- Email: [email protected]
Loss of Activity of Plasma Platelet-Activating Factor Acetylhydrolase Due to a Novel Gln281→Arg Mutation
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
236, 772–775 (1997)
RC977047
Loss of Activity of Plasma Platelet-Activating Factor Acetylhydrolase Due to a Novel Gln281rArg Mutation Yoshiji Yamada1 and Mitsuhiro Yokota* Department of Geriatric Research, National Institute for Longevity Sciences, Obu, Aichi, Japan; and *Department of Clinical Laboratory Medicine, Nagoya University School of Medicine, Nagoya, Japan
Received June 23, 1997
The prevalence of plasma platelet-activating factor (PAF) acetylhydrolase deficiency was investigated in 477 healthy Japanese individuals and 985 patients with various cardiovascular diseases. The genotype for this enzyme with regard to a G994rT mutation (MM, normal; Mm, heterozygote; mm, mutant homozygote) was determined by an allele-specific polymerase chain reaction in 80 subjects shown to have no or low plasma activity (õ10 nmol/min/ml). In 72 subjects, the genotype was consistent with plasma enzyme activity; 44 individuals with no activity were mm, and 28 with low activity were Mm. However, eight subjects with the MM genotype showed plasma enzyme activities of õ10 nmol/min/ml. Determination of the DNA sequence of exon 9 of the plasma PAF acetylhydrolase gene in these eight subjects revealed a previously unidentified A1001rG missense mutation, resulting in a Gln281rArg substitution, in a 72-year-old woman with coronary artery disease, essential hypertension, and no plasma enzyme activity. Site-directed mutagenesis in vitro showed that the corresponding recombinant mutant protein lacked PAF acetylhydrolase activity. Thus, the Gln281rArg substitution appears responsible for the loss of plasma PAF acetylhydrolase activity. q 1997 Academic Press
Platelet-activating factor (PAF) is a phospholipid with pro-inflammatory effects that result from its binding to a receptor (1) on target cells such as platelets, polymorphonuclear leukocytes, monocytes, macrophages, and smooth muscle cells (2). Activation of these cells by PAF can result in allergic, inflammatory, atherosclerotic, and thrombotic responses (2). A strongly oxidizing environment induces the fragmentation of polyunsaturated fatty acids of membrane phospholipids (3), and the resulting modified phospholipids are 1 To whom correspondence should be addressed. Fax: 81 (Japan)0562-44-6595.
0006-291X/97 $25.00
structurally similar to PAF and mimic its biological actions (4). The biological actions of PAF and of oxidatively fragmented phospholipids are abolished by hydrolysis of the sn-2 residue, a reaction that is catalyzed by PAF acetylhydrolase (5-8). PAF acetylhydrolase in plasma is tightly associated with low density lipoprotein (LDL) and high density lipoprotein; it may serve to protect LDL against oxidative modification (7-9), which is thought to be important in atherogenesis (10). The possibility that a reduced ability to degrade PAF or oxidatively fragmented phospholipids may result in pathological responses has been supported by the observation that the activity of PAF acetylhydrolase in plasma is decreased in individuals with such diseases as necrotizing enterocolitis (11), septic shock (12), and severe bronchial asthma in children (13). Stafforini et al. (14) showed that the gene for human plasma PAF acetylhydrolase is located at chromosome 6p12-21.1 and comprises 12 exons, spanning at least 45 kb of DNA. These researchers also detected a single point mutation (a G994rT transversion) in exon 9 in 14 Japanese families with a deficiency of plasma PAF acetylhydrolase activity. This nucleotide change results in a Val279rPhe substitution of the mature protein and is responsible for the loss of catalytic activity. In the present study, we investigated the prevalence and cause of plasma PAF acetylhydrolase deficiency in Japanese subjects with various cardiovascular diseases and in healthy individuals. METHODS Subjects. The study population consisted of 1462 Japanese subjects (837 men and 625 women) who had visited 14 participating hospitals between July 1994 and June 1995 either because they were experiencing various symptoms or for a medical checkup. A total of 985 subjects was diagnosed with various cardiovascular diseases, including essential hypertension, coronary artery disease, dilated cardiomyopathy, and hypertrophic cardiomyopathy. The population included 477 apparently healthy individuals (normal control group) who attended the medical centers for an annual checkup and did not
772
Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 7047
/
6933$$$941
07-10-97 13:22:22
bbrcg
AP: BBRC
Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
have any serious disorders. Informed consent for participation was obtained from each subject. Assay of plasma PAF acetylhydrolase activity. Venous blood was collected into tubes containing EDTA (disodium salt; final concentration, 50 mM) and was centrifuged at 1600 1 g for 15 min at 47C. Plasma samples were stored at 0307C until assayed. The activity of PAF acetylhydrolase was measured as previously described (5,8). Genotyping of G994rT mutation of plasma PAF acetylhydrolase gene. Venous blood (7 ml) was collected into tubes containing EDTA, and genomic DNA was prepared from isolated leukocytes with a DNA extraction kit (Biologica, Nagoya, Japan). The genotype of plasma PAF acetylhydrolase with regard to the G994rT mutation was determined by an allele-specific polymerase chain reaction (PCR) as previously described (14). Because the G994rT transversion produces a new restriction site for Mae II, genotypes were designated MM (normal), Mm (heterozygous), and mm (homozygous deficient). Identification of new DNA mutation. To identify the new DNA mutation, we amplified exon 9 of the plasma PAF acetylhydrolase gene by PCR. The reaction mixture (100 ml) contained 1 mg of genomic DNA, 100 pmol of each oligonucleotide primer, 0.2 mM each of dCTP, dTTP, dGTP, and dATP, 2 mM MgSO4 , 10 mM KCl, 10 mM (NH4)2SO4 , 0.1% Triton X-100, bovine serum albumin (0.1 mg/ml), 20 mM Tris-HCl (pH 8.8), and 5 U of Pfu DNA polymerase (Stratagene, La Jolla, CA). The thermocycling procedure was identical to that previously described (14). The PCR products were purified with Geneclean II (Bio101, La Jolla, CA) and cloned with pCR-Script Amp SK(/) (Stratagene) by blunt-end ligation. After transformation of competent Escherichia coli strain XL1-Blue MRF*, the plasmid DNA was purified with an automated isolation system (Kurabo, Osaka, Japan). The insert DNA was sequenced on both strands with a fluorescence-based automated DNA sequencer (Applied Biosystems, Foster City, CA). The amino acid-coding regions of other exons (exons 2 to 8 and 10 to 12) were also amplified by PCR with Pfu DNA polymerase; the primers and amplification cycles were as previously described (14). The PCR products were purified, cloned with pCRScript Amp SK(/), and sequenced on both strands. Site-directed mutagenesis. Total RNA was purified from U937 cells stimulated with phorbol 12-myristate 13-acetate (0.2 mM) for 3 days, at which time these cells had differentiated into macrophagelike cells and expressed plasma PAF acetylhydrolase activity. Firststrand cDNA was synthesized with Moloney murine leukemia virus reverse transcriptase and oligo(dT) primers (Stratagene), after which a 5-ml portion of the reaction mixture was subjected to PCR with Pfu DNA polymerase and sense I (5*-acgtgcggccgcatATACAAGTACTGATGGC-3*) and antisense II (5*-acgtgcggccgcctaATTGTATTTCTCTAT-3*) primers to ampify the plasma PAF acetylhydrolase gene (lowercase letters indicate additional primer sequence to generate Not I site for cloning of PCR products). The amplification protocol comprised initial denaturation at 947C for 5 min; 35 cycles of denaturation at 947C for 1 min, annealing at 567C for 1 min, and extension at 727C for 2 min; and a final extension at 727C for 5 min. PCR products were purified and cloned with both pCR-Script Amp SK(/) and the prokaryotic glutathione S-transferase (GST) gene fusion vector pGEX-5X-1 (Pharmacia, Uppsala, Sweden). The construct harboring the A1001rG mutation was prepared by sequential PCR as previously described (14), with minor modifications. Two sets of primers were used: sense I and antisense I (5*TAAGAGTCCGAATAACCGTTG-3*), and sense II (5*-ACGGTTATTCGGACTCTTAG-3*) and antisense II (underlines indicate DNA mutation). The initial PCR reactions were performed as described above. The predicted sizes of the PCR products were 740 and 506 bp for the first and second sets of primers, respectively. The products were purified with Geneclean II and used as the template (100 ng each) for PCR with sense I and antisense II primers as described above. The 1228-bp product was purified with Geneclean II, digested with Not I, and inserted into pGEX-5X-1. Sequence analysis revealed that the mutant contained the desired ArG substitution at position 1001.
The normal and mutant plasma PAF acetylhydrolase proteins were expressed in E. coli strain SF-8 by induction with isopropyl-b-Dthiogalactopyranoside. The GST fusion proteins were purified by affinity chromatography with glutathione sepharose 4B (Pharmacia). Both recombinant proteins were then assayed for PAF acetylhydrolase activity.
RESULTS The plasma activity of PAF acetylhydrolase was measured in 477 healthy subjects and 985 individuals with various cardiovascular diseases. The plasma activity in the total population was 27.36 { 11.28 nmol/ min/ml (mean { SD). We determined genotypes for the G994rT variant in exon 9 of the plasma PAF acetylhydrolase gene by allele-specific PCR in 80 subjects with a plasma activity of õ10 nmol/min/ml (mean 0 1.5SD). For 72 subjects, the genotype (MM, Mm, or mm) was consistent with the plasma enzyme activity; 44 individuals with no activity were mm and 28 with low activity (1 to 10 nmol/min/ml) were Mm. However, the genotypes (MM) in eight subjects were not consistent with their plasma activities (õ10 nmol/min/ml). We then determined the sequence of exon 9 of the plasma PAF acetylhydrolase gene in these eight subjects. A previously unidentified ArG transition at nucleotide position 1001, which results in a GlnrArg substitution at amino acid residue 281, was detected in a 72-year-old woman (Fig. 1, A and B). She was homozygous for this mutation and lacked plasma PAF acetylhydrolase activity. She had visited the hospital for chest discomfort on exertion and was diagnosed with coronary artery disease and essential hypertension. This mutation was not detected in the other seven subjects tested. We next determined the DNA sequences of the amino acid-coding regions of other exons (exons 2 to 8 and 10 to 12) of the plasma PAF acetylhydrolase gene in the subject but did not detect any additional missense mutation (data not shown). To confirm that the A1001rG mutation was responsible for the loss of plasma PAF acetylhydrolase activity in the affected subject, we prepared a mutant recombinant protein by in vitro site-directed mutagenesis. Whereas the recombinant wild-type proten exhibited marked PAF acetylhydrolase activity, the recombinant mutant protein lacked such activity (Fig. 2). DISCUSSION Tjoelker et al (15). isolated a cDNA encoding plasma PAF acetylhydrolase from human macrophages. The cDNA encodes a 441-amino acid protein that is cleaved between Lys41 and Ile42 to generate a mature enzyme with a calculated molecular mass of 45,388 daltons. The predicted protein contains the Gly-X-Ser-X-Gly consensus sequence for the catalytic site of lipases and esterases. The G994rT mutation in the ninth exon, which encodes the active site, detected by Stafforini et
773
AID
BBRC 7047
/
6933$$$941
07-10-97 13:22:22
bbrcg
AP: BBRC
Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
al. (14) and the associated Val279rPhe substitution of mature PAF acetylhydrolase result in a loss of catalytic activity. We measured the plasma PAF acetylhydrolase activity in 985 subjects with various cardiovascular diseases and 477 healthy individuals, and detected 46 sporadic cases of a deficiency of this enzyme. One of the eight subjects whose enzyme deficiency was not accounted for by homozygosity for the G994rT mutation was shown to be homozygous for a previous unidentified A1001rG transition, which results in a Gln281rArg substitution. With the use of site-directed mutagenesis, we confirmed that this mutation is responsible for the loss of plasma PAF acetylhydrolase activity in this subject.
FIG. 2. PAF acetylhydrolase activity of recombinant normal and mutant plasma PAF acetylhydrolase. Data are means of three experiments.
PAF acetylhydrolase catalyzes the degradation of PAF to biologically inactive lyso-PAF, which suggests that this enzyme protects against the pathological events mediated by PAF. This idea is supported by the observation that recombinant human PAF acetylhydrolase markedly inhibits the inflammation induced by PAF (15). This recombinant protein inhibited the activation of leukocytes induced by PAF in vitro and, in rats, reduced the PAF-induced vascular leakage in pleurisy and paw edema (15). The possibility that a reduction in PAF acetylhydrolase activity may result in pathological responses is also supported by clinical observations (11-13). In addition, PAF acetylhydrolase protects LDL against oxidation by hydrolyzing the oxidatively fragmented fatty acyl residues from the sn-2 position of phospholipids (6,9). This action may help prevent the recognition of LDL by the scavenger receptor of macrophages. Thus, PAF acetylhydrolase may be important in the defense against atherosclerosis. These observations suggest that individuals with a plasma PAF acetylhydrolase deficiency may be predisposed to develop inflammatory or atherosclerotic diseases. In conclusion, we detected an A1001rG missense mutation in exon 9 of the plasma PAF acetylhydrolase gene in a Japanese woman with coronary artery disease and essential hypertension. This mutation resulted in a Gln281rArg substitution and loss of catalytic activity. Determination of the plasma PAF acetylhydrolase genotype or enzyme activity in plasma may contribute to the prevention and management of inflammatory and atherosclerotic diseases. FIG. 1. Detection of a new missense mutation in exon 9 of the plasma PAF acetylhydrolase gene. DNA sequences of the sense (A) and antisense (B) strands reveal an ArG transition at nucleotide position 1001, resulting in a GlnrArg substitution at amino acid 281.
ACKNOWLEDGMENTS This work was supported in part by a grant from Kowa Life Science Funds. We thank Drs. S. Sato (Fujisawa Pharmaceutical Co., Osaka, Japan) and Y. Kaneko for technical assistance.
774
AID
BBRC 7047
/
6933$$$941
07-10-97 13:22:22
bbrcg
AP: BBRC
Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. Honda, Z., Nakamura, M., Miki, I., Minami, M., Watanabe, T., Seyama, Y., Okado, H., Toh, H., Ito, K., Miyamoto, T., and Shimizu, T. (1991) Nature 349, 342–346. 2. Zimmerman, G. A., Prescott, S. M., and McIntyre, T. M. (1992) in Inflammation: Basic Principles and Clinical Correlates (J. I. Gallin, I. M. Goldstein, and R. Snyderman, Eds.), pp. 149–176, Raven Press, New York. 3. Patel, K. D., Zimmerman, G. A., Prescott, S. M., and McIntyre, T. M. (1992) J. Biol. Chem. 267, 15168–15175. 4. Smiley, P. L., Stremler, K. E., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11104–11110. 5. Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1987) J. Biol. Chem. 262, 4223–4230. 6. Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095–11103. 7. Yamada, Y., Stafforini, D. M., Imaizumi, T., Zimmerman, G. A., McIntyre, T. M., and Prescott, S. M. (1994) Proc. Natl. Acad. Sci. USA 91, 10320–10324.
8. Stafforini, D. M., McIntyre, T. M., Carter, M. E., and Prescott, S. M. (1987) J. Biol. Chem. 262, 4215–4222. 9. Stafforini, D. M., Zimmerman, G. A., McIntyre, T. M., and Prescott, S. M. (1992) Trans. Assoc. Am. Physicians 105, 44–63. 10. Steinberg, D., Parthasarathy, S., Carew, T. E., Khoo, J. C., and Witztum, J. L. (1989) N. Engl. J. Med. 320, 915–923. 11. Caplan, M. S., Sun, X.-M., Hsueh, W., and Hageman, J. R. (1990) J. Pediatr. 116, 960–964. 12. Graham, R. M., Stephens, C. J., Silvester, W., Leong, L. L. L., Sturn, M. J., and Taylor, R. T. (1994) Crit. Care Med. 22, 204– 212. 13. Miwa, M., Miyake, T., Yamanaka, T., Sugatani, J., Suzuki, Y., Sakata, S., Araki, Y., and Matsumoto, M. (1988) J. Clin. Invest. 82, 1983–1991. 14. Stafforini, D. M., Satoh, K., Atkinson, D. L., Tjoelker, L. W., Eberhardt, C., Yoshida, H., Imaizumi, T., Takamatsu, S., Zimmerman, G. A., McIntyre, T. M., Gray, P. W., and Prescott, S. M. (1996) J. Clin. Invest. 97, 2784–2791. 15. Tjoelker, L. W., Wilder, C., Eberhardt, C., Stafforini, D. M., Dietsch, G., Schimpf, B., Hooper, S., Trong, H. L., Cousens, L. S., Zimmerman, G. A., Yamada, Y., McIntyre, T. M., Prescott, S. M., and Gray, P. W. (1995) Nature 374, 549–553.
775
AID
BBRC 7047
/
6933$$$941
07-10-97 13:22:22
bbrcg
AP: BBRC
236, 772–775 (1997)
RC977047
Loss of Activity of Plasma Platelet-Activating Factor Acetylhydrolase Due to a Novel Gln281rArg Mutation Yoshiji Yamada1 and Mitsuhiro Yokota* Department of Geriatric Research, National Institute for Longevity Sciences, Obu, Aichi, Japan; and *Department of Clinical Laboratory Medicine, Nagoya University School of Medicine, Nagoya, Japan
Received June 23, 1997
The prevalence of plasma platelet-activating factor (PAF) acetylhydrolase deficiency was investigated in 477 healthy Japanese individuals and 985 patients with various cardiovascular diseases. The genotype for this enzyme with regard to a G994rT mutation (MM, normal; Mm, heterozygote; mm, mutant homozygote) was determined by an allele-specific polymerase chain reaction in 80 subjects shown to have no or low plasma activity (õ10 nmol/min/ml). In 72 subjects, the genotype was consistent with plasma enzyme activity; 44 individuals with no activity were mm, and 28 with low activity were Mm. However, eight subjects with the MM genotype showed plasma enzyme activities of õ10 nmol/min/ml. Determination of the DNA sequence of exon 9 of the plasma PAF acetylhydrolase gene in these eight subjects revealed a previously unidentified A1001rG missense mutation, resulting in a Gln281rArg substitution, in a 72-year-old woman with coronary artery disease, essential hypertension, and no plasma enzyme activity. Site-directed mutagenesis in vitro showed that the corresponding recombinant mutant protein lacked PAF acetylhydrolase activity. Thus, the Gln281rArg substitution appears responsible for the loss of plasma PAF acetylhydrolase activity. q 1997 Academic Press
Platelet-activating factor (PAF) is a phospholipid with pro-inflammatory effects that result from its binding to a receptor (1) on target cells such as platelets, polymorphonuclear leukocytes, monocytes, macrophages, and smooth muscle cells (2). Activation of these cells by PAF can result in allergic, inflammatory, atherosclerotic, and thrombotic responses (2). A strongly oxidizing environment induces the fragmentation of polyunsaturated fatty acids of membrane phospholipids (3), and the resulting modified phospholipids are 1 To whom correspondence should be addressed. Fax: 81 (Japan)0562-44-6595.
0006-291X/97 $25.00
structurally similar to PAF and mimic its biological actions (4). The biological actions of PAF and of oxidatively fragmented phospholipids are abolished by hydrolysis of the sn-2 residue, a reaction that is catalyzed by PAF acetylhydrolase (5-8). PAF acetylhydrolase in plasma is tightly associated with low density lipoprotein (LDL) and high density lipoprotein; it may serve to protect LDL against oxidative modification (7-9), which is thought to be important in atherogenesis (10). The possibility that a reduced ability to degrade PAF or oxidatively fragmented phospholipids may result in pathological responses has been supported by the observation that the activity of PAF acetylhydrolase in plasma is decreased in individuals with such diseases as necrotizing enterocolitis (11), septic shock (12), and severe bronchial asthma in children (13). Stafforini et al. (14) showed that the gene for human plasma PAF acetylhydrolase is located at chromosome 6p12-21.1 and comprises 12 exons, spanning at least 45 kb of DNA. These researchers also detected a single point mutation (a G994rT transversion) in exon 9 in 14 Japanese families with a deficiency of plasma PAF acetylhydrolase activity. This nucleotide change results in a Val279rPhe substitution of the mature protein and is responsible for the loss of catalytic activity. In the present study, we investigated the prevalence and cause of plasma PAF acetylhydrolase deficiency in Japanese subjects with various cardiovascular diseases and in healthy individuals. METHODS Subjects. The study population consisted of 1462 Japanese subjects (837 men and 625 women) who had visited 14 participating hospitals between July 1994 and June 1995 either because they were experiencing various symptoms or for a medical checkup. A total of 985 subjects was diagnosed with various cardiovascular diseases, including essential hypertension, coronary artery disease, dilated cardiomyopathy, and hypertrophic cardiomyopathy. The population included 477 apparently healthy individuals (normal control group) who attended the medical centers for an annual checkup and did not
772
Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 7047
/
6933$$$941
07-10-97 13:22:22
bbrcg
AP: BBRC
Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
have any serious disorders. Informed consent for participation was obtained from each subject. Assay of plasma PAF acetylhydrolase activity. Venous blood was collected into tubes containing EDTA (disodium salt; final concentration, 50 mM) and was centrifuged at 1600 1 g for 15 min at 47C. Plasma samples were stored at 0307C until assayed. The activity of PAF acetylhydrolase was measured as previously described (5,8). Genotyping of G994rT mutation of plasma PAF acetylhydrolase gene. Venous blood (7 ml) was collected into tubes containing EDTA, and genomic DNA was prepared from isolated leukocytes with a DNA extraction kit (Biologica, Nagoya, Japan). The genotype of plasma PAF acetylhydrolase with regard to the G994rT mutation was determined by an allele-specific polymerase chain reaction (PCR) as previously described (14). Because the G994rT transversion produces a new restriction site for Mae II, genotypes were designated MM (normal), Mm (heterozygous), and mm (homozygous deficient). Identification of new DNA mutation. To identify the new DNA mutation, we amplified exon 9 of the plasma PAF acetylhydrolase gene by PCR. The reaction mixture (100 ml) contained 1 mg of genomic DNA, 100 pmol of each oligonucleotide primer, 0.2 mM each of dCTP, dTTP, dGTP, and dATP, 2 mM MgSO4 , 10 mM KCl, 10 mM (NH4)2SO4 , 0.1% Triton X-100, bovine serum albumin (0.1 mg/ml), 20 mM Tris-HCl (pH 8.8), and 5 U of Pfu DNA polymerase (Stratagene, La Jolla, CA). The thermocycling procedure was identical to that previously described (14). The PCR products were purified with Geneclean II (Bio101, La Jolla, CA) and cloned with pCR-Script Amp SK(/) (Stratagene) by blunt-end ligation. After transformation of competent Escherichia coli strain XL1-Blue MRF*, the plasmid DNA was purified with an automated isolation system (Kurabo, Osaka, Japan). The insert DNA was sequenced on both strands with a fluorescence-based automated DNA sequencer (Applied Biosystems, Foster City, CA). The amino acid-coding regions of other exons (exons 2 to 8 and 10 to 12) were also amplified by PCR with Pfu DNA polymerase; the primers and amplification cycles were as previously described (14). The PCR products were purified, cloned with pCRScript Amp SK(/), and sequenced on both strands. Site-directed mutagenesis. Total RNA was purified from U937 cells stimulated with phorbol 12-myristate 13-acetate (0.2 mM) for 3 days, at which time these cells had differentiated into macrophagelike cells and expressed plasma PAF acetylhydrolase activity. Firststrand cDNA was synthesized with Moloney murine leukemia virus reverse transcriptase and oligo(dT) primers (Stratagene), after which a 5-ml portion of the reaction mixture was subjected to PCR with Pfu DNA polymerase and sense I (5*-acgtgcggccgcatATACAAGTACTGATGGC-3*) and antisense II (5*-acgtgcggccgcctaATTGTATTTCTCTAT-3*) primers to ampify the plasma PAF acetylhydrolase gene (lowercase letters indicate additional primer sequence to generate Not I site for cloning of PCR products). The amplification protocol comprised initial denaturation at 947C for 5 min; 35 cycles of denaturation at 947C for 1 min, annealing at 567C for 1 min, and extension at 727C for 2 min; and a final extension at 727C for 5 min. PCR products were purified and cloned with both pCR-Script Amp SK(/) and the prokaryotic glutathione S-transferase (GST) gene fusion vector pGEX-5X-1 (Pharmacia, Uppsala, Sweden). The construct harboring the A1001rG mutation was prepared by sequential PCR as previously described (14), with minor modifications. Two sets of primers were used: sense I and antisense I (5*TAAGAGTCCGAATAACCGTTG-3*), and sense II (5*-ACGGTTATTCGGACTCTTAG-3*) and antisense II (underlines indicate DNA mutation). The initial PCR reactions were performed as described above. The predicted sizes of the PCR products were 740 and 506 bp for the first and second sets of primers, respectively. The products were purified with Geneclean II and used as the template (100 ng each) for PCR with sense I and antisense II primers as described above. The 1228-bp product was purified with Geneclean II, digested with Not I, and inserted into pGEX-5X-1. Sequence analysis revealed that the mutant contained the desired ArG substitution at position 1001.
The normal and mutant plasma PAF acetylhydrolase proteins were expressed in E. coli strain SF-8 by induction with isopropyl-b-Dthiogalactopyranoside. The GST fusion proteins were purified by affinity chromatography with glutathione sepharose 4B (Pharmacia). Both recombinant proteins were then assayed for PAF acetylhydrolase activity.
RESULTS The plasma activity of PAF acetylhydrolase was measured in 477 healthy subjects and 985 individuals with various cardiovascular diseases. The plasma activity in the total population was 27.36 { 11.28 nmol/ min/ml (mean { SD). We determined genotypes for the G994rT variant in exon 9 of the plasma PAF acetylhydrolase gene by allele-specific PCR in 80 subjects with a plasma activity of õ10 nmol/min/ml (mean 0 1.5SD). For 72 subjects, the genotype (MM, Mm, or mm) was consistent with the plasma enzyme activity; 44 individuals with no activity were mm and 28 with low activity (1 to 10 nmol/min/ml) were Mm. However, the genotypes (MM) in eight subjects were not consistent with their plasma activities (õ10 nmol/min/ml). We then determined the sequence of exon 9 of the plasma PAF acetylhydrolase gene in these eight subjects. A previously unidentified ArG transition at nucleotide position 1001, which results in a GlnrArg substitution at amino acid residue 281, was detected in a 72-year-old woman (Fig. 1, A and B). She was homozygous for this mutation and lacked plasma PAF acetylhydrolase activity. She had visited the hospital for chest discomfort on exertion and was diagnosed with coronary artery disease and essential hypertension. This mutation was not detected in the other seven subjects tested. We next determined the DNA sequences of the amino acid-coding regions of other exons (exons 2 to 8 and 10 to 12) of the plasma PAF acetylhydrolase gene in the subject but did not detect any additional missense mutation (data not shown). To confirm that the A1001rG mutation was responsible for the loss of plasma PAF acetylhydrolase activity in the affected subject, we prepared a mutant recombinant protein by in vitro site-directed mutagenesis. Whereas the recombinant wild-type proten exhibited marked PAF acetylhydrolase activity, the recombinant mutant protein lacked such activity (Fig. 2). DISCUSSION Tjoelker et al (15). isolated a cDNA encoding plasma PAF acetylhydrolase from human macrophages. The cDNA encodes a 441-amino acid protein that is cleaved between Lys41 and Ile42 to generate a mature enzyme with a calculated molecular mass of 45,388 daltons. The predicted protein contains the Gly-X-Ser-X-Gly consensus sequence for the catalytic site of lipases and esterases. The G994rT mutation in the ninth exon, which encodes the active site, detected by Stafforini et
773
AID
BBRC 7047
/
6933$$$941
07-10-97 13:22:22
bbrcg
AP: BBRC
Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
al. (14) and the associated Val279rPhe substitution of mature PAF acetylhydrolase result in a loss of catalytic activity. We measured the plasma PAF acetylhydrolase activity in 985 subjects with various cardiovascular diseases and 477 healthy individuals, and detected 46 sporadic cases of a deficiency of this enzyme. One of the eight subjects whose enzyme deficiency was not accounted for by homozygosity for the G994rT mutation was shown to be homozygous for a previous unidentified A1001rG transition, which results in a Gln281rArg substitution. With the use of site-directed mutagenesis, we confirmed that this mutation is responsible for the loss of plasma PAF acetylhydrolase activity in this subject.
FIG. 2. PAF acetylhydrolase activity of recombinant normal and mutant plasma PAF acetylhydrolase. Data are means of three experiments.
PAF acetylhydrolase catalyzes the degradation of PAF to biologically inactive lyso-PAF, which suggests that this enzyme protects against the pathological events mediated by PAF. This idea is supported by the observation that recombinant human PAF acetylhydrolase markedly inhibits the inflammation induced by PAF (15). This recombinant protein inhibited the activation of leukocytes induced by PAF in vitro and, in rats, reduced the PAF-induced vascular leakage in pleurisy and paw edema (15). The possibility that a reduction in PAF acetylhydrolase activity may result in pathological responses is also supported by clinical observations (11-13). In addition, PAF acetylhydrolase protects LDL against oxidation by hydrolyzing the oxidatively fragmented fatty acyl residues from the sn-2 position of phospholipids (6,9). This action may help prevent the recognition of LDL by the scavenger receptor of macrophages. Thus, PAF acetylhydrolase may be important in the defense against atherosclerosis. These observations suggest that individuals with a plasma PAF acetylhydrolase deficiency may be predisposed to develop inflammatory or atherosclerotic diseases. In conclusion, we detected an A1001rG missense mutation in exon 9 of the plasma PAF acetylhydrolase gene in a Japanese woman with coronary artery disease and essential hypertension. This mutation resulted in a Gln281rArg substitution and loss of catalytic activity. Determination of the plasma PAF acetylhydrolase genotype or enzyme activity in plasma may contribute to the prevention and management of inflammatory and atherosclerotic diseases. FIG. 1. Detection of a new missense mutation in exon 9 of the plasma PAF acetylhydrolase gene. DNA sequences of the sense (A) and antisense (B) strands reveal an ArG transition at nucleotide position 1001, resulting in a GlnrArg substitution at amino acid 281.
ACKNOWLEDGMENTS This work was supported in part by a grant from Kowa Life Science Funds. We thank Drs. S. Sato (Fujisawa Pharmaceutical Co., Osaka, Japan) and Y. Kaneko for technical assistance.
774
AID
BBRC 7047
/
6933$$$941
07-10-97 13:22:22
bbrcg
AP: BBRC
Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. Honda, Z., Nakamura, M., Miki, I., Minami, M., Watanabe, T., Seyama, Y., Okado, H., Toh, H., Ito, K., Miyamoto, T., and Shimizu, T. (1991) Nature 349, 342–346. 2. Zimmerman, G. A., Prescott, S. M., and McIntyre, T. M. (1992) in Inflammation: Basic Principles and Clinical Correlates (J. I. Gallin, I. M. Goldstein, and R. Snyderman, Eds.), pp. 149–176, Raven Press, New York. 3. Patel, K. D., Zimmerman, G. A., Prescott, S. M., and McIntyre, T. M. (1992) J. Biol. Chem. 267, 15168–15175. 4. Smiley, P. L., Stremler, K. E., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11104–11110. 5. Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1987) J. Biol. Chem. 262, 4223–4230. 6. Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095–11103. 7. Yamada, Y., Stafforini, D. M., Imaizumi, T., Zimmerman, G. A., McIntyre, T. M., and Prescott, S. M. (1994) Proc. Natl. Acad. Sci. USA 91, 10320–10324.
8. Stafforini, D. M., McIntyre, T. M., Carter, M. E., and Prescott, S. M. (1987) J. Biol. Chem. 262, 4215–4222. 9. Stafforini, D. M., Zimmerman, G. A., McIntyre, T. M., and Prescott, S. M. (1992) Trans. Assoc. Am. Physicians 105, 44–63. 10. Steinberg, D., Parthasarathy, S., Carew, T. E., Khoo, J. C., and Witztum, J. L. (1989) N. Engl. J. Med. 320, 915–923. 11. Caplan, M. S., Sun, X.-M., Hsueh, W., and Hageman, J. R. (1990) J. Pediatr. 116, 960–964. 12. Graham, R. M., Stephens, C. J., Silvester, W., Leong, L. L. L., Sturn, M. J., and Taylor, R. T. (1994) Crit. Care Med. 22, 204– 212. 13. Miwa, M., Miyake, T., Yamanaka, T., Sugatani, J., Suzuki, Y., Sakata, S., Araki, Y., and Matsumoto, M. (1988) J. Clin. Invest. 82, 1983–1991. 14. Stafforini, D. M., Satoh, K., Atkinson, D. L., Tjoelker, L. W., Eberhardt, C., Yoshida, H., Imaizumi, T., Takamatsu, S., Zimmerman, G. A., McIntyre, T. M., Gray, P. W., and Prescott, S. M. (1996) J. Clin. Invest. 97, 2784–2791. 15. Tjoelker, L. W., Wilder, C., Eberhardt, C., Stafforini, D. M., Dietsch, G., Schimpf, B., Hooper, S., Trong, H. L., Cousens, L. S., Zimmerman, G. A., Yamada, Y., McIntyre, T. M., Prescott, S. M., and Gray, P. W. (1995) Nature 374, 549–553.
775
AID
BBRC 7047
/
6933$$$941
07-10-97 13:22:22
bbrcg
AP: BBRC