Cloning and expression of bovine brain inositol monophosphatase.

Inositol monophosphatase is a key enzyme of the inositol phosphate second messenger signaling pathway. It is responsible for the provision of inositol required for synthesis of phosphatidylinositol and polyphosphoinositides and has been implicated as the pharmacological target for lithium action in brain. Using oligonucleotide probes based on partial amino acid sequence data for the bovine brain enzyme, several overlapping cDNA clones of 2-3 kilobases in length have been isolated. All contain an open reading frame encoding a 277-amino acid protein. No significant sequence homology was found with any known protein. The open reading frame was inserted into a bacterial expression vector in order to confirm the presumed identity of the protein. The expressed protein reacted with an anti-inositol monophosphatase monoclonal antibody. In addition, the protein was enzymically active and indistinguishable from the bovine brain enzyme with respect to Km values for substrate and Li+ sensitivity of inositol 1-phosphate hydrolysis.


Inositol
monophosphatase is a key enzyme of the inositol phosphate second messenger signaling pathway. It is responsible for the provision of inositol required for synthesis of phosphatidylinositol and polyphosphoinositides and has been implicated as the pharmacological target for lithium action in brain. Using oligonucleotide probes based on partial amino acid sequence data for the bovine brain enzyme, several overlapping cDNA clones of 2-3 kilobases in length have been isolated.
All contain an open reading frame en- In addition, the protein was enzymically active and indistinguishable from the bovine brain enzyme with respect to K,,, values for substrate and Li+ sensitivity of inositol l-phosphate hydrolysis.
The enzyme myo-inositol monophosphatase is a key component of the phosphoinositide cell signaling system. It hydrolyzes D-inositol l-phosphate (for review, see Majerus et al., 1988), D-inositol3-phosphate, and D-inositol4-phosphate and is responsible for the provision of inositol required for the synthesis of phosphatidylinositol and polyphosphoinositides. Its role in brain is particularly important since plasma inositol cannot traverse the blood-brain barrier to any appreciable extent (Spector, 1988). The enzyme is inhibited by Li' (Hallcher and Sherman, 1980) and there has been speculation that the blockade of inositol monophosphatase hydrolysis underlies the anti-manic and anti-depressant actions of Li+ (Berridge et al., 1982).
The enzyme has been purified from a number of sources, including rat (Takimoto et al., 1985) and bovine brain Attwood et al., 1888;Meek et al., 1988), and even from lily pollen (Gumber, 1984). In all cases the enzyme appears to be dimeric (-60 kDa) composed of similar, or identical, subunits. The enzyme has an absolute requirement for Mg2C (Hallcher and Sherman, 1980) and is also capable of hydrolyzing several non-inositol-containing substrates (Hallcher and Sherman, 1980;Takimoto et al., 1985;. Shute et al. (1988) have recently reported that Li' traps a phosphoryl enzyme intermediate preventing subsequent nucleophilic attack by water. Relatively little is known about the residues involved in substrate binding and catalysis.
In order to further our understanding of the molecular structure and mechanism of the enzyme, and for comparison with other phosphatase enzymes, we have isolated and sequenced a full length cDNA encoding the bovine brain enzyme. The cDNA has also been inserted into a bacterial expression vector and we have detected the expressed recombinant enzyme by immunological and enzymic methods. The properties of the recombinant and brain enzymes were found to be identical.

Inositol
Morwphosphutase Purification and Enzyme Assays The enzyme was purified from bovine brain as described previously . Enzyme activity was determined by measuring release of [Y$nositol from ["'Clinositol phosphate as described previously . One unit of enzyme activity represents 1 pmol of substrate hydrolyzed per min at 37 "C. Protein concentrations were determined by the method of Bradford (1976).

Protein Sequencing
Purified inositol monophosphatase was treated with CNBr in formic acid for 12 h followed by lyophilization (Spiess et al., 1979). Individual peptides were isolated by chromatography on a Vydac C-4 high pressure liquid chromatography column, eluted with a lo-70% acetonitrile gradient in 20 mM trifluoroacetic acid. Both the intact protein and the CNBr-derived peptides were subjected to gas phase sequencing using an Applied Biosystems gas phase sequenator (Hewick et al., 1981).
cDNA Cloning Total cellular RNA was isolated from bovine brain cortex by the guanidinium isothiocyanate-CsCl method (Chirgwin et al., 1979) followed by oligo(dT) cellulose chromatography (Maniatis et al., 1982). cDNA was synthesized by a modification of the procedure of Gubler and Hoffmann (1983) as described (Dixon et al., 1988). The EcoRldigested cDNA was size-fractionated to be greater than 1 kilobase in size by agarose-gel electrophoresis and ligated to X gtl0 arms. The cDNA library ( lo6 recombinants) was screened unamplified on Escherichia coli strain LE392 as described (Dixon et al., 1988 (Maniatis et al., 1982). Hybridization and washing conditions were as described (Dixon et al., 1988). Standard recombinant DNA procedures were used for the analysis of clones (Maniatis et al., 1982). Both strands of the sequence were determined in their entirety by the dideoxy chain termination method (Sanger et al., 1977;Hattori and Sakaki, 1983 an NdeI site at the start codon and a Hind111 site at the stop codon using polymerase chain reaction methodology (Saiki et al., 1988). Oligonucleotides 5'-CGCCGCCCTCATATGGCTGATCCTT-G-3' and 5'-TGTGTAAGCCGTCAAGCTTAATCTTCATC-3' were synthesized on an Applied Biosystems 380B instrument and purified using oligonucleotide purification cartridges (Applied Biosystems). Inositol monophosphatase cDNA (100 ng), in Bluescript Sk-(Stratagene), was linearized with KpnI and subjected to polymerase chain reaction under standard conditions (Saiki et al., 1988). Denaturation was performed at 94 "C for 2 min, the annealing at 55 "C for 2 min, and the polymerization at 72 "C! for 6 min. 20 cycles were performed, with the last polymerization step lasting 12 min. The polymerase chain reaction products were extracted with phenol/chloroform, ethanol-precipitated and digested with NdeI and HindIII, and the 837-base pair product of polymerase chain reaction subsequently purified by agarose gel electrophoresis. The T7 polymerase expression system used, pRSET5a  was derived from the pET3a vector (Rosenberg et al., 1987  . Additionally, Western blot analysis of recombinant protein was performed using a monoclonal antibody prepared to bovine inositol monophosphatase as described previously (Gee et al., 1989). For assay of enzyme activity, bacteria were pelleted in a microcentrifuge, and then resuspended in half the original culture volume of 25 mM Tris-HCl, pH 7.5, 50 mM NaCl, containing 1 mg/ ml lysozyme.
After a 5-min incubation the cells were sonicated (1 min, using a Ultrasonics Ltd. Soniprobe). Triton X-100 was then added to a final concentration of 0.5%, incubated for a further 15 min, and then pelleted in a microcentrifuge to remove insoluble material.
The insoluble material was resuspended in the same buffer by extensive vortexing. Both the soluble and insoluble fractions were then assayed for inositol monophosphatase activity as described above.

AND DISCUSSION
Cloning of Bovine Brain Inositol Monophosphatase-The inositol monophosphatase from bovine brain was purified to homogeneity as previously described . When the intact protein was subjected to amino acid sequence analysis the amino terminus was found to be blocked. The protein was then reacted with CNBr which released several peptide fragments. The amino termini of three of these peptides were determined and are indicated in Fig. 1B. Oligonucleotide probes were designed corresponding to each of the peptide coding sequences. These oligonucleotides were used as probes for bovine brain cDNA libraries. A large number of clones were detected which hybridized to several of the probes. Several of the clones were analyzed and found to have inserts of 2-3 kilobases with overlapping restriction maps. region and part of the untranslated region of one of these clones was sequenced (Fig. 1). The sequence of the coding region, encoding an open reading frame of 277 amino acids, is shown in Fig. 1B. The predicted amino acid sequence contains all of the peptide sequences obtained from the purified protein.
Analysis of the predicted protein sequence reveals that it is moderately hydrophilic with no unusual regions of hydrophobic character. No consensus sequences were identified for phosphorylation, Ca2+ binding sites, ATP binding sites, or any other recognizable regulatory sequence. Searches of the protein data base did not reveal significant homology with any known protein. Short regions of homology were noted between inositol monophosphatase and some ATPases, but were considered of too low homology to be significant.
Thus, inositol monophosphatase appears to be a novel enzyme. It will be interesting to determine if this enzyme has similarity to other enzymes in the phosphatidylinositol metabolizing pathway. There is no consensus sequence available for Li' binding, so this site will have to be determined directly.  Additionally, the specific activity of the inositol monophosphatase in the soluble fraction does not correct for the presence of 1 mg of lysozyme/ml. After correcting for the presence of lysozyme the specific activity of inositol monophosphatase in the soluble fraction of induced bacteria was 1460 milliunitsl mg, approximately 25% pure, assuming a specific activity of 5700 milliunits/mg for pure enzyme . The enzyme in the soluble fraction was further characterized. Hydrolysis of 0.1 mM inositol l-phosphate was inhibited by Li' with an I& of 2.0 mM. A superimposable inhibition curve was obtained if crude brain supernatant was used instead of bacterial extract. In addition, the K,,, for inositol l-phosphate for the recombinant enzyme was 0.11 mM, similar to that reported for the bovine brain enzyme . Thus, the recombinant and brain enzymes appear to be identical in all respects. In this context, there are no known post-translational modifications of the native enzyme which might result in a different behaviour, compared to the recombinant enzyme. The availability of recombinant inositol monophosphatase will allow future studies using mutagenesis and chemical modification techniques to further define the structure and function of this novel enzyme.  (Fig. 2B) confirmed that this M, 30,000 polypeptide was bovine inositol monophosphatase. The recombinant protein was further analyzed for inositol monophosphatase activity using a radiochemical enzyme assay . Data from a representative induction experiment are shown in Table I. Large amounts of enzyme were produced both with and without induction by IPTG. Induction did, however, result in an increase in both the amount of inositol monophosphatase produced, and the specific activity of the enzyme as would be predicted from the pattern of protein expression revealed by SDS-PAGE and Coomassie Blue staining ( Fig. 2A, lanes 2 and 3). Bacteria lacking the expression vector had no detectable inositol monophosphatase activity. A large proportion of the enzyme was produced in an insoluble form, presumably as inclusion bodies. As such, the enzyme activity of the insoluble fraction is probably an underestimate of the total inositol monophos-Note Added in Proof-We have recently found that the amino acid sequence of bovine inositol monophosphatase bears significant homology (-36% overall) to the QAX protein of Neurospora