EP0640093A1 - Crabp-i et crabp-ii humaines - Google Patents

Crabp-i et crabp-ii humaines

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Publication number
EP0640093A1
EP0640093A1 EP93910828A EP93910828A EP0640093A1 EP 0640093 A1 EP0640093 A1 EP 0640093A1 EP 93910828 A EP93910828 A EP 93910828A EP 93910828 A EP93910828 A EP 93910828A EP 0640093 A1 EP0640093 A1 EP 0640093A1
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Prior art keywords
crabp
human
nucleic acid
sequence
seq
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German (de)
English (en)
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EP0640093A4 (fr
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Anders 3070 Whisperwood Dr. Astrom
John J. Voorhees
Ulrika 3070 Whisperwood Dr. Petterson
Amir Tavakkol
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University of Michigan
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University of Michigan
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • 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
    • C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates generally to cellular retinoic acid binding proteins (CRABPs) and, more specifically, to human CRABP-I and CRABP-II and the sequences encoding them, and their use in various assay systems for screening and diagnostic applications and for therapeutic purposes.
  • CRABPs cellular retinoic acid binding proteins
  • Retinoids are essential regulators of epithelial cell growth and cellular differentiation, skin being a major target in both normal and pathological states.
  • Sporn M.B. et al., Cancer Res. 43:3034-3040 (1983); Kopan, R. et al., J. Cell Biol. 109:295-307 (1989); Asselineau, D. et al., Dev. Biol. 133:322-335 (1989); and Lippman, S.M. et al., Pharmacol. Ther. 40:107-122 (1989). It has been shown that retinoids prevent cancer in skin and have efficacy as agents in human malignant and premalignant cutaneous disorders. Asselineau, D.
  • retinoids cause growth inhibition in many hyper-proliferating cell lines, a feature that makes the compounds of fundamental interest as anti-tumor and anti-psoriatic agents.
  • Sporn M.B. et al., Cancer Res. 43:3034-3040 (1983); and Asselineau, D. et al., Dev. Biol. 133:322-335 (1989).
  • Retinoids also play fundamental roles in directing the spatial organization of cells during development and the generation of vertebrate limbs. Eichele, G. Trends Genet. 5:246-251 (1989); and Summerbell, D. et al., Trends Neurosci. 13:142-147 (1990).
  • retinoids in the complex biological processes involved in cell growth and differentiation requires the identification of the specific components of the retinoid signal transduction system as well as the genes directly regulated by this system.
  • retinoid-binding proteins include cellular retinol-binding proteins (CRBP), nuclear retinoic acid receptors (RAR), cellular retinoic acid binding proteins (CRABP) and, most recently, RXRs, also belonging to the nuclear receptor superfamily of genes. See Sundelin, J. et al., J. Biol. Chem. 260:6488-6493 (1985); U, E.
  • CRABP Cellular retinoic acid binding proteins
  • CRABP is expressed when a more differentiated phenotype is induced by growth to confluence in the presence of elevated extracellular calcium concentrations. Siegenthaler, G. et al., Exp. Cell Res. 178:114-126 (1988).
  • CRABP retinoic acid
  • RARs retinoic acid receptors
  • RXRs retinoid X receptors
  • CRABPs have been shown to bind RA with high affinity, but their function is poorly understood. However, it was recently demonstrated that CRABP may be involved in cytochrome P-450 metabolism of RA. Fiorella, P.D. et al., J. Biol. Chem. 266:16572-16579 (1991).
  • CRABP-I and CRABP-II The sequences encoding two isoforms of human cellular retinoic acid binding proteins, designated CRABP-I and CRABP-II, and the gene for CRABP-II, have been cloned and sequenced. Their nucleic acid and corresponding amino acid sequences are set forth in the sequence listing preceding the claims.
  • Expression of human CRABP-II, but not CRABP-I was markedly increased in human skin in vivo and in skin fibroblasts in vitro after treatment with retinoic acid (RA) and after treatments which induce keratinocyte differentiation. The importance of RA dependent mRNA stabilization for keying CRABP II message at induced levels once transcription has occurred is also described.
  • RA retinoic acid
  • Human CRABP provides the basis for the construction of human CRABP-I and II viral, prokaryotic and eukaryotic expression vectors and recombinant expression constructs.
  • Human CRABP can now also be produced synthetically or ex vivo (outside the human body), for example, through the production of fusion proteins in bacteria and later cleavage and purification of CRABP therefrom.
  • Ligand binding studies utilizing human CRABP sequences can determine ligand binding affinity and the interaction of human CRABP with other human retinoid-binding proteins, and can be used to better identify tissue-specific drugs for pathologies in which retinoids are implicated.
  • Various assay schemes including reporter assay systems, direct and competition hybridization and binding assays employ the nucleic and amino acid sequences herein described.
  • Antibodies or binding fragments thereof produced to human CRABP can also be used in immunoassays of patient tissues for CRABP levels for diagnosis and the monitoring of treament. Purified or synthetic human CRABP can also be used for supplementation therapy.
  • Figure 2 compares human, mouse, rat and chicken CRABP.
  • Panel A is a comparison of the amino acid sequences of human (h) and mouse (m) CRABP. Dashes represent sequence identity; the asterisk at residue 118 represents a gap introduced in the CRABP-i sequence for maximum alignment.
  • Panel B is a sequence comparison of the NH 2 -terminal ends of human (h), mouse (m), rat (r) and chicken (c) CRABP. Boxed residues represent those dissimilar to human CRABP-II.
  • Figure 3 is an autoradiogram of RNA blots derived from three individuals which illustrates induction of CRABP-II mRNA in human skin by topically applied retinoic acid (RA). Lane (a) represents no treatments; lane (b) RA vehicle; and lane (c) 0.1% RA cream in RA vehicle under occlusion.
  • RA retinoic acid
  • Figure 4 is a bar graph of the RNA blot hybridization results (quantitated by laser densitometry) of nine independent experiments involving five dermal fibroblast lines prepared from three individuals and three diploid human lung fibroblast lines. The results illustrate the induction of human CRABP-II mRNA in human dermal fibroblasts compared to lung fibroblasts. The inset shows the relevant autoradiographic bands from two representative experiments comparing dermal and lung fibroblasts.
  • Figure 5 illustrates the expression of CRABP-II mRNA in cultured keratinocytes under various conditions.
  • Figure 5A is a bar graph of the results obtained for seven independent keratinocyte strains and illustrates the effects of confluence on human CRABP-II mRNA levels.
  • Figure 5B shows the effects of RA and increased calcium concentration on human CRABP-II mRNA levels in postconfluent keratinocytes, with treatment for prolonged periods with low concentrations of RA (3x10 '9 M) having no detectable effect.
  • Figure 5C illustrates that prolonged treatment of postconfluent keratinocytes with higher concentrations of RA (3x1 O ⁇ M) reduced CRABP-II mRNA to undetectable levels.
  • Figure 6 is a schematic diagram of a retinoic acid receptor (RAR)-CRABP or RXR-
  • Figure 7 is a restriction map of a bacteriophage lambda clone ( ⁇ 2.1) isolated from a human placenta genomic library of the human CRABP-II gene is provided with the exons indicated as filled boxes numbered I to IV.
  • Figure 8 illustrates the nucleotide sequence and shown above the nucleotide sequence is the deduced amino acid sequence at the human CRABP-II gene.
  • Figure 9 illustrates the transcription start sites in the human CRABP-II gene as determined by primer extension analysis, with the major transcription site indicated by an arrow and the corresponding base in the sequence indicated by an asterisk.
  • Figure 10 illustrates a nuclear run-off assay using nuclei isolated from cultured human skin fibroblasts treated with retinoic acid.
  • Figure 11 illustrates the mRNA level and transcription rate of CRABP-II after quantitation by phosphorimaging and normalization to cyclophilin.
  • Figure 12 is an autoradiogram demonstrating the effects of cycloheximide or actinomycin D on the induction of CRABP-II mRNA expression.
  • Figure 13 is a bar graph demonstrating the effects of cycloheximide on CRABP-II transcription rates determined by nuclear run-on assays.
  • the cloning and sequencing of two human CRABP cDNAs revealed one with a predicted amino acid sequence 99.3% identical to the mouse and bovine CRABP-I, and a second with a predicted amino acid sequence, 93.5% identical to the mouse CRABP-II.
  • the CRABP-II described herein appears to be the human homolog to mouse CRABP-II, since both are expressed in adult skin, and is therefor designated as such.
  • the high amino acid homology seen between bovine, mouse and human CRABP-I indicates that this isoform has been more conserved throughout evolution than the CRABP-II ( Figure 2A described below). This was especially evident when the NH 2 -terminal sequence between rat, chicken, mouse and human CRABP-I and CRABP-II were compared. No differences were seen between the CRABP-ls, while several differences exist between the CRABP-lls.
  • CRABP-I transcripts were undetectable in adult human epidermis by R b.ot hybridization, while the CRABP-II cDNA probe detected an approximately 1.2 kiloba ⁇ e (Kb) mRNA transcript.
  • External application of 0.1% retinoic acid cream in vivo for 16 hours resulted in a 16-fold induction of CRABP-II, but not CRABP-I.
  • CRABP-II mRNA was also markedly increased (> 15-fold) by retinoic acid treatment of fibroblast cultured from human skin, whereas no significant induction of CRABP-II mRNA was observed in human lung fibroblast.
  • Human CRABP-II, but not CRABP-I mRNA was significantly induced by treatments which induce keratinocyte differentiation in vitro. Highly increased levels of
  • CRABP-II RNA were found in psoriatic epidermis when compared to normal epidermis for 10 different patients. CRABP-I message levels were on the other hand very low or undetectable in both normal and psoriatic skin (data not presented in Figures). Previous studies of the induction of CRABP by natural and synthetic retinoids and their increased levels during keratinocyte differentiation are consistent with the expression of CRABP-II message seen in the present investigation, but not with the expression of CRABP-I. These studies thus identify CRABP-II as the isoform likely to be expressed and regulated by RA in adult human skin.
  • Pillai S. et al., J. Cell. Physiol. 143:294-302 (1990). Moreover, these events are accelerated and began to occur prior to confluence when the extracellular calcium concentrations were increased to 1.2 or 2.4 mM. Pillai, S. et al., J. Cell. Physiol. 143:294-302 (1990). CRABP-II mRNA levels responded to confluence and external calcium in an identical fashion ( Figure 5 described below).
  • RA induction of the human CRABP-II gene occurs at the level of transcription and whetherthis regulation is mediated by specific nuclear receptors remains to be investigated, it has been shown that human skin as well as cultured human skin fibroblasts express RAR-y. Krust, A. et al., PNAS (USA). 86:5310-5314 (1989); and Elder, J.T. et al., J. Invest Dermatol. 96:425-433 (1991). However, this does not explain the lack of RA induction of CRABP-II mRNA seen in cultured human lung fibroblasts and keratinocytes, also known to express RAR- ⁇ . Elder, J.T. et al., J. Invest. Dermatol. 96:425-433 (1991). If the CRABP-II gene is regulated by the RARs, additional tissue or cell-specific factors may be required for RA induction.
  • TRAP apparently is forming a heterodimer with the thyroid hormone receptor on the responsive element. Such dimerization between nuclear receptors and other transcription factors could explain tissue-specific regulation. If there is a skin-specific factor forming a heterodimer with the RARs, that could explain why the CRABP-II gene is induced by RA in skin fibroblasts, but not lung fibroblasts.
  • CRABP-II is expressed in human skin in vivo and the CRABP-II gene appears regulated by RA in skin in vivo and in cultured skin fibroblasts in vitro.
  • CRABP-II was not, however, induced by RA in cultured lung fibroblasts, demonstrating cell-specific regulation of this gene.
  • CRABP-I does not appear regulated by RA and is found at very low or undetectable levels in human skin in vivo, as well as in keratinocytes and fibroblasts. This suggests that CRABP-II may participate in a regulatory feedback mechanism to control the action of RA on cell differentiation in skin.
  • the identification of human CRABPs, RARs and RXRs now allows studies, such as those described below, on interactions between members of these families in the complex molecular and cellular mechanisms of RA action.
  • the identification of the nucleic and amino acids sequences of human CRABP-I and CRABP-II provide the basis for a variety of recombinant products, including vectors carrying the human CRABP cDNA sequences and expression constructs cotransfected or infected with such vectors.
  • vectors carrying the human CRABP cDNA sequences and expression constructs cotransfected or infected with such vectors For example, plasmid or viral vectors carrying human CRABP cDNA have been constructed and are used to cotransfect or coinfect receptor-deficient CV-1 monkey kidney cells.
  • Reporter assay systems utilizing CV-1 recombinant expression constructs which include human CRABP cDNA, a reporter element containing a retinoid responsive element and a reporter gene, and preferably internal control sequences, such as generally described in Astr ⁇ m, A. et al., Biochem.
  • SV40 vector carrying a human RAR or RXR of interest and a ⁇ -galactosidase vector (pcH110) for an internal control are coinfected or cotransfected into CV-1 cells.
  • the recombinant CV-1 construct is exposed to the binding ligand (e.g. retinoid) of interest.
  • ligand e.g. retinoid
  • ligand is meant a molecule which binds to the receptor binding protein and induces the expression of the gene of interest.
  • induction of the reporter gene is used to assay for ligand binding to receptor protein. Absent any additional regulatory requirements, functional ligand would bind RAR or RXR, stimulating the expression (and translation) of the reporter gene (CAT).
  • the interaction between human CRABP and RAR or RXR or other binding receptors can be determined. For example, if CRABP sequesters RA in the cytoplasm, less ligand will reach the nucleus, thereby reducing RAR or RXR-mediated stimulation of the reporter gene.
  • the sequencing of human CRABP also allows the raising or production of antibodies or binding fragments, e.g. F'(ab), which can be used in immunoassays to further characterize binding or for diagnostic purposes and to monitor the course of patient treatment.
  • F'(ab) e.g. F'(ab)
  • patient tissue can be assayed for the presence and levels of human CRABP to diagnose particular conditions where retinoids are implicated and to monitor the effectiveness of drugs and other treatments in altering patient levels of CRABP.
  • CRABP purified directly from human tissue or cells, in culture or human CRABP produced synthetically or ex vivo will also provide CRABP for supplementation therapy where needed.
  • the gene for human cellular retinoic acid-binding protein II (CRABP-II) has been cloned and sequenced.
  • the sequence of the upstream region of the CRABP-II gene is rather GC rich and has a TATA box at -31 , and several possible binding sites for transcription factors. Of special interest is the presence of potential AP2 (CCC/GCA/GGGC) sites in the
  • AP2 has been shown to be identical to the transcription factor KER1 which has been suggested to be generally involved in epidermal gene regulation.
  • KER1 transcription factor 1
  • CRABP-II is predominantly expressed in the skin of adult mice and we have recently demonstrated that CRABP-II, but not CRABP-I is expressed in human skin. Giguere, V. et al., PNAS (USA) 87:6233-6237 (1990); Astr ⁇ m, A. et al., J. Biol. Chem. 266:17662-17666 (1991).
  • both AP2 and CRABP-II mRNA have been shown to be induced by RA. Astr ⁇ m, A. et al., J. Biol. Chem. 266:17662-17666 (1991); Luscher, B. et al., Genes Dev. 3: 1507-1517 (1989). Whether AP2 is involved in skin-specific expression and RA induction of the CRABP-II gene remains to be determined. As shown in Figure 8, the upstream region also contains a high affinity Sp1 binding site (GGGGCGGAGC) close to the TATA box, and two sequences (GCGGGGGCG) identical to Krox-24 binding sites. Kadonaga, J. T. et al., Trends Bichem. Sci.
  • the upstream region of the CRABP-II gene also contains a direct repeat (G/AGTTCA) spaced with one nucleotide with homology to the RARE found in the RAR- ⁇ 2 promoter, except that the RAR- ⁇ 2 RARE is spaced by five nucleotides.
  • G/AGTTCA direct repeat
  • two other members of the hydrophobic ligand-binding family of genes contain a RARE (CRBP-I) and a RXRE (CRBP-II) in their promoters. Smith, W.C. et al., EMBO J. 10:2223-2230 (1991 ); Mangelsdorf, D.J.
  • nucleotide and amino acid sequences of the present invention can include some variation from the sequences represented by and complementary to the sequences set forth in the Sequence Listing, but must be substantially represented by or complementary to those set forth therein. By “substantially represented by” or “substantially complementary to” is meant that any variation therein does not impair the functionality of the sequence to any significant degree.
  • SPECIFIC EXAMPLES .
  • the cDNA was used as a template for PCR (2 minutes denaturation at 92°C, 2 minutes, annealing at 42°C, 2 minutes amplification at 72°C and final extension for 10 minutes). After 40 cycles, the length of the amplified DNA was determined on a 1.5% agarose gel. The amplified 436-bp region of CRABP was isolated from the gel, subcloned into Bluescript phagemid and sequenced as described below. Screening and sequencing of cDNA clones
  • RNA was prepared from human skin as described in Elder, J.T. et al., J. Invest. Dermatol. 94:19-25 (1990) and used to prepare a cDNA library in Lambda Zapll (Stratagene Inc., La Jolla, CA). The library contained 1.0 x 10 7 primary recombinants. The 436-bp PCR product was labeled by random hexamer priming (Boeringer Mannheim, Indianapolis, IN) and used to screen the adult human whole skin cDNA library and a human skin fibroblast cDNA library in ⁇ gt11 (Clontech, Palo Alto, CA).
  • the filters were washed two times for 20 minutes in 0.2 x SSC, 0.2% SDS and one time for 20 minutes in 0.2 x SSC, 0.1% SDS at 55°C
  • Two positive clones were isolated from the skin library and five clones from the skin fibroblast library. The clones from the skin library were rescued, while the clones from the fibroblast library were subcloned into Bluescript phagemids (Stratagene Inc., La Jolla, CA).
  • DNA sequence analysis was performed on both strands by dideoxy chain termination as generally described in Sanger, F. et al., PNAS (USA) 74:5463-5467 (1977), using modified T7 polymerase (Sequenase, U.S. Biochemical Corp.) and synthetic oligonucleotides.
  • RNA was isolated from keratome biopsies and cultured cells by guanidinium isothiocyanate lysis and ultracentrifugation as previously described in Elder, J.T. et al., J. Invest. Dermatol.94:19-25 (1990).
  • 0.1% RA cream (Retin-A, Ortho Pharmaceutical Corp. Raritan, NJ) was applied once to skin and maintained under plastic wrap for 4 hours to 96 hours prior to biopsy. Adjacent sites were treated with Retin-A vehicle or left untreated. After 4 hours, 12 hours, 16 hours or 96 hours, keratome biopsies were obtained and used for RNA isolation.
  • RNA concentrations were determined by absorbance at 260 nm and verified by nondenaturing agarose gel electrophoresis and ethidium bromide staining as described in Thompson, CB. et al., Nature 314:363-366 (1985). Equal quantities of total RNA were electrophoretically separated in 1% formaldehyde-agarose gels containing 0.5 ⁇ g/ml ethidium bromide and transferred to derivatized nylon membranes (Zeta-Probe, BioRad, Richmond, CA) as described in Elder, J.T. et al., J. Invest. Dermatol. 94:19-25 (1990) and Maniatis, T.
  • Hybridization probes were prepared by random priming (see Feinberg, A.P. et al., Anal. Biochem. 132:6-13 (1983)) of low melting agarose-purified insert fragments from the human CRABP-I PCR product, the CRABP-I cDNA ( ⁇ fl.1) and rat cyclophilin. See Danielson, P.E. et al., DNA 7:261-267 (1988).
  • PCR and degenerate primers derived from bovine and mouse CRABP-I a 436 base pair (bp) product was obtained, subcloned and sequenced.
  • the predicted amino acid sequence of the PCR product was found to be 99.3% homologous to the mouse and bovine CRABP-I sequences. See Nilsson, M.H.L et al., Eur. J. Biochem. 173:45-51 (1988); and Stoner, CM. et al., Cancer Res. 49:1497-1504 (1989).
  • the PCR product was used to screen a human skin library and a human skin fibroblast library.
  • clones Five clones were isolated from the skin fibroblast library ( ⁇ f1.1, ⁇ f3.1, ⁇ f5.1, ⁇ f5.4 and ⁇ f5.6). Two clones ( ⁇ fl .1 , ⁇ f3.1) were sequenced and found to be identical. The predicted amino acid sequences of these clones were found to be 77.4% similar to human CRABP-I and 93.5 % similar to the recently cloned mouse CRABP-I. See Giguere, V. et al., PNAS (USA) 87:6233-6237 (1990). Because of the high homology to the mouse CRABP-II, clone ⁇ f1.1 was designated as human CRABP-II.
  • a third clone ⁇ f5.1 was partially sequenced and found to be a shorter clone, with a sequence identical to human CRABP-II.
  • No CRABP-I clones were isolated from the skin fibroblast library.
  • Two clones were isolated from the skin library and found to contain shorter inserts, one with a sequence identical to human CRABP-II ( ⁇ s2.1) and one with a sequence identical to human CRABP-II ( ⁇ s3.1).
  • SEQ ID NOS. 1 and 2 in the Sequence Listing represent the cDNA nucleotide and predicted amino acid sequences of human CRABP-II.
  • the translation initiation site was assigned to the first methionine codon corresponding to nucleotides 99-101.
  • An open-reading frame of 138 amino acids was found, predicting a polypeptide of M r 15,693.
  • the 3 * untranslated region was found to contain a polyadenylation signal (ATTAAA) and a poly(A) tract of 1 ( ⁇ f1.1) to 25 ( ⁇ f5.1).
  • SEQ ID NOS. 3 and 4 represent the cDNA nucleotide and predicted animo acid sequence of CRABP-i respectively. Comparison of amino acid sequences of CRABP
  • Panel A is a comparison of the amino acid sequences for human (h) and mouse (m) CRABP.
  • the predicted amino acid sequences of mouse and human CRABP-I and mouse CRABP-II were aligned with human CRABP-II.
  • Dashes (— ) represent identity to human CRABP-II.
  • One gap as indicated by an asterisk (*) was introduced in the human and mouse CRABP-I sequences for maximum alignment.
  • Panel B of Figure 2 is the sequence comparison of the NH 2 -terminal ends of human (h), mouse (m), rat (r), CRABP-I and CRABP-II. Residues dissimilar to human CRABP-II are boxed.
  • the amino acid sequence comparison presented in Figure 2, Panel A reveals a
  • Human and mouse CRABP-II displayed 9 amino acid differences with the following amino acids in the human sequence: residue 19 - Leu, 22 - Val, 27 - Val, 29 - Leu, 48 - Gly, 68 - Val, 91 - Giu, 99 - Lys and 111 - Thr.
  • residue 19 - Leu, 22 - Val, 27 - Val, 29 - Leu, 48 - Gly, 68 - Val, 91 - Giu, 99 - Lys and 111 - Thr In 6 of the 9 amino acid differences seen between human CRABP-II and mouse CRABP-II (residues 19, 29, 48, 68, 91 and 111), the human sequence was identical to CRABP-I.
  • RNA blot hybridization The autoradiograms of the RNA blots derived from three individuals are shown in Figure 3. Total RNA (40 ⁇ g per lane) was hybridized against the human CRABP-II or cyclophilin cDNA probes as described above.
  • Each volunteer was treated topically for 16 hours prior to biopsy as follows: (a) no treatment, (b) RA vehicle or (c) 0.1% RA cream in RA vehicle under occlusion with plastic wrap. Mobilities of ribosomal RNAs are indicated to the left of the blots.
  • CRABP-I transcripts were usually undetectable under exposure conditions sufficiently sensitive to detect single copy DNA sequences from 10 ⁇ g human genomic DNA.
  • CRABP-I and CRABP-II cDNA probes detected distinct band patterns using genomic DNA digested with BamHI. EcoRI, Hindlll and Pstl, demonstrating the specificity of these probes under our hybridization conditions (data not presented in Figures).
  • CRABP-II mRNA was not significantly induced by RA in three strains of human lung fibroblasts, suggesting that this response may be tissue specific. Induction of human CRABP-II transcripts by RA was dose-dependent over the range of 3 x 10 "10 to 3 x 10 "7 M RA (data not presented in Figures). In contrast, CRABP-I transcripts were undetectable in dermal and lung fibroblasts and were not induced by RA, whereas genomic DNA blots hybridized in parallel were positive. Expression of CRABP in human keratinocytes
  • FIG. 5 summarizes the results obtained for seven independent keratinocyte strains. Third passage normal adult human keratinocytes were grown in KGM containing 0.15 mM CaCI 2 , and medium was changed every other day. At the indicated number of days pre- or post-confluence, total RNA was prepared and analyzed for human CRABP-II mRNA by blot hybridization and densitometry.
  • CRABP-II mRNA was also markedly induced in subconfluent cultures by raising the calcium concentration in the medium from 0.15 mM to 2 mM, as illustrated for a representative strain of keratinocytes in Figure 5B. At 20-30% confluence, the medium was changed to KGM or KGM containing 2 mM CaCI 2 in the presence or absence of 3 x 10 ⁇ 9 M RA and maintained for the indicated number of days, with medium change every other day. Mobilities of 28S and 18S ribosomal RNAs are indicated to the left.
  • Figure 5C shows the effects of prolonged treatment with high concentrations of RA on CRABP-II mRNA levels.
  • FIG. 5B The same experiment described for Figure 5B was conducted above, except that cells were treated with or without 3 x 10 "6 M RA for 2 to 5 days, as indicated above the autoradiograms. Mobilities of 28 and 18S ribosomal RNAs are indicated to the left.
  • Figures 5A and B show the results representative of four independent experiments. As shown in Figure 5B, treatment of keratinocytes for prolonged periods of time with low concentrations of RA (3 x 10 "9 M) had no detectable effect on CRABP-II mRNA levels. However, as shown in Figure 5C, prolonged treatment of subconfluent keratinocytes with high concentrations of RA (3 x 10 "6 M) reduced CRABP-II mRNA to undetectable levels.
  • the CRABP-II cDNA probe was cloned by PCR from retinoic acid (RA) treated human skin using the CRABP-II degenerate primers shown in Figure 1B, derived from mouse CRABP-II mRNA sequence. See Giguere, V. et al., PNAS (USA) 87:6233-6237 (1990). BamHI restriction sites were included in the forward and reverse primers to aid in subsequent cloning.
  • the PCR product was further purified (Geneclean, BIO 101 Inc.), digested with BamHI and cloned into BamHI digested PGEM 3Z plasmid (Promega Inc., Madison, Wl).
  • the PCR fragment was initially identified as CRABP-II by digestion with Pstl restriction enzyme that cleaved the DNA at the same position as that described in the mouse CRABP-II mRNA.
  • the clone was sequenced and found to represent the major portion of the mature human analogue of the mouse CRABP-II.
  • the cDNA probe was hybridized to human RNA prepared from skin of human volunteers topically treated with RA for 4 and 12 hours.
  • CRABP-I was excised from the Bluescript phagemid (Stratagene) cloning vector and inserted between the Xbal and BamHI sites of the eukaryotic expression vector pSVL (Pharmacia) in forward orientation (pSVLCRABP-l).
  • CRABP-II was excised from the Bluescript phagemid (Stratagene) cloning vector and inserted into the EcoRI site of the eukaryotic expression vector pSG5 (Stratagene) in forward (pSG5CRABP-ll) or reverse orientation (pSG5CRABP-IIAS). The orientation of the insert was determined by restriction analysis.
  • CV-1 cells are grown in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal calf serum.
  • DMEM Dulbecco's modified Eagles medium
  • CV-1 are monkey kidney cells derived from the CV-1 cell line which do not express T cell antigen.
  • ChFCS charcoal treated fetal calf serum
  • Cells are contransfected using the calcium phosphate co-precipitation technique essentially as described in Rosenthal, N., Meth. Enzymol. 152:704-720 (1987) with CRABP-I (pSVLCRABP- I) or CRABP-II (pSGSCRABP-ll) expression vector.
  • cells are trypsinized and suspended in medium, pelleted and washed once with 40mM Tris-C1 , pH 7.6 containing 150 mM NaCI and 1 mM ⁇ DTA.
  • a cytosolic fraction is prepared on cell lysates by centrifugation at 100,000 x g for I hour.
  • CRABP-I the nucleotide sequence of CRABP-I is changed at position 9 T to G) and 13 (C to T) to create a Stul site, using synthetic oligonucleotides and the polymerase chain reaction.
  • CRABP-II the nucleotide sequence of CRABP-II is changed at position 100 (T to G) and 104 (C to T) to create a Stul site, using synthetic oligonucleotides and the polymerase chain reaction.
  • the mutated CRABP cDNAs are then cut with Stul and ligated into the Stul and EcoRI sites of the bacterial expression vector pMAL-c (New England Biolabs).
  • MBP-CRABP fusion protein Bacteria is transformed and the maltose-binding protein (MBP)-CRABP fusion proteins are expressed in large quantities.
  • the MBP-CRABP fusion protein is purified by affinity chromatography on any amylose column (New England Biolabs). CRABP lacking the first methionine is released from MBP by digestion with factor Xa.(New England Biolabs) and purified from MBP by a second passage over an amylose column.
  • Spectrofluorimetric methods are used to study the affinities and binding stoichiometries of purified human CRABP-I and CRABP-II for a variety of ligands.
  • the capacity of ligands to bind to CRABP-I or CRABP-II is assessed by monitoring their ability to quench the native fluorescence of this protein.
  • SPECIFIC EXAMPLE 6 Production of Antibodies
  • Peptides corresponding to amino acids 94 to 104 in the CRABP-I and CRABP-II proteins have been synthesized using the multiple antigenic peptide method as generally desbribed in Posnett, D.N. etal., ⁇ fetf ⁇ . Enzymol. 178:739-746 (1989), eliminating the need for conjugation to a carrier protein.
  • This region of the two CRABPs has a low homology and is most probably situated on the outside of the proteins based on hydrophobic'rty and surface probability calculations.
  • the peptides were injected into chickens for production of egg IgY.
  • the resulting antibodies if not specific, are adsorbed to peptides (CRABP-I antibodies to CRABP-II peptides and the reverse) immobilized on a sepharose gel to enhance specificity.
  • CRABP-I antibodies to CRABP-II peptides and the reverse
  • the expression and regulation of CRABP-I and CRABP-II proteins in human skin and skin cells is examined quantitatively by Western blot analysis and semi-quantitatively by immunocytochemistry.
  • the pattern of expression and regulation of CRABP-II (and CRABP-I) by RA in normal and psoriatic skin will provide insight into the function of CRABPs.
  • Antibodies to CRABP-I and CRABP-II, or binding fragments (e.g. F'(ab)) thereof, and the purified proteins obtained as described above can be used to monitor levels of CRABP-I and CRABP-II in normal and pathological states by using immunological techniques known to those skilled in the art. For example, patient skin tissue can be assayed with antibody specific for human CRABP-I or CRABP-II to determine the presence and levels of CRABP by ELISA, Western blot analysis or immunochemistry essentially as described in Busch, C et al., Meth. Enzymol. 189:315-324 (1990). SPECIFIC EXAMPLE 7. Reporter Assay System
  • CV-1 cells are grown in Dulbecco's modified eagles medium (DMEM) containing 10% fetal calf serum. The day before transfection, cells are seeded on tissue cultured dishes in DMEM containing 10% charcoal treated fetal calf serum (CHFCS). Cells are cotransfected using the calcium phosphate co-precipitation technique with 0.6 mg of human retinoic acid receptor (hRAR) expression vectors (hRAR ⁇ O, hRAR ⁇ O or hRAR ⁇ O), a reporter plasmid and a ⁇ -galactosidase expression vector (pcH110, Pharmacia) used as an internal control to normalize for variations in transfection efficiency essentially as described in Astr ⁇ m, A.
  • DMEM Dulbecco's modified eagles medium
  • CHFCS charcoal treated fetal calf serum
  • Cells are also cotransfected with CRABP-I (pSVLCRABP-l) or CRABP-II (pSG5CRABP-ll) expression vectors of pSVL (Pharmacia) as a control.
  • CRABP-I pSVLCRABP-l
  • CRABP-II pSG5CRABP-ll
  • the reporter plasmid (TRE) 3 -#r-CAT is constructed by ligating synthetic oligonucleotides encoding three palindromic thyroid hormone responsive elements (TRE) ((TCAGGTCATGACCTGAJg) flanked by Hindlll and BamHI sites on the 5' and 3' ends respectively and cloned into the Hindlll - BamHI cloning sites of the plasmid pBLCAT2. 24 hours after transfection, cells are washed once with DMEM, and medium (DMEM, 10%ChFCS) containing different concentrations of ligands are added to the cells.
  • DMEM DMEM, 10%ChFCS
  • two mutually dependent viruses one containing a receptor transcription unit and the second containing a gene responsive element are coinfected into receptor- deficient cells such as CV-1.
  • receptor- deficient cells such as CV-1.
  • two mutually dependent adenoviruses one containing a human glucocorticoid receptor transcription unit and the other a glucocorticoid responsive element linked to the firefly luciferase gene, or one containing rat thyroid hormone receptor a and the other the luciferase gene, can be utilized in the practice of the assay. Hormone-induced transcription is then quantitated after infection from cells coinfected with the complementary virus pair.
  • SPECIFIC EXAMPLE 8 Ligand Binding Assays
  • Ligands for testing include, but are not limited to didehydro-RA, RA and its metabolites, 4-hydroxy-RA, 4-oxo-RA, and 5,6- epoxy-RA as well as compounds previous reported not to bind to CRABP, e.g. CD-394. Darmon, M. et al., Skin Pharmacol. 1:161-175 (1988).
  • CRABP amino-acid sequences or proteins provide the means to select more tissue-specific drugs for repair of photoaging skin, psoriasis, acne, skin cancer, leukemia, diseases of keratinization, osteoperosis, rheumatoid sclerosis, and other conditions.
  • Soluble protein from CV-1 ceils transfected with CRABP-I (pSVLCRABP-l) or CRABP-II (pSG5CRABP-ll) expression vectors obtained as described above is incubated with ⁇ H] retinoic acid at 4°C overnight. Free ligand is separated from bound ligand by size fractionation on a GF 250 column (DuPont Pharmaceuticals) connected to a FPLC system (Pharmacia). The amount of specific binding is determined by incubating samples with an excess of cold retinoic acid. The affinity of CRABP-II for retinoic acid can be determined by titrating with increasing amounts of ⁇ H] Retinoic acid.
  • RNA is isolated from tissue biopsies and cultured cells by guanidinium isothiocyanate lysis and ultracentrifugation as previously described in Elder, et al., J. Invest. Dermatol. 94:19-25 (1990). Equal quantities of total RNA can be separated on 1% formaldehyde-agarose gels and transferred to nylon membranes. After baking for 2 hours at 80°C, filters can be hybridized to agarose-purified CRABP-I or CRABP-II cDNAs labeled by random priming.
  • CRABP-I and II oligonucleotides (typically 8-15 residues long) may also be utilized as probes in hybridization assays if of a sufficient length to bind complementary sequences.
  • SPECIFIC EXAMPLE 10 Examination of Function of Human CRABP Receptor assay
  • CRABP transports RA to the nucleus (Takase, S. et al., Arch. Biochem. Biophys.247:328-334 (1986)) or that CRABP remains in the cytoplasm, thereby preventing RA from moving to the nucleus. Maden, M. et al., Nature 335:733-735 (1988).
  • One way of addressing this issue is to express increasing amounts of CRABP-I and CRABP-II in CV-1 cells together with the RARs and a reporter gene containing a retinoic acid responsive element, as described above. If CRABP transports RA to the nucleus an enhancement of reporter gene activity at low concentrations of RA may be seen. On the other hand, if CRABP sequesters RA a decrease in response will be seen. Overexpression
  • CRABP-I and CRABP-II is overexpressed in fibroblasts and keratinocytes.
  • Cells will be transfected with CRABP expression vectors (pSVLCRABP-l or pSG5CRABP-ll) as described above, using Lipofectin (BRL) essentially as described by the manufacturer.
  • BRL Lipofectin
  • fibroblasts the effect of CRABP overexpression on induction of the RAR- ⁇ gene by RA is studied. It has been shown that RAR- ⁇ mRNA is induced by RA in skin fibroblasts, and it is known that this gene is directly regulated by the RARs. See DeThe, H. et al., Nature 343:177-180 (1990).
  • the RAR- ⁇ gene acts as an "endogenous reporter gene" for RAR induction.
  • proliferating keratinocytes the effect of overexpression of CRABPs on markers of differentiation is studied. Translation blocking
  • CRABP-II Translation of CRABP-II is blocked in fibroblasts and keratinocytes by tran ' sfecting the cells with an expression vector construct having the CRABP-II cDNA in a reverse orientation. This produces an anti-sense mRNA that is able to hybridize to the endogenous CRABP-II mRNA, thereby blocking translation.
  • the effect of antisense expression in fibroblasts and keratinocytes is studied the same way as described for overexpression. Effect of CRABP on RA metabolism
  • CRBP cellular retinol binding protein
  • CRABP may function by increasing the interaction of RA with cytochrome P- 450.
  • RA is not the natural ligand for CRABP-II, but rather one of the metabolites. If CRABP-II for example, binds 4-hydroxy-RA (as determined by ligand- binding studies) with a higher affinity than RA, it is possible that CRABP-II increases cytochrome P-450 mediated metabolism by decreasing product concentration.
  • CRABPS are involved in RA metabolism or not is tested by incubating skin microsomes with increasing amounts of RA and of expressed and purified CRABP-I or CRABP-II. The effects of CRABPs on RA metabolism is assayed by HPLC.
  • the sequence of the gene was determined on both strands after subcloning into M13 vectors by dideoxy chain termination using modified T7 polymerase (Sequenase, U.S. Biochemical Corp.) and synthetic oligonucleotides. Sanger, F. et al., PAWS (USA) 74:5463-5467 (1977). Exon positions were determined by restriction mapping and sequencing.
  • RNA concentrations were determined by absorbance at 260 nm and equal quantities of total RNA were electrophoreticaliy separated in 1 % formaldehyde-agarose gels containing 0.5 ⁇ g/ml ethidium bromide. The RNA was transferred to nylon membranes (Zeta-Probe, BioRad, Richmond, CA) as described.
  • the filters were then washed twice for 15 minutes in 2 x SSC, 0.5% SDS at 42°C and once for 30 minutes in 0.5 x SSC, 0.5% SDS at 42°C A final wash was carried out in 0.1 x SSC, 0.1 % SDS for 30 minutes at 55°C
  • the amount of radioactivity present in each slot was determined using a phosphorimager (Molecular Dynamics) after over-night exposure and autoradiograms were exposed for 4-5 days at - 70°C with intensifying screens.
  • oligonucleotide primer complimentary to position 80-104, a predominant reaction product was identified when mRNA from untreated cultured human skin fibroblasts were used, see Figure 9. Comparison of the extension product with sequencing reactions originating from the same primer indicated that the major transcription initiation site should be assigned to the A residue 137 bp upsteam of the ATG. When fibroblasts were treated with 1 ⁇ M RA for 24 hours an increase in initiation at the A residue could be seen. In addition a second initiation product with the same intensity could be seen at -1.
  • TATAAA TATA box
  • Several potential regulatory elements including two potential AP2 binding sites at -631 and -402, and one potential SP1 site at -89 as demonstrated in Figure 8.
  • two sequences can be found that exactly match a binding site for the early growth response gene Krox-24 (Egr-1, zif268 or NGFI-A) at -579 and -116. Lemaire, P. et al., Mol. Cell. Biol. 10:3456-3467 (1990).
  • a direct repeat spaced by one bp can be found in the upstream region of the gene at -454, with homology to the retinoic acid responsive element found in the RAR- ⁇ 2 gene.
  • CRABP Retinoic Acid-Proteins
  • CCTCGTGGAC CAGAGAACTG ACCAACGATG GGGAACTGAT CCTGACCATG ACGGCGGATG 480
  • CRABP Retinoic Acid-Proteins
  • CRABP Retinoic Acid-Proteins
  • CAAGCCGCAC GTGGAGATCC GCCAGGACGG GGATCAGTTC TACATCAAGA CATCCACCAC 180
  • Tavakkol Amir Pettersson, Ulrika Cromie, Matthew Elder, James T. Voorhees, John J.
  • CRABP Retinoic Acid-Proteins
  • CA CA) AUTHORS: Astrom, Anders Tavakkol, Amir Elder, James T. Pettersson, Ulrika Cromie, Matthew Voorhees, John J.
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Homo sapiens
  • F TISSUE TYPE: Placenta
  • IMMEDIATE SOURCE
  • CB TITLE: Structure of the human cellular retinoic acid-binding protein II (CRABP-II) gene: Early transcriptionai regulation by retinoic acid
  • CTGCAGGAAG CCGTGCCCTC CTCCCACCCT CTTTGATCTC CCGTTTCAAA GCCGCTCTCC 60
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ORGANISM Homo sapiens
  • F TISSUE TYPE: Placenta
  • TITLE Structure of the human cellular retinoic-acid binding protein (CRABP-II) gene: Early transcriptionai regulation by retinoic acid
  • TTCACTGCCC CCTCCGTCCC
  • ACCCCCTCCT TCTAGGATAG CGCTCCCCTT ACCCCAGTCA 1320
  • GTAGCCTATA CAGTTTAGAATATTTATTTG TTAATTTTAT TAAAATGCTTTAAAAAAATA 1620

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Abstract

Les séquences codant deux isoformes de protéines de liaison d'acide rétinoïque cellulaire humaines, CRABP-I et CRABP-II, ont été clonées et séquencées, et sont présentées avec leurs séquences d'amino-acides correspondantes dans les identifications de séquences Nº 1-4. L'identification de séquences d'amino-acides et d'acide nucléique de CRABP humaine constitue la base d'élaboration de structures d'expression et de vecteurs de CRABP humaine recombinés. La CRABP humaine peut également être synthétisée ou produite ex vivo, par exemple dans des systèmes de production bactériens ou autres. Des méthodes de détection de liaison par ligand, ainsi que des dosages de gènes rapporteur et récepteur chimères recombinés et des dosages par hybridation directe et compétitive, utilisant les séquences de CRABP humaines décrites, peuvent être utilisés pour tester des médicaments par rapport à l'induction de rétinoïdes et à la spécificité tissulaire pour des états pathologiques impliquant les rétinoïdes. Des dosages immunologiques utilisant des anticorps ou des fragments de liaison produits et dirigés contre la CRABP humaine peuvent également être utilisés pour tester des tissus d'un patient par rapport à la présence et aux niveaux de CRABP afin d'émettre un diagnostic et de surveiller le traitement. L'identification des séquences d'aminoacides et d'acide nucléique pour les CRABP-I et CRABP-II humaindes contribue également à l'élucidation de la fonction et de l'interaction de protéines de liaison des rétinoïdes. Le gène de CRABP-II, isolé à partir d'une bibliothèque génomique du placenta humain, recouvre 6 kilobases et comprend 4 exons. Un site initiateur majeur de transcription a été cartographié sur un résidu A de 137 nucléotides en amont du codon initiateur ATG. L'ARN messager de CRABP-II a été rapidement induit en 2 à 6 heures, mis en culture avec l'acide rétinoïque, principalement en raison d'un rythme de transcription accru qui nécessitait une synthèse continue. Le gène de CRABP humaine est ainsi apparemment régulé de manière transcriptionnelle par une protéine régulatrice récemment synthétisée. Une fois la CRABP-II produite, la stabilisation du message peut permettre de maintenir des niveaux de CRABP-II élevés dans l'ARN messager.
EP93910828A 1992-04-28 1993-04-27 Crabp-i et crabp-ii humaines. Withdrawn EP0640093A4 (fr)

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US6092619A (en) * 1996-05-09 2000-07-25 Honda Giken Kogyo Kabushiki Kaisha Steering assist system in a vehicle

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US6861238B1 (en) 1996-06-21 2005-03-01 Queen's University At Kingston Retinoid metabolizing protein
US6063606A (en) * 1996-06-21 2000-05-16 Queen's University Kingston Retinoid metabolizing protein
EP0935676A2 (fr) 1996-06-21 1999-08-18 Queen's University At Kingston Procede d'identification du cytochrome p450
US5955305A (en) * 1997-04-28 1999-09-21 Incyte Pharmaceuticals, Inc. Human retinoid binding protein
WO2005015220A1 (fr) * 2003-08-04 2005-02-17 Roche Diagnostics Gmbh Utilisation de la proteine crabp-i en tant que marqueur pour le cancer du sein
EP2380909A1 (fr) 2010-04-26 2011-10-26 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Protéine PTK-7 impliquée dans le cancer du sein
LT2731973T (lt) 2011-07-13 2018-02-26 Ucb Biopharma Sprl Bakterijų padermė, ekspresuojanti rekombinantinį dsbc

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EP0325849A2 (fr) * 1987-12-02 1989-08-02 The Salk Institute For Biological Studies Composition de récepteur de l'acide rétinoique et procédé d'identification de ligands

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EP0325849A2 (fr) * 1987-12-02 1989-08-02 The Salk Institute For Biological Studies Composition de récepteur de l'acide rétinoique et procédé d'identification de ligands

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 173, no. 1, 30 November 1990 ORLANDO, FL US, pages 339-345, ANDERS ASTR\M ET AL. 'Retinoic acid and synthetic analogs differentially activate retinoic acid receptor dependent transcription' *
EXP CELL RES 199(2). 1992. 328-336. CODEN: ECREAL ISSN: 0014-4827, ELLER M S ET AL 'THE MOLECULAR CLONING AND EXPRESSION OF TWO CRABP CDNAS FROM HUMAN SKIN.' *
J. BIOL. CHEM. (1991), 266(26), 17662-6 CODEN: JBCHA3;ISSN: 0021-9258, ASTROM, ANDERS ET AL 'Molecular cloning of two human cellular retinoic acid-binding proteins ( CRABP ). Retinoic acid-induced expression of CRABP -II but not CRABP -I in adult human skin in vivo and in skin fibroblasts in vitro' *
JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 267, no. 35, 15 December 1992 MD US, pages 25251-25255, ANDERS ASTR\M ET AL. 'Structure of the human cellular retinoic acid-binding protein II gene' *
See also references of WO9322331A1 *

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US6092619A (en) * 1996-05-09 2000-07-25 Honda Giken Kogyo Kabushiki Kaisha Steering assist system in a vehicle

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