cDNA CLONING OF INOSITOL MONOPHOSPHATASE

Abstract: The present invention relates to the cloning of novel cDNA sequences encoding human and rat inositol monophosphatase (IMP); to the preparation of IMP enzyme by incorporation of the cDNAs into an expression vector and the expression thereof in recombinant host cells; and to the use of the enzyme thereby obtained in designing and developing medicaments which are inhibitors of human or rat IMP.

Description: CDNA CLONING OF INOSITOL MONOPHOSPHATASE The present invention relates to cDNA encoding inositol monophosphatase (IMP) which is isolated from brain cells. As used herein, the abbreviation IMP refers to an enzyme which can specifically liberate inositol (Ins) from the naturally occurring substrates Ins(l)P, Ins(3)P and Ins(4)P. IMP is also capable of hydrolyzing certain non-inositol-containing substrates including but not limited to those disclosed in Hallcher and Sherman, (1980), J. Biol. Che . , 224. pp. 10896-10901; Ta imoto et al.. (1985), J. Biochem, (Tokyo), 8_, pp. 363-370; and Gee et al., (1988), Biochem. J., 249. pp. 883-889. The a ino acid and cDNA sequence of bovine IMP is known [Diehl et al. , (1989), J. Biol. Chem., 265, pp. 5946-5949]. Mammalian cells capable of producing IMP include, but are not limited to, brain tissue cells. Transformed mammalian cell lines which may produce IMP include, but are not limited to, brain derived cell lines such as those available from the American Type Culture Collection listed in the Catalogue of Cell lines & Hvbridomas. 7th Edition, 1992. The preferred cells for the present invention include normal human brain-derived tissue cells. Other cells and cell lines may also be suitable for use in isolating IMP cDNA. Selection of suitable cells may be done by screening for IMP activity in cell extracts or conditioned medium. Methods for detecting IMP activity are well known in the art [Ragan et aJL. , (1988), Biochem. J. , 249, pp. 143-148], and measure the liberation of 1 C-labelled inositol from a substrate. Cells which possess IMP activity in this assay may be suitable for the isolation of IMP cDNA. Any of a variety of procedures may be used to molecularly clone human IMP cDNA. These methods include, but are not limited to, direct functional expression of the human IMP cDNA following the construction of a human IMP containing cDNA library in an appropriate expression vector system. Another method is to screen a human IMP- containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labelled oligonucleotide probe designed from the amino acid sequence of the purified IMP protein or from the DNA sequence of bovine IMP cDNA. The preferred method consists of screening a human IMP-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a 3 p- labelled cDNA oligonucleotide-primed fragment of the bovine IMP cDNA [Diehl et al. , (1989), J. Biol. Chem., 265. pp. 5946-5949]. The preferred cDNA library is a commercially available human hippocampal cDNA library in lambdaZAP (Stratagene) . It is readily apparent to those skilled in the art that other types of libraries, as well as libraries constructed from other cells or cell types, may be useful for isolating IMP-encoding DNA. Other types of libraries include, but are not limited to, cDNA libraries derived from other tissues, cells or cell lines other than human hippocampal cells, and genomic DNA libraries. It is readily apparent to those skilled in the art that suitable cDNA libraries may be prepared from cells or cell lines which have IMP activity. The selection of cells or cell lines for use in preparing a cDNA library to isolate IMP cDNA may be done by first measuring cell-associated IMP activity using the 1 C- labelled inositol substrate cleavage assay described above [Ragan et al. , supra] . Preparation of cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found, for example, in Maniatis, T. , Fritsch, E.F., Sambrook, J., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, New York, 2nd edition, 1989) . It is also readily apparent to those skilled in the art that DNA encoding IMP may also be isolated from a suitable genomic DNA library. Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techiques can be found in Maniatis et al.. supra. Using the preferred method, cDNA clones encoding human IMP were isolated by cDNA library screening. 32 P-radiolabelled oligonucleotide-primed fragments of the bovine IMP cDNA served as probes for the isolation of full length human IMP cDNA from a commercially available lambdaZAP cDNA library (Stratagene) derived from human hippocampal cells. Three positively hybridising phage, designated lambdaHIMPl-3, were detected using the bovine IMP cDNA- derived probe. These cDNA clones contained an insert of about 2 kilobases (kb) in length and had a single open reading frame of about 277 amino acids. Following in vivo excision according to the standard protocol as supplied by the manufacturer (Stratagene) , three plasmids were obtained, designated pHIMPl-3 respectively. These positive cDNA clones were bi-directionally sequenced by the dideoxy chain termination method (Sanger et al. , P.N.A.S. USA, 74., 5463, 1977). The sequence for the full-length cDNA encoding human brain IMP is shown in Table 1, and was derived from clone pHIMP3. The deduced amino acid sequence of human IMP from the cloned cDNA is shown alongside the cDNA sequence in Table 1. Inspection of the cDNA sequence reveals the presence of a single, large open reading frame of 277 amino acids. A comparison of the bovine IMP amino acid sequence with the human IMP amino acid sequence shows about 85% sequence identity. TABLE 1 CTC CGA CTC AAG ATA TTT GAA TGC ATG GAT TAT GCA E C M D Y A 2 GAA GCT ATA AAA AAT GAA E A I K N E 4 GTA ACT GCT ACG GAC CAA V T A T D Q 6 AAG TAT CCA TCT CAC AGT K Y P S H S 7 AGT ATC TTA ACC GAC AAC S I L T D N 9 AAC TTT GTA CAT AGA TTT N F V H R F 11 AAA AAG ATA GAA TTT GGA K K I E F G 13 GCC AGA AAA GGA AAA GGG A R K G K G 15 CAA GAA GAT ATT ACC AAA TCT CTC TTG GTG ACT GAG TTG GGC TCT TCT AGA ACA Q E D I T K S L L V T E L G S S R T 16 CCA GAG ACT GTG AGA ATG GTT CTT TCT AAT ATG GAA AAG CTT TTT TGC ATT CCT P E T V R M V L S N M E K L F C I P 18 GTT CAT GGG ATC CGG AGT GTT GGA ACA GCA GCT GTT AAT ATG TGC CTT GTG GCA V H G I R S V G T A A V N M C L V A 20 ACT GGC GGA GCA GAT GCA TAT TAT GAA ATG GGA ATT CAC TGC TGG GAT GTT GCA T G G A D A Y Y E M G I H C W D V A 2 GGA GCT GGC ATT ATT GTT ACT GAA GCT GGT GGC GTG CTA ATG GAT GTT ACA GGT G A G I I V T E A G G V L M O V T G 2 GGA CCA TTT GAT TTG ATG TCA CGA AGA GTA ATT GCT GCA AAT AAT AGA ATA TTA G P F D L M S R R V I A A N N R I L 2 GCA GAA AGG ATA GCT AAA GAA ATT CAG GTT ATA CCT TTG CAA CGA GAC GAC GAA A E R I A K E I Q V I P L Q R D D E 2 GAT TAA TTA AGG CAG CTC ATA GTC ATC CAG TTG D END In addition to the isolation and cloning of human brain IMP cDNA, rat brain IMP cDNA was also isolated and cloned from a cDNA library. A commercially available rat brain cDNA library (Clontech) constructed in lambdaGTll was screened using 32 P-radiolabelled oligonucleotide-primed fragments of the bovine IMP cDNA as probes. Plaques were plated and two positive clones were identified. Both clones contained DNA inserts of approximately 2.1 kb which were sequenced. The DNA inserts were identical and contained an open reading frame of 270 amino acids beginning from amino acid residue 8 of the human brain IMP DNA sequence (cf. Table 1) . The sequence of this cDNA insert, together with the deduced amino acid sequence corresponding thereto, is shown in Table 2. TABLE 2 27 54 TGC ATG GAT TAT GCA GTG ATC CTC GCA AGA CAA GCT GGA GAG ATG ATT CGA GTT C M O Y A V I U A R Q A G E M I R V βl lOΘ GCT CTA AAA AAT AAG ATG GAT GTC ATG ATT AAA AGT TCT CCA GCC GAC TTG GTA c A L K N K M D V I K S S P A D L V 135 162 ACA GTT ACT GAC CAA AAA GTT GAA AAA ATG CTT ATG TCT TCT ATA AAG GAA AAA T V T O O K V E K M L M S S I K E K 1Θ9 216 TAC CCA TAT CAC AGT TTC ATT GGT GAA GAA TCT GTG GCA GCC GGG GAA AAG ACA Y P Y H S F I G E E Ξ V A A G E K T 243 270 0 GTC TTC ACA GAG CAG CCC ACG TGG ATC ATT GAT CCC ATT GAC GGG ACG ACC AAC V F T E G P T I I D P I D G T T N 297 324 TTT GTG CAC CGG TTT CCC TTT GTA GCT GTT TCG ATT GGC TTC GTT GTA AAT AAA F- V H R F P F V A V S I G F V V N K 351 378 GAG ATG GAG TTT GGA GTT GTA TAC AGC TGT GTG GAA GAT AAG ATG TAT ACG GGC E M E F G V V Y S C V E D K M Y T G 15 405 432 AGG AAA GGA AAA GGC GCC TTT TGT AAC GGT CAG AAG CTT CGG GTC TCG CAG CAG R K G K G A F C N G Q K R V S α α ■459 4β6 GAA GAC ATT ACC AAA TCA CTC TTG GTG ACC GAG CTG GGA TCG TCC AGA AAG CCG E D I T K S L L V T E L- G S S R K P 513 540 GAG ACT TTG CGG ATT GTT CTC TCC AAC ATG GAA AGG CTT TGC TCC ATT CCT ATC « n E T L R I V L S tsl M E R L C S I P I 567 594 CAT GGA ATC CGG AGT GTT GGG ACA GCG GCT GTT AAT ATG TGC CTT GTG GCA ACG H G I R S V G T A A V N C U V A T 621 648 GGA GGA GCG GAT GCC TAT TAC GAG ATG GGG ATC CAC TGC TGG GAC ATG GCT GGA G G A D A Y Y E M G I H C W D M A G 675 702 25 GCT GGC ATC ATC GTC ATA GAG GCT GGC GGA GTG CTG CTG GAT GTG ACA GGT GGA A G I I V I E A G G V L L. D V T G G 729 756 CCA TTC GAT TTG ATG TCT CGG AGA ATA ATT GCT GCA AGT AAT ATA GCA TTA GCA P F D M S R R I I A A S N I A L A 783 ΘIO GAA AGA ATA GCC AAA GAA CTT GAG ATA ATA CCT TTA CAA CGA GAC GAC GAA AGT E R I A K E L E I I P L Q R D D E S 30 837 864 TAG GCA CGT AGA ACC GCA TCC AGC TCC GTC ACA CCT GCT CTC CCT GGG ATG TTT End 891 AAA GAT GTA TGA TGT CAC TGA TTT AAA TTT AAC TTT GCA GTC CTG SUBSTITUTE SHEET IMP in substantially pure form derived from natural sources is found to be an association of two IMP polypeptides apparently encoded by a single mRNA [Gee et al.. Biochem. J.. 1988, 249_, 883-889]. The two IMP polypeptides are found to have an apparent molecular weight of about 30 kDa. The cloned IMP cDNA obtained through the methods described above may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and then transferring the expression vector into prokaryotic or eukaryotic host cells to produce recombinant IMP. Molecular cloning of cDNAs into an expression vector can be achieved by a variety of standard techniques; in the present context, polymerase chain reaction (PCR) methodology [Saiki et al.. Science. 1988, 239, 487-491] is preferably employed. Alternative conventional techniques for such manipulations are fully described in Maniatis et al. , supra. and are well known in the art. Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. A variety of mammalian expression vectors may be used to express recombinant IMP in mammalian cells. Commercially available mammalian expression vectors which may be suitable for recombinant IMP expression include, but are not limited to, pRSETδa [Schoepfer et al.. FEBS Lett.. 1990, 257, 393-399], pMClneo (Stratagene), pXTl (Stratagene) , pSG5 (Stratagene) , EB0-pSV2-neo (ATCC 37593), pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146) , pUCTag (ATCC 37460) , and gZD35 (ATCC 37565) . DNA encoding IMP cloned into an expression vector may then be transferred to a recombinant host cell for expression. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria, yeast, mammalian cells (including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin) , and insect cells (including but not limited to drosophila derived cell lines) . A preferred bacterial cell line is BL21-DE3 [Schoepfer et al.. FEBS Lett.. 1990, 257. 393-399]. Cell lines derived from mammalian species which may be suitable and which are commercially available include, but are not limited to, CV-1 (ATCC CCL 70) , COS-1 (ATCC CRL 1650) , COS-7 (ATCC CRL 1651), CH0-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616) , BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171) . It will be appreciated that, if the cDNA is to be inserted into the expression vector pRSETSa, then this expression vector must be used in conjunction with a recombinant host containing the T7 polymerase gene. One such host is the bacterial strain BL21-DE3. Thus, a preferred method in the present case is to insert the cDNA into the expression vector pRSETSa and express this vector in the bacterial strain BL21-DE3. The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation. The expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce IMP protein. Identification of IMP expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti-IMP antibodies, and the presence of host cell- associated IMP activity. Expression of IMP DNA may also be performed using in vitro produced synthetic mRNA. Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes. To determine the IMP cDNA sequence(s) that yield(s) optimal levels of enzymatic activity and/or IMP protein, IMP cDNA molecules including but not limited to the following can be constructed: the full-length open reading frame of the IMP cDNA (base 1 to base 834; Table 1) and constructs containing portions of the cDNA encoding enzymatically active protein. All constructs can be designed to contain none, all or portions of the 5' and 3" untranslated region of IMP cDNA. A particular construct is one which is substantially free of the 5' untranslated region of IMP cDNA. IMP activity and levels of protein expression can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells. Following determination of the IMP cDNA cassette which yields optimal expression, this IMP cDNA construct may be transferred to a variety of expression host cells, including but not limited to mammalian cells, baculovirus-infected insect cells, E. coli. and the yeast S. cerevisiae. Expression of IMP in a recombinant host cell affords IMP protein in active form, capable of enzymatically liberating inositol from naturally occurring IMP substrates. Several IMP purification procedures are available and suitable for use. Recombinant IMP may be purified from cell lysates and extracts, or from conditioned culture medium, by various combinations of, or individual application of, salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography. The preferred method is ion-exchange chromatography utilizing a column of HR5/5 Mono Q matrix (LKB Pharmacia) . In addition, recombinant IMP can be separated from other cellular proteins by use of an immuno-affinity column made with monoclonal or polyclonal antibodies specific for IMP, or polypeptide fragments of IMP. The preparation and purification of monoclonal or polyclonal antibodies specific for IMP or polypeptide fragments thereof can be accomplished by conventional techniques well known in the art. Typical procedures include those described, for example, by Maniatis et al. in Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press, 2nd edition, 1989, Chapter 18. This invention provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes IMP, preferably human IMP, so as to prevent translation of the mRNA molecule. The antisense oligonucleotide may have a sequence capable of binding specifically with any sequences of the cDNA molecule whose sequence is shown in Table 1 or the cDNA molecule whose sequence is shown in Table 2. A particular example of an antisense oligonucleotide is an antisense oligonucleotide comprising chemical analogues of nucleotides. This invention provides a transgenic nonhuman mammal expressing DNA encoding IMP, preferably human IMP. This invention also provides a transgenic nonhuman mammal expressing DNA encoding IMP, preferably human IMP, so mutated as to be incapable of normal receptor activity, and not expressing native IMP. This invention further provides a transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding IMP, preferably human IMP, so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding IMP and which hybridizes to mRNA encoding IMP thereby reducing its translation. The DNA may additionally comprise an inducible promoter or additionally comprise tissue specific regulatory elements, so that expression can be induced, or restricted to specific cell types. Examples of DNA are DNA or cDNA molecules having a coding sequence substantially the same as the coding sequence shown in Table 1, or the coding sequence shown in Table 2. An example of a transgenic animal is a transgenic mouse. Examples of tissue specificity-determining regions are the metallothionein promotor (Low et al.. Science. 1986, 231. 1002-1004) and the L7 promotor (Oberdick et al.. Science. 1990, 248., 223-226). The cloned IMP enzyme in accordance with the invention, when isolated, may be utilised to generate structural data, in particular X-ray crystallographic data. Knowledge of, for example, the X-ray crystal structure of the IMP enzyme, especially human IMP, may facilitate in designing drugs which are inhibitors of this enzyme. The present invention accordingly provides the use of the cloned IMP enzyme in screening for and designing medicaments which are inhibitors of IMP. The following non-limiting Examples illustrate the present invention. Example 1 Cloning of Human and Rat Brain Inositol Monophosphatase A human hippocampal cDNA library constructed in lambdaZAP (Stratagene) was screened using a 3 p- radiolabelled oligonucleotide-primed fragment of bovine inositol monophosphatase cDNA as a probe [Diehl et al.. , (1990), J. Biol. Chem., 2£5, PP« 5946-5949]. Phage (100,000) were plated and three independent cDNA clones were isolated. All three contained an insert of approximately 2 kb which were subsequently shown to have identical sequences. One of these clones was characterized further and found to contain a 277 amino acid open reading frame (Table 1) . The predicted amino acid sequence (Table 1) is very similar to that of the bovine enzyme and has an estimated subunit M r of approximately 30,000, as does the bovine enzyme [Gee et al.. (1988), Biochem. J., 249. pp. 883-889]. Additionally, a rat brain cDNA library constructed in lambdaGTll (Clontech) was screened using 32 P-radiolabelled oligonucleotide-primed fragments of bovine inositol monophosphatase cDNA as a probe as described above. Phage were plated and two independent cDNA clones were isolated. Each contained an insert of approximately 2.1 kb which were subsequently shown to have identical sequences. One clone was characterized further and found to contain a 270 amino acid open reading frame beginning with amino acid 8 of the human brain IMP cDNA. The rat brain IMP cDNA sequence, together with the deduced amino acid sequence corresponding thereto, is shown in Table 2. Example 2 Expression of recombinant human inositol monophosphatase in bacteria The T7 polymerase bacterial expression system (pRSETδa) was used as described previously [Diehl et aJL. , supra] . The coding region of the human inositol monophosphatase cDNA was reconstructed to contain an Ndel site at the start codon and a PstI site just downstream of the stop codon using PCR methodology [Saiki et al. , (1988), Science, 239. pp. 487-491]. Oligonucleotides 5 1 - AATATTTTCAGCATATGGCTGATCCTTG-3' and 5 1 - ATGACTATGAGCTGCAGTAATTAATCTTC-3' were synthesized on an Applied Biosystems 380B instrument. Inositol monophosphatase cDNA (100 ng) in pBluescript II SK (Stratagene Ltd., U.K.) was subjected to PCR under standard conditions [Saiki, et al. , supra] . denaturation at 94°C for 2 minutes, annealing at 55"C for 2 minutes and polymerisation at 72"C for 6 minutes. Twenty cycles were performed with the last polymerisation step lasting 12 minutes. The Ndel/PstI-digested PCR product was cloned into Ndel-PstI-digested pRSETSa and transformed into Escherichia coli strain DH5α competent cells. Positive clones were identified by restriction analysis and DNA sequencing. For subsequent expression studies, the expression vector was transformed into competent BL21-DE3 cells. Bacterial cells containing the expression vector were induced by isopropylthio-3-D- galactoside and analysed by both SDS/PAGE and enzyme assay. Recombinant bacteria expressed a prominent polypeptide of about M r 30,000 which was absent from bacteria not containing the expression vector. Bacterial lysates contained significant amounts of enzyme activity, which was further purified by heat treatment, centrifugation and ion-exchange chromatography (Example 3). Example 3 Purification of recombinant human inositol monophosphatase E. coli bacteria (strain BL21-DE3) containing the IMP expression vector (Example 2) were grown and induced by isopropylthio-J-D-galactoside, and analysed by SDS/PAGE and enzyme assay as described previously [Ragan et al., supra] . After induction, cells were pelleted and frozen until required for purification. The bacterial pellets (1-2 g/litre of fermentation mixture) were thawed, resuspended in 10 volumes of 20 mM-TRIS/HCl/1 mM- EGTA buffer, pH 7.8 (buffer A) and sonicated on ice (3 x 1 in) . The homogenate was then centrifuged at 100,000 x g for 20 minutes and the resultant supernatant was loaded, at a flow rate of 1 ml/min, on an HR5/5 Mono Q column (LKB Pharmacia) , previously equilibrated with buffer A. The column was eluted at 1 ml/min with a gradient of 0-300 mM NaCl in buffer A, and 1 ml fractions were collected. Portions (5 μl) of each fraction were subjected to SDS/PAGE on 12% gels according to the method of Laemmli, (1970), Nature, 227. pp. 680-685. Gels were stained with Coomassie Blue and the appropriate inositol monophosphatase-containing fractions were pooled for subsequent analysis. SDS/PAGE of a typical purification shows a single band of about M r 30,000. Table 3 shows a 5.4-fold purification of t * -e enzyme, demonstrating that recombinant inosi ol monophosphatase constituted nearly 20% of the original bacterial protein. This level of expression enables the isolation of large amounts of pure enzyme for X-ray crystallography and other studies. The biochemical properties of the human enzyme were further examined. Table 3 Step Bacterial homogenate 751,100 214 3500 100 Bacterial supernatant 481,000 90 5300 64 Heat-treated supernatant 264,000 22 12,000 35 Mono Q 177,000 19,700 24 Example 4 Kinetic properties of recombinant human inositol monophosphatase In agreement with previous work on the rat and bovine enzyme [Takimoto et al., (1985), J. Biochem., 18. pp. 363-370; Gee et al. , supra] we found that human brain IMP activity was totally dependent on Mg + . Under standard assay conditions, the apparent affinity of the human enzyme for Mg 2+ was approximately 2-fold greater than that of the bovine enzyme, and the human enzyme was also more sensitive to inhibition by high Mg + concentrations. In the absence of KCl, activation by Mg 2+ occurred at approximately 2-fold lower concentrations, but maximum enzyme activity was independent of KCl. As is shown in Table 4, the recombinant human brain enzyme has similar kinetic properties to the native human platelet-derived enzyme as well as both the native and recombinant bovine enzymes [Gee et al. , supra; Diehl et al. , supra1. The K m for DL- Ins(l)P was lower for the human enzyme, suggesting that it may have a higher affinity for substrate than the bovine enzyme. These data also suggest that there is no significant post-translational modification of the native human enzyme that might change its behaviour compared with the recombinant enzyme. Table 4 Kinetic properties of Inositol monophosphatases K m for V max (μmol/min Enzyme DL-Ins t l . P .mM) per mg of protein) Human recombinant 0.075 + 0.003 36.8 + 1 Human platelet 0.108 + 0.003 ND Bovine recombinant 0.12 + 0.007 ND Bovine brain 0.16 + 0.02 13.3 + 0.9 Initial rates determined with several substrate concentrations were fitted to a Michaelis-Menten expression by non-linear least-squares regression analysis. Values for K m and V max were determined in the present study and are given as means + S.E.M. (n=3) . Bovine brain values are as reported in Gee et al.. , supra f but confirmed here. Example 5 Inhibition of recombinant human inositol monophosphatase Pi inhibited recombinant human IMP competitively with a Ki value of 0.14 mM (cf. bovine enzyme, 0.5 mM) , and Li + was an unco petitive inhibitor with an apparent Ki value of 0.3 mM (cf. bovine enzyme 0.26 mM) . The competitive inhibitor lS-phosphoryloxy- 2R,4S-dihydroxycyclohexane [Baker et al. , (1990), J. Chem. Soc. Chem. Comm. , pp. 462-464] inhibited with an apparent Ki value of 2.7 μM (cf. bovine enzyme, 1.1 μM) . All comparisons are from data obtained in parallel experiments carried out in this study. Overall, these data confirm that the human enzyme is similar to, but not identical with, the bovine enzyme. The availability of multiple protein sequences and recombinant human enzyme will assist future studies using site-directed mutagenesis and chemical-modification techniques to characterize important residues for the structure and function of this enzyme. IMP Enzyme assays Enzyme activity was determined by measuring the release of [ 14 C]inositol from DL-Ins(l)P containing L-[U- C]Ins(l)P as label as described previously [Gumber et al. , (1989), Plant Physiol., 76., pp. 40-44]. One unit of enzyme activity represents 1 μmol of substrate hydrolysed/min, at 37°C. Protein concentrations were determined by the method of Bradford [Bradford, M. , (1976), Anal. Biochem., 7_2., pp. 248-252]. Kinetic analyses were performed as described previously [Gee et al. , supra] . SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Merck Sharp & Dohme Limited (B) STREET: Hertford Road (C) CITY: Hoddesdon (D) STATE: Hertfordshire (E) COUNTRY: England (F) POSTAL CODE (ZIP) : EN11 9BU (ii) TITLE OF INVENTION: cDNA Cloning of Inositol Monophosphatase (iϋ) NUMBER OF SEQUENCES: 6 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO) (Vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: GB 9212261.3 (B) FILING DATE: 10-JUN-1992 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 897 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 37..871 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Is CTCCGACTCA AGATATTTGT CAAATATTTT CAGAAG ATG GCT GAT CCT TGG CAG 54 Het Ala Asp Pro Trp Gin 1 5 GAA TGC ATG GAT TAT GCA GTA ACT CTA GCA AGA CAA GCT GGA GAG GTA 102 Glu Cys Het Asp Tyr Ala Val Thr Leu Ala Arg Gin Ala Gly Glu Val 10 15 20 GTT TGT GAA GCT ATA AAA AAT GAA ATG AAT GTT ATG CTG AAA AGT TCT 150 Val Cys Glu Ala lie Lys Asn Glu Het Asn Val Het Leu Lys Ser Ser 25 30 35 CCA GTT GAT TTG GTA ACT GCT ACG GAC CAA AAA GTT GAA AAA ATG CTT 198 Pro Val Asp Leu Val Thr Ala Thr Asp Gin Lys Val Glu Lys Het Leu 40 45 50 ATC TCT TCC ATA AAG GAA AAG TAT CCA TCT CAC AGT TTC ATT GGT GAA 246 lie Ser Ser lie Lys Glu Lys Tyr Pro Ser His Ser Phe lie Gly Glu 55 60 65 70 GAA TCT GTG GCA GCT GGG GAA AAA AGT ATC TTA ACC GAC AAC CCC ACA 294 Glu Ser Val Ala Ala Gly Glu Lys Ser He Leu Thr Asp Asn Pro Thr 75 80 85 TGG ATC ATT GAC CCT ATT GAT GGA ACA ACT AAC TTT GTA CAT AGA TTT 342 Trp He He Asp Pro He Asp Gly Thr Thr Asn Phe Val His Arg Phe 90 95 100 CCT TTT GTA GCT GTT TCA ATT GGC TTT GCT GTA AAT AAA AAG ATA GAA 390 Pro Phe Val Ala Val Ser He Gly Phe Ala Vat Asn Lys Lys He Glu 105 110 115 TTT GGA GTT GTG TAC AGT TGT GTG GAA GGC AAG ATG TAC ACT GCC AGA 438 Phe Gly Val Val Tyr Ser Cys Val Glu Gly Lys Het Tyr Thr Ala Arg 120 125 130 AAA GGA AAA GGG GCC TTT TGT AAT GGT CAA AAA CTA CAA GTT TCA CAA 486 Lys Gly Lys Gly Ala Phe Cys Asn Gly Gin Lys Leu Gin Vat Ser Gin 135 140 145 150 CAA GAA GAT ATT ACC AAA TCT CTC TTG GTG ACT GAG TTG GGC TCT TCT 534 Gin Glu Asp He Thr Lys Ser Leu Leu Val Thr Glu Leu Gly Ser Ser 155 160 165 AGA ACA CCA GAG ACT GTG AGA ATG GTT CTT TCT AAT ATG GAA AAG CTT 582 Arg Thr Pro Glu Thr Vat Arg Het Val Leu Ser Asn Het Glu Lys Leu 170 175 180 TTT TGC ATT CCT GTT CAT GGG ATC CGG AGT GTT GGA ACA GCA GCT GTT 630 Phe Cys He Pro Val His Gly He Arg Ser Val Gly Thr Ala Ala Val 185 190 195 AAT ATG TGC CTT GTG GCA ACT GGC GGA GCA GAT GCA TAT TAT GAA ATG 678 Asn Het Cys Leu Val Ala Thr Gly Gly Ala Asp Ala Tyr Tyr Glu Het 200 205 210 GGA ATT CAC TGC TGG GAT GTT GCA GGA GCT GGC ATT ATT GTT ACT GAA 726 Gly He His Cys Trp Asp Val Ala Gly Ala Gly He He Val Thr Glu 215 220 225 230 GCT GGT GGC GTG CTA ATG GAT GTT ACA GGT GGA CCA TTT GAT TTG ATG 774 Ala Gly Gly Val Leu Het Asp Val Thr Gly Gly Pro Phe Asp Leu Het 235 240 245 TCA CGA AGA GTA ATT GCT GCA AAT AAT A.GA ATA TTA GCA GAA AGG ATA 822 Ser Arg Arg Val He Ala Ala Asn Asn Arg He Leu Ala Gtu Arg He 250 255 260 GCT AAA GAA ATT CAG GTT ATA CCT TTG CAA CGA GAC GAC GAA GAT TAA T 871 Ala Lys Glu He Gin Val He Pro Leu Gin Arg Asp Asp Glu Asp * 265 270 275 TAAGGCAGCT CATAGTCATC CAGTTG 897 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 278 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Het Ala Asp Pro Trp Gin Glu Cys Het Asp Tyr Ala Val Thr Leu Ala 1 5 10 15 Arg Gin Ala Gly Glu Val Val Cys Glu Ala He Lys Asn Glu Het Asn 20 25 30 Val Het Leu Lys Ser Ser Pro Val Asp Leu Val Thr Ala Thr Asp Gin 35 40 45 Lys Vat Glu Lys Het Leu He Ser Ser He Lys Glu Lys Tyr Pro Ser 50 55 60 His Ser Phe He Gly Glu Glu Ser Val Ala Ala Gly Glu Lys Ser He 65 70 75 80 Leu Thr Asp Asn Pro Thr Trp He He Asp Pro He Asp Gly Thr Thr 85 90 95 Asn Phe Val His Arg Phe Pro Phe Val Ala Val Ser He Gly Phe Ala 100 105 110 Val Asn Lys Lys He Glu Phe Gly Val Val Tyr Ser Cys Val Glu Gly 115 120 125 Lys Het Tyr Thr Ala Arg Lys Gly Lys Gly Ala Phe Cys Asn Gly Gin 130 135 140 Lys Leu Gin Val Ser Gin Gin Glu Asp He Thr Lys Ser Leu Leu Val 145 150 155 160 Thr Glu Leu Gly Ser Ser Arg Thr Pro Glu Thr Val Arg Het Val Leu 165 170 175 Ser Asn Het Glu Lys Leu Phe Cys He Pro Val His Gly He Arg Ser 180 185 190 Val Gly Thr Ala Ala Val Asn Het Cys Leu Val Ala Thr Gly Gly Ala 195 200 205 Asp Ala Tyr Tyr Glu Het Gly He His Cys Trp Asp Val Ala Gly Ala 210 215 220 Gly He He Val Thr Glu Ala Gly Gly Val Leu Het Asp Val Thr Gly 225 230 235 240 Gly Pro Phe Asp Leu Met Ser Arg Arg Val He Ala Ala Asn Asn Arg 245 250 255 He Leu Ala Glu Arg He Ala Lys Glu He Gin Vat He Pro Leu Gin 260 265 270 Arg Asp Asp Glu Asp * 275 ( 2 ) INFORMATION FOR SEQ ID NO : 3 : ( i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 909 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1. .813 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 TGC ATG GAT TAT GCA GTG ATC CTC GCA AGA CAA GCT GGA GAG ATG ATT 48 Cys Met Asp Tyr Ala Val He Leu Ala Arg Gin Ala Gly Glu Het He 1 5 10 15 CGA GTT GCT CTA AAA AAT AAG ATG GAT GTC ATG ATT AAA AGT TCT CCA 96 Arg Val Ala Leu Lys Asn Lys Met Asp Val Met He Lys Ser Ser Pro 20 25 30 GCC GAC TTG GTA ACA GTT ACT GAC CAA AAA GTT GAA AAA ATG CTT ATG 144 Ala Asp Leu Val Thr Val Thr Asp Gin Lys Val Glu Lys Met Leu Met 35 40 45 TCT TCT ATA AAG GAA AAA TAC CCA TAT CAC AGT TTC ATT GGT GAA GAA 192 Ser Ser He Lys Glu Lys Tyr Pro Tyr His Ser Phe He Gly Glu Glu 50 55 60 TCT GTG GCA GCC GGG GAA AAG ACA GTC TTC ACA GAG CAG CCC ACG TGG 240 Ser Val Ala Ala Gly Glu Lys Thr Vat Phe Thr Glu Gin Pro Thr Trp 65 70 75 80 ATC ATT GAT CCC ATT GAC GGG ACG ACC AAC TTT GTG CAC CGG TTT CCC 288 He He Asp Pro He Asp Gly Thr Thr Asn Phe Val His Arg Phe Pro 85 90 95 TTT GTA GCT GTT TCG ATT GGC TTC GTT GTA AAT AAA GAG ATG GAG TTT 336 Phe Val Ala Val Ser He Gly Phe Val Val Asn Lys Gtu Met Glu Phe 100 105 110 GGA GTT GTA TAC AGC TGT GTG GAA GAT AAG ATG TAT ACG GGC AGG AAA 384 Gly Val Val Tyr Ser Cys Val Glu Asp Lys Het Tyr Thr Gly Arg Lys 115 120 125 GGA AAA GGC GCC TTT TGT AAC GGT CAG AAG CTT CGG GTC TCG CAG CAG 432 Gly Lys Gly Ala Phe Cys Asn Gly Gin Lys Leu Arg Val Ser Gin Gin 130 135 140 GAA GAC ATT ACC AAA TCA CTC TTG GTG ACC GAG CTG GGA TCG TCC AGA 480 Glu Asp He Thr Lys Ser Leu Leu Val Thr Glu Leu Gly Ser Ser Arg 145 150 155 160 AAG CCG GAG ACT TTG CGG ATT GTT CTC TCC AAC ATG GAA AGG CTT TGC 528 Lys Pro Glu Thr Leu Arg He Val Leu Ser Asn Het Glu Arg Leu Cys 165 170 175 TCC ATT CCT ATC CAT GGA ATC CGG AGT GTT GGG ACA GCG GCT GTT AAT 576 Ser He Pro He His Gly He Arg Ser Val Gly Thr Ala Ala Val Asn 180 185 190 ATG TGC CTT GTG GCA ACG GGA GGA GCG GAT GCC TAT TAC GAG ATG GGG 624 Met Cys Leu Val Ala Thr Gly Gly Ala Asp Ala Tyr Tyr Glu Met Gly 195 200 205 ATC CAC TGC TGG GAC ATG GCT GGA GCT GGC ATC ATC GTC ATA GAG GCT 672 He His Cys Trp Asp Het Ala Gly Ala Gly He He Val He Glu Ala 210 215 220 GGC GGA GTG CTG CTG GAT GTG ACA GGT GGA CCA TTC GAT TTG ATG TCT 720 Gly Gly Val Leu Leu Asp Val Thr Gly Gly Pro Phe Asp Leu Het Ser 225 230 235 240 CGG AGA ATA ATT GCT GCA AGT AAT ATA GCA TTA GCA GAA AGA ATA GCC 768 Arg Arg He He Ala Ala Ser Asn He Ala Leu Ala Glu Arg He Ala 245 250 255 AAA GAA CTT GAG ATA ATA CCT TTA CAA CGA GAC GAC GAA AGT TAGGCACGTA 820 Lys Glu Leu Glu He He Pro Leu Gin Arg Asp Asp Glu Ser 260 265 270 GAACCGCATC CAGCTCCGTC ACACCTGCTC TCCCTGGGAT GTTTAAAGAT GTATGATGTC 880 ACTGATTTAA ATTTAACTTT GCAGTCCTG 909 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 270 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Cys Het Asp Tyr Ala Val He Leu Ala Arg Gin Ala Gly Glu Met He 1 5 10 15 Arg Val Ala Leu Lys Asn Lys Met Asp Val Met He Lys Ser Ser Pro 20 25 30 Ala Asp Leu Val Thr Val Thr Asp Gin Lys Val Glu Lys Met Leu Het 35 40 45 Ser Ser He Lys Glu Lys Tyr Pro Tyr His Ser Phe He Gly Glu Glu 50 55 60 Ser Val Ala Ala Gly Glu Lys Thr Val Phe Thr Glu Gin Pro Thr Trp 65 70 75 80 He He Asp Pro He Asp Gly Thr Thr Asn Phe Val His Arg Phe Pro 85 90 95 Phe Val Ala Val Ser He Gly Phe Val Val Asn Lys Glu Met Glu Phe 100 105 110 Gly Val Val Tyr Ser Cys Val Glu Asp Lys Het Tyr Thr Gly Arg Lys 115 120 125 Gly Lys Gty Ala Phe Cys Asn Gly Gin Lys Leu Arg Val Ser Gin Gin 130 135 140 Glu Asp He Thr Lys Ser Leu Leu Val Thr Glu Leu Gly Ser Ser Arg 145 150 155 160 Lys Pro Glu Thr Leu Arg He Val Leu Ser Asn Het Glu Arg Leu Cys 165 170 175 Ser He Pro He His Gly He Arg Ser Val Gly Thr Ala Ala Val Asn 180 185 190 Het Cys Leu Val Ala Thr Gly Gly Ala Asp Ala Tyr Tyr Glu Het Gly 195 200 205 He His Cys Trp Asp Het Ala Gly Ala Gly He He Val He Glu Ala 210 215 220 Gly Gly Val Leu Leu Asp Val Thr Gly Gly Pro Phe Asp Leu Met Ser 225 230 235 240 Arg Arg He He Ala Ala Ser Asn He Ala Leu Ala Glu Arg He Ala 245 250 255 Lys Glu Leu Glu He He Pro Leu Gin Arg Asp Asp Glu Ser 260 265 270 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: AATATTTTCA GCATATGGCT GATCCTTG 28 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ATGACTATGA GCTGCAGTAA TTAATCTTC 29

Claims: CLAIMS: 1. Recombinant human inositol monophosphatase substantially free of other human protein. 2. A protein corresponding to human inositol monophosphatase and comprising the amino acid sequence depicted in Table 1 herein. 3. Recombinant rat inositol monophosphatase substantially free of other rat protein. 4. A protein corresponding to rat inositol monophosphatase and comprising the amino acid sequence depicted in Table 2 herein. 5. A DNA molecule encoding human inositol monophosphatase comprising all or a portion of the nucleotide sequence depicted in Table 1 herein, or a degenerate variation thereof. 6. A DNA molecule encoding rat inositol monophosphatase comprising all or a portion of the nucleotide sequence depicted in Table 2 herein, or a degenerate variation thereof. 7. A recombinant expression vector comprising the nucleotide sequence as claimed in claim 5 or claim 6 together with additional sequences capable of directing the synthesis of inositol monophosphatase. 8. A recombinant host cell containing the expression vector as claimed in claim 7. 9. A host cell as claimed in claim 8 which consists of the bacterial cell line BL21-DE3 containing the recombinant expression vector pRSETSa. 10. A process for preparing recombinant inositol monophosphatase which comprises inserting the nucleotide sequence as claimed in claim 5 or claim 6 into an expression vector, incorporating the vector into a suitable host cell, and growing the host cell under conditions suitable for the expression of inositol monophosphatase. 11. A process for preparing a nucleotide sequence as claimed in claim 5 or claim 6 which comprises: (A) constructing an inositol monophosphatase containing cDNA library in an appropriate expression vector system and functionally expressing the inositol monophosphatase cDNA directly; or (B) screening an inositol monophosphatase containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labelled oligonucleotide probe designed from the amino acid sequence of the purified inositol monophosphatase protein or from the DNA sequence of bovine inositol monophosphatase cDNA. 12. An antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes human or rat inositol monophosphatase so as to prevent translation of the mRNA molecule. 13. An antisense oligonucleotide having a sequence capable of binding specifically with any sequences of the DNA molecule as claimed in claim 5 or claim 6. 14. A transgenic nonhuman mammal expressing DNA encoding human inositol monophosphatase. 15. The use of the enzyme as claimed in any one of claims 1 to 4 in screening for and designing medicaments which are inhibitors of human or rat inositol monophosphatase.

Inventors: MCALLISTER GEORGE, WHITING PAUL JOHN, MCALLISTER, GEORGE, WHITING, PAUL, JOHN
Applicants: MCALLISTER GEORGE, MERCK SHARP & DOHME, WHITING PAUL JOHN, MERCK SHARP & DOHME LIMITED
Application year: 1993
Main classification: C12N15
IPC codes: A01K67, C12N1, C12N9, C12N15, C12Q1, C12R1