©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Identification of Amino Acid Residues of Human Phospholipase C1 Essential for Catalysis (*)

(Received for publication, December 14, 1994; and in revised form, December 29, 1994)

Hwei-Fang Cheng (1) Meei-Jyh Jiang (2) Chih-Lin Chen (2) Su-Min Liu (2) Li-Ping Wong (2) Jon W. Lomasney (3) Klim King (2)(§)

From the  (1)Department of Health, National Laboratories of Foods and Drugs, Executive Yuan, Taipei 115, Taiwan, the (2)Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, Republic of China, and the (3)Feinberg Cardiovascular Research Institute, Northwestern University Medical School, Chicago, Illinois 60611-3008

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C1 (PLC1) isolated from a human aorta cDNA library. Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme. Exceptions were mutants with the conversion of Arg to Leu (R338L), Glu to Gly (E341G), or His to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP(2)) with an affinity similar to that of wild type enzyme. Western blotting analysis of trypsin-treated enzyme-PIP(2) complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC1 was preincubated with 7.5 µM PIP(2), whereas if it was preincubated with 80 µM PIP(2), the size of major protein surviving was comparable to that of intact enzyme. However, mutant enzyme R338L was not protected from trypsin degradation by PIP(2) binding. These observations suggest that PLC1 can recognize PIP(2) through a high affinity and a low affinity binding site and that residues Glu and His are not involved in either high affinity or low affinity PIP(2) binding but rather are essential for the Ca-dependent cleavage activity of PLC.


INTRODUCTION

Phospholipase C hydrolyzes inositol phospholipids into diacylglycerol and inositol 1,4,5-trisphosphate (IP(3)), (^1)a process that constitutes a major pathway for receptor-coupled signaling at the plasma membrane of most eukaryotic cells. Both diacylglycerol and IP(3) function as important second messengers that activate protein kinase C and mobilize intracellular Ca, respectively, thus triggering multiple enzymatic cascades to regulate cellular functions including cell growth and neuronal activity(1) . Phospholipase C exists as isoenzymes (PLCbeta, PLC, and PLC)(2) . PLC is activated following phosphorylation by nonreceptor or receptor protein tyrosine kinase activities(3, 4) , whereas PLCbetas are regulated by alpha subunits of G proteins (Gq family) (5, 6, 7) or by beta subunits(8, 9, 10) . How PLC1 is regulated still remains to be determined.

Although PLC isozymes differ in the way they are regulated, they have similar enzymatic properties(2) . All three members of the PI-PLC family are able to recognize phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP(2)) and to carry out the Ca-dependent hydrolysis of these inositol phospholipids. Comparison of the amino acid sequences of all three isoforms reveals that PI-PLCs are highly conserved in two distinct regions designated X and Y(2) . Structural integrity of the highly conserved X and Y region is essential for a functional catalytic core, as partial deletion of either the X or Y region sequence in PLC1 (11, 12) or PLC (13, 14) inactivates the enzyme. The intervening peptide connecting the X and Y regions is not essential for the hydrolytic properties of either PLC or PLC1, as partial deletion of these sequences in PLC1 (14) or PLC2 (13) or trypsin cleavage of this sequence in PLC1 (11) does not inactivate the truncated enzymes. The first 60 NH(2)-terminal residues of the PLC1 sequence are not essential for Ca-dependent catalysis, but are required for the enzyme to hydrolyze PIP(2) in a processive manner (15) . A subfamily-specific sequence of 400 amino acid residues in the family is located between the X and Y regions and is characterized by src homology domains (SH2 and SH3), which are essential for the activation of PLC by tyrosine protein kinases(3) . Partial deletion of either the NH(2) terminus or COOH terminus of PLCbeta does not affect its catalytic activity, but the enzyme loses its ability to be activated by G proteins(10, 16) . These observations strongly indicate that residues essential for specific substrate recognition and Ca-dependent cleavage can be identified among those conserved residues located in the X and Y regions.

To identify specific amino acid residues involved in catalysis, we isolated cDNA of the smallest PLC from human aorta, expressed it in Escherichia coli, and purified the protein. To minimize structural disturbance of the enzyme and to facilitate genetic analysis of the role of PLC1 in cellular function, we used single amino acid residue substitution mutagenesis to evaluate the contribution of each conserved residue in PI-PLC to its catalytic function. To narrow down the number of potential residues involved in catalysis, only those residues containing a functional side chain and which are invariant in both prokaryotic and eukaryotic PI-PLC were subjected to base substitution mutagenesis. The mutant enzymes were assayed for their abilities to hydrolyze PI and PIP(2), to bind inositol phospholipid, and to form trypsin-resistant enzyme-substrate complexes. We demonstrate that His and Glu, located in the X conserved region of PLC1, are essential for the Ca-dependent hydrolysis of PIP(2) rather than for substrate binding.


EXPERIMENTAL PROCEDURES

Isolation and Sequencing of cDNA Clones

A 0.6-kb DNA fragment spanning from nucleotide 1194 to 1841 of the rat C6 glioma cell PLC1 gene was obtained using forward primer 5`-CAGCACGGAGGCCTACATCC-3` and reverse primer 5`-ACAAAACCATTTCCTGATTCTTG-3` to amplify cDNA of C6 glioma cells(17) . This 0.6-kb fragment was [P]dCTP labeled by the random priming DNA method and used to screen 10^6 phage plaques from a human aortic gt11 cDNA library (CLONTECH). Duplicate nitrocellulose filter papers were hybridized at 42 °C in 50% formamide, 1% dextran sulfate and washed at 55 °C in 1 times SSC. DNA sequences were determined by the dideoxy chain termination method, using a primer-directed sequencing strategy and dye terminator method on purified double-stranded gt11 DNA template.

Subcloning of PLC1

The PLC1 coding gene was isolated from gt11 recombinant phage and subcloned into the EcoRI site of pTZ19R. EcoRI digestion of the gt11 PLC1 cDNA clones results in 2.0- and 0.7-kb fragments corresponding to cDNA sequences coding for the NH(2) terminus and COOH terminus of PLC1. These two fragments were subcloned separately into the EcoRI site of pTZ19R to obtain constructs pTZ19R5`PLC and pTZ19R3`PLC, respectively.

Construction of Expression Vector

To remove the 5`-untranslated region and construct a BamHI sequence 5` to the initiation codon, a set of oligo DNA primers was used to perform PCR using the cloned cDNA as template. The forward primer (5`-GGGGGATCCATGGACTCGGGCCGGGACTT-3`) contained a BamHI sequence (GGATCC) followed by a sequence identical to the PLC1 coding sequence spanning from nucleotide 95 to 114. The reverse primer spans from nucleotide 624 to 599 (5`-TCCACCTGGATGTTGAGCTCCTTCAG-3`). This set of primers was used to amplify a PLC1 cDNA fragment that covers the first 529 base pairs (from nucleotide 95 to 624) of human PLC1 cDNA coding sequence including the BglII site. The amplified PCR product was blunt ended with T4 DNA polymerase and inserted into the blunt-ended EcoRI site of pTZ19R to obtain pTZ19B5`500plc. To construct pTZ19RB5`PLC which harbors the PLC1 coding sequence from the initiation codon to the end of its internal EcoRI site (nucleotide 1945) with a BamHI sequence 5` to the initiation codon, the BglII/HindIII DNA fragment from pTZ19R5`plc was isolated and inserted into the BglII/HindIII-digested pTZ19R600plc. To obtain the full-length PLC1 gene without its 5`-untranslated region, the EcoRI fragment containing the remaining part of the COOH-terminal coding sequence and its 3`-untranslated sequence was isolated from pTZ19R3`plc and inserted into the EcoRI site of pTZ19RB5`plc. The BamHI fragment from pTZ19RhPLC1 covers the entire coding region, and the 3`-untranslated sequence of human PLC1 was inserted into the BamHI site in the polylinker region of pRSETA expression vector (Invitrogen) for expressing the cloned PLC1 in bacteria E. coli BL21 (DE3) pLys. The orientation of the insertion was determined by restriction mapping with EcoRI/BglII. In this vector (pRSETAplc), the transcription of PLC1 cDNA in E. coli BL21 (DE3) pLys is directed by the T7 promoter.

Expression of Human PLC1 in E. coli

E. coli BL21 (DE3) pLys cells harboring pRSETAplc plasmid were grown at 30 °C in 2 liters of LB medium containing 100 µg/ml ampicillin. When the OD of the culture reached 1.0, 20 ml of 0.1 M isopropyl 1-thio-beta-D-galactopyranoside was added, and the culture was incubated for an additional 2 h. Cells were harvested by centrifugation and resuspended in 10 ml of ice-cold lysis buffer containing 50 mM sodium phosphate, pH 8.0, 0.1 M KCl, 5 mM EDTA, 5 mM EGTA, 10 µM phenylmethylsulfonyl fluoride, and 0.1% Tween 20. All subsequent steps were performed at 4 °C. Cells were lysed by five cycles of 20-s pulses sonication with 40-s cooling intervals in between in an MSE sonicator. The cell lysate was then cleared by centrifugation at 15,000 times g for 30 min, and the supernatant fraction (10 ml of 33.8 mg/ml protein) was applied directly onto a column of 1 ml of Ni-NAT agarose (Qiagen) equilibrated with a buffer of 50 mM sodium phosphate, pH 8.0, 0.1 M KCl, 0.1% Tween 20, and 10 µM phenylmethylsulfonyl fluoride (buffer A). The column was washed with 40 ml of buffer A containing 15 mM imidazole, then eluted with 10 ml of the same buffer containing 100 mM imidazole; eluent was collected as 0.5 ml/fraction. The active fractions were identified by PI hydrolysis assay and then pooled. The partially purified PLC1 from the Ni-NAT column was concentrated by an Amicon Centriprep-30 concentrator, diluted 10-fold with lysis buffer, and then applied directly to 1 ml of a heparin-Sepharose CL-6B column (Pharmacia Biotech Inc.) preequilibrated with lysis buffer. After being washed with 10 ml of the same buffer, PLC1 was eluted by a 50-ml linear salt gradient from 0.1 to 0.5 M KCl in 50 mM sodium phosphate, pH 8.0, 0.1% Tween 20, 5 mM EDTA, and 5 mM EGTA. The active fractions were identified by PI hydrolysis activity or Western blotting and were pooled.

Protein Determination

The protein concentration was determined by the method of Bradford(18) , and bovine serum albumin (BSA) was used to calibrate the assays.

Immunoblotting Analysis

PLC1 protein was separated in 10% SDS-polyacrylamide gels and then electrophoretically transferred to nitrocellulose membrane in blotting buffer (48 mM Tris-HCl, pH 9.2, 39 mM glycine, 0.037% SDS, and 20% methanol). The membrane was blocked with 5% skim milk in 20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 0.2% Tween 20 (TBS-T buffer) for 1 h at room temperature and then rinsed with two changes of TBS-T followed by one 15- and two 5-min washes in the same buffer. Membrane was then incubated with mixed monoclonal anti-PLC1 antibody (0.2 µg/ml in TBS-T) at 4 °C for 14 h, rinsed and washed as before, and then incubated with peroxidase-labeled sheep anti-mouse antibody (0.055 µg/ml in TBS-T) for 1 h at room temperature. The membrane was washed and rinsed as before, and PLC1 proteins were detected by Amersham ECL detection system. The quantity of PLC1 protein was determined by reading the density of bands on a densitometer.

RNA Analyses

Multiple human tissue blots were obtained from CLONTECH. [P]dCTP-labeled BamHI fragment coding for full-length human PLC1 was used to probe the amount of transcripts. Hybridization was carried out as recommended by the supplier.

Construction of Point Mutations in Human PLC1

Site-directed point mutagenesis by a two-stage PCR method was used(19) , with a slight modification as shown in Fig. 6. Primary PCR steps consisted of two separate PCRs for each point mutation. One of the PCRs was initiated by using a mutagenic primer and one of the outer primers; the other was initiated by using an internal primer and the other outer primer. Both reactions used pRESTAplc as template. The PCR took place in a reaction volume of 100 µl containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.01% gelatin (w/v), 1.5 mM Mg, 200 µM dNTPs, 10 pM template (pRESTAplc), 1 µM primers, and 2.5 units of Taq polymerase. The reaction mixture was prewarmed to 55 °C for 5 min; the three-step PCR reaction (94 °C for 40 s, 55 °C for 1 min, and 72 °C for 2 min) for 30 cycles was then initiated by adding 0.5 µl (2.5 units) of Taq polymerase. The products of primary PCRs were separated electrophoretically and isolated in a low melting point agarose gel. The purified primary PCR products in low melting agarose were incubated at 70 °C for 15 min and then diluted with distilled H(2)O to a final concentration of 5 µg/ml. Equal volumes (20 µl) of two diluted primary PCR products were mixed, boiled to 100 °C for 5 min, and cooled slowly to 30 °C. The secondary PCR reaction was carried out in a volume of 100 µl, containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.01% gelatin (w/v), 1.5 mM Mg, 200 mM dNTPs, 50 ng of template (the mixed and annealed primary PCR products), a 1 µM concentration of two outer primers, and 2.5 units of Taq polymerase. Amplified mutant DNA fragments from the secondary PCR reaction were electrophoretically purified in low melting agarose followed by phenol/chloroform extraction and ethanol precipitation. The purified mutant DNA fragment was then digested with SphI and SacII restriction endonuclease and was used to replace the corresponding restriction fragment of the wild type pRSETAplc. The desired point mutation and sequence flanking by SphI and SacII sites were confirmed by DNA sequence analysis from 10 independently isolated pRSETAplc-containing mutant DNA fragments. For mutagenesis of Lys, the mutagenic PCR products from the secondary PCR were digested with SphI and EcoRI and cloned into the SphI/EcoRI-cleaved pRSETAplc(RI), whose EcoRI in the polylinker region had been destroyed by T4 DNA polymerase filling in and the subsequent self-ligation.


Figure 6: Schematic presentation of two-stage PCR method to construct point mutations in PLC1. The first stage of mutagenesis consists of two independent primary PCRs. One reaction uses the mismatch primer (mutagenic primer) paired with one of the external primers flanking one of the unique restriction sites; the other uses the internal primer paired with another external primer flanking the other unique restriction site. Both reactions used pRSETAplc plasmid as template, and the PCRs were carried out as described under ``Experimental Procedures.'' The primary PCR products were isolated and purified, mixed, and reannealed. The hybrid template was first extended with Taq polymerase and followed by using the same pair of external primers for the secondary stage PCR as described under ``Experimental Procedures.'' The final mutagenic DNA fragment was cleaved with a pair of unique restriction endonuclease (SphI/SacII or SphI/EcoRI) and used to replace the corresponding fragment in wild type pRSETAplc.



Measurement of PLC Activity

Hydrolysis of PIP(2) by PLC1 was determined exactly as described previously (7) in an assay volume of 60 µl of 50 mM HEPES, pH 7.2, 3 mM EGTA, 0.2 mM EDTA, 0.83 mM MgCl(2), 20 mM NaCl, 30 mM KCl, 1 mM dithiothreitol, 0.1 mg/ml BSA, 0.16% sodium cholate, 1.5 mM CaCl(2) containing 50 µM PIP(2) (8,000 cpm) and 500 µM PE. The reaction was carried out at 30 °C for 2-15 min and terminated by adding 0.2 ml of 10% ice-cold trichloroacetic acid and 0.1 ml of BSA (10 mg/ml). After incubating on ice for 15 min, the unhydrolyzed [^3H]PIP(2) (pellet) was separated from [^3H]IP(3) (supernatant) by centrifugation at 2,000 times g for 10 min. Radioactivity in the supernatant was measured by liquid scintillation counting. The activity of PIP(2) hydrolysis is expressed as µmol of InsP(3)/min/mg of protein; usually 0.5-10 ng of partially purified recombinant PLC1 was used per assay. Determination of PI hydrolysis activity was essentially the same as described by Hofmann and Majerus(20) . The reaction was carried out in a volume of 200 µl of 50 mM HEPES, pH 7.0, 3 mM CaCl(2), 1 mM EGTA, 0.1% sodium cholate containing 30,000 cpm of [^3H]PI (300 µM). After incubation at 37 °C for 5-15 min, the reaction was terminated by adding 1 ml of chloroform/methanol/HCl (100:100:0.6) followed by 0.3 ml of 1 N HCl containing 5 mM EGTA. The aqueous and organic phases were separated by centrifugation, and a 400-µl portion of upper aqueous phase was counted by liquid scintillation.

Preparation of Phospholipid Vesicles

Phospholipid vesicles PE/PC (8:2 molar ratio) containing the indicated concentration of PI or PIP(2) were prepared followed Mueller and Chien (21) with slight modifications. A dry phospholipid film was formed by slowly blowing a 0.25-ml solution of chloroform/methanol (2:1, v/v) containing mixing lipids (320 nmol of PE, 80 nmol of PC, and the indicated amount of PIP(2) or PI) under a stream of nitrogen followed by freeze-drying under vacuum for 4 h. The phospholipid film was hydrated under nitrogen with 1 ml of nitrogen-aerated 0.18 M sucrose solution for 18 h at 4 °C followed by mixing with an equal volume of 100 mM HEPES, pH 7.0, 200 mM KCl, and 10 mM EGTA. Vesicles were isolated from the pellet by centrifuging the hydrated phospholipids at 1,200 times g, 4 °C for 20 min. The phospholipid vesicles were washed once with 1 ml of 50 mM HEPES, pH 7.0, 100 mM KCl, 5 mM EGTA, 200 µg/ml BSA (binding buffer) and resuspended in 1 ml of the same buffer.

Binding Assay

The binding of PLC1 to phospholipid vesicles was estimated by centrifugation assay(22) . The final assay volume was 0.2 ml containing 50 mM HEPES, pH 7.0, 100 mM KCl, 5 mM EGTA, and 200 µg/ml BSA (binding buffer) plus the indicated concentration of phospholipid vesicles and 1 µg of enzyme. To perform the assay, 1 µg of enzyme in 20 µl of binding buffer was incubated with 180 µl of mixed phospholipid vesicles in the same buffer at 30 °C for 15 min. The free and bound PLC1 were separated by sedimentation of the samples at 100,000 times g for 30 min. The amounts of the free PLC1 (supernatant) and the bound enzyme (pellet) were determined by PI hydrolysis assay and Western blotting analysis.

Trypsin Digestion of PLC1

Digestion of 0.2 µg of PLC1 with 0.1 µg of trypsin was carried out in a reaction volume of 40 µl of 12.5 mM sodium phosphate, 37.5 mM HEPES, pH 7.0, 5 mM EGTA, 3.75 mM EDTA, 75 mM NaCl, 25 mM KCl, 0.025% Tween 20, 0.075% sodium cholate, 375 µg/ml BSA containing 400 µM PE, and the indicated concentration of PIP(2). (This is the minimal concentration of trypsin which will degrade more than 95% of PLC1 protein within 5 min when incubated with phospholipid micelles free of PIP(2).) To carry out the reaction, PLC1 was diluted to 0.02 mg/ml with the ice-cold buffer containing 50 mM sodium phosphate, pH 7.0, 0.1 M KCl, 0.1% Tween 20, 5 mM EDTA, and 5 mM EGTA. To prepare the substrate solution, 0.44 mg of PE and various amounts of PIP(2) in chloroform/methanol (2:1, v/v) were first dried down under a stream of nitrogen gas, and the phospholipids were solubilized by resuspending the dry lipid with 0.9 ml of 50 mM HEPES, pH 7.0, 5 mM EGTA, 100 mM NaCl, and 0.1% sodium cholate. The mixture was sonicated in a water bath sonicator for 10 min. One hundred µl of BSA stock (5 mg/ml) in the same buffer was then added to make a final concentration of 0.5 mg/ml. The proteolysis reaction was initiated by mixing 10 µl of diluted PLC1 with 30 µl of 37 °C warmed trypsin buffer containing 50 mM HEPES, pH 7.0, 5 mM EGTA, 100 mM NaCl, 0.1% sodium cholate, 0.5 mg/ml BSA, 0.1 µg of trypsin, 400 µM PE, and the indicated concentration of PIP(2). The reaction was proceeded at 37 °C for an indicated period and was stopped by adding trypsin inhibitor to a final concentration of 2.5 µg/ml. The residual PLC1 was determined by PI hydrolysis activity and Western blotting analysis.


RESULTS

Isolation and Sequencing of Human Aortic PLC1 cDNA and Its Homology to Other PI-specific Phospholipase Cs

A human aortic smooth muscle cDNA library was screened with a probe derived from the rat C6 glioma cell PLC1 cDNA gene(17) . This resulted in the isolation of 23 positive cDNA clones. Through direct DNA sequence analysis using the purified recombinant gt11 phage DNA as template, two of the clones were found to contain a 2.8-kb insert with an open reading frame of 2.3 kb, which encoded a protein of 756 amino acids with an estimated molecular mass of 87 kDa. The human PLC1 cDNA had 95% homology to the rat PLC1 gene at the level of deduced amino acid sequence (Fig. 1). Like members of the PLC subfamily, human PLC1 lacks the COOH-terminal tail immediately after the Y domain.


Figure 1: Nucleotide and deduced amino acid sequence of human PLC1 cDNA. Panel A, the structure of human PLC1 cDNA (2.6 kb) is schematically shown with the coding region (open box) flanked by untranslated sequences (solid line). The 2040- and 591-base pair (bp) EcoRI fragments from the phage clone, corresponding to the sequence coding for the NH(2)-terminal and COOH-terminal region of PLC1, respectively, were subcloned further into the EcoRI site of pTZ19R to generate pTZ19R5`plc and pTZ19R3`plc. Panel B, the nucleotide sequence of the 2.6-kb full-length human phospholipase C1 cDNA was determined as described under ``Experimental Procedures.'' The nucleotide residue numbers are given to the right of each line. The deduced amino acid sequence encoded by the longest open reading frame (beginning at nucleotide 95 ATG) is shown using the single letter amino acid code. Deduced amino acid residues are numbered beginning with the initiation methionine, and the residue numbers are shown to the left of each line. Amino acid residues different from those of rat enzyme are shown. Conserved X and Y regions of PI-PLC are underlined.



Fig. 2compares the deduced amino acid sequence of the most conserved X and Y regions of human PLC1 with those of the previously described PI-specific PLCs. Within these regions, human PLC1 has 40 and 38% sequence identity to rat PLCbeta1, 35 and 43% sequence identity to rat PLC, 40 and 38% sequence identity to the Drosophila NorpA gene, and 38 and 32% sequence identity to yeast PLC1. PI-specific PLC from Bacillus cereus only contains the X domain, with which the human clone shares 30% similarity.


Figure 2: Comparison of the primary structures of the conserved regions of PLC isoenzymes from rat, human, Drosophila, yeast, and bacillus. Amino acid sequence comparison of human PLC1 with the conserved X and Y regions of rat PLCbeta1(17) , PLCbeta3(42) , PLC1(43) , PLC2(13) , human PLCbeta2 (44) , yeast PLC(45) , Drosophila norpA (Dro PLCbeta)(46) , and the X region of bacillus PI-PLC(41) . Organisms, PI-PLC isoenzyme classes, and the starting position in each protein sequence of the residues shown are indicated on the far left. The boxed areas denote positions at which amino acids in seven or more sequences are identical or represent conservative substitutions grouped as follows: A and G; T and S; I, L, M and V; K, H, and R; W, Y, and F; D and E; N and Q. Gaps introduced to optimize the alignment are indicated by hyphens. Identical residues containing functional side chain in the X region of PI-PLC from human to bacillus are indicated by an asterisk and were subjected to base substitution mutagenesis study.



Tissue Distributions of PLC1 mRNA

Since the cDNA clone of human PLC1 was initially isolated from a human aorta cDNA library, we examined further the level of expression of PLC1 mRNA in various human tissues by Northern hybridization. Polyadenylated RNA was prepared from various human tissues and hybridized with a P-labeled PLC1 cDNA probe. As shown in Fig. 3, PLC1 mRNA is ubiquitously expressed with a predominant length of 3.8 kb in all human tissues we examined. However, the level of mRNA varied significantly depending on tissues, with the 3.8-kb transcript most abundantly expressed in lung, heart, pancreas, skeletal muscle, and kidney. Hybridization of mRNA prepared from skeletal muscle showed a 7.5-kb hybridizing species in addition to the 3.8-kb band.


Figure 3: Northern analysis of RNA from various human tissues. Human multiple tissue Northern blot obtained from CLONTECH was hybridized with full-length human PLC1 cDNA washed and exposed as suggested by the supplier. Lane 1, pancreas; lane 2, kidney; lane 3, skeletal muscle; lane 4, liver; lane 5, lung; lane 6, placenta; lane 7, brain; and lane 8, heart.



Expression of Human PLC1 in E. coli

To confirm that the human PLC1 cDNA gene encodes a protein comparable to that of rat, we expressed the cloned human cDNA in E. coli and purified the recombinant protein by two-step column chromatography (Table 1). The BamHI DNA fragment containing the full coding length of PLC1 cDNA was inserted into the BamHI site of expression vector pRSETA to obtain pRSETAplc; upon induction with isopropyl-1-thio-beta-D-galactopyranoside, E. coli BL21 harboring PRSETAplc would express a fusion human PLC1 with 34 amino acids (derived from enterokinase cleavage site, phage T7 gene 10 sequences, and six consecutive histidine tags) at its NH(2) terminus. This modification would allow us to remove most bacterial proteins by one-step Ni-NAT affinity column chromatography, while the hydrolysis activity was not affected. Trace amounts of bacterial contaminants in this PLC1 preparation were further removed by heparin-Sepharose column chromatography. The homogeneity of the recombinant human PLC1 was examined by SDS-polyacrylamide gel electrophoresis (Fig. 4). PLC1 expressed from E. coli was used to hydrolyze PI and PIP(2), and the specific activities were determined to be 64 and 37 µmol/min/mg, respectively. When using the phospholipid micelle substrate with a 1:10 molar ratio of PIP(2) to PE, we found that the specific activity of the recombinant PLC1 was highly dependent on the free calcium concentration. As shown in Fig. 5, the specific activity increased 17-fold as the free calcium concentration increased from 0.1 to 5.6 µM.




Figure 4: SDS-polyacrylamide gel electrophoresis and immunoblots of human PLC1 expressed from E. coli BL21. Panel A, 100 µg of crude extracts from E. coli BL21 carrying pRSETAplc (lane a), vector pRSET (lane b), or 0.1 µg of heparin-Sepharose column chromatography-purified PLC1 (lane c) were separated on 10% SDS-polyacrylamide gels and further detected with mixed monoclonal anti-PLC1 antibodies. Panel B, 5 µg of heparin-Sepharose column chromatography-purified PLC1 (lane a), 100 µg of crude extracts from E. coli BL21 carrying pRSETAplc (lane b), and 10 µg of Ni-NAT agarose column-purified PLC1 (lane c) were separated on 10% SDS-polyacrylamide gels and stained with Coomassie Blue.




Figure 5: Ca dependence of PLC1. The PIP(2) hydrolysis activity of purified PLC1 expressed from E. coli BL21 was assayed as described under ``Experimental Procedures,'' and the free calcium concentrations were calculated according to Fabiato and Fabiato(47) . The values are expressed relative to the activity at a free Ca concentration of 95 nM.



Amino Acid Residues Essential for Catalytic Activity of PLC1

To identify residues potentially involved in cleavage activity of PI-PLC, 9 amino acids with a functional side chain which were invariant in PI-PLCs from human to bacteria (Fig. 2) were subjected to residue substitution mutagenesis. Since the prokaryotic enzyme lacks the conserved Y region, we believe that these residues located in the X region of eukaryotic enzymes play a critical role in the catalysis of PI-PLC. To test this possibility, these 9 residues were individually substituted by in vitro mutagenesis of the PLC1 cDNA. A mutant restriction DNA fragment containing a base substitution at the corresponding amino acid codon was generated by PCR (Fig. 6) and used to replace the corresponding restriction fragment in the native pRSETAplc expression construct. Crude extract derived from the E. coli BL21 strain harboring a mutant expression construct was passed through Ni-NAT agarose followed by heparin-Sepharose column chromatography, and the homogeneity of mutant protein was examined by SDS-polyacrylamide gel electrophoresis and Western blotting analysis (data not shown). The purified mutant was tested for the characteristic inositol phospholipid hydrolysis using PI or PIP(2) as substrate. This analysis allowed us to categorize the present mutants into at least three classes (Table 2). In the first class, the mutant enzymes bear no detectable PIP(2) or PI hydrolysis activity, e.g. R338L, E341G, and H356L; the second class of mutants is partially active in inositol phospholipid hydrolysis, e.g. S381A, K434Q, and K441Q; the inositol phospholipid hydrolysis activities of the third group are similar to that of wild type, e.g. E327G, S388A, and K440Q. Only the first class of mutant enzymes (R338L, E341G, and H356L) were subjected to further characterization in the present report.



Binding of PLC1 to Phospholipid Vesicle

Since no PI or PIP(2) hydrolysis activity was detectable by mutant enzymes R338L, E341G, and H356L at free Ca concentrations of 0.1 µM to 3 mM and at PIP(2) concentrations of 50-500 µM (data not shown), these mutant enzymes might be defective either in cleavage or in substrate binding. To distinguish between these possibilities and to identify the role of residues Arg, Glu, and His in the catalysis, equilibrium binding of the wild type and mutant forms of PLC1 to phospholipid vesicles was examined by centrifugation assay. As shown in Fig. 7A, wild type and all three cleavage-defective mutant forms of PLC1 were unable to bind vesicles of PE/PC and bound weakly (45% were bound) to PE/PC/PI vesicles (molar ratio 4:1:2.5). In contrast, more than 90% of the wild type and mutant enzymes were bound to PE/PC vesicles containing 5% (molar ratio) PIP(2). The binding of PLC1 to PIP(2) phospholipid vesicles was highly dependent on vesicle concentration and could be saturated as shown in Fig. 7B. Fifty percent of the wild type protein was bound when incubated with 1.25 µM PIP(2), and the binding was saturated when the PIP(2) reached a concentration of 10 µM. Although mutant enzymes R338L, E341G, and H356L are not able to hydrolyze PI or PIP(2), all of these three cleavage-defective mutant forms of PLC1 can bind phospholipid vesicles containing 5% (molar ratio) PIP(2) in a concentration-dependent and saturable fashion with affinities comparable to that of the wild type enzyme.


Figure 7: Centrifugation assay of PLC1 binding to lipid vesicles. Panel A, binding of wild type PLC1 and cleavage-defective mutants to phospholipid vesicles of different lipid content: 400 µM phospholipid vesicles with a PE/PC molar ratio of 4:1 (box), 600 µM phospholipid vesicles of PE/PC/PI with a molar ratio of 4:1:2.5 (&cjs2108;), and 420 µM (box) phospholipid vesicles of PE/PC/PIP(2) with a molar ratio of 4:1:0.25 (). Panel B, dose-dependent binding of PE/PC/PIP(2) (4:1:0.25) lipid vesicles to wild type and cleavage-defective mutants. The concentration of PIP(2) (µM) was a fraction of phospholipid vesicle containing PE/PS/PIP(2) with a molar ratio of 4:1:0.25. All of the centrifugation assays were carried out in a 0.2-ml total volume using a Beckman TL-100 table top ultracentrifuge and TLA-100 rotor (see ``Experimental Procedures''). The unbound enzyme fractions (supernatant) were quantified by PI hydrolysis assay and immunoblotting using mixed monoclonal antibodies (for cleavage-defective mutant). The bound enzyme fractions (pellets) were dissolved in 0.05 ml of phosphate-buffered saline buffer then quantified by Western blotting analysis.



PIP(2) Protection of PLC1 from Trypsin Digestion

To study further specific protein-PIP(2) interaction and the structure of the enzyme-PIP(2) complex, we compared the sensitivity of free enzyme and enzyme-phospholipid complexes to trypsin digestion. As shown in Fig. 8A, digestion of PLC1 (0.2 µg) with trypsin (0.005 µg) gave one major 80-kDa proteolytic fragment concomitant with two minor degraded fragments of about 60 kDa. When the trypsin concentration was increased to 0.1 µg, more than 90% of PLC1 protein (0.2 µg) was degraded within 5 min. The rapid degradation of PLC1 protein by trypsin correlated with loss of PI hydrolysis activity, as shown in Fig. 8B; the specific activity of the PLC1 was reduced to 50% within less than 1 min at 37 °C by trypsin and was reduced to 50% in less than 2 min when the enzyme was preincubated with 400 µM PE or PI. In contrast, this rapid inactivation of PLC1 by trypsin was prevented by preincubating the enzyme with its specific substrate PIP(2). As shown in Fig. 8B, the half-life of PLC1 was prolonged to 7.8 min when the enzyme was preincubated with 200 µM specific substrate (PIP(2)) prior to trypsin digestion. The loss of PI hydrolysis activity by PLC1 as a result of trypsin digestion was prevented by PIP(2) in a substrate concentration-dependent and saturable manner (Fig. 8C). The activity of PLC1 was protected by as much as 65% as the PIP(2) concentration increased from 20 to 500 µM. The saturation concentration of PIP(2) for protecting PI hydrolysis activity from the action of trypsin was 80 µM, and half-maximal protection occurred at 34 µM PIP(2). Although PE also protected PLC1 activity from the action of trypsin in a concentration-dependent manner, this nonspecific phospholipid did not protect as efficiently as PIP(2), and the protection was not saturable even when the concentration was increased to 500 µM.


Figure 8: Effect of PIP(2) on the trypsin cleavage of PLC1. Panel A, Western blotting analysis of PLC1 (0.2 µg) digested with 0 (lane 1), 0.005 (lane 2), 0.01 (lane 3), 0.05 (lane 4), and 0.1 µg (lane 5) of trypsin for 5 min in the presence of 400 µM PE. Panel B, kinetic analysis of PLC1 inactivation by the proteolytic action of trypsin. 0.2 µg of the PLC1 was preincubated with PE/PIP(2) micelles containing 200 µM PIP(2) and 400 µM PE (bullet); PE micelles containing 600 µM PE (circle) or in the absence of phospholipid (box). At each indicated time point, an aliquot of incubation mixture was diluted and used to determine the residual PI hydrolysis activity. Panel C, the residual activity of PLC1 after a 5-min trypsin digestion in the presence of PE/PIP(2) micelles containing 400 µM PE and the indicated concentration of PIP(2) (circle) or PE only (box).



The ability of PIP(2) to protect PLC1 against trypsin-inflicted loss of PI hydrolysis activity was correlated to the extent of degradation of PLC1 by trypsin. As shown in Fig. 9A, incubating PLC1 with PE/PIP(2) micelles prior to trypsin digestion markedly reduced the rate of trypsin cleavage; at least 50% of the PLC1 protein still remained intact after a 5-min trypsin digestion, and this was reduced to less than 10% as the enzyme-PIP(2) complexes were digested further with trypsin for a total period of 15 min. However, 90% of the enzyme was degraded by trypsin within 5 min when the enzyme was preincubated with lipid micelles containing PE only. The pattern and extent of digestion of PLC1bulletPIP(2) complexes by trypsin were highly dependent on the concentration of PIP(2) used to form the specific complexes. As the concentration of PIP(2) was increased to 7.5 and 35 µM, the major protein species surviving trypsin cleavage was a 67-kDa fragment. As the PIP(2) concentration increased to 200 µM, the size of protein surviving trypsin cleavage was similar to that of the intact enzyme. The proportion of the 67-kDa fragment and intact protein surviving the action of trypsin was dependent on the concentration of PIP(2). Trypsin digestion of PLC1 preincubated with low concentrations of PIP(2) yielded a major 67-kDa proteolytic fragment. As the concentration of PIP(2) increased, the size of the major protein species surviving the trypsin digestion was 89 kDa. We were not able to detect the 80-kDa fragment, which was the major proteolytic product when free PLC1 was digested with the reduced level of trypsin, suggesting that both the sensitivity and specificity of the enzyme to trypsin cleavage were changed as a result of specific binding of PIP(2). This observation shows that the site of enzyme-PIP(2) complex and its sensitivity to trypsin cleavage were highly dependent on the concentration of PIP(2) used to form the specific enzyme-PIP(2) complex.


Figure 9: Western blotting analysis of trypsin cleavage of PLC1. Panel A, trypsin (0.1 µg) cleavage of PLC1 for 0, 5, and 15 min in the presence of 200 µM or absence of PIP(2) (control). Panel B, trypsin (0.1 µg) cleavage of PLC1 (0.2 µg) for 5 min in the presence of PE/PIP(2) micelles containing the indicated concentration of PIP(2). All of the trypsin cleavage reactions were carried out at 37 °C in 40 µl of 12.5 mM sodium phosphate, 37.5 mM HEPES, pH 7.0, 5 mM EGTA, 3.75 mM EDTA, 75 mM NaCl, 25 mM KCl, 0.025% Tween 20, 0.075% sodium cholate, 375 µg/ml BSA containing 400 µM PE and the indicated concentration of PIP(2).



Interaction of E341G, H356L, and R338L with PIP(2)

To determine whether these three cleavage-defective mutant PLC1s could interact with PIP(2) like the wild type enzyme does, we compared the sensitivity and specificity of trypsin cleavage of these mutants preincubated with PIP(2). The rates of degradation of H356L and E341G mutants by trypsin were significantly reduced if the mutant enzymes were allowed to preform enzyme-substrate complexes by preincubating them with 200 µM PIP(2) (Fig. 10A). In contrast, PIP(2) did not affect the rate of trypsin cleavage of mutant PLC1 which has Arg replaced by Leu; 90% of this mutant enzyme was degraded by trypsin within 5 min whether or not it was preincubated with 200 µM PIP(2).


Figure 10: Analysis of the trypsin sensitivity of mutant forms of PLC1 in the presence or absence of PIP(2). Panel A, trypsin (0.1 µg) cleavage of 0.2 µg of mutant PLC1 E341G, H356L, and R338L proteins in the presence of 200 µM PIP(2) or in the absence of PIP(2) (control) for 0, 5, 10, and 15 min. Panel B, trypsin (0.1 µg) cleavage of 0.2 µg of mutant PLC1 E341G, H356L, and R338L proteins for 5 min in the presence of the indicated concentration of PIP(2). All of the trypsin cleavage reactions were carried out in a buffer (40 µl) containing 12.5 mM sodium phosphate, 37.5 mM HEPES, pH 7.0, 5 mM EGTA, 3.75 mM EDTA, 75 mM NaCl, 25 mM KCl, 0.025% Tween 20, 0.075% sodium cholate, 375 µg/ml BSA containing 400 µM PE and the indicated concentrations of PIP(2). At the indicated time point, the reactions were stopped and analyzed by Western blotting as described under ``Experimental Procedures.''



The cleavage site(s) of preformed substrate-enzyme complexes by trypsin was also highly dependent on the PIP(2) concentration. For mutants H356L and E341G, enzyme-substrate complexes formed at a low concentration of PIP(2) (7.5 µM) were easily cleaved to a 67-kDa fragment by trypsin, whereas enzyme-substrate complexes formed at a saturating concentration of PIP(2) (200 µM) were relatively resistant to trypsin cleavage (Fig. 10B). Preincubating mutant R338L with increasing concentrations of PIP(2) from 7.5 to 200 µM did not protect it from trypsin cleavage (Fig. 10B). The saturation concentration of PIP(2) required to protect H356L and E341G from trypsin digestion was estimated to be 100 µM, and the half-maximal protection concentration of PIP(2) was estimated to be 35 µM. This result demonstrated that according to their sensitivity to trypsin cleavage, mutant E341G and H356L are able to form at least two types of complex with PIP(2) in a way similar to that of the wild type enzyme. This indicates that Glu and His may play a role in the step of cleavage rather than in the steps of substrate binding.


DISCUSSION

At least four isoforms of PLC have been identified and cloned: 1, 2, 3(23, 24) , and 4(25) . Judged by the slight deviation of its amino acid sequence from those of rat and bovine isoforms, the present PLC isoform we isolated from a human aorta library is classified as 1 type. Patterns of expression of PLC1 from various tissues of rats and bovines examined by immunoanalysis (26) and Northern analysis (27) reveal that although the level of expression is relatively lower than for other isoforms of PLC, PLC1 is widespread among most tissues. We found that transcripts of PLC1 are also present in almost all human tissues, implying that the enzyme may play a role in some fundamental cellular process. Several lines of evidence agree with this implication. First, in an attempt to select mutants from mutagenized Chinese hamster lung fibroblasts defective in thrombin-induced mitogenesis, several of these mutants were later shown to lack PLC1 protein(28) . Second, disruption of a PLC-like (PLC1) gene in yeast resulted in slow growth or death of the cells, which could be rescued by exogenous expression from a cloned rat PLC1 cDNA(29) . Third, a mutation in PLC1 leading to an 8-fold increase in specific activity has been identified in the spontaneous hypertensive rat(30, 31) . Finally, our laboratory has recently found that suppression of PLC1 in a rat cell line arrests cell growth and diminishes mitogen-induced intracellular calcium mobilization. (^2)

In at least two structural aspects, the present recombinant version of PLC1 differs from purified forms. First, a 34-amino acid region including 6 consecutive histidine residues was fused to the NH(2) terminus of the recombinant enzyme. Second, post-translational modification of PLC1 expressed in E. coli may be quite different from that of PLC1 expressed in mammalian cells. Since we have demonstrated here that recombinant human PLC1 expressed from E. coli displays PI and PIP(2) hydrolyzing activity similar to that of the purified forms(7, 32) , this version of recombinant human PLC1 should be suitable for in vitro structure-function analyses.

Centrifugation binding assays show that recombinant human PLC1 expressed from E. coli can form specific complexes with PIP(2) vesicles, with an affinity comparable to that of the purified form(22, 33) . The structure of the enzyme-substrate complex differs from that of the free enzyme, as the enzyme-PIP(2) complex is much more resistant to trypsin cleavage than its free form. Moreover, the site and sensitivity of the enzyme-PIP(2) complex to trypsin cleavage are highly dependent on the concentrations of PIP(2) used to bind PLC1. One of the cleavage-defective mutants PLC1 (R338L) bound PIP(2) vesicles as tightly as the wild type enzyme did, but its complex was cleaved by trypsin as readily as the free protein. Therefore, these changes in the sites and sensitivities to trypsin cleavage are not caused by hindrance of the phospholipid moiety but rather reflect a temporal rearrangement of potential trypsin-sensitive sites during binding.

At least two types of specific PLC1bulletPIP(2) complexes were demonstrated in these experiments; complexes formed at a low concentration of PIP(2) were readily degraded to a 67-kDa fragment, whereas specific complexes formed at a higher concentration of PIP(2) micelles were relatively resistant to trypsin cleavage. The simplest interpretation is that there are at least two types of PIP(2) binding sites in PLC1: a high affinity site and a low affinity site. When the PIP(2) substrate concentration is low, PLC1 will bind to PIP(2) through the high affinity site to form a complex that is cleaved readily by trypsin to give a 67-kDa fragment. As the concentration of PIP(2) increases, another binding site with lower affinity will be saturated and cause formation of a complex much more resistant to trypsin digestion. Because the 67-kDa fragment was retained in a Ni-NAT gel (data not shown), the present results also suggest that the COOH-terminal sequences of PLC1, although required for catalysis(11, 12) , are not involved in the high affinity binding of PIP(2). Two lines of evidence are consistent with this interpretation that at least two PIP(2) binding sites exist in PLC1. Deletion of the first 60 amino acids of bovine PLC1 does not eliminate its PIP(2) hydrolysis activity but dramatically reduces the binding of the truncated enzyme to PIP(2) lipid vesicles(15) . Furthermore, a peptide corresponding to residues 30-43 of PLC1 binds to IP(3)(12) . These observations indicate that in addition to the PIP(2) cleavage site, PLC1 can bind to PIP(2) via a noncatalytic PIP(2) binding site in the NH(2) terminus of the protein. Indeed, residues 16-134 in the NH(2) terminus of PLC1 have been found to share homologous sequences with pleckstrin (PH domain)(34, 35, 36) , which can specifically bind to PIP(2)(37) . The binding of PIP(2) to the noncleavage site of PLC1 can either allow the enzyme to hydrolyze the phospholipid in a processive manner (15) or may be required for other regulatory process(38, 39) .

All the eukaryotic PI-PLC identified so far can be structurally divided into conserved X and Y regions(2, 17, 40) . Deletion mutagenesis studies have shown that both X and Y region sequences are essential for the catalytic activity(11, 12, 13, 14) . Consistent with this observation, conversion of several highly conserved amino acid residues in the Y region causes the enzyme to lose its catalytic functions (data not shown). In particular, conversion of Arg to Gly (R549G) makes the enzyme selectively defective in the cleavage of PIP(2), whereas this mutant enzyme can retain 20% of the ability to hydrolyze PI. We are investigating further the contribution of these conserved residues to the catalytic function of PLC1.^2

Prokaryotic PI-PLC does not contain a Y conserved region(41) . Its catalytic activity is independent of Ca and does not recognize PIP or PIP(2). However, structural comparison of the X region sequences between eukaryotic and prokaryotic PI-PLC reveals a high degree of similarity. This supports the hypothesis that catalytic sites of PLCs might reside in the X region. Within this region, 9 amino acid residues are invariant from prokaryotic to human PLCs. Our preliminary in vitro mutagenesis and enzymatic characterization of the mutant human PLC1 show that side chain switching of most of these residues does not cause significant loss of catalytic activity of the mutant enzyme. It is unlikely that switching their functional side chains does not alter catalytic function of PLC1. We would rather believe that these mutant enzymes might be defective in other functions not detected in the present in vitro enzymatic assay.

However, in agreement with the hypothesis that catalytic residues could be localized in the X region, we found that conversion of Glu to Gly, His to Leu, or Arg to Leu totally eliminated the catalytic activity of PLC1. Although these three cleavage-defective mutant enzymes can bind to PIP(2) vesicles as tightly as the wild type enzyme does, our substrate protection experiments demonstrated that the structures of specific PIP(2)-protein complexes of these three mutants are not quite the same. Both the sites of and sensitivity to trypsin digestion of the specific PIP(2)-protein complexes of H356L and E341G are similar to those of the specific complexes of the wild type enzyme, whereas binding of R338L to PIP(2) does not affect its site or sensitivity to trypsin digestion. The simplest interpretation is that upon sequential binding of PIP(2), both mutant PLC1 E341G and H356L can adopt structural adjustments similar to those of the wild type enzyme, thus Glu and His are not involved in either high affinity or low affinity interactions with PIP(2). Although R338L is still able to bind tightly to PIP(2) vesicles, its structural adjustment upon binding to PIP(2) is distinct from that of the wild type enzyme.

In conclusion, this study shows that Glu and His are not involved in specific interaction with PIP(2). Since Ca is not essential for the PIP(2) binding activity of PLC but is required for cleavage, this implies that Glu and His may be involved either in the binding of Ca or in the cleavage step. However, His and Glu are also invariant in prokaryotic PI-PLC, whose activity is independent of Ca. Therefore, these two residues are most likely involved in the hydrolysis of PIP(2) once the specific substrate-enzyme complex has been formed.


FOOTNOTES

*
This work was supported by the National Sciences Council of the Republic of China Grants NSC83-0203-B-001-006. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U09117[GenBank].

§
To whom correspondence should be addressed. Fax: 886-2-785-3569.

(^1)
The abbreviations used are: IP(3), inositol 1,4,5-trisphosphate; PLC, phospholipase C; PI-PLC, phosphoinositide-specific phospholipase C; PIP(2), phosphatidylinositol 4,5-bisphosphate; PIP, phosphatidylinositol 4-phosphate; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; BSA, bovine serum albumin; PCR, polymerase chain reaction; kb, kilobase(s).

(^2)
H.-F. Cheng, M.-J. Jiang, C.-L. Chen, S.-M. Liu, L.-P. Wong, J. W. Lomasney, and K. King, manuscript in preparation.


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