Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6472—Cysteine endopeptidases (3.4.22)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/12—Ophthalmic agents for cataracts
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• the invention relates to a new calpain protease called calpain protease 12 and functional analogs thereof (hereinafter referred to as Capnl2); nucleic acids coding therefor; recombinant vectors containing these coding sequences; microorganisms transfected therewith; Process for recombinant production of Capnl2; as well as various applications of Capnl2 and nucleic acids coding therefor.
• Capnl2 calpain protease 12 and functional analogs thereof
• Calpaine are a family of cytosolic cysteine proteases.
• the classic calpains consist of an isoform-specific large subunit (80 kDa) and an invariable small subunit (30 kDa), which is called Capn4.
• the large subunit of classic calpains has a four-domain structure, comprising a domain with protease activity and a C-terminal, calmodulin-like domain that can bind calcium.
• Calpaine cleave numerous substrates (Carafoli and Molinari, 1998) and have been associated with a variety of processes, including apoptosis (ang, 2000), cell division (Mellgren, 1997), modulation of integrin cytoskeleton interactions (S ⁇ hoenwalder et al ., 1997) and synaptic plasticity (Chan and Mattson, 1999). They have also been associated with numerous pathological conditions, such as Alzheimer's, cataract, demyelination, cardiac ischemia, inflammation and traumatic brain injury (review: Carafoli and Molinari, 1998; Sorimachi et al., 1997; Wang and Yuen, 1997).
• Capn3 Mutations in the Capn3 gene are responsible for type 2A pelvic girdle muscular dystrophy (Richard et al., 1995). Due to the diverse physiological and pathological functions of calpains, the task was to provide new homologs of the gene family of the large calpain subunit. In this way, for example, new active substances or new active substance targets can be found or developed which can be used in the diagnosis, therapy and / or prophylaxis of disease states in which calpains and their substrates or substances acting on them are involved. This task was surprisingly achieved by the provision of a new calpain protease, the calpain protease 12 (Capnl2) and functional equivalents thereof.
• the Capnl2 is characterized in that it has an amino acid sequence comprising amino acids 1 - 342 of SEQ ID NO: 1.
• the invention also relates to functional equivalents of this partial sequence.
• Preferred variants thereof are characterized in that they have an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
• SEQ ID NO: 1 stands for the amino acid sequence of the splice variant Capnl2A
• SEQ ID NO: 2 for the amino acid sequence of the splice variant Capnl2B
• SEQ ID NO: 3 for the amino acid sequence of the splice variant Capnl2C
• SEQ ID NO: 4 for the Amino acid sequence of Capnl2 from clone 914413 of the mouse EST database.
• the amino acid sequences SEQ ID NO: 1 to 4 in the N-terminal section of amino acids 1 - 342 are identical to one another.
• the predicted protein corresponding to the Spli ⁇ e variant Capnl2A has 720 amino acids and a molecular weight of 80.5 kDa.
• the invention also relates to the functional equivalents of Capnl2 or the specifically disclosed amino acid sequences.
• Functional equivalents comprise amino acid sequences which can be derived from the specific sequences and in which, in comparison, one or more amino acids are substituted, deleted, inverted or added, without the cysteine protease activity and / or at least Another characteristic of the Capnl2 can be essentially influenced. Additional Capnl2 characteristics are described in later sections.
• the specifically disclosed amino acid sequences represent amino acid sequences of Spli ⁇ e variants of Capnl2, which were determined from a mouse EST database.
• the invention also relates to all Capnl2 homologs of eukaryotic species, ie the evertebrates and vertebrates, in particular mammals, for example rats, cats, dogs, pigs, sheep, cattle, horses, monkeys and, with particular preference, humans and other naturally occurring variants.
• all developmental and organ or tissue-specific expressed Capnl2 forms and artificially generated homologs are also included, which have the specified structural and / or functional properties.
• Capnl2 according to the invention is particularly characterized in that it has cysteine protease activity. In its amino acid sequence, it has the amino acids Cys, His and Asn (in the Spli ⁇ e variants Capnl2A, B and C: Cysl05, His259 and
• Capnl2A also has a distinctly acidic region and a Calmodulin-like, presumably Ca 2+ -binding region.
• the Capnl2 according to the invention is also characterized in that the mouse coding gene is located on chromosome 7 between the markers D7Mit72 (10.4 ⁇ M) and D7Mit267 (11.0 cM).
• the Capnl2 according to the invention is also characterized in that it e.g. in the mouse, in the cortex of the hair follicle of the skin.
• Capnl2 according to the invention is characterized in that it is expressed in the anagen phase of the hair cycle.
• the invention also relates to calpain proteins which are characterized in that they have at least one Capn12 according to the invention.
• a calpain protein preferably has, besides the Capnl2 as a large subunit, a Capn4 as a small protein subunit.
• no other protein subunits such as regulatory subunits or subunits that mediate the localization of the protein in defined cell compartments, can be included.
• the invention also comprises polynucleotides which code for a Capn12 according to the invention, and their functional equivalents and polynucleotides which can be hybridized or complementary thereto, comprising single and double-stranded DNA and RNA sequences. quences.
• polynucleotides can be found when genomic or DNA libraries are patterned and, if appropriate, can be amplified therefrom with suitable primers by means of PCR and then isolated, for example, with suitable probes.
• Another possibility is the transformation of suitable microorganisms with polynucleotides or vectors according to the invention, the multiplication of the microorganisms and thus the polynucleotides and their subsequent isolation.
• polynucleotides according to the invention can also be synthesized by ⁇ hemis ⁇ hem ways.
• the invention also relates to polynucleotides with a nucleic acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, Seq ID NO: 7 or SEQ ID NO: 8.
• SEQ ID NO: 5 stands for the nucleic acid sequence of ⁇ DNA of the Spli ⁇ e variant Capnl2A
• SEQ ID NO: 6 for the nucleic acid sequence of the ⁇ DNA of Capnl2B
• SEQ ID NO: 7 for the nucleic acid sequence of the ⁇ DNA of Capnl2C
• SEQ ID NO: 8 for the genomic nucleic acid sequence of the Capnl2 of the mouse, comprising all exons - and intron sequences.
• the predicted genomic sequence of the mouse Capnl2 comprises 21 exons and a genomic separation of 13116 base pairs.
• Functional equivalents of polynucleotides according to the invention include sequences derived by degeneration of the genetic code and thus silent nucleotide substitutions (i.e. without changes in the resulting amino acid sequence) and conservative nucleotide substitutions (i.e. the amino acid in question is replaced by an amino acid of the same charge, size, polarity and / or solubility).
• Functional equivalents of polynucleotides according to the invention thus have a sequence changed by nucleotide substitution, deletion, inversion or addition, but also code for a functionally equivalent Capnl2, such as e.g. with the same or comparable cysteine protease activity.
• polynucleotides which are suitable according to the invention comprise at least one of the partial sequences which code for characteristic amino acid sequences of the Capn12.
• the invention also relates to the primer sequences SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, which can hybridize or are complementary to polynucleotides according to the invention and can be used, for example, for their amplification by RT-PCR or PCR.
• the property of being able to hybridize to polynucleotides means the ability of a poly- or oligonucleotide to bind to an almost complementary sequence under stringent conditions, while under these conditions unspecific bindings between no complementary partners.
• the sequences must be 70-100%, preferably 90-100%, complementary.
• the property of complementary sequences of being able to specifically bind to one another is exploited, for example, in Northern or Southern blot technology or in primer binding in PCR or RT-PCR. Usually oligonucleotides from a length of 30 base pairs are used.
• Stringent conditions mean, for example, in Northerner blot technology the use of a 50-70 ° C, preferably 60-65 ° C warm water solution, for example 0.1x SSC buffer with 0.1% SDS (20x SSC: 3M NaCl, 0.3M Na citrate, pH 7.0) for the elution of unspecific hybridized ⁇ DNA probes or oligonucleotides.
• a 50-70 ° C, preferably 60-65 ° C warm water solution for example 0.1x SSC buffer with 0.1% SDS (20x SSC: 3M NaCl, 0.3M Na citrate, pH 7.0) for the elution of unspecific hybridized ⁇ DNA probes or oligonucleotides.
• SDS 3M NaCl, 0.3M Na citrate, pH 7.0
• the invention also relates to expression cassettes which comprise at least one polynucleotide according to the invention which is operatively linked to at least one regulatory nucleic acid sequence.
• a promoter sequence is preferably located 5 'upstream from the polynucleotide according to the invention and in this way enables controlled expression of the Capn12.
• An operative link is understood to mean the sequential arrangement of regulatory and coding sequences, such as of promoter, coding sequence, terminator and possibly further regulatory elements such that each of the regulatory elements can perform its function as intended before, during or after expression of the coding sequence.
• regulatory and coding sequences such as of promoter, coding sequence, terminator and possibly further regulatory elements such that each of the regulatory elements can perform its function as intended before, during or after expression of the coding sequence.
• further operatively linkable sequences are targeting sequences, translation enhancers, enhancers, polyadenylation signals and the like.
• Usable regulatory elements also include selectable markers, amplification signals, origins of replication and the like.
• the natural regulatory sequence may be present before the actual structural gene. This natural regulation can possibly be withheld by genetic modification and the expression of the genes increased or decreased.
• the expression cassette can, however, have a more simple structure, ie no additional regulation signals are inserted in front of the structural gene and the natural promoter with its regulation is not removed. Instead, for example, the natural regulatory sequence are mutated in such a way that regulation no longer takes place and gene expression is increased or decreased.
• the nucleic acid sequences can be contained in the expression cassette in one or more copies.
• brewable promoters are: ⁇ os-, ta ⁇ -, trp-, tet-, trp-tet-, lpp-, la ⁇ -, lpp-la ⁇ -, la ⁇ lq-, T7-, T5-, T3-, gal-, tr ⁇ -, ara, SP6, ⁇ -PR or in the ⁇ -PL promoter, which are advantageously used in gram-negative bacteria; as well as the gram-positive promoters amy and SP02, the yeast promoters ADCl, MFa, AC, P-60, CYC1, GAPDH or the plant promoters CaMV / 35S, SSU, OCS, lib4, usp, STLS1, B33, nos or the Ubiquitin or phaseolin promoter.
• inducible promoters such as, for example, light-inducible and in particular temperature-inducible promoters, such as the P r P ⁇ promote
• the regulatory sequences mentioned are intended to enable the targeted expression of the nucleic acid sequences and the protein expression. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction, or that it is immediately expressed or overexpressed. These regulatory elements can also be used to express tissue, cell or development-specific elements if the vector is introduced into a higher organism, such as an animal or a plant.
• the regulatory sequences or factors can preferably have a positive influence on the expression and thereby increase or decrease it.
• the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers".
• an increase in translation is also possible, for example by increasing the stability of the mRNA.
• Enhancers are to be understood, for example, as DNA sequences which bring about increased expression via an improved interaction between RNA polymerase and DNA.
• An expression cassette according to the invention is produced by fusion of a suitable promoter with a suitable polynucleotide encoding Capn12, and a terminator or polyadenylation signal. Common recombination and cloning techniques are used for this, such as insertion via restriction enzyme sections or how they are used. for example in T. Maniatis, EF Frits ⁇ h and J.
• the invention also relates to recombinant vectors for transforming eukaryotic or prokaryotic hosts which carry a polynucleotide according to the invention or an expression cassette according to the invention. These vectors allow the expression of Capnl2 in a suitable host organism. Vectors are well known to the person and can be found, for example, in "Cloning Ve ⁇ tors" (Pouwels P.H. et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).
• vectors are also understood to mean all other vectors known to the person, such as phages, viruses, such as SV40, CMV, Baululovirus and adenovirus, transposons, IS elements, plasmids, cosmids, and linear or circular DNA. These vectors can be replicated autonomously in the host organism or chromosomally.
• the invention also relates to microorganisms which contain a vector according to the invention or which express the Capnl2 endogenously. These can be used to produce recombinant
• Capnl2 can be used.
• the recombinant expression cassettes according to the invention described above are advantageously incorporated and expressed as part of an expression vector in a suitable host system.
• Common cloning and transfection methods known to the man are preferably used, such as, for example, co-precipitation, protoplast fusion, electroporation, retroviral transfection and the like, in order to express the nucleic acids mentioned in the respective expression system.
• Suitable systems are described, for example, in Current Proto ⁇ ols in Molecular Biology, F. Ausubel et al., Ed., Wiley Inters ⁇ ien ⁇ e, New York 1997 and in J. Sambrook, E.F. Frits ⁇ h and T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1980).
• host organisms for transformation with vectors according to the invention.
• Host organisms mean, for example, bacteria, fungi, yeasts, plant or animal cells.
• Preferred organisms are bacteria, such as those of the genera Es ⁇ heri ⁇ hia, such as eg Escheri ⁇ hia ⁇ oli, Streptomy ⁇ es, Bacillus or Pseudomonas, eukaryotic microorganisms, in particular yeasts such as Sa ⁇ haromy- ⁇ es ⁇ erevisiae, Aspergillus, higher eukaryotic cells from animals or plants, for example Sf9 or CHO cells.
• the gene product can also be used for expression in transgenic organisms such as transgenic animals, such as, in particular, mice, ports or transgenic plants.
• the transgenic organisms can also be so-called knock-out animals or plants in which the corresponding endogenous gene has been kept, such as by mutation or partial or complete deletion.
• Successfully transformed organisms can be selected using marker genes which are also contained in the vector or in the expression cassette.
• marker genes are genes for antibiotic resistance and for enzymes which catalyze a color-giving reaction which stains the transformed cell. These can then be selected using automatic cell sorting.
• Microorganisms transformed successfully with a vector and carrying an appropriate antibiotic resistance gene e.g. G418 or hygromy ⁇ in
• an appropriate antibiotic resistance gene e.g. G418 or hygromy ⁇ in
• Marker proteins which are presented on the cell surface, can be used for selection by means of affinity chromatography.
• the combination of the host organisms and the vectors suitable for the organisms form an expression system.
• the term “expression system” means the combination of mammalian cells, such as CHO cells, and vectors, such as p ⁇ DNA3neo vector, which are suitable for mammalian cells.
• the gene product can advantageously also be found in transgenic animals, e.g. Mice, ports or transgenic plants are brewed for expression. It is also possible to program cell-free translation systems with the RNA derived from the nucleic acid.
• the invention furthermore relates to processes for producing a Capnl2 according to the invention, in which a Capnl2-producing microorganism is cultivated, where appropriate the expression of the Capnl2 is induced and the Capnl2 is isolated from the culture.
• the Capnl2 can thus also be produced on an industrial scale if this is desired.
• the microorganism can be cultivated and fermented by known methods. Bacteria can be propagated, for example, in TB or LB medium and at a temperature of 20 to 40 ° C and a pH of 6 to 9. Suitable cultivation conditions are described in detail, for example, in T. Maniatis, E.F. Frits ⁇ h and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).
• Capnl2 is not secreted into the culture medium, the cells are then disrupted and the Capnl2 is obtained from the lysate by known protein isolation methods.
• the cells can optionally have high frequency ultrasound, high pressure, e.g. in a Fren ⁇ h pressure cell, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by homogenizers or by a combination of several of the listed processes.
• a purification of the Capnl2 can be achieved with known chromatographic processes, such as molecular sieve chromatography (gel filtration), such as Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, as well as with other conventional processes such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis.
• chromatographic processes such as molecular sieve chromatography (gel filtration), such as Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, as well as with other conventional processes such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis.
• Suitable methods are described, for example, in Cooper, F.G., Bio ⁇ hemis ⁇ he Working Methods, Verlag Walter de Gruyter, Berlin, New York or in S ⁇ opes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.
• vector systems or oligonucleotides which extend the ⁇ DNA by certain nucleotide sequences and thus code for modified polypeptides or fusion proteins which serve for easier purification.
• suitable modifications are, for example, so-called “tags” which act as anchors, such as, for example, the modification known as hexa-histidine anchors or epitopes which can be recognized as antigens of antibodies (described, for example, in Harlow, E. and Lane , D., 1988, Antibody: A Laboratory Manual, Cold Spring Harbor (NY) Press).
• anchors can be used to attach the proteins to a solid support, such as a polymer matrix, for example, which can be filled in a chromatography column, or can be used on a microtiter plate or on another support. At the same time, these anchors can also be used to recognize the proteins.
• a solid support such as a polymer matrix, for example, which can be filled in a chromatography column, or can be used on a microtiter plate or on another support.
• these anchors can also be used to recognize the proteins.
• conventional markers such as fluorescent dyes, enzyme markers, which form a detectable reaction product after reaction with a substrate, or radioactive markers, alone or in combination with the anchors, can be used to derivatize the proteins.
• the invention also relates to the use of a Capnl2 according to the invention or a calpain protein according to the invention as a cysteine protease.
• a Capnl2 according to the invention or a calpain protein according to the invention as a cysteine protease.
• the use in connection with natural substrates of Capnl2 is preferred, however, all substrates can be used which bind to the active center of Capnl2 and are split there.
• the Capnl2 can thus be used, for example, as a cysteine protease in molecular biological and chemical processes.
• the invention also relates to pharmaceutical compositions which contain a Capn12 according to the invention, a calpain protein according to the invention or a recombinant vector according to the invention, and at least one pharmaceutically acceptable carrier or diluent.
• the Capnl2 according to the invention, the calpain protein according to the invention or the vector can be administered as such, but preferably together with a carrier or diluent.
• This carrier can be in solid or liquid form depending on the dosage form desired.
• Suitable pharmaceutical agents can also contain, in addition to a Capn12 according to the invention, a calpain protein according to the invention or a recombinant vector according to the invention, if desired, no further pharmaceutical active ingredients in a mixture or separately in a combination preparation.
• active substances can enhance the action of the contained Capnl2, the calpain protein or the vector, have a different action mechanism and thus have an additive effect or improve the overall constitution of the patient.
• the invention furthermore relates to the use of a Capnl2 according to the invention, a calpain protein according to the invention or a vector according to the invention for the manufacture of a medicament for the treatment of diseases or disease states which are associated with inadequate expression of the
• Capnl2 stand.
• a treatment according to the invention includes the prevention of the development of the disease in a patient with a corresponding predisposition or the therapy of an already existing disease by slowing down or even improving the condition of the patient, possibly until it heals completely.
• several substitution methods can be used.
• a Capnl2 or a calpain protein according to the invention can be applied directly or by gene therapy in the form of its coding nucleic acids (DNA or RNA) in a medicament according to the invention.
• Any vehicle for example both viral (retroviral transfection) and also non-viral vehicles (for example liposome transfection), can be used for gene therapeutic use.
• Suitable vehicles can bind to precisely defined target cells via suitable receptor molecules or the same specific ones and transform them in a targeted manner.
• the transfection can take place in the patient's body or removed cells are transfected in vitro and subsequently applied to the patient again.
• Suitable methods are described, for example, by Strauss and Barranger in Con ⁇ epts in Gene Therapy (1997), Walter de Gruyter, ed.
• Another method for Capnl2 substitution is the stimulation of the endogenous, endogenous gene.
• the turn-over or the inactivation of Capnl2 according to the invention for example by proteases, can be blocked in order to achieve an increased number of active Capn12 molecules.
• agonists of Capnl2 can be used to increase the activity of existing Capnl2 molecules. With reduced Capnl2 expression, this can only be a therapy-supporting procedure.
• Pharmaceutical agents or medicaments according to the invention can be in the form of tablets, granules, powders, dragées, pastilles, pellets, capsules, suppositories, solutions, emulsions and suspensions for enteral and parenteral administration.
• Pharmaceutical compositions according to the invention can preferably be contained in gels, lotions and creams for cutaneous application.
• the respective dosage of pharmaceutical agents or medications according to the invention and the respective dosage schedule are the decision of the treating doctor.
• the patient will select a suitable dose and a suitable dosing schedule.
• the pharmacologically active substances can be administered to a mammal (human and animal) in doses of approximately 0.5 mg to 100 mg per kg of body weight per day. They can be administered in single dose or in multiple doses.
• the areas of application include diseases and disease states which are associated with an inadequate Capn12 expression.
• the invention also relates to the use of a Capn12 according to the invention or a calpain protein according to the invention for screening for calpain protease E fectors.
• Calpain protease effectors are understood to mean, for example, substances which can influence the activity of Capnl2 and / or other calpaines, such as activators or inhibitors, or those which can act on the substrates of Capnl2 during enzymatic catalysis or Capn12- binding molecules, such as immunoglobulins or low-molecular Capnl2-binding molecules, which can also modulate the biological function of Capnl2.
• Capnl2-binding molecules are understood to mean all natural and synthetic ligands and interaction partners of Capnl2.
• the Capnl2 or a calpain protein according to the invention is incubated with an analyte which contains an effector of a physiological or pathological Capn12 activity, for example the cysteine protease activity, and the activity of the Capnl2, if appropriate determined by adding substrates and cosubstrates.
• an analyte which contains an effector of a physiological or pathological Capn12 activity, for example the cysteine protease activity, and the activity of the Capnl2, if appropriate determined by adding substrates and cosubstrates.
• the Capnl2 or the calpain protein according to the invention if appropriate after corresponding derivatization, can be immobilized on a support and brought into contact with an analyte in which at least one Capnl2 binding partner is suspected.
• the components of the analyte bound to the immobilized Capn12 or the immobilized calpain protein according to the invention can then optionally be eluted, determined and characterized after an incubation phase.
• the analyte can also be immobilized and then linked to
• Capnl2 molecules or bindable Capn12 fragments are examined for constituents of the analyte.
• the invention furthermore relates to immunoglobulins with specificity for a Capn12 according to the invention.
• immunoglobulins include mono- or polyclonal antibodies that can bind to characteristic epitopes of Capnl2, as well as their fragments.
• Anti-Capnl2 immunoglobulins are produced in a manner familiar to the company. Immunoglobulins mean both polyclonal, monoclonal, optionally human or humanized antibodies or fragments thereof, single- ⁇ hain antibodies or also synthetic antibodies, and antibody fragments such as Fv, Fab and F (from ') 2 .
• peptides can be synthesized which can be used individually or in combination as antigens for the production of monoclonal or polyclonal antibodies.
• the invention also relates to the use of immunoglobulins according to the invention or polynucleotides according to the invention for the diagnosis of diseases or disease states which are related to Capnl2 expression.
• the amount, activity and distribution of the Capnl2 or its underlying mRNA in the human body can be determined.
• the Capn12 concentration can be determined in biological samples, e.g. Determine cells or body fluids.
• polynucleotides according to the invention for example by means of Northern blot technology or RT-PCR, the expression can be assessed at the mRNA level and, for example, a lower expression can be detected and a disease associated therewith diagnosed.
• FIG. 1 shows a sequence comparison of the predicted amino acid sequence of Capnl2 with representative members of the vertebrate gene family of the large calpain subunit:
• Capnl has a classic calmodulin-like, C-terminal domain
• Capn5 Capn7 and CapnlO have C-terminal domains, which are labeled N, T and X, respectively.
• Hyphens indicate gaps that have been inserted for comparison and thus for the best possible comparison of the sequences.
• the three conserved amino acids that are part of the active center of the calpaine are marked with arrows.
• the Cal ⁇ ium-binding EF hand domains of Capnl (Lin et al., 1997; Blan ⁇ hard et al., 1997) are highlighted by a bar above the respective sequence and are only numbered in sections.
• the calpain domains predicted from the crystal structure (Hosfield et al., 1999) are also characteristic.
• the exceptionally acidic region in domain III which can interact with calcium and possibly acts as an "electrostatic holder" of the protease activity, is known by circles above the relevant sequence.
• FIG. 2 shows the genomic structure of the Capnl2 gene:
• A Chemical diagram of the intron / exon structure of the Capnl2 gene.
• the black rectangles represent exons of Capnl2. These are numbered consecutively.
• the checkered Re ⁇ hte ⁇ k identifies the outermost 3 'end exon of A ⁇ tn4.
• the dotted Re ⁇ hte ⁇ k identifies the exon sequence that Capnl2 and A ⁇ tn4 share.
• the arrows indicate the transcription direction of both genes. The location of the repeats that were discovered in the sequence are given above.
• the Spli ⁇ e event between exons 9 and 20, from which the mRNA transcript of clone 914413 resulted, and the partial sequences surrounding the Spli ⁇ e donor and Spli ⁇ e acceptor site of exons 9 and 20 of Capnl2 are also given. Large letters identify the coding sequence and small letters the intron sequence.
• a schematic diagram of exons 11, 12 and 13 shows the alternative splice variants A, B and C.
• the sequence of the common exon 11 is shown on the left and the associated exon used in the respective splice variant will be on the right.
• the predicted amino acid sequence is given under the corresponding nucleotide sequence.
• the last two nucleotides of the Spli ⁇ e acceptor in exon 12, AG, which are used in Spli ⁇ e variant B, are shown in bold.
• the table shows the Spli ⁇ e events of the individual exons with the nucleotide sequence surrounding the respective Spli ⁇ e donor and Spli ⁇ e acceptor. Spli ⁇ e donor and Spli ⁇ e acceptor are shown in bold. The size of the respective exons and introns is indicated.
• FIG. 3 shows the phylogenetic family tree of the mammalian gene family of the large calpain subunit:
• Capnl AF021847)
• Capn2 Y10139
• Capn3 X92523
• Capn5 Y10656
• Capn ⁇ Y12582
• Capn7 AJ012475
• Rat Capn ⁇ D14480
• human CAPN9 AF022799
• CapnlO AF126867
• human CAPN11 AJ242832
• FIG. 4 shows an mRNA expression analysis of A ⁇ tn4 and Capnl2:
• A. Expression of A ⁇ tn4 in Different Mouse Tissues A 32 P-labeled sample, which corresponds to the 3 'end of the mouse A ⁇ tn4 ⁇ DNA, was hybridized to a Clonte ⁇ h mouse master blot. The location of the RNAs on the filters is shown on the right side. The blot was stripped and hybridized with a mouse Hprt probe (in the middle) to check the RNA loading. The exposure time was 48 hours.
• a 32 P-labeled sample was hybridized to a Northern filter that carried RNAs from the skin of mice of the specified age. The blot was then hybridized once more with a ß- ⁇ tin- ⁇ DNA sample to check the applied RNA levels. The positions of the 28S and 18S rRNAs are marked and the specific Capnl2 RNA band marked with an arrow. The exposure time for Capnl2 was 144 hours and 2 hours for ß-A ⁇ tin.
• FIG. 5 shows an in-situ hybridization on skin tissue sections from mouse embryos:
• Capnl2 is selectively expressed in the cortex of the hair follicle (irs: inner root sheath; ors: outer root sheath; ⁇ o: ⁇ ortex).
• a cosmid library created by cloning partially digested Sau3A mouse 129 / Sv DNA into the cosmid vector pSuper-Cos (Stratagene), was analyzed by PCR using the Capnl2-specific primer 5 '-gaatgg ⁇ gagtgg ⁇ aacaggaag-3' ( SEQ ID NO: 9) and 5 '-tgggg ⁇ t ⁇ ag ⁇ a ⁇ aaaa ⁇ t ⁇ at-3' (SEQ ID NO: 10). The cosmid DNA was purified using the Qiagen Plasmid Midi Kit according to the manufacturer's instructions.
• RNA Five micrograms of total RNA were transcribed into ⁇ DNA using AMV reverse transcriptase using the Promega reverse transcription system.
• the PCRs were in 50 ul reaction volume, the 50 mM KC1, 10 itiM Tris-HCl, pH 9, 0.1% Triton X-100, 2 units Taq DNA polymerase, 50 pmol both the forward and the reverse primer and contained 0.1 ng of cDNA using a Thermo ⁇ y ⁇ ling protocol of 35 cycles comprising 15 s at 94 ° C, 30 s at 55 ° C and 1 min at 72 ° C.
• Capnl2 forward and backward primer sequences for RT-PCR were 5'-tt ⁇ aaga ⁇ ttt ⁇ t ⁇ a ⁇ g-3 '(SEQ ID NO: 11) and 5' -t ⁇ g ⁇ ttgagtttattctga-3 '(SEQ ID NO: 12).
• the Hprt forward and backward primer sequences were 5'-atg ⁇ gac ⁇ g ⁇ agt ⁇ ag ⁇ g-3 '(SEQ ID NO: 13) and 5'-gg ⁇ tttgtatttgg ⁇ tttt ⁇ -3' (SEQ ID NO: 14).
• a 20 ⁇ l reaction mixture containing 8 ⁇ l BigDye reaction mix (Perkin-Elmer Biosystems), 500 ng purified DNA and 10 pmol primer was used for 30 cycles, including 15 s at 94 ° C, 15 s at 50 ° C and Incubated for 2 min at 60 ° C.
• the reaction products were separated by polyacrylamide gel electrophoresis using an ABI 377 DNA sequencer and the sequence was dye-terminated by fluorescence using the Perkin-Elmer Biosystems sequence analysis software version 3.3. Further sequencing with synthesized oligonucleotides expanded the DNA sequences. The sequences were put together using the SeqMan from the DNASTAR program series to form a contig.
• DNA and amino acid sequences were homologated with the non-redundant nucleotide, protein, and EST databases of the National Center for Biotechnology Information (http://www.nabi.nlm.nih.gov) using the BLAST program series ( Alts ⁇ hul et al., 1990).
• the sequence comparison and the comparison of amino acid sequences was carried out with CLUSTAL W (Thompson et al., 1994).
• the prediction of the exons was possible using the FGNENESH program, which is available on the Sanger Center web server (www.sanger.aa.uk). Repetitive sequences were identified using the "RepeatMasker” (http: // repeatmasker.genome.washington.edu).
• the phylogenetic analysis was carried out with the CLUSTREE program, available from the HUSAR server of the German Cancer Research Center, Heidelberg (www.dkfz-heidelberg.de).
• RNA from mouse tissues was isolated using the guanidine isothiocyanate method (Chomzynski and Sa ⁇ hi, 1987). 10 ⁇ g of total RNA were separated by electrophoresis in a 1.4% (w / v) agarose sail which, as already described, contained 2.2 M formaldehyde (Sambrook et al., 1989), and was separated onto a Hybond according to the manufacturer's instructions -N-nylon membrane (Amersham) blotted. The blot was hybridized with a 32 P-labeled ⁇ DNA fragment which corresponded to the nucleotides 33-852 cDNA sequence of the Capnl2 in Expresshyb hybridization solution (Clonte ⁇ h). The conditions of hybridization and highly stringent washing were determined according to the Manufacturer information selected. In a second step, the blot was hybridized with a ⁇ DNA sample of ß-a ⁇ tin in order to check the RNA loading.
• Capnl2 cDNA which was used as a template for the synthesis of RNAs corresponding to nucleotides 33-852 of the described sequence, was cloned into the E ⁇ oRV site of the pBlues ⁇ ript. This ⁇ DNA fragment does not overlap with the Actn4 gene.
• RNAs were obtained by in vitro transcription of restriction enzyme linearized plasmid DNA in a reaction volume of 12.5 ⁇ l, the 1 x transcription buffer (buffer for T7 and T3 RNA polymerases, from Statagene), 200 ⁇ M ATP, CTP, GTP, 40 ⁇ Ci ⁇ - 33 P-UTP (Amersham), 10 mM DTT, 1 ⁇ g linearized plasmid DNA, 40 units RNAsin (Promega) and 10 units RNA polymerase , After 2 hours of incubation at 37 ° C, the template DNA was removed by adding 2 units of DNAasel (Boehringer Mannheim) followed by a 30-minute incubation at 37 ° C.
• the reaction mixture was extracted, precipitated with ethanol and resuspended in 26 ⁇ l DEPC-treated dH 2 0.
• the embryos were fixed in 4% (w / v) paraformaldehyde in PBS and the 5 ⁇ m tissue sections obtained were transferred to pre-cleaned SuperFrost Plus slides (Menzel-Glaeser).
• the hybridization and washing conditions were as already described (Dressler and Gruss, 1989). A hybridization temperature of 55 ° C was chosen.
• the DNAs of the T31 radiation hybrid mapping plate were PCR by means of two, the Capnl2 [Set 1: 5'-gggagggccaggacaagga ⁇ t-3 '(SEQ ID NO: 15), 5'-agggaagg ⁇ tggaa ⁇ aatggagaa-3' (SEQ ID NO: 15) 16), Set 2: 5'-gaatgg ⁇ gagtgg ⁇ aa ⁇ aggaag-3 '(SEQ ID NO: 17), 5'- ⁇ tgggg ⁇ tcag ⁇ a ⁇ aaaa ⁇ t ⁇ at-3' (SEQ ID NO: 18)] and the Capn5 (Set 1: 5'- ⁇ ggtga ⁇ tg ⁇ g SEQ ID NO: 19), 5 '-aag ⁇ g ⁇ tg ⁇ agagca ⁇ tgtgg-3' (SEQ ID NO: 20); Set 2: 5'- ⁇ gggagtgga ⁇ ggg ⁇ c ⁇ tg-3 '(SEQ ID NO: 15) 16), Set 2
• the PCRs were in a reaction volume of 20 ul, containing 50 mM KC1, 10 mM Tris-HCl, pH 9, 1.5 mM MgCl 2 , 1 Unit Taq DNA polymerase and 25 ng DNA, with a Thermo ⁇ y- ⁇ ling protocol of 35 cycles, comprising 15 s at 94 ° C, 30 s at 60 ° C and 1 min at 72 ° C.
• the raw data were submitted for analysis to the mouse radiation hybrid database in the Ja ⁇ kson Laboratory (www.jax.org/resour ⁇ es/do ⁇ uments/ ⁇ mdata/rhmap/).
• the publicly accessible EST databases were used to identify new Calpaingen genes. Using the data obtained previously, a new member of the mammalian gene family of the large calpain subunit, which is characterized by a cell-specific expression pattern, was found and characterized.
• the mouse EST database was screened with protein sequences from known vertebrate calpains using the TBLAST algorithm (Alts ⁇ hul et al., 1990).
• the translated protein of a 3'EST, AA1314413 was typical of the large calpain subunit family.
• the ⁇ DNA clone, 914413 which corresponds to this EST clone, was sequenced in its entirety.
• the cDNA has a polyA tail and contains an open reading frame, the predicted protein of which shows homology to domains I and II of the large subunit of classic Calpaine. However, the predicted sequence deviates from the classic Calpaine in the connection to domain II and ends shortly thereafter (FIG. 1).
• the ⁇ DNA clone 914413 appears to be the result of an atypical or faulty RNA splicing event that has deleted the exons of this calpain gene coding for domains III and IV.
• a genomic DNA cosmid clone was isolated and sequenced. A continuous sequence (SEQ ID NO: 8) of 13116 bp was obtained.
• the gene prediction software FGE-NESH identified a potential gene with 21 exons, with an exon / intron structure that is typical of the Calpaine gene family. These include exons with pronounced homology to domains III and IV of classic Calpaine.
• mRNA isolated from the skin was analyzed using RT-PCR.
• the software predicted 20 of the 21 exons, allowing some errors in the position of the donor or acceptor splice site.
• the intron / exon limits of the complete gene are shown in Figure 2A and summarized in Table 1.
• the mouse genome nomenclature committee named this gene Capnl2.
• Four simple repeats and 16 SINES short interspersed repeats; 4 Bl, 1 B2, 3 B4 and 8 ID; Fig. 2A) were found in the Capnl2 intron sequence.
• the ⁇ DNA clone 914413 appears to be the result of an incorrect splice event, because both the splice donor and the splice acceptor site are atypical and most of the Exons of domains III and IV will then be deleted.
• the splice takes place between exons 9 and 20 within a 5-base pair region, CACTG, which is common to these two exons (FIG. 2A).
• Capnl2 mRNA identified (here called Capnl2A, Capnl2B and Capnl2C).
• the Spli ⁇ e variant A has an open reading frame, which presumably encodes a protein of 720 amino acids (M r 80.5 kDa).
• the proposed start methionine (cgaATGg) corresponds to the minimal consensus sequence of the translation start site (Kozak, 1996).
• a further 5 'starting points are excluded by a TAA stop o-don in the reading frame, which is located 39 nucleotides upstream of this ATG.
• the predicted amino acid sequence shows similarities to members of the family of the large calpain subunit and can be divided into the four domains I to IV typical for calpains (FIG. 1).
• Domain II of the Capn12 according to the invention has the three amino acid residues (Cysl05, His259 and Asn283) which are essential for the active center of cysteine proteases (Berti and Storer, 1995). Accordingly, the Capn12 according to the invention, like most classic calpains, has cysteine protease activity.
• Capn2 Each of the five Ca 2+ -binding sequences described for Capn2 (Blan ⁇ hard et al., 1997; Lin et al., 1997) is conserved to a certain extent in the amino acid sequence of Capnl2 (FIG. 1).
• the crystal structure of Capn2 revealed an extremely acidic region in domain III, which interact with Ca 2+ and could act as an "electrostatic switch" of protease activity (Strobl et al., 2000). The authors suspect that the large number of acidic residues in this region could reduce the Ca 2+ concentration required for activation.
• the corresponding region of the Capnl2 is also markedly acidic (DEEEDDDDEE; Fig. 1).
• transcripts of the Spli ⁇ e variants A and B are examined in the Spli ⁇ e acceptor of exon 12, while in variant C the exon
• the predicted proteins of the alternative splice variants B and C thereby show a different amino acid sequence in domain III and due to a shift in the reading frame the translation ends within this domain (FIG. 2B). As a result, they may also lack the calmodulin-like
• Spli ⁇ e variant B is more abundant than that of the Spli ⁇ e variant A.
• a Capnl2 protein which lacks a Ca 2+ -binding domain is probably a substantial part of the Capnl2 protein pool.
• the RT-PCR product of the Spli ⁇ e variant C was the least abundant.
• the complete amino acid sequences of representative members of all known large calpain subunits of the mammals were subjected to phylogenetic analysis. This enabled the calpaine to be divided into three main groups 0 (Fig. 3).
• the first group (A) is represented by Capnl, Capn2, Capn3, Capn8 and Capn9 and the second group (B) by Capn5, Capn ⁇ , Capn7, CapnlO and Capnl2.
• Capnl1 a very different calpain (Dear et al., 1999), does not fit in any of the groups.
• Group (A) contains all calpains with a calmodulin-like, 5 C-terminal domain, while group (B) contains all those "atypical"
• Calpaine contains which presumably lack the ability to bind Ca 2+ .
• An exception is the Capnl2, which in general is more similar to group (B) except that it has a calmodulin-like C-terminal domain.
• the phylogenetic analysis suggests that Capnl2 is the oldest member of this group.
• a predecessor of the Capnl2 gene could consequently be the founder of the genes of the atypical large calpain subunit, the Capn12 possibly serving as a source of both classic and also atypical proteins via alternative splicing.
• the location of the Capnl2 gene on mouse chromosomes was determined by PCR analysis of the T31 radiation hybrid mapping plate using primers that bind within intron 1 of the Capnl2 gene.
• the raw data were analyzed using the radiation hybrid map of the mouse genome (Van Etten et al., 1999) and the associated World Wide Web server.
• the highest LOD value was 16 in connection with the marker D7Mit72.
• Other high LODs were 14.4 in connection with D7Mitll6, 14.4 in connection with D7Mit77, and 13.9 in connection with D7Mit267. These markers were located on chromosome 7 at 9.4 (D7Mit77), 10.7 (D7Mitll ⁇ ), 10.4 (D7Mit72), and 11.0 cM (D7Mit267).
• the most logical order is: proximal - D7Mit77 - D7Mitll ⁇ - D7Mit72 - Capnl2 - D7Mit267 - distal.
• the region is orthologous to human chromosome 19ql3.
• the mouse Capn5 gene was also recently localized on mouse chromosome 7 using a radiation hybrid mapping plate of the chromosomes of somatic cells of the mouse (Matena et al., 1998). In order to determine the exact distance between Capn5 and Capnl2 on chromosome 7, the T31 plate was analyzed with mouse Capn5-specific PCR primers. The highest LOD value was 13.4 in connection with D7Mit321.
• a ⁇ tn4 must also be on mouse chromosome 7. Since the human A ⁇ tn4 gene was located on chromosome 19ql3 (Kaplan et al., 2000), the human Capnl2-0rtholog is also very likely to be found in this region.
• the sequence of overlapping ESTs formed a sequence with an open reading frame, which codes for the mouse ortholog of ⁇ -A ⁇ tinin-4 (A ⁇ tn4).
• the RT-PCR of various mouse tissue RNAs confirmed the sequence.
• the predicted mouse protein is 98.9% identical to human Actn4.
• the last exon overlaps the last exon of the Capnl2 gene by 330 bp, but in the opposite orientation (FIG. 2). It has recently been shown that mutations in the human Actn4 gene can cause familial segmental herbal nephretitis (Kaplan et al., 2000).
• RNA dot blot analysis and in situ hybridization using a specific sample showed that the A ⁇ tn4 gene is expressed ubiquitously (FIG. 4).
• the 914413 ⁇ DNA clone was isolated from a mammary gland cDNA library, Northern blot and RT-PCR analysis showed no significant level of expression in this tissue.
• Only a more precise RT-PCT analysis in connection with an in situ hybridization on mouse embryos of the stages dElO, 5 to dE18.5 and various adult tissues showed that Capnl2 is expressed exclusively in the skin.
• Capnl2 is expressed in the cortex of the hair follicle (Fig. 5).
• Hair undergoes a cycle that lasts about 25 days in the mouse (Chase, 1965).
• the cycle is roughly divided into three phases: the anagen (proliferation), the catagen (regression) and the telogen phase (resting phase).
• the back skin of the adult mouse contains hair follicles in all phases of the hair cycle.
• mice have been different Samples were taken from the back skin at times after birth and the extracted RNAs were examined by Northern blot hybridization.
• the first hair cycle of the mouse runs synchronously (Chase, 1965) and thus relatively pure hair follicle populations 5 of a specific cycle phase can be examined.
• Capnl2 mRNA of approximately 3.5 kb can be detected in the anagen phase (approximately P1-P16) but not in the telogen phase (P19-P25) (Fig. 4B).
• the mRNA reaches its highest level of expression around day P12, in the middle of the anagen phase.
• An RT-PCR-10 analysis of the same skin samples confirmed this result (Fig. 4C).
• Capnl2 thus shows a high-specific mRNA expression pattern.
• the gene family of the large calpain subunit can be any gene family of the large calpain subunit.
• Capnl, Capn2, Capn7 and CapnlO are to be ubiquitously expressed, while the other Calpains are differentiated
• Capn9 is predominantly expressed in the intestine and stomach, but is also detectable in other tissues (Li et al., 1998). In contrast, Capnll is apparently only expressed in certain cells of the testis (Dear and Boehm,
• Capn5 is expressed in embryonic thymus in the T cell precursors, while after birth, expression in the thymus is down-regulated (Dear and Boehm, 1999).
• CAPN 8 isolation of a new ouse ⁇ alpain isoenzyme. Bio ⁇ hem. Biophys. Res. Commun. 260: 671-675
• CAPN11 A calpain with high mRNA levels in testis and lo ⁇ ated on ⁇ hromosome 6. Genomi ⁇ s 59: 243-247
• Capn7 A highly divergent vertebrate ⁇ alpain with a novel C-terminal do-main. Mamm. Genome 10: 318-321
• Genomi ⁇ organization of mouse Capn5 and Capn6 genes ⁇ onfirms that they are a distin ⁇ t ⁇ alpain subfamily. Genomi ⁇ s 48: 117-120

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