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WO1993007269A1 - ISOLATION D'UNE PROTEINE DE LIAISON FK506 DE Mr52000 ET CLONAGE MOLECULAIRE D'UN ADNc HUMAIN CORRESPONDANT - Google Patents

ISOLATION D'UNE PROTEINE DE LIAISON FK506 DE Mr52000 ET CLONAGE MOLECULAIRE D'UN ADNc HUMAIN CORRESPONDANT Download PDF

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WO1993007269A1
WO1993007269A1 PCT/US1992/008667 US9208667W WO9307269A1 WO 1993007269 A1 WO1993007269 A1 WO 1993007269A1 US 9208667 W US9208667 W US 9208667W WO 9307269 A1 WO9307269 A1 WO 9307269A1
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fkbp52
protein
binding
human
sequence
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PCT/US1992/008667
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Debra A. Peattie
Matthew W. Harding
David J. Livingston
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Vertex Pharmaceuticals Incorporated
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Publication of WO1993007269A1 publication Critical patent/WO1993007269A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones

Definitions

  • FK506 and rapamycin are structurally related macrolides that block distinct steps in intracellular signalling pathways.
  • Both are potent immunosuppressants, and drug action is mediated in part by binding to members of the immunophilin protein family.
  • FKBP FK506 binding protein
  • FKPB12 binds FK506, this activity is inhibited.
  • the present invention relates to the isolation of an FK506 binding protein (FKBP) of mammalian origin of approximate size (M r ) 52,000 and to the molecular cloning of a corresponding human cDNA from a human placental cDNA library.
  • FKBP52 FK506 binding protein
  • M r 52,000 protein hereinafter referred to as FKBP52
  • FKBP52 is a cytosolic protein isolated from bovine thy us by FK506 affinity chromatography and is a new member of a class of immunosuppressant FK506 binding proteins that play a key role in regulating immune responses.
  • Partial amino acid sequence of FKBP52 (approximately 30% of the complete protein sequence) is presented herein. The remaining sequence can be subsequently determined using known methods, such as those used to determine the partial sequence.
  • the human cDNA clone which is the subject of the present invention was isolated by screening a human placental cDNA library with a DNA probe whose sequence predicted a consensus amino acid sequence present in five FKBP12 sequences (human, urine, bovine,
  • Saccharomyces cerevisiae and Neurospora crassa and in the human FKBP13 sequence, another recently identified FK506 binding protein.
  • a clone identified in this manner contained a cDNA insert of approximately 2.2 kilobases.
  • the cDNA insert was purified and sequenced in its entirety.
  • the nucleotide sequence of the coding strand (2167 bases) including the ATG initiation codon and the TAG stop codon for the deduced protein product (the correct open reading frame) , is presented herein.
  • the amino acid sequence of the protein product of the open reading frame of the human cDNA clone was deduced.
  • the deduced protein has 459 amino acids and an M of 51,810, which is essentially the same M r as that of FKBP52.
  • the present invention includes a M p 52,000 FK506 binding protein (FKBP52) of mammalian origin, particularly a bovine and human M p 52,000 protein, DNA or RNA encoding FKBP52, and nucleic acid probes which hybridize with DNA or RNA encoding FKBP52.
  • FKBP52 FKBP52 binding protein
  • the present invention also includes FKBP52 homo- logues or equivalents (i.e., proteins which have amino acid sequences substantially similar, but not identical, to that of FKBP52 and exhibit FK506 binding characteristics) .
  • This invention further includes peptides (FKBP52 fragments which retain FK506 binding affinity, yet are less than the entire FKBP52 amino acid sequence) , monoclonal and polyclonal antibodies specific for FKBP52, and uses for the nucleic acid sequences, FKBP52, FKBP52 equivalents, and FKBP52 specific antibodies.
  • FKBP52 fragments which retain FK506 binding affinity, yet are less than the entire FKBP52 amino acid sequence
  • monoclonal and polyclonal antibodies specific for FKBP52 and uses for the nucleic acid sequences, FKBP52, FKBP52 equivalents, and FKBP52 specific antibodies.
  • These uses include methods of screening for new immunosuppressive compounds, methods of measuring the parent compound and/or metabolites in biological samples obtained from individuals taking immunosuppressive drugs, methods of identifying natural intracellular rapamycin-like and FK506-like substances (i.e., molecules or compounds) which function in regu ⁇ lation of cellular metabolism, and methods of identifying natural intracellular substrates which are potential targets for other novel immunosuppressive agents.
  • FKBP52 is associated with the 90kDa heat shock protein (hsp90) in untransformed steroid receptor complexes. Therefore, FKBP52 may also be useful in mediating steroidal hormone receptor transformation.
  • Figure 1 is the partial amino acid sequence of the M r 52,000 protein (FKBP52) .
  • Figure 1A is the N-terminal sequence of the bovine M r 52,000 FKBP52 (SEQ ID NO: 1).
  • Figure IB is the internal sequence data determined after endoproteinase Lysine C cleavage (SEQ ID NOS: 2- 11).
  • Figure 2 depicts the deduced sequence of hFKBP52 (SEQ ID NO: 12) and 133 chemically determined residues of bFKBP52 (SEQ ID NOS: 2-11) and shows that they align well with other known FKBPs (SEQ ID NOS: 12-21), polypeptides encoded by the GenBank murine cDNAs X17068 (SEQ ID NO: 22) and X17069 (SEQ ID NO: 23), and p59 (SEQ ID NO: 24) a defined component of untransformed steroid receptor complexes.
  • hFKBP52 shares 51 residues (above alignment) with hFKBP12, conserving 12 (dots) of the 14 residues involved in hydrogen-binding or hydrophobic interactions between hFKBP12 and FK506 or rapamycin.
  • residues all except Arg42, Phe46, and Glu54.
  • Asterisk (*) denotes an ambiguous residue; hyphen (-) denotes a gap.
  • Figure 3 depicts the 2167 bp sequence of the hFKBP52 cDNA that contains 99 bp 5' untranslated region (UTR) , 1377 bp ORF, and a 691 bp 3 , UTR (SEQ ID NO: 25).
  • the deduced hFKBP52 sequence (below ORF) contains 459 residues and predicts a 51.8 kDa protein (SEQ ID NO: 26) . Nucleotide and residue positions are on the left, with the initiating ATG as position 1.
  • the TAG stop codon is identified by 3 asterisks (***) , and the consensus polyadenylation-cleavage sequence AATAAA (38) is underlined.
  • the hFKBP52 cDNA sequence has been assigned GenBank accession number M88279.
  • a cytosolic protein of mammalian origin of M 52,000 has been isolated by the Applicant on the basis of its affinity for FK506, and its partial amino acid sequence has been determined.
  • a corresponding human cDNA has been cloned from a human placental cDNA library, its nucleic acid sequence has been determined and the amino acid sequence of the encoded protein has been deduced.
  • This M r 52,000 protein is referred to herein as FKBP52 and is a member of a novel class of FK506 binding proteins of varying size and binding capabilities.
  • FK506 affinity matrix was performed to isolate FK506 binding proteins from mammalian tissue samples (specifically, a bovine thymus cytosolic preparation) .
  • SDS-PAGE analysis of the eluate revealed that several proteins including the M r 52,000 protein were retained on the FK506 matrix and released by FK506 in solution.
  • FKBP52 the novel immunophilin described herein will be termed FKBP52 and referred to as having an M r 52,000.
  • FKBP52 is similar to other recently identified members of the FKBP family in this respect.
  • the other FKBPs identified include FKBP12, FKBP13 (Jim, Y.L. , et ah, Proc. Natl. Acad. Sci. USA. 88:6677-6681 (1991)), and FKBP25 (Galat, A., et al..
  • FKBP52 is consistent with prior convention for naming FKBPs according to their calculated M p s.
  • N-terminal amino acid sequencing of this M p 52,000 protein was performed after electrotransfer of the protein to a PVDF membrane, according to the method described by Matsudaira (Matsudaira, P., J. Biol. Chem. 262:10035-10038 (1987)).
  • internal sequence data SEQ ID NOS: 2-11 obtained by digestion of nitrocellulose membrane-bound peptide with an appropriate endopeptidase, such as Lysine C, followed by isolation of the resulting peptide fragments using microbore HPLC techniques described by Matsudaira in A PRACTICAL GUIDE TO PROTEIN AND PEPTIDE PURIFICATION FOR MICROSEOUENCING. Academic Press (San Diego, CA, 1989)) .
  • 133 amino acids of the sequence of the M_ 52,000 protein have been determined by chemical sequencing. This represents approximately 30% of the complete amino acid sequence.
  • Enzymatic properties of the M r 52,000 protein (FKBP52) eluted from the FK506 affinity matrix were assessed using known methods.
  • the assay of Harrison and Stein can be used to measure peptidyl prolyl cis-trans isomerization (PPIase) activity of
  • FKBP52 Also as described in Example 2, the ability of FK506 to inhibit isomerase activity of FKBP52 was assessed, using standard techniques.
  • FKBP52 is an active catalyst of the PPIase reaction.
  • the specific activity of FKBP52 is approximately 10% that of recombinant human FKBP12 (rhFKBP12) , measuring 3.9 x 10 5 M ' V for FKBP52 and 4.3 x lO ⁇ ' V for FKBP12 at 15°C.
  • Both FKBPs have similar selectivities for tetrapeptides differing at the P 1 position, with both immunophilins most efficiently catalyzing isomerization of peptides with large hydrophobic residues, such as leucine or phenylalanine, at P.,, as shown in Table 1.
  • Table 1 Characterization of hFKBP52 and hFKBP12 as PPIase catalysts of the isomerization of Suc-Ala-Pl-Pro-Phe-pNA substrates
  • the PPIase activity of hFKBP52 is potently inhibited by FK506 and rapamycin; both drugs are tight- binding inhibitors, with K j S of 10 nM and 8 nM, respectively (vs 0.6 nM and 0.25 nM, respectively, for hFKBP12) .
  • K j S 10 nM and 8 nM, respectively (vs 0.6 nM and 0.25 nM, respectively, for hFKBP12) .
  • the high affinity of FKBP52 for FK506 and rapamycin reasonably implies that FKBP52 could bind to these ligands at the systemic concentrations (blood levels) achieved during clinical use of these drugs, and that the well-documented spectrum of immunosuppressive effects and/or side- effects of FK506 therapy results, in part, from FKBP52- mediated actions.
  • DNA probes were designed as described in Example 3.
  • a computer search was used to screen the GenPept library for peptide sequences matching a consensus pattern derived from five known FKBP12 sequences and the human FKBP13 sequence. Two murine peptides were identified in this manner. Two DNA oligomers with sequences corresponding to part of the murine cDNA coding for the two peptides identified by the computer search were synthesized.
  • DNA oligomers were then used as polymerase chain reaction primers to amplify the DNA fragment.
  • This fragment was then cloned into a cloning vector and its DNA sequence determined.
  • This DNA fragment was then excised from the vector, radiolabeled with 32 P, and used to screen a human placental cDNA library (Stratagene, Catalog #936203) .
  • a human cDNA clone containing an approximately 2.2 kb insert which hybridizes with a DNA fragment encoding a consensus amino acid sequence present in both FKBP-12 and FKBP-13, has been identified, purified, and sequenced in its entirety.
  • the sequence of the coding strand which is 2167 bases, is presented in Figure 3 (SEQ ID NO: 25).
  • the correct open reading frame of the 2.2 kb cDNA sequence was identified (see Example 5) and the deduced amino acid sequence, from amino terminus to carboxyl terminus, is shown in Figure 3.
  • the deduced protein has 459 amino acids and an M r of 51,819 (SEQ ID NO: 26) .
  • hFKBP52 open reading frame was expressed in J__. coli and cleaved and uncleaved proteins were analyzed by gel electrophoresis to confirm the identity of hFKBP52.
  • This recombinant protein migrated with an apparent M 55,000, just as native bovine FKBP52.
  • the deduced hFKBP52 sequence is 79% identical to the X17069 polypeptide (452 residues) and 63% identical to the X17068 polypeptide (560 residues) , the lower percentage resulting from a 107 amino acid extension at the carboxyl terminus of the X17068 polypeptide ( Figure 2) .
  • hFKBP52 amino terminus is identical to the amino termini of two partially characterized proteins, p56 (Sanchez, E.R., et al.. Biochem.. 291:5145-5152 (1990)), now termed hsp 56 (Sanchez, E.R. , J. Biol. Chem. r 265:22067-22070 (1990); Yem, A.W., et al.. J. Biol. Chem.. 267:2868-2871 (1992)), and a reported 59 kDA immunophilin (Tai, P.- K.K., et al.. Science.
  • hFKBP52 sequence is 91% identical to the predicted sequence (458 residues, in Figure 2) of p59 (Lebeau, A.-C, et al. , J. Biol. Chem.. 267:4281-4284 (1992)), a 59kDa protein that associates with hsp90 in the untransformed rabbit androgen, estradiol, glucocorticoid, and progesterone receptors.
  • hFKBP52 and p59 sequences reflect the complete sequence of the 56-60 kDA protein found in untransformed mammalian and avian steroid hormone receptor complexes.
  • Steroid hormones bind to their respective steroid hormone receptors, and transform, or activate, the receptor to a DNA-binding form.
  • the untransformed, (in-active, non-DNA-binding) steroid hormone receptor typically comprises a receptor polypeptide associated with a number of heat shock proteins (hsps) , with a sedimentation coefficient of approximately 9S.
  • the glucocorticoid receptor is a heterotetramer with one receptor polypeptide, two hsp 90 molecules and on hsp 59 molecule.
  • Rexin M. et al.. J. Biol. Chem.. 266:24601-24605 (1990).
  • the 9S complex Upon binding of steroid hormone to untransformed receptor, the 9S complex dissociates to a -4-6S form, which then binds to DNA.
  • FKBP52 could be involved in stabilizing, or blocking, the inactive receptor and this could affect conversion of the receptor to its active, DNA binding, state by binding FK506 and/or rapamycin.
  • the deduced hFKBP52 sequence reveals a core consensus region when aligned with FKBP12 and other FKBPs.
  • This consensus region lies within the amino terminal portion of hFKBP52, between residues 41- 134, and contains 51 residues of conserved identity and position ( Figure 2) .
  • the key residues contributing to the high-affinity interaction between hFKBP12 and FK506 corroborate this FKBP12-like core of hFKBP52 and reasonably predict that residues 41-134 define the FK506- and rapamycin-binding domain of hFKBP52.
  • a pattern search alignment algorithm and secondary structure analysis also corroborate the hFKBP52 homology alignment.
  • Pattern searching built around the positions and identities of hFKBP12 residues that interact with FK506 and are conserved in different FKBP12 sequences, aligned the FKBP12, p59, and X17069 polypeptide sequences.
  • Secondary structure analysis of the hFKBP52 sequence predicted that the first one-third of the protein contains the FKBP12-like domain.
  • the Trp59 residue of FKBP12 in Van der aals contact with the pipecolinic moiety of FK506 and completely conserved in all FKBPs ( Figure 2) , was a particularly useful benchmark of the latter analysis. In all known members of the FKBP family, this conserved Trp residue is found near the beginning of a short ⁇ -helix that follows a short ⁇ - sheet.
  • HCA Hydrophobic cluster analysis
  • HBI hsp binding immunophilins
  • FKBP52 associates directly with hsp90 (Renoir, J.-M., et al. , J. Biol. Chem.. 265:10740-10745 (1990), Rexin, A., et al.. J. Biol. Chem.. 266: 24601-24605 .(1991)), and that FK506 and rapamycin bind directly to FKBP52, it is reasonable to predict the FKBP52 will have at least two structural domains to accommodate these distinct functions.
  • the FKBP12-like consensus region in the first one-third of FKBP52 reasonably defines the immunosuppressant binding domain of the protein, while the remaining residues reasonably constitute the putative hsp90 binding site.
  • the deduced hFKBP52 sequence contains a variety of consensus motifs that reflect possible post- translational modification(s) and/or functional characteristics of the protein. Consensus motifs typical of asparagine-linked glycoproteins, protein kinase phosphorylation sites, and calmodulin binding domains are present. Moreover, fourteen protein kinase phosphorylation site elements, representing five classes of motifs, are present in the deduced hFKBP52 sequence.
  • L 317 RLAS * H a multifunctional calmodulin-dependent protein kinase II or S6 kinase II element (XRXXS * X) ; I 25 S * PK and G 117 S * PP, a proline- dependent protein kinase motif (XS * PX) ; G 114 SAGS * P, W 25 EMNS * E, L 00 EYES * S, E 393 SSFS * N, L 346 ELDS * N, A 427 EASS * G and E 42 EQKS * N, casein kinase I phosphorylation sites (XS(P)XXS * X or XEXXS * X) ; and V 297 S * WLEY, F 306 S * NEEH, D 39 S * NNEK, and Q 452 S * QVET, sites of casein
  • This human cDNA clone can be used to produce an FKBP52 in vitro, such as by introducing the insert into an appropriate expression vector (e.g., pKK223, pOP, pRK5B) and expressing the encoded product in host cells (bacterial, yeast, or mammalian) containing the expression vector.
  • an appropriate expression vector e.g., pKK223, pOP, pRK5B
  • host cells bacterial, yeast, or mammalian
  • This expressed FKBP52 can be used for a number of diagnostic and therapeutic purposes.
  • the FKBP52 can be used in screening assays for detection of new naturally occurring immunosuppressant compounds.
  • FKBP52 could be used to screen fermentation broths, produced by known techniques, for compounds that bind to it and, thus, are potential immunosuppressant candidates.
  • FKBP52 can be used to screen existing synthetic compounds for binding affinity and subsequent immunosuppressant evaluation. It is reasonable to expect that a compound which binds FKBP52 will be FK506-like and, thus, have immunosuppressive capabilities.
  • FKBP52 can also be used as the basis for design of FK506-like molecules by determining and characterizing the active binding site(s) of FKBP52, designing a molecule which binds to it (them) and assessing its ability to suppress an immune response.
  • FKBP52 can be affixed to a solid support using a variety of chemical coupling techniques which link amino acid residues, such as methionine, lysine, cystine, and tryptophan to inert matrixes, such as Affigel (BioRad) or cyanogen bromide-treated Sepharose (Pharmacia) .
  • the FKBP52 bearing solid support is then contacted with tissue extracts or body fluids, such as blood and urine, from individuals receiving FK506 immunosuppressant treatment. Detection and/or quantitation of the parent compound FK506, or its metabolites, can be carried out using known methods, such as spectrophotometric measurement or scintillation counting.
  • FKBP52 it is also possible to use FKBP52 to identify natural, intracellular FK506-like substances (i.e., molecules or compounds) that function in intrinsic regulatory events in cellular immunity and metabolism.
  • FK506-like substances are defined herein as substances which bind FKBP52 to a similar extent as FK506 under the same conditions under which FK506 binds with
  • FKBP52 can be used to identify natural intracellular substances that may be targets for other novel immunosuppressive agents.
  • FKBP52 can also be modified in such a way as to enhance its binding capability, and/or other immunosuppressive characteristics. Such modifications (e.g., truncating sequence length) can be carried out using known methods, such as site directed mutagenesis. Finally, FKBP52 can be used to modify the transformation of steroid hormone receptors. As discussed herein, FKBP52 is common to several vertebrate species and is associated with the 90kDa heat shock protein (hsp90) in untransformed steroid hormone receptors. Thus, it is reasonable to predict that FKBP52 plays a critical role in the transformation of steroid hormone receptors.
  • hsp90 heat shock protein
  • FK506 binds tightly to FKBP52.
  • FKBP52 also binds rapamycin, another immunosuppressive agent
  • certain immunosuppressive treatments e.g. , cyclosporin, which binds to the immunophilin, cyclophilin
  • result in unpleasant side- effects which can be attributed to an increase in steroid hormone levels.
  • FK506 binds to FKBP52, which, in turn, transforms a steroid hormone receptor by causing dissociation of the FKBP52 molecule from a steroid hormone receptor complex, such as the androgen receptor. This transformation of the steroid receptor could lead to unwanted side-effects, such as an increase in body hair growth.
  • An antibody to FKBP52 can be co-administered to an individual receiving FK506 therapy to block binding of FK506 to FKBP52 and consequently block the steroid hormone receptor transformation.
  • an FK506-like substance can be used as an antagonist to block FK506 binding to FKBP52.
  • the extract was clarified by centrifugations at 40,000 x g and then 100,000 x g. Cytosol extract was then passed over a 5 ml FK506 affinity column containing an amino acid derivative of FK506 at the C32 position. Flow rate was 0.2 ml/min. The column was washed extensively with phosphate buffered saline containing 0.1% Tween 20 detergent and eluted sequentially with FK506 (200 ⁇ g/ l in phosphate buffer) and then 6 M guanidine hydrochloride. Eluted proteins were dialyzed extensively against 10 mM Tris, pH 7.0, and aliquots were lyophilized.
  • Approximate molecular weight was determined by SDS-PAGE on a 12%% acrylamide gel using lysozyme (M.W. 14,400), ⁇ -chymotrypsin (M.W. 21,500), carbonic anhydrase (M.W. 31,000), ovalbumin (M.U. 45,000) and bovine serum albumin (M.W. 66,000) to calibrate relative migration.
  • Proteins were visualized by Coomassie blue or silver staining or electroblotted onto either Immobilon-P (0.45 ⁇ m pore size, Millipore) or nitrocellulose (Schleicher and Scheull) .
  • the proteins transferred to Immobilin-P were visualized by Coomassie blue and used for N-terminal sequencing, described below. Proteins transferred to nitrocellulose were visualized with Ponceau S and used for in situ digestion.
  • N-terminal amino acid sequencing was performed after electrotransfer to a PVDF membrane as described by Matsudaira, P., J. Biol. Chem. , 262:10035 (1987).
  • a band of protein with M r - 55,000 band was excised from the Immobilin " P membrane and loaded directly into an automated sequencer (Applied Biosystems) for amino terminal sequencing.
  • Applied Biosystems automated sequencer
  • peptide fragments were generated by digest M r ⁇ 55,000 band (on nitrocellulose) with endoproteinase Lysine C (Wako Chemicals, USA) and then separating them by an HPLC system (Hewlett Packard) equipped with a variable wavelength detector and a Vydac C18 2.1 x 250 mm column.
  • the peptidyl-prolyl isomerization rate was determined by coupling isomerization of a prolyl- containing peptide to trans substrate hydrolysis by chymotrypsin (Fisher, G. , et al. , Nature 337:476-478 (1989) ) .
  • the assay was performed according to Harrison and Stein (Biochem.. 29:3813-3816 (1980) with modifications described by Park S. T. , et al.. J. Biol. Chem. 267:3316-3324 (1992).
  • PI Phe, Val, Ala, Gly, Glu or Lys
  • FKBP52 (60 nM final) was added to a reaction mixture containing substrate (27 ⁇ M final) in 0.1 M Tris-HCl, pH 7.8 at 15°C, and the solution was incubated in a 2 ml cuvette for 5 min at 15°C (950 ⁇ l final) before adding chymotrypsin (100 ⁇ g ml "1 final) to start reaction.
  • the final FKBP52 concentration was adjusted so the K obs was at least four-fold hig -*her than knon- exertenz.
  • Inhibition data were fit to an equation for tight-binding competitive inhibitors using KineTicTM software (BioKin, Ltd.) running on a Macintosh Ilex computer.
  • EXAMPLE 3 Identification of Murine cDNA Sequences A computer search was undertaken to identify protein sequences that contain a consensus pattern of conserved residues derived from five FKBP12 sequences (human, murine, bovine, Saccharomvces cerevisiae and Neurospora crassa) and the human FKBP13 sequence. This consensus pattern (SEQ ID NO: 32) is as follows:
  • the residues indicated in upper case letters are specific amino acids, defined by the single letter amino acid code. Each dash (-) indicates a gap introduced into one or more of the protein sequences for optimal alignment. A cross mark (x) represents any amino acid. Of the thirty-one conserved amino acids defined by the consensus pattern, nine Y26, F36, D37, V55, 156, W59, Y82, 191 and F99 (the upper case letter is the amino acid and the number is the position of the residue within human FKBP12) are residues known to interact with FK506 in the human FKBP12/FK506 co-complex (Van Duyne, G.D. et a_l. , Science 251:839 (1991) ) .
  • the predicted protein products from X17068 and X17069 were also identified by searching the GenPept database directly within the human FKBP12 amino acid sequence (SEQ ID NO: 13).
  • the alignment of the human FKBP12 (hFKBP12) sequence with the homologous portions of the predicted protein products of X17068 and X17069 is shown below:
  • Two short DNA oligomers each selected from a 1300 bp region of identity within X17068 and X17069 cDNAs, were synthesized as PCR primers.
  • the oligomers SEQ ID NO: 27, forward primer, and SEQ ID NO: 28, reverse primer) were constructed on a DNA synthesizer (Applied Biosystems) and used to amplify an approximate 500 bp fragment from a ⁇ ZAPII mouse thymus cDNA library (Stratagene Cloning Systems) .
  • Applied Biosystems Applied Biosystems
  • 2 ⁇ l of the library was heated at 80°C in 33.7 ⁇ l of water for 15 min.
  • the fragment was cloned into pCRlOOO (Invitrogen Corporation) , and competent K ⁇ coli DH5 ⁇ was transformed and plated.
  • the cloned insert (positive colony identified by hybridization) , corresponding exactly to a 534 bp portion of the murine cDNAs (nucleotides 40-573 in X17068 and X17069) , was sequenced with a Sequenase Version 2.0 DNA sequencing kit (US Biochemicals) .
  • the fragment was excised with EcoR I and Hind III • (all restriction enzymes from New England BioLabs) , radiolabeled with 32 P dCTP, and used as a hybridization probe for library screening.
  • Eighteen clones were selected by screening 4 x lo 5 plaques of a human placenta ⁇ ZAPII cDNA library (Stratagene) under stringent conditions. Fifteen clones were rescreened, and the inserts of twelve were excised to produce pBluescript (Stratagene) subclones for sequence analysis. Purified DNA from each clone was digested with Sac I and Kpn I, and insert sizes were determined by agarose gel electrophoresis.
  • Partial nucleotide sequences of each insert were determined with universal sequencing primers and the Sequenase kit.
  • a human FKBP52 (hRKBP52) cDNA clone containing an approximate 2.2 kilobase (kb) insert with 73% identity to the X17068 and X17069 nucleotide sequences was purified and sequenced in its entirety.
  • the sequence of the coding strand of the human cDNA clone, from 5' to 3', is shown in Figure 3 (SEQ ID NO: 25) .
  • the sequence is 2167 bases in length.
  • the ATG initiation codon and the TAG stop codon for the deduced protein product are underlined.
  • the correct open reading frame of the human cDNA sequence was identified by comparing the possible translation products to (1) the determined peptide sequences from the bovine thymus M r 52,000 protein and 2) the deduced amino acid sequences of the murine cDNAs identified by computer search.
  • the deduced amino acid sequence, from amino terminus to carboxyl terminus, of the human protein is shown in Figure 3 (SEQ ID NO: 26) .
  • EXAMPLE 6 Expression and Purification of Human FKB52 From E.
  • the hFKBP52 open reading frame (ORF) was expressed in E__ coli with a vector (pQE8, Qiagen Inc.) that expresses recombinant proteins with an amino terminal histidine tag that facilitates protein purification via Ni 2+ affinity chromatography.
  • pQE8, Qiagen Inc. a vector that expresses recombinant proteins with an amino terminal histidine tag that facilitates protein purification via Ni 2+ affinity chromatography.
  • Factor Xa to remove the tag and cleavage site from the recombinant hFKBP52 (rhFKBP52) .
  • Synthetic oligomers were used as PCR primers to modify and amplify the ORF.
  • the forward primer, SEQ ID NO: 30, included a BamHI site (GGATCC) , nucleotides encoding the Factor Xa cleavage site (ATCGAGGGTAGA to encode Ile-Glu-Gly-Arg) , and the first nineteen nucleotides of the hFKBP52 ORF (ATGACAGCCGAGGAGATGA) .
  • the ORF was amplified from the hFKBP52 insert by 10 rounds of PCR (5 min denaturation at 94°C for 1 min, 72°C for 2 min, final extension at 72°C for 10 min) in a thermocycler (Perkin Elmer Corporation) , and the resultant DNA fragment was digested with BamH I and
  • the cells were lysed by stirring for 1 hr at room temperature in 6 M guanidine HC1, 0.1 M NaH ⁇ O ⁇ ,, 10 mM Tris adjusted to pH 8.0 with NaOH, and the lysate was cleared by centrifugation (10,000 x g, 15 min, 4°C) and applied to an 8 ml Ni 2+ -NTA-agarose (Qiagen Inc.) affinity column.
  • rhFKBP52 was eluted from the column according to the manufacturer's instructions and was refolded by dialysis against Factor Xa buffer (0.1M NaCl, 50 mM Tris-HCl, pH 8.0, l mM CaCl 2 ) for 3 hr at 4°C.
  • the amino terminal tag was removed by dissolving -30 ⁇ g of lyophilized Factor Xa (Boehringer Mannheim

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Abstract

Protéine de liaison FK506 d'origine mammifère d'une taille exprimée en masse moléculaire d'environ 52000, isolée par la chromatographie d'affinité à FK506, et ADNc humain correspondant d'une taille approximative de 2,2 Kb, isolé par triage dans une banque d'ADNc de placenta humain à l'aide d'une sonde d'ADN dont la séquence prédit une séquence consensuelle d'acides aminés présente dans cinq séquences FKBP12 et dans la séquence FKBP13 humaine.
PCT/US1992/008667 1991-10-11 1992-10-09 ISOLATION D'UNE PROTEINE DE LIAISON FK506 DE Mr52000 ET CLONAGE MOLECULAIRE D'UN ADNc HUMAIN CORRESPONDANT WO1993007269A1 (fr)

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Publication number Priority date Publication date Assignee Title
EP0482189A4 (fr) * 1990-05-09 1994-03-30 Children's Research Institute
GB2279349A (en) * 1993-06-25 1995-01-04 Merck & Co Inc Human FKBP-38 protein
US5457182A (en) * 1994-02-15 1995-10-10 Merck & Co., Inc. FK-506 cytosolic binding protein, FKBP12.6
EP0672756A4 (fr) * 1992-08-12 1996-10-09 Fujisawa Pharmaceutical Co Anticorps monoclonal reconnaissant la proteine se liant au fk506, procede de titrage du niveau de proteine se liant au fk506 et trousse de titrage.
US5696135A (en) * 1995-06-07 1997-12-09 Gpi Nil Holdings, Inc. Inhibitors of rotamase enzyme activity effective at stimulating neuronal growth
US5763590A (en) * 1991-10-11 1998-06-09 Vertex Pharmaceuticals, Inc. Isolation of an Mr 52,000 FK506 binding protein and molecular cloning of a corresponding human cDNA
US5786378A (en) * 1996-09-25 1998-07-28 Gpi Nil Holdings, Inc. Heterocyclic thioesters
US5795908A (en) * 1995-06-07 1998-08-18 Gpi Nil Holdings, Inc. Small molecule inhibitors of rotamase enzyme activity
US5798355A (en) * 1995-06-07 1998-08-25 Gpi Nil Holdings, Inc. Inhibitors of rotamase enzyme activity
US5801197A (en) * 1995-10-31 1998-09-01 Gpi Nil Holdings, Inc. Rotamase enzyme activity inhibitors
US5801187A (en) * 1996-09-25 1998-09-01 Gpi-Nil Holdings, Inc. Heterocyclic esters and amides
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DE19907598A1 (de) * 1999-02-22 2000-08-24 Schulz Burkhard DNA Sequenz kodierend ein FKBP ähnliches Protein, Plasmide, Bakterien, Hefen und Pflanzen enthaltend dieses Protein sowie Mutanten in Arabidopsis für dieses Gen, die in der Ausprägung der Pflanzenarchitektur, der Reaktion gegenüber Brassinosteroiden und ihren Verbindungen und durch Ethylen vermittelten Gravitropismus der Wurzel defekt sind
US6172087B1 (en) 1998-06-03 2001-01-09 Gpi Nil Holding, Inc. N-oxide of heterocyclic ester, amide, thioester, or ketone hair growth compositions and uses
US6187796B1 (en) 1998-06-03 2001-02-13 Gpi Nil Holdings, Inc. Sulfone hair growth compositions and uses
US6187784B1 (en) 1998-06-03 2001-02-13 Gpi Nil Holdings, Inc. Pipecolic acid derivative hair growth compositions and uses
US6218423B1 (en) 1998-08-14 2001-04-17 Gpi Nil Holdings, Inc. Pyrrolidine derivatives for vision and memory disorders
US6218424B1 (en) 1996-09-25 2001-04-17 Gpi Nil Holdings, Inc. Heterocyclic ketone and thioester compounds and uses
WO2001049738A1 (fr) * 1999-12-29 2001-07-12 Fudan University Nouveau polypeptide, proteine fkbp humaine 11, et polynucleotide codant pour ce polypeptide
US6271244B1 (en) 1998-06-03 2001-08-07 Gpi Nil Holdings, Inc. N-linked urea or carbamate of heterocyclic thioester hair growth compositions and uses
US6274602B1 (en) 1998-06-03 2001-08-14 Gpi Nil Holdings, Inc. Heterocyclic thioester and ketone hair growth compositions and uses
US6274617B1 (en) 1998-06-03 2001-08-14 Gpi Nil Holdings, Inc. Heterocyclic ester and amide hair growth compositions and uses
US6313264B1 (en) 1994-03-08 2001-11-06 American Home Products Corporation Effector proteins of Rapamycin
US6333340B1 (en) 1998-08-14 2001-12-25 Gpi Nil Holdings, Inc. Small molecule sulfonamides for vision and memory disorders
US6335348B1 (en) 1998-08-14 2002-01-01 Gpi Nil Holdings, Inc. Nitrogen-containing linear and azepinyl/ compositions and uses for vision and memory disorders
US6337340B1 (en) 1998-08-14 2002-01-08 Gpi Nil Holdings, Inc. Carboxylic acids and isosteres of heterocyclic ring compounds having multiple heteroatoms for vision and memory disorders
US6339101B1 (en) 1998-08-14 2002-01-15 Gpi Nil Holdings, Inc. N-linked sulfonamides of N-heterocyclic carboxylic acids or isosteres for vision and memory disorders
US6376517B1 (en) 1998-08-14 2002-04-23 Gpi Nil Holdings, Inc. Pipecolic acid derivatives for vision and memory disorders
US6384056B1 (en) 1998-08-14 2002-05-07 Gpi Nil Holdings, Inc. Heterocyclic thioesters or ketones for vision and memory disorders
US6395758B1 (en) 1998-08-14 2002-05-28 Gpi Nil Holdings, Inc. Small molecule carbamates or ureas for vision and memory disorders
US6399648B1 (en) 1998-08-14 2002-06-04 Gpi Nil Holdings, Inc. N-oxides of heterocyclic ester, amide, thioester, or ketone for vision and memory disorders
US6462072B1 (en) 1998-09-21 2002-10-08 Gpi Nil Holdings, Inc. Cyclic ester or amide derivatives
US6506788B1 (en) 1998-08-14 2003-01-14 Gpi Nil Holdings, Inc. N-linked urea or carbamate of heterocyclic thioesters for vision and memory disorders
US6734211B1 (en) 1999-07-09 2004-05-11 Oregon Health & Sciences University Compositions and methods for promoting nerve regeneration
EP1044212A4 (fr) * 1998-01-09 2004-11-24 Human Genome Sciences Inc Proteines de liaisons fk506 humaines
US6852496B1 (en) 1997-08-12 2005-02-08 Oregon Health And Science University Methods of screening for agents that promote nerve cell growth
US6943187B2 (en) 1997-06-04 2005-09-13 Gpi Nil Holdings, Inc. Pyrrolidine derivative hair growth compositions and uses
WO2005109004A3 (fr) * 2004-05-06 2006-03-02 Leuven K U Res & Dev Agregation de proteines associee a une maladie
US7056935B2 (en) 1995-06-07 2006-06-06 Gpi Nil Holdings, Inc. Rotamase enzyme activity inhibitors
US7338976B1 (en) 1998-08-14 2008-03-04 Gpi Nil Holdings, Inc. Heterocyclic esters or amides for vision and memory disorders
EP3334424A4 (fr) * 2015-08-13 2019-04-24 River Town Therapeutics, Inc. Compositions et procédés pour le traitement de l'alopécie
EP3432987A4 (fr) * 2016-03-21 2019-12-25 David Weinstein Procédés de cicatrisation de plaies et de prévention de cicatrices

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EP0482189A4 (fr) * 1990-05-09 1994-03-30 Children's Research Institute
US5763590A (en) * 1991-10-11 1998-06-09 Vertex Pharmaceuticals, Inc. Isolation of an Mr 52,000 FK506 binding protein and molecular cloning of a corresponding human cDNA
EP0672756A4 (fr) * 1992-08-12 1996-10-09 Fujisawa Pharmaceutical Co Anticorps monoclonal reconnaissant la proteine se liant au fk506, procede de titrage du niveau de proteine se liant au fk506 et trousse de titrage.
US5576183A (en) * 1992-08-12 1996-11-19 Fujisawa Pharmaceutical Co., Ltd. Monoclonal antibody recognizing FK506-binding protein, method for assaying FK506-binding protein level, and kit therefor
US5846981A (en) * 1993-05-28 1998-12-08 Gpi Nil Holdings Inc. Inhibitors of rotamase enzyme activity
GB2279349A (en) * 1993-06-25 1995-01-04 Merck & Co Inc Human FKBP-38 protein
US5457182A (en) * 1994-02-15 1995-10-10 Merck & Co., Inc. FK-506 cytosolic binding protein, FKBP12.6
US6313264B1 (en) 1994-03-08 2001-11-06 American Home Products Corporation Effector proteins of Rapamycin
US6713607B2 (en) 1994-03-08 2004-03-30 Wyeth Effector proteins of Rapamycin
US5859031A (en) * 1995-06-07 1999-01-12 Gpi Nil Holdings, Inc. Small molecule inhibitors of rotamase enzyme activity
US5798355A (en) * 1995-06-07 1998-08-25 Gpi Nil Holdings, Inc. Inhibitors of rotamase enzyme activity
US5843960A (en) * 1995-06-07 1998-12-01 Gpi Nil Holdings, Inc. Inhibitors of rotamase enzyme activity
US5795908A (en) * 1995-06-07 1998-08-18 Gpi Nil Holdings, Inc. Small molecule inhibitors of rotamase enzyme activity
US7056935B2 (en) 1995-06-07 2006-06-06 Gpi Nil Holdings, Inc. Rotamase enzyme activity inhibitors
US7282510B2 (en) 1995-06-07 2007-10-16 Gliamed, Inc. Small molecule inhibitors of rotamase enzyme activity
US7960570B2 (en) 1995-06-07 2011-06-14 Gliamed, Inc. Small molecule inhibitors of rotamase enzyme activity
US6500843B2 (en) 1995-06-07 2002-12-31 Gpi Nil Holdings, Inc. Inhibitors of rotamase enzyme activity
US5696135A (en) * 1995-06-07 1997-12-09 Gpi Nil Holdings, Inc. Inhibitors of rotamase enzyme activity effective at stimulating neuronal growth
US6022878A (en) * 1995-06-07 2000-02-08 Gpi Nil Holdings, Inc. Inhibitors of rotamase enzyme activity
US7652060B2 (en) 1995-06-07 2010-01-26 Glia Med, Inc Small molecule rotamase enzyme inhibitors
US6140357A (en) * 1995-06-07 2000-10-31 Gpi Nil Holdings, Inc. Small molecule inhibitors of rotamase enzyme activity
US5801197A (en) * 1995-10-31 1998-09-01 Gpi Nil Holdings, Inc. Rotamase enzyme activity inhibitors
US5786378A (en) * 1996-09-25 1998-07-28 Gpi Nil Holdings, Inc. Heterocyclic thioesters
US6417209B2 (en) 1996-09-25 2002-07-09 Gpi Nil Holdings, Inc. Heterocyclic ketone and thioester compounds and uses
US5990131A (en) * 1996-09-25 1999-11-23 Gpi Nil Holdings Inc. Heterocyclic thioesters and ketones
US5801187A (en) * 1996-09-25 1998-09-01 Gpi-Nil Holdings, Inc. Heterocyclic esters and amides
US6218424B1 (en) 1996-09-25 2001-04-17 Gpi Nil Holdings, Inc. Heterocyclic ketone and thioester compounds and uses
US6984639B2 (en) 1996-09-25 2006-01-10 Gpi Nil Holdings, Inc. Heterocyclic ketone and thioester compounds and uses
US6486151B2 (en) 1997-02-28 2002-11-26 Gpi Nil Holdings Inc. N-oxides of heterocyclic esters, amides, thioesters, and ketones
US5846979A (en) * 1997-02-28 1998-12-08 Gpi Nil Holdings, Inc. N-oxides of heterocyclic esters, amides, thioesters, and ketones
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US6004993A (en) * 1997-06-04 1999-12-21 Gpi Nil Holdings, Inc. N-linked sulfonamide of heterocyclic thioester hair growth compounds and uses
US6943187B2 (en) 1997-06-04 2005-09-13 Gpi Nil Holdings, Inc. Pyrrolidine derivative hair growth compositions and uses
US6194440B1 (en) 1997-06-04 2001-02-27 Gpi Nil Holdings, Inc. Small molecule carbamate or urea hair growth compositions and uses
US6191125B1 (en) 1997-06-04 2001-02-20 Gpi Nil Holdings, Inc. Small molecule pipecolic acid derivative hair growth compositions and uses
US6187806B1 (en) 1997-06-04 2001-02-13 Gpi Nil Holdings N-linked sulfone of heterocyclic thioester hair growth compositions and uses
US6177455B1 (en) 1997-06-04 2001-01-23 Gpi Nil Holdings, Inc. Pyrrolidine derivative hair growth compositions and uses
US6852496B1 (en) 1997-08-12 2005-02-08 Oregon Health And Science University Methods of screening for agents that promote nerve cell growth
US7282340B2 (en) 1997-08-12 2007-10-16 Oregon Health Sciences University Methods for identifying an analog that promotes nerve regeneration
US6641810B2 (en) 1997-10-24 2003-11-04 Oregon Health & Science University Methods of using geldanamycin and FK506 to treat peripheral nerve damage
US5968921A (en) * 1997-10-24 1999-10-19 Orgegon Health Sciences University Compositions and methods for promoting nerve regeneration
US6210974B1 (en) 1997-10-24 2001-04-03 Oregon Health Sciences University Compositions and methods for promoting nerve regeneration
US6881409B2 (en) 1997-10-24 2005-04-19 Oregon Health And Science University Compositions and methods for promoting nerve regeneration
EP1044212A4 (fr) * 1998-01-09 2004-11-24 Human Genome Sciences Inc Proteines de liaisons fk506 humaines
US6187796B1 (en) 1998-06-03 2001-02-13 Gpi Nil Holdings, Inc. Sulfone hair growth compositions and uses
US6274602B1 (en) 1998-06-03 2001-08-14 Gpi Nil Holdings, Inc. Heterocyclic thioester and ketone hair growth compositions and uses
US6172087B1 (en) 1998-06-03 2001-01-09 Gpi Nil Holding, Inc. N-oxide of heterocyclic ester, amide, thioester, or ketone hair growth compositions and uses
US6187784B1 (en) 1998-06-03 2001-02-13 Gpi Nil Holdings, Inc. Pipecolic acid derivative hair growth compositions and uses
US6271244B1 (en) 1998-06-03 2001-08-07 Gpi Nil Holdings, Inc. N-linked urea or carbamate of heterocyclic thioester hair growth compositions and uses
US6274617B1 (en) 1998-06-03 2001-08-14 Gpi Nil Holdings, Inc. Heterocyclic ester and amide hair growth compositions and uses
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US6218423B1 (en) 1998-08-14 2001-04-17 Gpi Nil Holdings, Inc. Pyrrolidine derivatives for vision and memory disorders
US6333340B1 (en) 1998-08-14 2001-12-25 Gpi Nil Holdings, Inc. Small molecule sulfonamides for vision and memory disorders
US7338976B1 (en) 1998-08-14 2008-03-04 Gpi Nil Holdings, Inc. Heterocyclic esters or amides for vision and memory disorders
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US6399648B1 (en) 1998-08-14 2002-06-04 Gpi Nil Holdings, Inc. N-oxides of heterocyclic ester, amide, thioester, or ketone for vision and memory disorders
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US6462072B1 (en) 1998-09-21 2002-10-08 Gpi Nil Holdings, Inc. Cyclic ester or amide derivatives
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US6734211B1 (en) 1999-07-09 2004-05-11 Oregon Health & Sciences University Compositions and methods for promoting nerve regeneration
WO2001049738A1 (fr) * 1999-12-29 2001-07-12 Fudan University Nouveau polypeptide, proteine fkbp humaine 11, et polynucleotide codant pour ce polypeptide
WO2005109004A3 (fr) * 2004-05-06 2006-03-02 Leuven K U Res & Dev Agregation de proteines associee a une maladie
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EP3432987A4 (fr) * 2016-03-21 2019-12-25 David Weinstein Procédés de cicatrisation de plaies et de prévention de cicatrices

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