Kyte-Doolittle), Sekundärstruktur (Chou-Fasman), Oberflächen lokalisation (Robson-Garnier) und Flexibilität, wodurch die Antigenität von Teilpeptiden errechnet wird. Das Prinzip der T-Zellepitop- Voraussage erfolgte im Prinzip nach demThe derived amino acid sequence of the allergens provides the prerequisite for the prediction of B and T cell epitopes using suitable computer programs. With these studies, specific T and B cell epitopes can be defined that have the ability, e.g. To stimulate T lymphocytes and stimulate proliferation, but also to put the cells (at a precisely defined dose) into a state of tolerance or non-reactivity (anergy) (Rothbard et al. 1991). The specific epitopes are given in the description of the recombinant protein in separate figures. The search for B cell epitopes was carried out using the GCG program (Genetics Computer Group). The determination is based on a weighing of the parameters hydrophilicity (Kyte-Doolittle), secondary structure (Chou-Fasman), surface localization (Robson-Garnier) and flexibility, whereby the antigenicity of partial peptides is calculated. The principle of the T cell epitope prediction was in principle based on the
Algorithmus von Margali t et al. (1987). Das Prinzip besteht in der Suche nach amphipathischen Helices laut Primärsequenz des zu bestimmenden Proteins, flankiert von hydrophilen Bereichen. Der berechnete Score muß für relevante T-Zellepitope größer als 10 sein. Bei MHC II assoziierten Peptiden kann kein Konsensus, weder der Sequenz noch der Länge des Peptids nach, wie bei HLA-A2 (human leucocyte antigen) (MHC I) assoziierten definiert werden. Bei HLA-A2 assoziierten Peptiden beträgt die Länge des Peptids 10 Aminosäuren, wobei die 2. Aminosäure ein Tyrosin und die letzte Aminosäure ein Leucin darstellt (Rammensee et al. 1993). Die berechneten Epitope werden bei der Beschreibung der einzelnen allergenen Sequenzen getrennt angeführt.Margali t et al. (1987). The principle consists in the search for amphipathic helices according to the primary sequence of the protein to be determined, flanked by hydrophilic areas. The calculated score must be greater than 10 for relevant T cell epitopes. No consensus can be defined for MHC II-associated peptides, neither in terms of the sequence nor the length of the peptide, as associated with HLA-A2 (human leucocyte antigen) (MHC I). In the case of HLA-A2-associated peptides, the length of the peptide is 10 amino acids, the second amino acid being a tyrosine and the last amino acid being a leucine (Rammenee et al. 1993). The calculated epitopes are listed separately in the description of the individual allergenic sequences.
Im folgenden Kapitel werden nun die cDNA Sequenzen und die mit ihnen durchgeführten Analysen der Reihe nach angeführt. Die Computerauswertung der nachfolgenden Sequenzen wurden auf einer Ultrix-DEC 5000 Workstation unter Zuhilfename des GCG-Softwarepaketes (= Wisconsin Paket: die Algorithmen dieses Paketes wurden von der "University of Wisconsin" entwickelt) durchgeführt.In the following chapter the cDNA sequences and the analyzes carried out with them are listed in order. The computer evaluation of the following sequences was carried out on an Ultrix-DEC 5000 workstation using the GCG software package (= Wisconsin package: the algorithms of this package were developed by the "University of Wisconsin").
Die erfindungsgemäßen DNA-Moleküle weisen daher eine Nucleinsäuresequenz auf, die mit den nachfolgenden Sequenzen (1-6), oder mit Teilbereichen dieser Sequenzen in homoiger Weise übereinstimmen, bzw. Nucleinsäuresequenzen, die mit den genannten Nucleinsäuresequenzen unter stringenten Bedingungen hybridisieren. Der Grad der Stringenz ist durch 0, 1 x SSC definiert. Der Grad der Homologie soll mehr als 60% betragen.The DNA molecules according to the invention therefore have a nucleic acid sequence which coincidentally matches the subsequent sequences (1-6) or with partial regions of these sequences, or nucleic acid sequences which hybridize with the nucleic acid sequences mentioned under stringent conditions. The degree of stringency is defined by 0.1 x SSC. The degree of homology is said to be more than 60%.
A. Alt a 45 Die nachfolgende Sequenz 1 zeigt die vollständige cDNA-Sequenz von Alt a 45 beginnend mit dem Start- ATG. Die Länge der cDNA beträgt 1302bp, was einem berechneten Molekulargewicht von 45904 Dalton entspricht. Die beobachtete Bande imA. Alt a 45 Sequence 1 below shows the complete cDNA sequence of Alt a 45 starting with the start ATG. The length of the cDNA is 1302 bp, which corresponds to a calculated molecular weight of 45904 daltons. The observed gang in the
Westernblot bei 45kD korrelieπ somit dem Molekulargewicht nach mit dem klonierten und sequenzierten Allergen. Dem reifen Protein dürfte nach bisheπger Analyse keinWestern blot at 45 kD thus correlates in molecular weight with the cloned and sequenced allergen. According to previous analysis, the mature protein should not
Signalpeptid voranstehen.Precede signal peptide.
Sequenz 1: AI 45 45904 Dalton (1) ANGABEN ZU SEQ ID NO: lSequence 1: AI 45 45904 Dalton (1) INFORMATION ON SEQ ID NO: l
(i) SEQUENZKENNZEICHEN: (A) LÄNGE: 1302 Basenpaare / 434 Aminosäurereste 5 (B) ART: Nukleinsäure / Protein(i) SEQUENCE LABEL: (A) LENGTH: 1302 base pairs / 434 amino acid residues 5 (B) TYPE: nucleic acid / protein
(C) STRANGFORM: ds(C) STRANDFORM: ds
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ü) ART DES MOLEKÜLS: cDNA zu mRNA / Protein (iii) HYPOTHETISCH: nein (iv) ANTISENSE: nein 10 (v) ART DES FRAGMENTS: Gesamtsequenz (vi) URSPRÜNGLICHE HERKUNFT: (A) ORGANISMUS: Alternaria alternata (C) ENTWICKLUNGSSTADIUM: Sporen und vegetative Hyphen(ü) TYPE OF MOLECULE: cDNA to mRNA / protein (iii) HYPOTHETICAL: no (iv) ANTISENSE: no 10 (v) TYPE OF FRAGMENT: total sequence (vi) ORIGINAL ORIGIN: (A) ORGANISM: Alternaria alternata (C) DEVELOPMENT LEVEL : Spores and vegetative hyphae
15 DNA sequence 1302 b.p. ATGACCAAGCAG . . . GACGAGTTGTAA l inear15 DNA sequence 1302 b.p. ATGACCAAGCAG. . . GACGAGTTGTAA l inear
1 / 1 31 / 111/31/11
ATG ACC AAG CAG GCT CTC CCC GCC GTC TCC GAA GTC ACC AAG GAC ACA QC GAG GAG TTC met thr lys gln ala leu pro ala val ser glu val thr lys asp thr leu glu glu pheATG ACC AAG CAG GCT CTC CCC GCC GTC TCC GAA GTC ACC AAG GAC ACA QC GAG GAG TTC met thr lys gln ala leu pro ala val ser glu val thr lys asp thr leu glu glu phe
61 / 21 91 / 3161/21 91/31
AAG ACC GCC GAC AAG GTC GTC CTC GTC GCC TAC TTC GCC GCC GAC GAC AAG GCC TCC AAC lys thr ala asp lys val val leu val ala tyr phe ala ala asp asp lys ala ser asnAAG ACC GCC GAC AAG GTC GTC CTC GTC GCC TAC TTC GCC GCC GAC GAC AAG GCC TCC AAC lys thr ala asp lys val val leu val ala tyr phe ala ala asp asp lys ala ser asn
20121 / 41 151 / 5120121/41 151/51
GAG ACC πC ACC TCG GTC GCC AAC GGT CTC CGT GAC AAC TTC CTC TTC GGT GCC ACC AAC glu thr phe thr ser val ala asn gly leu arg asp asn phe leu phe gly ala thr asnGAG ACC πC ACC TCG GTC GCC AAC GGT CTC CGT GAC AAC TTC CTC TTC GGT GCC ACC AAC glu thr phe thr ser val ala asn gly leu arg asp asn phe leu phe gly ala thr asn
181 / 61 211 / 71181/61 211/71
GAC GCT GCT CTG GCC AAG GCT GAG GGT GTC AAG CAG CCC GGT CTC GTC TGT ACA AGT CCT asp ala ala leu ala lys ala glu gly val lys gln pro gly leu val cys thr ser proGAC GCT GCT CTG GCC AAG GCT GAG GGT GTC AAG CAG CCC GGT CTC GTC TGT ACA AGT CCT asp ala ala leu ala lys ala glu gly val lys gln pro gly leu val cys thr ser pro
241 / 81 271 / 91241/81 271/91
TCG ACG ACG GCA AGG ACG TCT TCA CCG AGA CCT TCG ATG CGG ACG TAT CCG CGA CTT CGC ser thr thr ala arg thr ser ser pro arg pro ser met arg thr tyr pro arg leu argTCG ACG ACG GCA AGG ACG TCT TCA CCG AGA CCT TCG ATG CGG ACG TAT CCG CGA CTT CGC ser thr thr ala arg thr ser ser pro arg pro ser met arg thr tyr pro arg leu arg
25301 / 101 331 / 11125301/101 331/111
AAG GTC GCC TCC ACA CCC CTC ATT GGT GAG GTT GGC CCC GAG ACC TAC GCC GGA TAC ATG lys val ala ser thr pro leu ile gly glu val gly pro glu thr tyr ala gly tyr metAAG GTC GCC TCC ACA CCC CTC ATT GGT GAG GTT GGC CCC GAG ACC TAC GCC GGA TAC ATG lys val ala ser thr pro leu ile gly glu val gly pro glu thr tyr ala gly tyr met
361 / 121 391 / 131361/121 391/131
GCC GCT GGC ATT CCC CTC GCA TAC ATC TTC GCC GAG ACT CCC GAG GAA CGT GAG GAG TTT ala ala gly ile pro leu ala tyr ile phe ala glu thr pro glu glu arg glu glu pheGCC GCT GGC ATT CCC CTC GCA TAC ATC TTC GCC GAG ACT CCC GAG GAA CGT GAG GAG TTT ala ala gly ile pro leu ala tyr ile phe ala glu thr pro glu glu arg glu glu phe
421 / 141 451 / 151421/141 451/151
GCC AAG GAG CTG AAG CCC CTC GCT CTC AAG CAC AAG GGC GAG ATC AAC T C GCT ACC ATC ala lys glu leu lys pro leu ala leu lys his lys gly glu ile asn phe ala thr ileGCC AAG GAG CTG AAG CCC CTC GCT CTC AAG CAC AAG GGC GAG ATC AAC T C GCT ACC ATC ala lys glu leu lys pro leu ala leu lys his lys gly glu ile asn phe ala thr ile
30481 / 161 511 / 17130481/161 511/171
GAC GCC AAG TCC TTC GGC CAG CAC GCT GGC AAC CTT AAC CTC AAG GTC GGC ACC TGG CCC asp ala lys ser phe gly gln his ala gly asn leu asn leu lys val gly thr trp pro 541 / 181 571 / 191GAC GCC AAG TCC TTC GGC CAG CAC GCT GGC AAC CTT AAC CTC AAG GTC GGC ACC TGG CCC asp ala lys ser phe gly gln his ala gly asn leu asn leu lys val gly thr trp pro 541/181 571/191
GCT ΠC GQ ATC CAG CGC ACC GAG AAG AAC GAG AAG TTC CCT ACG AAC CAG GAG GCC AAG ala phe ala ile gln arg thr glu lys asn glu lys phe pro thr asn gln glu ala lysGCT ΠC GQ ATC CAG CGC ACC GAG AAG AAC GAG AAG TTC CCT ACG AAC CAG GAG GCC AAG ala phe ala ile gln arg thr glu lys asn glu lys phe pro thr asn gln glu ala lys
601 / 201 631 / 211601/201 631/211
ATC ACC GAG AAG GAG ATT GGC AAG TTC GTT GAC GAC TTC CTC GCT GGC AAG ATT GAC CCT ile thr glu lys glu ile gly lys phe val asp asp phe leu ala gly lys ile asp proATC ACC GAG AAG GAG ATT GGC AAG TTC GTT GAC GAC TTC CTC GCT GGC AAG ATT GAC CCT ile thr glu lys glu ile gly lys phe val asp asp phe leu ala gly lys ile asp pro
661 / 221 691 / 231661/221 691/231
AGC ATC AAG TCT GAG CCC ATT CCC GAA TCC AAT GAC GGT CCC GTA ACT GTC GTC GTT GCC - ser i le lys ser glu pro i le pro glu ser asn asp gly pro val thr val val val al aAGC ATC AAG TCT GAG CCC ATT CCC GAA TCC AAT GAC GGT CCC GTA ACT GTC GTC GTT GCC - ser i le lys ser glu pro i le pro glu ser asn asp gly pro val thr val val val al a
721 / 241 751 / 251721/241 751/251
CAC AAC TAC AAG GAT GTC GTC ATT GAC AAC GAC AAG GAC GTT CTC GTT GAG TTC TAC GCC his asn tyr lys asp val val ile asp asn asp lys asp val leu val glu phe tyr alaCAC AAC TAC AAG GAT GTC GTC ATT GAC AAC GAC AAG GAC GTT CTC GTT GAG TTC TAC GCC his asn tyr lys asp val val ile asp asn asp lys asp val leu val glu phe tyr ala
781 / 261 811 / 271781/261 811/271
CCC TGG TGC GGT CAC TGC AAG GCT CTT GCT CCC AAG TAC GAG GAG CTC GGC CAG CTC TAC pro trp cys gly hi s cys lys ala leu ala pro lys tyr glu glu leu gly gln leu tyrCCC TGG TGC GGT CAC TGC AAG GCT CTT GCT CCC AAG TAC GAG GAG CTC GGC CAG CTC TAC pro trp cys gly hi s cys lys ala leu ala pro lys tyr glu glu leu gly gln leu tyr
841 / 281 871 / 291841/281 871/291
GCT TCC GAC GAG CTC TCC AAG TTG GTG ACC ATT GCC AAG GTT GAC GCT ACT CTC AAC GAC ιυ ala ser asp glu leu ser lys leu val thr ile ala lys val asp ala thr leu asn aspGCT TCC GAC GAG CTC TCC AAG TTG GTG ACC ATT GCC AAG GTT GAC GCT ACT CTC AAC GAC ιυ ala ser asp glu leu ser lys leu val thr ile ala lys val asp ala thr leu asn asp
901 / 301 931 / 311901/301 931/311
GTT CCC GAC GAG ATC CAA GGT TTC CTA CCA TCA AGC CTC TTC CCG CTG GCA AGA AGG ATG val pro asp glu ile gln gly phe leu pro ser ser leu phe pro leu ala arg arg metGTT CCC GAC GAG ATC CAA GGT TTC CTA CCA TCA AGC CTC TTC CCG CTG GCA AGA AGG ATG val pro asp glu ile gln gly phe leu pro ser ser leu phe pro leu ala arg arg met
961 / 321 991 / 331961/321 991/331
CCC CAG TCG ACT ACT CTG CTT CCG CAC TGT CGA GGA TCT CGT CCA GTT CAT CGA AGA GAA pro gln ser thr thr leu val pro hi s cys arg gly ser arg pro val hi s arg arg gluCCC CAG TCG ACT ACT CTG CTT CCG CAC TGT CGA GGA TCT CGT CCA GTT CAT CGA AGA GAA pro gln ser thr thr leu val pro hi s cys arg gly ser arg pro val hi s arg arg glu
1021 / 341 1051 / 3511021/341 1051/351
CGG CTC ACA CAA GCT AGC GCC AGC GTT GGC GAA GCT CTT GAA GAT GCT ACC GAG TCC GCC 1-) arg leu thr gln ala ser ala ser val gly glu ala val glu asp al a thr glu ser alaCGG CTC ACA CAA GCT AGC GCC AGC GTT GGC GAA GCT CTT GAA GAT GCT ACC GAG TCC GCC 1 - ) arg leu thr gln ala ser ala ser val gly glu ala val glu asp al a thr glu ser ala
1081 / 361 Uli / 3711081/361 Uli / 371
AAG GCC AGT GCC TCT TCC GCC ACA GAC TCT GCT GCC TCA GCT GTA TCA GAA GGC ACC GAG lys ala ser ala ser ser ala thr asp ser al a al a ser al a val ser glu gly thr gluAAG GCC AGT GCC TCT TCC GCC ACA GAC TCT GCT GCC TCA GCT GTA TCA GAA GGC ACC GAG lys ala ser ala ser ser ala thr asp ser al a al a ser al a val ser glu gly thr glu
1141 / 381 1171 / 3911141/381 1171/391
ACG GTC AAG TCT GGT GCG TCT GTC GCT TCC GAC TCA GCC TCT TCC GCC GCT TCC GAG GCT thr val lys ser gly ala ser val ala ser asp ser ala ser ser al a al a ser glu alaACG GTC AAG TCT GGT GCG TCT GTC GCT TCC GAC TCA GCC TCT TCC GCC GCT TCC GAG GCT thr val lys ser gly ala ser val ala ser asp ser ala ser ser al a al a ser glu ala
1201 / 401 1231 / 4111201/401 1231/411
ACC AAG TCT GTC AAG TCT GCC GCG TCC GAG GTT ACC AAC TCT GCC TCG TCG GCT GCG TCA ^υ thr lys ser val lys ser ala ala ser glu val thr asn ser al a ser ser ala ala serACC AAG TCT GTC AAG TCT GCC GCG TCC GAG GTT ACC AAC TCT GCC TCG TCG GCT GCG TCA ^ υ thr lys ser val lys ser ala ala ser glu val thr asn ser al a ser ser ala ala ser
1261 / 421 1291 / 4311261/421 1291/431
GAG GCT TCA GCT TCG GCC TCA AGC GTC AAG GAC GAG TTG TAA glu ala ser al a ser ala ser ser val lys asp glu leu OCHGAG GCT TCA GCT TCG GCC TCA AGC GTC AAG GAC GAG TTG TAA glu ala ser al a ser ala ser ser val lys asp glu leu OCH
Homologiesuchen mit Alt a 45 in der SWISSPROT-Proteindatenbank ergaben, 25 daß es sich bei Alt a 45 um eine Protein-Disulphidisomerase (PDI) handelt. Ein "multiple Alignment" mit PDI-Sequenzen von mehreren Organismen spiegelte die hohe Homologie von Alt a 45 zu PDI-Sequenzen wieder. Der ermittelte Konsensus zeigt Identitäten von Aminosäuren quer durch alle Organismen. Zentrales Motiv der Homologie ist durch die Sequenz "EFYAPWCGHCK" gegeben. Die Funktion der Proteindisulfidisomerase (PDI) liegt in der Unterstützung beim Faltvorgang von 30 Proteinen. Der exakte Mechanismus ist noch nicht bekannt. Die aktive Stelle von PDI ähnelt der von Thioredoxm. Der hohe Konsensus zwischen den homologen Proteinen findet sich von Bakterien zu Pflanzen und weiter zu höheren Säugern. Das PDI-Protein findet man im Lumen des Endoplasmatischen Retikulums.Homology searches with Alt a 45 in the SWISSPROT protein database showed 25 that Alt a 45 is a protein disulphide isomerase (PDI). A "multiple alignment" with PDI sequences from several organisms reflected the high homology of Alt a 45 to PDI sequences. The consensus found shows identities of amino acids across all organisms. The central motif of homology is given by the sequence "EFYAPWCGHCK". The function of protein disulfide isomerase (PDI) is to support the folding process of 30 proteins. The exact mechanism is not yet known. The active site of PDI is similar to that of Thioredoxm. The high consensus between the homologous proteins is found from bacteria to plants and further to higher mammals. The PDI protein is found in the lumen of the endoplasmic reticulum.
Sequenz 2: Alt a 45: B-ZeliepitopeSequence 2: Alt a 45: B cell epitopes
(1) ANGABEN ZU SEQ ID NO:2(1) INFORMATION ON SEQ ID NO: 2
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: einzeln angeführt(A) LENGTH: listed individually
(B) ART: Protein(B) ART: protein
(ii) ART DES MOLEKÜLS: Peptide (üi) HYPOTHETISCH: nein(ii) TYPE OF MOLECULE: Peptides (üi) HYPOTHETICAL: no
(v) ART DES FRAGMENTS: N-Terminus bis C-Terminus (vi) URSPRÜNGLICHE HERKUNFT: (A) ORGANISMUS: Alternaria alternata(v) TYPE OF FRAGMENT: N-terminus to C-terminus (vi) ORIGINAL ORIGIN: (A) ORGANISM: Alternaria alternata
(C) ENTWICKLUNGSSTADIUM: Sporen und vegetative Hyphen(C) DEVELOPMENT STAGE: spores and vegetative hyphae
Gly Glu Val Thr Lys Asp Thr Leu Gly Glu Gly Glu Phe Lys Thr Ala Asp Lys (11-25)Gly Glu Val Thr Lys Asp Thr Leu Gly Glu Gly Glu Phe Lys Thr Ala Asp Lys (11-25)
Phe Ala Ala Asp Asp Lys Ala Ser Asn Gly Glu Thr Phe Thr Ser Val Ala (32-47)Phe Ala Ala Asp Asp Lys Ala Ser Asn Gly Glu Thr Phe Thr Ser Val Ala (32-47)
Ser Pro Ser Thr Thr Ala Arg Thr Ser Ser Pro Arg Pro Ser Met Arg Thr Tyr Pro Arg Leu Arg Lys Val Ala (79-103)Ser Pro Ser Thr Thr Ala Arg Thr Ser Ser Pro Arg Pro Ser Met Arg Thr Tyr Pro Arg Leu Arg Lys Val Ala (79-103)
Phe Ala Gly Glu Thr Pro Gly Glu Gly Glu Arg Gly Glu Gly Glu Phe Ala Lys Gly Glu Leu Lys Pro Leu Ala Leu (130-149)Phe Ala Gly Glu Thr Pro Gly Glu Gly Glu Arg Gly Glu Gly Glu Phe Ala Lys Gly Glu Leu Lys Pro Leu Ala Leu (130-149)
Gln Arg Thr Gly Glu Lys Asn Gly Glu Lys Phe Pro Thr Asn Gln Gly Glu Ala Lys Ile Thr Gly Glu Lys Gly Glu Ile Gly Lys Phe Val Asp Asp (185-212)Gln Arg Thr Gly Glu Lys Asn Gly Glu Lys Phe Pro Thr Asn Gln Gly Glu Ala Lys Ile Thr Gly Glu Lys Gly Glu Ile Gly Lys Phe Val Asp Asp (185-212)
Asp Pro Ser Ile Lys Ser Gly Glu Pro Ile Pro Gly Glu Ser Asn Asp Gly Pro Val Thr (219-236)Asp Pro Ser Ile Lys Ser Gly Glu Pro Ile Pro Gly Glu Ser Asn Asp Gly Pro Val Thr (219-236)
Ala Arg Arg Met Pro Gln Ser Thr Thr Leu Val Pro His Cys (317-330)Ala Arg Arg Met Pro Gln Ser Thr Thr Leu Val Pro His Cys (317-330)
Arg Gly Ser Arg Pro Val His Arg Arg Gly Glu Arg Leu Thr Gln Ala Ser Ala Ser Val Gly Gly Glu Ala (331-352) Sequenz 3: Predicted amphipathatic segments = T-ZellepitopeArg Gly Ser Arg Pro Val His Arg Arg Gly Glu Arg Leu Thr Gln Ala Ser Ala Ser Val Gly Gly Glu Ala (331-352) Sequence 3: Predicted amphipathatic segments = T cell epitopes
Flags Midpoints Angles ScoreFlags Midpoints Angles Score
K 9:22 85:120 33.4 VSEVTKDTLEEF TK 9:22 85: 120 33.4 VSEVTKDTLEEF T
* 41:50 85:105 23.4* 41:50 85: 105 April 23
K P 66:71 125:135 11.9 KAEGVKK P 66:71 125: 135 11.9 KAEGVK
P 82:85 115:125 6.3P 82:85 115: 125 6.3
K P 96:101 115:120 • 16.0 YPRLRKP 96: 101 115: 120 • 16.0 YPRLR
P 110:114 80:90 6.8P 110: 114 80:90 6.8
P 116:120 85:105 10.4P 116: 120 85: 105 10.4
K P 141:145 100:125 9.4 AKELKK P 141: 145 100: 125 9.4 AKELK
P 178:182 130:135 8.1P 178: 182 130: 135 8.1
K P 204:216 80:100 31.5 KEIGKFVDDFLAGK P 204: 216 80: 100 31.5 KEIGKFVDDFLAG
K P 257:267 95:110 28.8 EFYAPWCGHCKK P 257: 267 95: 110 28.8 EFYAPWCGHCK
275:281 110:125 15.2275: 281 110: 125 2/15
283:293 90:130 25.6 DELSKLVTIAK283: 293 90: 130 25.6 DELSKLVTIAK
P 295:309 85:105 39.9 DATLNDVPDEIQGFLP 295: 309 85: 105 39.9 DATLNDVPDEIQGFL
K 345:361 90:125 45.9 ASASVGEAVEDATESAKK 345: 361 90: 125 45.9 ASASVGEAVEDATESAK
K 364:385 85:135 49.3 ASSATDSAASAVSEGTETVKSGK 364: 385 85: 135 49.3 ASSATDSAASAVSEGTETVKSG
K 391:413 80:115 73.2 DSASSAASEATKSVKSAASEVTNK 391: 413 80: 115 73.2 DSASSAASEATKSVKSAASEVTN
(1) ANGABEN ZU SEQ ID NO:3(1) INFORMATION ON SEQ ID NO: 3
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: einzeln angeführt(A) LENGTH: listed individually
(B) ART: Protein(B) ART: protein
(ii) ART DES MOLEKÜLS: Peptide (iii) HYPOTHETISCH: nein(ii) MOLECULE TYPE: Peptides (iii) HYPOTHETICAL: no
(v) ART DES FRAGMENTS: N-Terminus bis C-Terminus (vi) URSPRÜNGLICHE HERKUNFT: (A) ORGANISMUS: Alternaria alternans(v) TYPE OF FRAGMENT: N-terminus to C-terminus (vi) ORIGINAL ORIGIN: (A) ORGANISM: Alternaria alternans
(C) ENTWICKLUNGSSTADIUM: Sporen und vegetative Hyphen(C) DEVELOPMENT STAGE: spores and vegetative hyphae
Val Ser Gly Glu Val Thr Lys Asp Thr Leu Gly Glu Gly Glu Phe Lys Thr (9-22) Lys Ala Gly Glu Gly Val Lys (66-71)Val Ser Gly Glu Val Thr Lys Asp Thr Leu Gly Glu Gly Glu Phe Lys Thr (9-22) Lys Ala Gly Glu Gly Val Lys (66-71)
Tyr Pro AArrgg LLee •uu AArrgg Lys (96-101)Tyr Pro AArrgg LLee • uu AArrgg Lys (96-101)
Ala Lys GGllyy GGlluu LLeeuu Lys (141-145)Ala Lys GGllyy GGlluu LLeeuu Lys (141-145)
Lys Gly GGlluu 1I1lee GGllyy Lys Phe Val Asp Asp Phe Leu Ala Gly (204-216)Lys Gly GGlluu 1I1lee GGllyy Lys Phe Val Asp Asp Phe Leu Ala Gly (204-216)
Gly Glu PPhhee TT>y -rr AAllaa Pro Trp Cys Gly His Cys Lys (257 267)Gly Glu PPhhee TT> y -rr AAllaa Pro Trp Cys Gly His Cys Lys (257 267)
Asp Gly GGlluu LLeeιuu SSeerr Lys Leu Val Thr Ile Ala Lys (283-293)Asp Gly GGlluu LLeeιuu SSeerr Lys Leu Val Thr Ile Ala Lys (283-293)
Asp Ala TThhrr l L .eeuu AA;sn Asp Val Pro Asp Gly Glu Ile Gln Gly Phe LeuAsp Ala TThhrr l L .eeuu AA; sn Asp Val Pro Asp Gly Glu Ile Gln Gly Phe Leu
(295-309)(295-309)
Ala Ser Ala Se ;r Val Gly Gly Glu Ala Val Gly Glu Asp Ala Thr Gly Glu SerAla Ser Ala Se; r Val Gly Gly Glu Ala Val Gly Glu Asp Ala Thr Gly Glu Ser
Ala Lys (345- 361) Ala Ser Ser Ala Thr Asp Ser Ala Ala Ser Ala Val Ser Gly Glu Gly Thr GlyAla Lys (345- 361) Ala Ser Ser Ala Thr Asp Ser Ala Ala Ser Ala Val Ser Gly Glu Gly Thr Gly
Glu Thr Val Lys Ser Gly (364-385)Glu Thr Val Lys Ser Gly (364-385)
Asp Ser Ala Ser Ser Ala Ala Ser Gly Glu Ala Thr Lys Ser Val Lys Ser AlaAsp Ser Ala Ser Ser Ala Ala Ser Gly Glu Ala Thr Lys Ser Val Lys Ser Ala
Ala Ser Gly Glu Val Thr Asn (391-413)Ala Ser Gly Glu Val Thr Asn (391-413)
Die T-Zellepitope errechnen sich aus den Aminosäurepositionen der Midpoints, die N-terminal von einem Lysin (K), C-terminal von einem Prolin (P) flankiert werden (=Flags). Es sind nur dann potentielle T-Zellepitope vorhanden, wenn der "Score-Index" größer als 10 ist.The T cell epitopes are calculated from the amino acid positions of the midpoints, which are flanked N-terminally by a lysine (K), C-terminally by a proline (P). Potential T cell epitopes are only present if the "score index" is greater than 10.
B: Alt a 12B: Alt a 12
Die folgende Sequenz 4 zeigt die vollständige cDNA-Sequenz von Alt a 12 und der von ihr abgeleiteten Aminosäuresequenz. Der offene Leserahmen umfaßt 333bp bzw. 111 Aminosäuren. Das berechnete Molekulargewicht bertägt 11728 Dalton und entspricht somit dem llkD großen antigenen Protein, das im Westemblot von 8% der Patienten erkannt wird.Sequence 4 below shows the complete cDNA sequence of Alt a 12 and the amino acid sequence derived from it. The open reading frame comprises 333bp or 111 amino acids. The calculated molecular weight is 11,728 daltons and thus corresponds to the llkD-sized antigenic protein, which is recognized by 8% of the patients in western emblot.
Sequenz 4: Alt a 12 11728 DaltonSequence 4: Alt a 12 11728 Dalton
(1) ANGABEN ZU SEQ ID NO:4(1) INFORMATION ON SEQ ID NO: 4
(i) SEQUENZKENNZEICHEN: (A) LÄNGE: 333 Basenpaare / 111 Aminosäurereste (B) ART: Nukleinsäure / Protein(i) SEQUENCE LABEL: (A) LENGTH: 333 base pairs / 111 amino acid residues (B) TYPE: nucleic acid / protein
(C) STRANGFORM: ds(C) STRANDFORM: ds
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: cDNA zu mRNA / Protein (iii) HYPOTHETISCH: nein (iv) ANTISENSE: nein (v) ART DES FRAGMENTS: Gesamtsequenz (vi) URSPRÜNGLICHE HERKUNFT: (A) ORGANISMUS: Alternaria alternata (C) ENTWICKLUNGSSTADIUM: Sporen und vegetative Hyphen(ii) TYPE OF MOLECULE: cDNA to mRNA / protein (iii) HYPOTHETICAL: no (iv) ANTISENSE: no (v) TYPE OF FRAGMENT: total sequence (vi) ORIGINAL ORIGIN: (A) ORGANISM: Alternaria alternata (C) DEVELOPMENT STAGE: Spores and vegetative hyphae
DNA sequence 333 b.p. ATGTCTACCTCC ... CTCTTCGACTAA linear 1 / 1 31 / 11DNA sequence 333 b.p. ATGTCTACCTCC ... CTCTTCGACTAA linear 1/31/11
ATG TCT ACC TCC GAG CTC GCC ACC TCT TAC GCC GCT CTC ATC CTC GCT GAT GAC GGT GTC met ser thr ser glu leu ala thr ser tyr ala ala leu ile leu ala asp asp gly val 61 / 21 91 / 31ATG TCT ACC TCC GAG CTC GCC ACC TCT TAC GCC GCT CTC ATC CTC GCT GAT GAC GGT GTC met ser thr ser glu leu ala thr ser tyr ala ala leu ile leu ala asp asp gly val 61/21 91/31
GAC ATC ACT GCC GAC AAG CTT CAA TCC CTC ATC AAG GCC GCA AAG ATC GAG GAG GTC GAG asp i le thr al a asp lys leu gln ser leu i le lys ala ala lys i le glu glu val gluGAC ATC ACT GCC GAC AAG CTT CAA TCC CTC ATC AAG GCC GCA AAG ATC GAG GAG GTC GAG asp i le thr al a asp lys leu gln ser leu i le lys ala ala lys i le glu glu val glu
121 / 41 151 / 51121/41 151/51
CCC ATC TGG ACG ACC CTG TTC GCC AAG GCT CTT GAG GGC AAG GAT GTC AAG GAC CTG CTA pro i le trp thr thr leu phe al a lys ala leu glu gly lys asp val lys asp leu leuCCC ATC TGG ACG ACC CTG TTC GCC AAG GCT CTT GAG GGC AAG GAT GTC AAG GAC CTG CTA pro i le trp thr thr leu phe al a lys ala leu glu gly lys asp val lys asp leu leu
181 / 61 211 / 71181/61 211/71
CTG AAC GTC GGC TCA GGC GGC GGT GCT GCC CCG CTG CCG GAG gCg cTG CTC CTG CGC TGGCTG AAC GTC GGC TCA GGC GGC GGT GCT GCC CCG CTG CCG GAG gCg cTG CTC CTG CGC TGG
-* leu asn val gly ser gly gly gly ala ala pro leu pro glu ala leu leu leu arg trp- * leu asn val gly ser gly gly gly ala ala pro leu pro glu ala leu leu leu arg trp
241 / 81 271 / 91241/81 271/91
CGT GCT GCT GAT GCC GCA CCA GCT GCT GAG GAG AAG AAG GAG GAG GAG AAG GAG GAG TCG arg ala al a asp al a al a pro ala ala glu glu lys lys glu glu glu lys glu glu serCGT GCT GCT GAT GCC GCA CCA GCT GCT GAG GAG AAG AAG GAG GAG GAG AAG GAG GAG TCG arg ala al a asp al a al a pro ala ala glu glu lys lys glu glu glu lys glu glu ser
301 / 101 331 / 111301/101 331/111
GAC GAG GAC ATG GGC TTC GGT CTC TTC GAC TAA asp glu asp met gly phe gly leu phe asp OCHGAC GAG GAC ATG GGC TTC GGT CTC TTC GAC TAA asp glu asp met gly phe gly leu phe asp OCH
Homologiesuchen in der SWISSPROT-Proteindatenbank ergaben hier Homologien zu ribosomalen Proteinen. Das allergene Protein Alt a 12 ist nicht nur wegen seiner Eigenschaft als Allergen von Altemaria alternata interessant. Ribosomale Proteine, hier im speziellen die humanen ribosomalen Proteine Pl und P2, sind in der Literatur als Autoantigene beschrieben worden (Francoeur et al. 1985, Rieh et al. 1987 , Hines et el. 1991). 20% der Patienten mit Lupus erythematosus besitzen Autoantikörper (ami-rRNP) gegen Komponenten der Ribosomen, im speziellen Autoantikörper gegen die ribosomalen Proteine PO (38kD), Pl (16kD) und P2 (15kD). Die humanen Autoantikörper kreuzreagieren mit ähnlichen Proteinen, was heißt, daß Epitope erkannt werden die in der Evolution stark konserviert wurden. Die Basis der immunologischen Kreuzreaktivität bildet die 17 Aminosäurereste lange carboxyterminale Region KEESEESD(D/E)DMGFGLFD. Ob eine in der Kindheit und 0Homology searches in the SWISSPROT protein database revealed homologies to ribosomal proteins. The allergenic protein Alt a 12 is not only interesting because of its property as an allergen from Altemaria alternata. Ribosomal proteins, here in particular the human ribosomal proteins Pl and P2, have been described in the literature as autoantigens (Francoeur et al. 1985, Rieh et al. 1987, Hines et el. 1991). 20% of patients with lupus erythematosus have autoantibodies (ami-rRNP) against components of the ribosomes, in particular autoantibodies against the ribosomal proteins PO (38kD), Pl (16kD) and P2 (15kD). The human autoantibodies cross-react with similar proteins, which means that epitopes are recognized that have been highly conserved in evolution. The basis of the immunological cross-reactivity is the 17 amino acid residue carboxy-terminal region KEESEESD (D / E) DMGFGLFD. Whether one in childhood and 0
Jugend erfolgte Sensibilisierung durch Alt a 12 mit einem im Erwachsenenalter auftretenden Autoimmunkrankheit korreliert, bedarf einer genauen Prüfung.Youth sensitization by Alt a 12 correlated with an autoimmune disease occurring in adulthood requires careful examination.
Applikationen von ribosomalen Proteinen konnten allerdings in Mäusen keineHowever, applications of ribosomal proteins were not possible in mice
Autoimmunkrankheit erzeugen (Hines et al. 1991). Andere ribosomale Proteine (Cla hGenerate autoimmune disease (Hines et al. 1991). Other ribosomal proteins (Cla h
11 und Alt a 11) wurden ebenfalls schon als Allergene identifiziert (Achatz et al.11 and Alt a 11) have also already been identified as allergens (Achatz et al.
1994). 5 Die gezeigten B-Zellepitope in der nächsten Sequenz 16 sind unter1994). 5 The B cell epitopes shown in the next sequence 16 are below
Berücksichtigung von Sekundärstruktur, Oberflächenlage, Hydrophilität, Flexibilität etc. berechnet worden.Consideration of secondary structure, surface position, hydrophilicity, flexibility etc. have been calculated.
0 Sequenz 5: Alt a 12: B-Zellepitope0 Sequence 5: Alt a 12: B cell epitopes
(1) ANGABEN ZU SEQ ID NO:5(1) INFORMATION ON SEQ ID NO: 5
(i) SEQUENZKENNZEICHEN: (A) LÄNGE: einzeln angeführt(i) SEQUENCE LABEL: (A) LENGTH: listed individually
(B) ART: Protein(B) ART: protein
(ii) ART DES MOLEKÜLS: Peptide (iii) HYPOTHETISCH: nein(ii) MOLECULE TYPE: Peptides (iii) HYPOTHETICAL: no
(v) ART DES FRAGMENTS: N-Terminus bis C-Terminus (vi) URSPRÜNGLICHE HERKUNFT: (A) ORGANISMUS: Altemaria alternata(v) TYPE OF FRAGMENT: N-terminus to C-terminus (vi) ORIGINAL ORIGIN: (A) ORGANISM: Altemaria alternata
(C) ENTWICKLUNGSSTADIUM: Sporen und vegetative Hyphen(C) DEVELOPMENT STAGE: spores and vegetative hyphae
Ala Asp Asp Gly Val Asp (16-21)Ala Asp Asp Gly Val Asp (16-21)
Thr Ala Asp Lys Leu Gln Ser Leu (23-30)Thr Ala Asp Lys Leu Gln Ser Leu (23-30)
Ala Lys Ile Gly Glu Gly Glu Val Gly Glu Pro Ile Trp Thr (34-44)Ala Lys Ile Gly Glu Gly Glu Val Gly Glu Pro Ile Trp Thr (34-44)
Ala Leu Gly Glu Gly Lys Asp Val Lys Asp (50-58)Ala Leu Gly Glu Gly Lys Asp Val Lys Asp (50-58)
Val Gly Ser Gly Gly Gly Ala Ala (63-70) Trp Arg Ala Ala Asp Ala (80-85)Val Gly Ser Gly Gly Gly Ala Ala (63-70) Trp Arg Ala Ala Asp Ala (80-85)
Ala Pro Ala Ala Gly Glu Gly Glu Lys Lys Gly Glu Gly Glu Gly Glu Lys GlyAla Pro Ala Ala Gly Glu Gly Glu Lys Lys Gly Glu Gly Glu Gly Glu Lys Gly
Glu Gly Glu Ser Asp Gly Glu Asp Met Gly (105)Glu Gly Glu Ser Asp Gly Glu Asp Met Gly (105)
Sequenz 6: Predicted amphipathatic segments = T-ZellepitopeSequence 6: Predicted amphipathatic segments = T cell epitopes
Flags Midpo-Lnts Anglos ScoreFlags Midpo-Lnts Anglos Score
K P 26:37 90:135 27.6 KLQSLIKAA IEK P 26:37 90: 135 27.6 KLQSLIKAA IE
39:49 80:115 22.0 VEPIWTTLFAK39:49 80: 115 22.0 VEPIWTTLFAK
K 52:55 80:135 6.4K 52:55 80: 135 6.4
(1) ANGABEN ZU SEQ ID NO: 6(1) INFORMATION ON SEQ ID NO: 6
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: einzeln angeführt(A) LENGTH: listed individually
(B) ART: Protein(B) ART: protein
(ii) ART DES MOLEKÜLS: Peptide (iii) HYPOTHETISCH: nein(ii) MOLECULE TYPE: Peptides (iii) HYPOTHETICAL: no
(v) ART DES FRAGMENTS: N-Terminus bis C-Terminus (vi) URSPRÜNGLICHE HERKUNFT: (A) ORGANISMUS: Altemaria altemans(v) TYPE OF FRAGMENT: N-terminus to C-terminus (vi) ORIGINAL ORIGIN: (A) ORGANISM: Altemaria altemans
(C) ENTWICKLUNGSSTADIUM: Sporen und vegetative Hyphen Lys Leu Gln Ser Leu Ile Lys Ala Ala Lys Ile Gly Glu (26-37) Val Gly Glu Pro Ile Trp Thr Thr Leu Phe Ala Lys (39-49)(C) DEVELOPMENT STAGE: spores and vegetative hyphae Lys Leu Gln Ser Leu Ile Lys Ala Ala Lys Ile Gly Glu (26-37) Val Gly Glu Pro Ile Trp Thr Thr Leu Phe Ala Lys (39-49)
Die T-Zellepitope errechnen sich aus den Aminosäurepositionen der Midpoints, die N-terminal von einem Lys (K), C-terminal von einem Prolin (P) flankiert werden (=Flags). Es sind nur dann potentielle T-Zellepitope vorhanden, wenn der "Score-Index" größer als 10 ist. The T cell epitopes are calculated from the amino acid positions of the midpoints, which are flanked N-terminally by a Lys (K), C-terminally by a proline (P) (= flags). Potential T cell epitopes are only present if the "score index" is greater than 10.
6. Literatur6. Literature
Achatz. G. , Oberkofler, H., Lechenauer, E., Simon, B. , Unger, A., Kandier, D., Ebner, C, Prillinger, H., Kraft, D., Breitenbach, M. (1994). Molecular cloning of major and minor allergens of Altemaria alternata and Cladosporium herbarum. Mol. Immunol. in press.Achatz. G., Oberkofler, H., Lechenauer, E., Simon, B., Unger, A., Kandier, D., Ebner, C, Prillinger, H., Kraft, D., Breitenbach, M. (1994). Molecular cloning of major and minor allergens of Altemaria alternata and Cladosporium herbarum. Mol. Immunol. in press.
Birkner, T., Rumpold, H., Jarolim, E. Ebner, H., Breitenbach, M., Skarvil, F., Scheiner, O., Kraft, D. (1990).Birkner, T., Rumpold, H., Jarolim, E. Ebner, H., Breitenbach, M., Skarvil, F., Scheiner, O., Kraft, D. (1990).
Evaluation of immunotherapy-induces changes in specific IgE, IgG and IgG subclasses in birch pollen allergic patients by means of immunoblotting. Correlation with clinical response. Allergy 45, 418.Evaluation of immunotherapy-induces changes in specific IgE, IgG and IgG subclasses in birch pollen allergic patients by means of immunoblotting. Correlation with clinical response. Allergy 45, 418.
Bousquet, J. , Becker, W.M., Hejjaoudi, A. (1991).Bousquet, J., Becker, W.M., Hejjaoudi, A. (1991).
Differences in clinical and immunologic reactivity of patients allergic to grass pollens and to multiple-pollen species. II. Efficacy of a double blind, placebo-controlled, specific immunotherapy with standardized extracts. J. Allergy Clin. Immunol. 88, 43.Differences in clinical and immunologic reactivity of patients allergic to grass pollens and to multiple-pollen species. II. Efficacy of a double blind, placebo-controlled, specific immunotherapy with standardized extracts. J. Allergy Clin. Immunol. 88, 43.
Francoeur, A.M., Peebles, C.L., Heckman, K.J. , Lee, J.C., Tan, E.M. (1985). Identification of ribosomal protein autoantigens. J- Immunol. 135, 1767.Francoeur, A.M., Peebles, C.L., Heckman, K.J. , Lee, J.C., Tan, E.M. (1985). Identification of ribosomal protein autoantigens. J-Immunol. 135, 1767.
Hines, J.J. , Weissbach, H., Brot, N., Elton, K. (1991).Hines, J.J. , Weissbach, H., Brot, N., Elton, K. (1991).
Anti-P autoantibody production requires P1/P2 as immunogens but is not driven by exogenous self-antigen in mrl mice.Anti-P autoantibody production requires P1 / P2 as immunogens but is not driven by exogenous self-antigen in mrl mice.
J. Immunol.. 146, 3386.J. Immunol. 146, 3386.
Margalit, H., Spogue, J.L., Comette, J.L., Cease, K.B., Delisi, C, Berzofsky, J.A.Margalit, H., Spogue, J.L., Comette, J.L., Cease, K.B., Delisi, C, Berzofsky, J.A.
(1987).(1987).
Prediction of immunodominant Helper T cell antigenic sites from the primary sequence.Prediction of immunodominant Helper T cell antigenic sites from the primary sequence.
J. Immunol. 138, 2213.J. Immunol. 138, 2213.
Rammensee, H.G., Falk, K., Rötzschke, O. ( 1993). MHC molecules as peptide receptors. Current Opinion in Immunol. 5, 35.Rammenee, HG, Falk, K., Rötzschke, O. (1993). MHC molecules as peptide receptors. Current opinion in Immunol. 5, 35.
Rieh, B.E., Steitz, J.A. (1987).Rieh, B.E., Steitz, J.A. (1987).
Human acidic ribosomal phosphoproteins PO, Pl and P2: analysis of cDNA clones, in vitro synthesis and assembly. Mol. Cell. Biol. 7, 4065.Human acidic ribosomal phosphoproteins PO, Pl and P2: analysis of cDNA clones, in vitro synthesis and assembly. Mol. Cell. Biol. 7, 4065.
Rothbard, J.B., Gefter, M.L. (1991).Rothbard, J.B., Gefter, M.L. (1991).
Interactions between immunogenic peptides and MHC proteins. Ann. Rev. Immunol. 9, 527.Interactions between immunogenic peptides and MHC proteins. Ann. Rev. Immunol. 9, 527.
Sanger, F., Nicklen, S., Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Naü. Acad. Sei. USA 74, 5463-5468 Sanger, F., Nicklen, S., Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Well. Acad. Be. USA 74, 5463-5468

Claims

Patentansprüche claims
1. Rekombinante DNA Moleküle, die für Polypeptide kodieren, die die Antigenität der Allergene Alt a 45 und Alt a 12 besitzen oder für Peptide, die mindestens ein Epitop dieser Allergene aufweisen, dadurch gekennzeichnet, daß sie Nukleinsäuresequenzen aufweisen, die mit den Sequenzen (1-6), oder mit Teilbereichen dieser Sequenzen in homologer Weise übereinstimmen, bzw. Nucleinsäuresequenzen, die mit den genannten Nucleinsäuresequenzen unter stringenten Bedingungen hybridisieren.1. Recombinant DNA molecules which code for polypeptides which have the antigenicity of the allergens Alt a 45 and Alt a 12 or for peptides which have at least one epitope of these allergens, characterized in that they have nucleic acid sequences which correspond to the sequences (1 -6), or agree with partial regions of these sequences in a homologous manner, or nucleic acid sequences which hybridize with the nucleic acid sequences mentioned under stringent conditions.
2. Rekombinante DNA-Moleküle nach Anspruch 1, dadurch gekennzeichnet, daß sie Nukleinsäuresequenzen aufweisen, die durch Degeneration aus den Sequenzen2. Recombinant DNA molecules according to claim 1, characterized in that they have nucleic acid sequences which by degeneration from the sequences
1 bis 6 ableitbar sind.1 to 6 can be derived.
3. Rekombinante DNA Moleküle nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß sie Nukleinsäuresequenzen aufweisen, die für Polypeptide kodieren, die als Antigene kreuzreaktiv mit den Allergenen Alt a 45 und Alt a 12 sind und zu diesen eine hohe Homologie aufweisen. 3. Recombinant DNA molecules according to claim 1 or 2, characterized in that they have nucleic acid sequences which code for polypeptides which are cross-reactive as antigens with the allergens Alt a 45 and Alt a 12 and have a high homology to these.
4. Rekombinante DNA-Moleküle nach Ansprüchen 1 bis 3. dadurch gekennzeichnet, daß sie funktionell mit einer Expressionskontrollsequenz zu einem Expressionskonstrukt verbunden sind.4. Recombinant DNA molecules according to claims 1 to 3, characterized in that they are functionally linked to an expression control sequence to form an expression construct.
5. Wirtssystem zur Expression von Polypeptiden, dadurch gekennzeichnet, daß es mit einem rekombinanten Expressionskonstrukt nach Anspruch 4 transformiert ist.5. Host system for the expression of polypeptides, characterized in that it is transformed with a recombinant expression construct according to claim 4.
6. Aus einem DNA-Molekül nach einem der Ansprüche 1 bis 3 abgeleitetes rekombinantes oder synthetisches Protein oder Polypeptid, dadurch gekennzeichnet, daß es die Antigenität von Alt a 45 oder Alt a 12, oder zumindest von einem Epitop dieser Proteine aufweist.6. Recombinant or synthetic protein or polypeptide derived from a DNA molecule according to one of claims 1 to 3, characterized in that it has the antigenicity of Alt a 45 or Alt a 12, or at least one epitope of these proteins.
7. Rekombinantes oder synthetisches Protein oder ein Polypeptid nach Anspruch 6, dadurch gekennzeichnet, daß es eine Aminosäuresequenz aufweist, die mit den gezeigten Sequenzen 1-6 zur Gänze oder teilweise entspricht. 7. Recombinant or synthetic protein or a polypeptide according to claim 6, characterized in that it has an amino acid sequence which corresponds to the sequences 1-6 shown in whole or in part.
8. Rekombinantes oder synthetisches Protein oder Polypeptid nach8. Recombinant or synthetic protein or polypeptide after
Patentanspruch 6 oder 7, dadurch gekennzeichnet, daß es ein Fusionsprodukt darstellt, das die Antigenität der Allergene Alt a 45 oder Alt a 12, oder zumindest eines Epitops davon aufweisen und einen zusätzlichen Polypeptidanteil besitzt, wobei das gesamte Fusionsprodukt von der DNA eines Expressionskonstrukts gemäß Anspruch 4 kodiert wird. Claim 6 or 7, characterized in that it represents a fusion product which has the antigenicity of the allergens Alt a 45 or Alt a 12, or at least one epitope thereof and has an additional polypeptide fraction, the entire fusion product being the DNA of an expression construct according to claim 4 is encoded.
9. Rekombinantes oder synthetisches Protein oder Polypeptid nach Patentanspruch 8, dadurch gekennzeichnet, daß der besagte zusätzliche Polypeptidanteil ß-Galaktosidase oder ein anderes zur Fusion geeignetes Polypeptid ist.9. Recombinant or synthetic protein or polypeptide according to claim 8, characterized in that said additional polypeptide portion is β-galactosidase or another polypeptide suitable for fusion.
10. Diagnostisches oder therapeutisches Reagens, dadurch gekennzeichnet, daß es ein synthetisches Protein oder Polypeptid gemäß einem der Ansprüche 6 bis 9 enthält.10. Diagnostic or therapeutic reagent, characterized in that it contains a synthetic protein or polypeptide according to one of claims 6 to 9.
11. Verfahren, zum in vitro -Nachweis der Allergie eines Patienten gegen die Allergene Alt a 45 oder Alt a 12, dadurch gekennzeichnet, daß die Reaktion der IgE Antikörper im Serum des Patienten mit einem rekombinanten oder sythetischen Protein oder Polypeptid nach einem der Anprüche 6 bis 9 gemessen wird.11. A method for in-vitro detection of a patient's allergy to the allergens Alt a 45 or Alt a 12, characterized in that the reaction of the IgE antibodies in the patient's serum with a recombinant or synthetic protein or polypeptide according to one of Claims 6 to 9 is measured.
12. Verfahren, zum in vitro - Nachweis der zellulären Reaktion auf die Allergene Alt a 45 oder Alt a 12, dadurch gekennzeichnet, daß ein rekombinantes oder synthetisches Protein oder Polypeptid nach einem der Ansprüche 6 bis 9 zur Stimulierung oder Hemmung der zellulären Reaktion eingesetzt wird. 12. A method for in vitro detection of the cellular response to the allergens Alt a 45 or Alt a 12, characterized in that a recombinant or synthetic protein or polypeptide according to one of claims 6 to 9 is used to stimulate or inhibit the cellular response .
EP95935287A 1994-11-02 1995-10-31 Recombinant alternaria alternata allergens Withdrawn EP0787184A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AT2038/94 1994-11-02
AT0203894A AT401181B (en) 1994-11-02 1994-11-02 RECOMBINANT DNA MOLECULES
PCT/AT1995/000212 WO1996014407A1 (en) 1994-11-02 1995-10-31 Recombinant alternaria alternata allergens

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EP0787184A1 true EP0787184A1 (en) 1997-08-06

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EP (1) EP0787184A1 (en)
JP (1) JPH10508474A (en)
AT (1) AT401181B (en)
AU (1) AU3736795A (en)
CA (1) CA2204362A1 (en)
FI (1) FI971894A0 (en)
NO (1) NO971836L (en)
WO (1) WO1996014407A1 (en)

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GB201209862D0 (en) 2012-06-01 2012-07-18 Circassia Ltd Cladosporium peptides
CN116098124A (en) * 2022-12-16 2023-05-12 中国医学科学院北京协和医院 Allergic asthma mice to Alt a1 method for establishing subcutaneous specific immunotherapy model

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Title
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WO1996014407A1 (en) 1996-05-17
CA2204362A1 (en) 1996-05-17
ATA203894A (en) 1995-11-15
NO971836L (en) 1997-06-25
NO971836D0 (en) 1997-04-21
JPH10508474A (en) 1998-08-25
AT401181B (en) 1996-07-25
AU3736795A (en) 1996-05-31
FI971894A (en) 1997-05-02
FI971894A0 (en) 1997-05-02

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