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Novel Priming Compounds of Cystathionine Metabolites on Superoxide Generation in Human Neutrophils
Biochemical and Biophysical Research Communications 269, 297–301 (2000) doi:10.1006/bbrc.1999.1970, available online at http://www.idealibrary.com on
BREAKTHROUGHS AND VIEWS Novel Priming Compounds of Cystathionine Metabolites on Superoxide Generation in Human Neutrophils Hiroyuki Kodama, Jianying Zhang, and Kazunori Sugahara Department of Chemistry, Kochi Medical School, Oko-cho, Nankoku-shi, Kochi, 783-8505, Japan
Received November 22, 1999
Human peripheral blood polymorphonuclear leukocytes were preincubated with cystathionine and cystathionine metabolites found in the urine of patients with cystathioninuria. Among the cystathionine metabolites, cystathionine ketimine and N-acetyl-S-(3oxo-3-carboxy-n-propyl) cysteine (NAc-OCPC) significantly enhanced the N-formylmethionylleucylphenylalanine (fMLP)-induced superoxide generation, but cystathionine, NAc-cystathionine, and cyclothionine did not enhance the superoxide generation. Cystathionine ketimine and NAc-OCPC also enhanced superoxide generation induced by opsonized zymosan (OZ) but not that induced by arachidonic acid (AA) and phorbol 12-myristate 13-acetate (PMA). Superoxide generation induced by cystathionine ketimine and NAc-OCPC was inhibited by genistein, an inhibitor of tyrosine kinase, and was enhanced by 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C. Cystathionine ketimine and NAcOCPC markedly also increased phosphorylation of 45kDa protein in human neutrophils and the phosphorylation depended on the concentrations of cystathionine ketimine and NAc-OCPC. The phosphorylation of 45-kDa protein induced by cystathionine ketimine and NAc-OCPC was inhibited by genistein and herbimycin A, inhibitors of tyrosine kinase, but was not inhibited by H-7 and staurosporine, inhibitors of protein kinase C. Cystathionine metabolites and L-cystathionine sulfoxides were separated into two diastereoisomers, CS-I and CS-II. CS-I enhanced the superoxide generation induced by AA and PMA but not that induced by fMLP and OZ. In contrast, CS-II enhanced the superoxide generation induced by fMLP and OZ, but not that induced by AA and PMA. © 2000 Academic Press
Polymorphonuclear leukocytes (PMN) play critical roles in the defense mechanism against microorganisms (1). When PMN are exposed to various stimuli,
one-electron reduction of molecular oxygen by NADPHoxidase leading to “respiratory burst” is induced (2, 3). N-Formylmethionylleucylphenylalanine (fMLP), opsonized zymosan (OZ), arachidonic acid (AA), and phorbol 12-myristate 13-acetate (PMA) are known as the stimuli (4). Although the responsibility of PMN to agonist is low in the basal condition, preincubation of PMN with non-stimulatory concentrations of agonists or some pharmacological agents and hypotonic treatment of the cells accelerate and potentiate the respiratory burst induced by a second stimulus (5–12). This phenomenon, termed “priming,” may account for the exaggerated physiological responses of human peripheral blood polymorphonuclear leukocytes (HPPMN). Priming and stimulation of PMN have been proposed to occur through different mechanisms (13, 14). Recently, several reports described that various cytokines and hypotonic condition enhanced the tyrosyl phosphorylation of specific proteins in PMN in the primed stage, suggesting the contribution of tyrosine kinase (TK) to the regulatory mechanism of priming in neutrophils (11, 14 –16). We found that the iminodipeptides such as pro-pro enhanced the O 2⫺ generation induced by fMLP or OZ, but not that induced by AA or PMA and that tyrosyl phosphorylation of 45 kDa protein occurred in parallel with the iminodipeptidedependent enhancement of O 2⫺ generation in neutrophils (17). The results also suggest that TK may play critical role in priming and activation of NADPHoxidase. However, the precise mechanism and the role of tyrosyl phosphorylation in priming and activation of neutrophils are unknown yet. Cystathioninuria is an autosomal recessive hereditary disorder and the phenotypical homozygotes lead to persistent excretion of large amounts of cystathionine in the urine owing to cystathionine ␥-lyase deficiency (18). We previously identified various sulfur-containing amino acids as cystathionine metabolites in the urine of patients with cystathioninuria (19 –24), and
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FIG. 1.
New metabolic pathway of cystathionine in a patient with cystathioninuria.
proposed a new pathway of cystathionine as shown in Fig. 1. Cystathionine is monodeaminated by a transaminase (25) exhibiting the properties described for glutamine transaminase, and monodeaminated cystathionine, mono-oxo-acids cyclize non-enzymatically producing cystathionine ketimine (CK) (26). CK is reduced enzymatically in mammalian tissues (27, 28), and produced cyclothionine (CT). CK was detected in bovine brain cerebellum (27) and human urine (29). However, the biochemical and physiological relevance of cystathionine and these sulfur-containing amino acids has not yet been clarified. The presence of these compounds in central nervous system led us to investigate their specific functions. In a series of our investigations to clarify the physiological function of cystathionine and cystathionine metabolites, we found that cystathionine metabolites, cystathionine ketimine, cystathionine sulfoxide and N-acetyl-S-(3-oxo-3-carboxyn-propyl)cysteine (NAc-OCPC) had a priming effect on O 2⫺ generation in human neutrophils. RESULTS 1. Effect of Cystathionine and Cystathionine Metabolites on the Stimulus-Coupled Responses of Human Neutrophils We examined the effect of cystathionine and cystathionine metabolites on O 2⫺ generation by HPPMN (30,
33). Incubation of the cells with cystathionine or the metabolites at 30 M did not induce the O 2⫺ generation. However, when the cells were preincubated with the cystathionine metabolites for 3 min, an enhancement of fMLP-induced O 2⫺ generation was observed. Among the cystathionine metabolites examined, CK, NAcOCPC, HCEHC and HCPC showed a stimulative effect on the fMLP-induced O 2⫺ generation by HPPMN, while cystathionine and other cystathionine metabolites gave no effect (Table 1). The rates of enhancement by the effective cystathionine metabolites followed in the order: CK Ⰷ NAc-OCPC ⬎ HCEHC ⬎ HCPC. The fMLP-induced O 2⫺ generation was enhanced by CK or NAc-OCPC in a concentration-dependent manner. CK and NAc-OCPC enhanced not only the fMLP-induced O 2⫺ generation but also the OZ-induced O 2⫺ generation, while the stimulative effect was not observed on the O 2⫺ generation induced by PMA or AA. These results indicate that CK and NAc-OCPC are typical priming factors for agonist-mediated respiratory burst of the neutrophils. To determine whether protein kinase C (PKC) and TK participate in the mechanism for priming of HPPMN, the effect of protein kinase inhibitors on the fMLP-induced O 2⫺ generation was examined. As shown in Fig. 2, the fMLP-induced O 2⫺ generation by CK-treated HPPMN was inhibited by genistein, an inhibitor of TK in a concentrationdependent manner, whereas H-7, an inhibitor of PKC,
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TABLE 2
Effect of Cystathionine and Cystathionine Metabolites on the fMLP-Induced O 2⫺ Generation in HPPMN
Effect of Inhibitors of Tyrosine Kinase and Protein Kinase C on Cystathionine Ketimine-Dependent Tyrosyl Phosphorylation of 45-kDa Protein in HPPMN
Sulfur amino acids (30 M)
% of control mean ⫾ SD a 100.0 ⫾ 0.00 102.1 ⫾ 1.86 175.2 ⫾ 9.18 98.5 ⫾ 1.33 274.3 ⫾ 11.91 99.3 ⫾ 1.32 99.3 ⫾ 4.68 144.4 ⫾ 3.50 99.9 ⫾ 4.69 116.8 ⫾ 9.95
Control b NAc-Cysta NAc-OCPC Cysta CK CT CMHC HCEHC -CEC HCPC
% of phosphorylation by cystathionine ketimine
Compounds Without compounds Cystathionine ketimine Cystathionine ketimine Cystathionine ketimine Cystathionine ketimine Cystathionine ketimine
Note. The neutrophils were preincubated with 30 M priming compound for 3 min at 37°C prior to the assay of fMLP-induced O 2⫺ generation. a n ⫽ 3. b Without sulfur amino acid.
⫺ 2
rather enhanced the O generation. The similar results were also obtained by using NAc-OCPC-treated HPPMN. We found new priming factors, CK and NAcOCPC for fMLP-induced O 2⫺ generation by HPPMN
FIG. 2. Effect of protein kinase inhibitors on fMLP-induced O 2⫺ generation by CK-treated HPPMN. The cells were preincubated with 30 M CK in the presence or absence of 20 M of a protein kinase inhibitor prior to addition of fMLP. The inset shows the relative amount of the O 2⫺ generation at 3 min after the addition of fMLP in the presence of the protein kinase inhibitor. F genistein; E H-7.
⫹ ⫹ ⫹ ⫹
H-7 staurosporine genistein herbimycin A
69.8 ⫾ 9.36 100 113.0 ⫾ 11.17 103.4 ⫾ 1.55 74.3 ⫾ 3.59 86.9 ⫾ 0.86
Note. The results are expressed as the mean ⫾ SD from three independent experiments. The cells were incubated with cystathionine ketimine in the presence of protein kinase inhibitor, and the tyrosyl phosphorylation of 45-kDa protein was estimated. Cystathionine ketimine, H-7, staurosporine, genistein, and herbimycin A used were 50 M, 20 M, 35 nM, 25 M and 20 M, respectively.
and obtained the results suggesting that TK participates in the fMLP-mediated O 2⫺ generation by CK or NAc-OCPC-treated HPPMN. 2. Effect of Cystathionine and Cystathionine Metabolites on the Phosphorylation of Tyrosine Residues in Human Neutrophils The stimulation of O 2⫺ generation by CK or NAcOCPC was inhibited by genistein, an inhibitor of TK, and was enhanced by H-7, an inhibitor of protein kinase C. The results suggested that the priming mechanism of HPPMN by CK or NAc-OCPC might involve TK (31). The contribution of TK to the priming of HPPMN has also been reported on several priming compounds (17). fMLP-induced phosphorylation of tyrosine residues of HPPMN protein has been detected by immunoblotting with anti-phosphotyrosine antibody. Using this method, we examined the effect of CK or NAc-OCPC on the phosphorylation of tyrosine residue of HPPMN protein. The phosphorylation of tyrosyl residues of 45 kDa protein was increased by preincubation with CK or NAc-OCPC, but was not by preincubation with cystathionine, NAc-cystathionine and CT, a reduced product of CK. This enhancement of tyrosyl phosphorylation consisted with that observed for the stimulus-specific effect of CK or NAc-OCPC on the fMLP-induced O 2⫺ generation by neutrophils. To determine whether TK or PKC participate in the CK or NAc-OCPC-stimulated phosphorylation of 45 kDa protein in HPPMN, the effect of protein kinase inhibitors on the phosphorylation of 45 kDa protein was examined. The phosphorylation process of tyrosine residue of 45 kDa protein was inhibited by genistein and herbimycin A, inhibitors of TK, but not by H-7 and staurosporine, inhibitors of PKC (Table 2).
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FIG. 3. The stimulus-specific enhancing effect of CS-I and CS-II on the O 2⫺ generation in human neutrophils. The cells were preincubated with CS-I or CS-II for 3 min prior to the assay of each stimulus-induced O 2⫺ generation. The final concentration of fMLP (A) and PMA (B) used were 12.5 and 1 nM, respectively. The O 2⫺ generation for the 100% was 0.163, 0.209, 0.184, and 0.174 nmol/10 6 cells for CS-I (A), CS-II (A), CS-I (B), and CS-II (B), respectively.
3. Different Effect of Diastereoisomers of L-Cystathionine Sulfoxide on the Stimulus-Coupled Responses of Human Neutrophils The synthetic L-cystathionine sulfoxide consists of two diastereoisomers, CS-I and CS-II. The presence of diastereoisomers was also reported on the L-cystathionine sulfoxides obtained from the urine samples of patients with cystathioninuria (21). The fMLP-induced O 2⫺ generation was enhanced by CS-II but not by CS-I (Fig. 3A). On the contrary, the PMA-induced O 2⫺ generation was enhanced by CS-I but not by CS-II (Fig. 3B). To determine whether PKC and TK participate in the priming mechanisms of CS-I and CS-II, the effect of protein kinase inhibitors on the O 2⫺ generation induced by PMA and fMLP was examined. The PMA-induced O 2⫺ generation by CS-I-treated cells was suppressed by H-7, an inhibitor of protein kinase C, showing a dependency on H-7 concentration, whereas genistein, an inhibitor of tyrosine kinase, rather enhanced the O 2⫺ generation. On the contrary, the fMLP-induced O 2⫺ generation by CS-II-treated cells was suppressed by genistein, showing a dependency on genistein concentration, whereas H-7 rather enhanced the O 2⫺ generation. The similar results were obtained by using other inhibitors; staurosporine for PKC and herbimycin A for TK. These results suggest that CS-I and CS-II enhance the O 2⫺ generation in neutrophils via activation of PKC and TK, respectively. DISCUSSION We have identified various sulfur-containing amino acids as cystathionine metabolites in the urine of the patients with cystathioninuria. However, the biochem-
ical and physiological relevance of these sulfur-containing amino acids has not yet been clarified. In the present study, we examined the effect of these sulfurcontaining amino acids on O 2⫺ generation by HPPMN. We found some cystathionine metabolites are new priming factors, for fMLP-induced O 2⫺ generation by HPPMN and obtained the results suggesting that TK participated in the fMLP-mediated O 2⫺ generation by CK or NAc-OCPC-treated HPPMN. The phosphorylation of tyrosine residues in 45 kDa protein in CK or NAc-OCPC-treated HPPMN markedly increased with time. The increase of O 2⫺ generation and the phosphorylation depended on the concentrations of these compounds. It has been widely appreciated that phosphorylation of proteins is involved in metabolic as well as morphological responses of the cell to external stimulation. There are several reports about phosphorylation of neutrophil protein with a molecular weight around 45 kDa. It was reported that 47 kDa protein was phosphorylated in human neutrophils after stimulation with fMLP or PMA on a bases of SDS-PAGE (23). Yamaguchi et al. reported that many stimuli, which cause respiratory burst in guinia pig neutrophils, commonly phosphorylated 46 kDa protein. They mentioned that 46 kDa protein is probably an equivalent to p47-phox (24), one of the cytosolic factors for activation of NADPH oxidase in human neutrophils. It is generally accepted, however, p47-phox is phosphorylated on serine/threonine by PKC-like enzymes. Furthermore, in our experiment, the 45 kDa protein noticed in our study was distinguishable from p47-phox (33). Stefano et al. reported that tyrosyl phosphorylation of 43 kDa protein was induced by fMLP and that of 45 kDa protein was induced by PMA (25). They men-
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tioned these two proteins are mitogen-activated protein kinase (MAPK) isoforms; p43 MAPK and p45 MAPK. At present, whether the 45 kDa protein found in our experiment is equivalent to p45 MAPK remains to be investigated. Enhancement of tyrosyl phosphorylation of 45 kDa protein in HPPMN by CK or NAcOCPC might seem to underlie the triggering mechanism for priming dependent respiratory burst. Priming of neutrophils results in the rapid mobilization of such cellular factors to the cell surface to increase the number of some receptor function (26). This change in function can alter the ligand binding properties of the receptor, and can allow the receptor become coupled to a new signalling pathway that leads the cells to activation. At present, the mechanism for activation of TK by CK or NAc-OCPC and whether CK or NAc-OCPC alters the property of the fMLP-receptor, by direct binding or indirectly by changing the membrane environment, are unknown. Comparing the structures of NAcOCPC, NAc-cysta, cystathionine sulfoxide, CK and cystathionine, we suggest it may be necessary for a primer to have one or more electronegative atoms and double bonds. Biological functions of the 45 kDa protein and the mechanism of TK activation in HPPMN priming should be studied further. As some patients with cystathioninuria have mental retardation, their mental retardation might be caused by the enhancement of O 2⫺ generation in neutrophils by cystathionine metabolites such as NAc-OCPC, cystathionine sulfoxide and CK. The studies on the mechanisms of O 2⫺ generation and tyrosyl phosphorylation of 45 kDa protein in HPPMN primed by NAc-OCPC, cystathionine sulfoxide and CK may make it possible to clarify the relation between the physiological roles and clinical symptom of NAc-OCPC, cystathionine sulfoxide and CK in the patients with cystathioninuria. REFERENCES 1. Stossal, T. P. (1974) N. Engl. J. Med. 290, 717–723. 2. Goldstein, I. M., Roos, D., Kaplan, H. B., and Weissmanm, G. (1975) J. Clin. Invest. 56, 1155–1163. 3. Rossi, F. (1986) Biochim. Biophys. Acta 853, 65– 89. 4. Takahashi, R., Edashige, K., Sato, E. F., Inoue, M., Matsuno, T., and Utsumi, K. (1991) Arch. Biochem. Biophys. 285, 325–330. 5. McCall, C. E., Bass, D. A., Dechatelt, L. R., Link, Jr., A. S., and Mann, M. (1979) J. Infect. Dis. 140, 277–286. 6. McPhail, L. C., Clayton, C. C., and Snyderman, R. (1983) J. Biol. Chem. 259, 5768 –5775. 7. Perianin, A., Snyderman, R., and Malfroy, B. (1989) Biochem. Biophys. Res. Commun. 161, 520 –524. 8. Combadiere, C., Hakin, J., Giroud, J. P., and Periain, A. (1990) Biochem. Biophys. Res. Commun. 168, 65–70.
9. Utsumi, T., Klostergaard, J., Akimaru, K., Sato, E. F., Yoshioka, T., and Utsumi, K. (1992) Physiol. Chem. Phys. Med. NMR 24, 77– 88. 10. Yuo, A., Kitagawa, S., Ohsaka, A., Saito, M., and Takaku, F. (1990) Biochem. Biophys. Res. Commun. 171, 491– 497. 11. Edashige, K., Watanabe, Y., Sato, E. F., Takehara, Y., and Utsumi, K. (1993) Arch. Biochem. Biophys. 302, 343–347. 12. Miyahara, M., Watanabe, Y., Edashige, K., and Yagyu, K. (1993) Biochem. Biophys. Acta 1177, 61–70. 13. Bass, D. A., Garard, C., Olbrantz, P., Wilson, J., McCall, C. E., and McPhai, L. C. (1987) J. Biol. Chem. 262, 6643– 6649. 14. Utsumi, T., Klostergaard, J., Akimaru, K., Edashige, K., Sato, E. F., and Utsumi, K. (1992) Arch. Biochem. Biophys. 294, 271–278. 15. McColl, S. R., Dipersio, J. F., Caon, A. C., Ho, P., and Naccache, P. H. (1991) Blood 78, 1842–1852. 16. Azuma, E. K., Kitagawa, S., Yuo, A., Mizoguchi, H., Umezawa, K., Takaku, F., and Saito, M. (1993) Biochim. Biophys. Acta 1179, 213–223. 17. Watanabe, Y., Sagara, Y., Sugahara, K., and Kodama, H. (1994) Biochem. Biophys. Res. Commun. 205, 758 –764. 18. Mudd, H. S., Levy, H. S., and Skobhy, F. (1989) in The Metabolic Basis of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., Eds.), 6th ed., Vol. 1, McGraw–Hill, New York. 19. Kodama, H., Yao, K., Kobayashi, K., Hirayama, K., Fujii, Y., Mizuhara, S., Haraguchi, H., and Hirosawa, M. (1969) Physiol. Chem. Phys. 1, 72–76. 20. Kodama, H., Ohomori, S., Suzuki, M., and Mizuhara, S. (1970) Physiol. Chem. Phys. 2, 287–292. 21. Kodama, H., Ishimoto, Y., Shimomura, M., Hirota, T., and Ohomori, S. (1975) Physiol. Chem. Phys. 7, 147–152. 22. Watanabe, H., Fujita, Y., Sugahara, K., Kodama, H., and Ohomori, S. (1991) Biol. Mass Spectrom. 220, 602– 608. 23. Okada, T., Takechi, T., Wakiguchi, H., Kurashige, T., Sugahara, K., and Kodama, H. (1993) Arch. Biochem. Biophys. 305, 385–391. 24. Zhang, J., Sugahara, K., Sagara, Y., Hashimoto, K., Masuoka, N., and Kodama, H. (1996) Metabolism 45, 1312–1316. 25. Costa, M., Pensa, B., Di Costanzo, B., Coccia, R., and Cavallini, D. (1987) Neurochem. Int. 10, 377–382. 26. Ricci, T., Santoro, L., Achili, M., Matarese, M. R., Nardini, M., and Cavallini, D. (1983) J. Biol. Chem. 258, 1599 –1602. 27. Ricci, G., Vesci, L., Matarese, M. R., Antonucci, A., Maggio, A., Pecci, L., and Cavallini, D. (1990) J. Neurochem. 55, 1599 –1602. 28. Nardini, M., Ricci, G., Caccuri, M. A., Solinas, P. S., Vesci, L., and Cavallini, D. (1988) Eur. J. Biochem. 173, 689 – 694. 29. Pecci, L., Antonucci, A., Nardini, M., and Cavallini, D. (1988) Biochem. Int. 17, 877– 883. 30. Zhang, J., Sugahara, K., Sagara, Y., Fontana, M., Dupre`, S., and Kodama, H. (1996) Biochem. Biophys. Res. Commun. 218, 371–376. 31. Ito, O., Zhang, J., Zhang, M., Sagara, Y., Masuoka, N., Ubuka, T., and Kodama, H. (1997) Clin. Chim. Acta 266, 93–104. 32. Zhang, J., Sagara, Y., Fontana, M., Dupre`, S., Cavallini, D., and Kodama, H. (1996) Biochem. Biophys. Res. Commun. 224, 849 – 854. 33. Shimazaki, Y., Zhang, J., Wakiguchi, H., Kurashige, T., Sagara, Y., Masuoka, N., Ohta, J., Ubuka, T., and Kodama, H. (1998) Biochem. Biophys. Res. Commun. 247, 387–391.
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BREAKTHROUGHS AND VIEWS Novel Priming Compounds of Cystathionine Metabolites on Superoxide Generation in Human Neutrophils Hiroyuki Kodama, Jianying Zhang, and Kazunori Sugahara Department of Chemistry, Kochi Medical School, Oko-cho, Nankoku-shi, Kochi, 783-8505, Japan
Received November 22, 1999
Human peripheral blood polymorphonuclear leukocytes were preincubated with cystathionine and cystathionine metabolites found in the urine of patients with cystathioninuria. Among the cystathionine metabolites, cystathionine ketimine and N-acetyl-S-(3oxo-3-carboxy-n-propyl) cysteine (NAc-OCPC) significantly enhanced the N-formylmethionylleucylphenylalanine (fMLP)-induced superoxide generation, but cystathionine, NAc-cystathionine, and cyclothionine did not enhance the superoxide generation. Cystathionine ketimine and NAc-OCPC also enhanced superoxide generation induced by opsonized zymosan (OZ) but not that induced by arachidonic acid (AA) and phorbol 12-myristate 13-acetate (PMA). Superoxide generation induced by cystathionine ketimine and NAc-OCPC was inhibited by genistein, an inhibitor of tyrosine kinase, and was enhanced by 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C. Cystathionine ketimine and NAcOCPC markedly also increased phosphorylation of 45kDa protein in human neutrophils and the phosphorylation depended on the concentrations of cystathionine ketimine and NAc-OCPC. The phosphorylation of 45-kDa protein induced by cystathionine ketimine and NAc-OCPC was inhibited by genistein and herbimycin A, inhibitors of tyrosine kinase, but was not inhibited by H-7 and staurosporine, inhibitors of protein kinase C. Cystathionine metabolites and L-cystathionine sulfoxides were separated into two diastereoisomers, CS-I and CS-II. CS-I enhanced the superoxide generation induced by AA and PMA but not that induced by fMLP and OZ. In contrast, CS-II enhanced the superoxide generation induced by fMLP and OZ, but not that induced by AA and PMA. © 2000 Academic Press
Polymorphonuclear leukocytes (PMN) play critical roles in the defense mechanism against microorganisms (1). When PMN are exposed to various stimuli,
one-electron reduction of molecular oxygen by NADPHoxidase leading to “respiratory burst” is induced (2, 3). N-Formylmethionylleucylphenylalanine (fMLP), opsonized zymosan (OZ), arachidonic acid (AA), and phorbol 12-myristate 13-acetate (PMA) are known as the stimuli (4). Although the responsibility of PMN to agonist is low in the basal condition, preincubation of PMN with non-stimulatory concentrations of agonists or some pharmacological agents and hypotonic treatment of the cells accelerate and potentiate the respiratory burst induced by a second stimulus (5–12). This phenomenon, termed “priming,” may account for the exaggerated physiological responses of human peripheral blood polymorphonuclear leukocytes (HPPMN). Priming and stimulation of PMN have been proposed to occur through different mechanisms (13, 14). Recently, several reports described that various cytokines and hypotonic condition enhanced the tyrosyl phosphorylation of specific proteins in PMN in the primed stage, suggesting the contribution of tyrosine kinase (TK) to the regulatory mechanism of priming in neutrophils (11, 14 –16). We found that the iminodipeptides such as pro-pro enhanced the O 2⫺ generation induced by fMLP or OZ, but not that induced by AA or PMA and that tyrosyl phosphorylation of 45 kDa protein occurred in parallel with the iminodipeptidedependent enhancement of O 2⫺ generation in neutrophils (17). The results also suggest that TK may play critical role in priming and activation of NADPHoxidase. However, the precise mechanism and the role of tyrosyl phosphorylation in priming and activation of neutrophils are unknown yet. Cystathioninuria is an autosomal recessive hereditary disorder and the phenotypical homozygotes lead to persistent excretion of large amounts of cystathionine in the urine owing to cystathionine ␥-lyase deficiency (18). We previously identified various sulfur-containing amino acids as cystathionine metabolites in the urine of patients with cystathioninuria (19 –24), and
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0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 269, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1.
New metabolic pathway of cystathionine in a patient with cystathioninuria.
proposed a new pathway of cystathionine as shown in Fig. 1. Cystathionine is monodeaminated by a transaminase (25) exhibiting the properties described for glutamine transaminase, and monodeaminated cystathionine, mono-oxo-acids cyclize non-enzymatically producing cystathionine ketimine (CK) (26). CK is reduced enzymatically in mammalian tissues (27, 28), and produced cyclothionine (CT). CK was detected in bovine brain cerebellum (27) and human urine (29). However, the biochemical and physiological relevance of cystathionine and these sulfur-containing amino acids has not yet been clarified. The presence of these compounds in central nervous system led us to investigate their specific functions. In a series of our investigations to clarify the physiological function of cystathionine and cystathionine metabolites, we found that cystathionine metabolites, cystathionine ketimine, cystathionine sulfoxide and N-acetyl-S-(3-oxo-3-carboxyn-propyl)cysteine (NAc-OCPC) had a priming effect on O 2⫺ generation in human neutrophils. RESULTS 1. Effect of Cystathionine and Cystathionine Metabolites on the Stimulus-Coupled Responses of Human Neutrophils We examined the effect of cystathionine and cystathionine metabolites on O 2⫺ generation by HPPMN (30,
33). Incubation of the cells with cystathionine or the metabolites at 30 M did not induce the O 2⫺ generation. However, when the cells were preincubated with the cystathionine metabolites for 3 min, an enhancement of fMLP-induced O 2⫺ generation was observed. Among the cystathionine metabolites examined, CK, NAcOCPC, HCEHC and HCPC showed a stimulative effect on the fMLP-induced O 2⫺ generation by HPPMN, while cystathionine and other cystathionine metabolites gave no effect (Table 1). The rates of enhancement by the effective cystathionine metabolites followed in the order: CK Ⰷ NAc-OCPC ⬎ HCEHC ⬎ HCPC. The fMLP-induced O 2⫺ generation was enhanced by CK or NAc-OCPC in a concentration-dependent manner. CK and NAc-OCPC enhanced not only the fMLP-induced O 2⫺ generation but also the OZ-induced O 2⫺ generation, while the stimulative effect was not observed on the O 2⫺ generation induced by PMA or AA. These results indicate that CK and NAc-OCPC are typical priming factors for agonist-mediated respiratory burst of the neutrophils. To determine whether protein kinase C (PKC) and TK participate in the mechanism for priming of HPPMN, the effect of protein kinase inhibitors on the fMLP-induced O 2⫺ generation was examined. As shown in Fig. 2, the fMLP-induced O 2⫺ generation by CK-treated HPPMN was inhibited by genistein, an inhibitor of TK in a concentrationdependent manner, whereas H-7, an inhibitor of PKC,
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TABLE 2
Effect of Cystathionine and Cystathionine Metabolites on the fMLP-Induced O 2⫺ Generation in HPPMN
Effect of Inhibitors of Tyrosine Kinase and Protein Kinase C on Cystathionine Ketimine-Dependent Tyrosyl Phosphorylation of 45-kDa Protein in HPPMN
Sulfur amino acids (30 M)
% of control mean ⫾ SD a 100.0 ⫾ 0.00 102.1 ⫾ 1.86 175.2 ⫾ 9.18 98.5 ⫾ 1.33 274.3 ⫾ 11.91 99.3 ⫾ 1.32 99.3 ⫾ 4.68 144.4 ⫾ 3.50 99.9 ⫾ 4.69 116.8 ⫾ 9.95
Control b NAc-Cysta NAc-OCPC Cysta CK CT CMHC HCEHC -CEC HCPC
% of phosphorylation by cystathionine ketimine
Compounds Without compounds Cystathionine ketimine Cystathionine ketimine Cystathionine ketimine Cystathionine ketimine Cystathionine ketimine
Note. The neutrophils were preincubated with 30 M priming compound for 3 min at 37°C prior to the assay of fMLP-induced O 2⫺ generation. a n ⫽ 3. b Without sulfur amino acid.
⫺ 2
rather enhanced the O generation. The similar results were also obtained by using NAc-OCPC-treated HPPMN. We found new priming factors, CK and NAcOCPC for fMLP-induced O 2⫺ generation by HPPMN
FIG. 2. Effect of protein kinase inhibitors on fMLP-induced O 2⫺ generation by CK-treated HPPMN. The cells were preincubated with 30 M CK in the presence or absence of 20 M of a protein kinase inhibitor prior to addition of fMLP. The inset shows the relative amount of the O 2⫺ generation at 3 min after the addition of fMLP in the presence of the protein kinase inhibitor. F genistein; E H-7.
⫹ ⫹ ⫹ ⫹
H-7 staurosporine genistein herbimycin A
69.8 ⫾ 9.36 100 113.0 ⫾ 11.17 103.4 ⫾ 1.55 74.3 ⫾ 3.59 86.9 ⫾ 0.86
Note. The results are expressed as the mean ⫾ SD from three independent experiments. The cells were incubated with cystathionine ketimine in the presence of protein kinase inhibitor, and the tyrosyl phosphorylation of 45-kDa protein was estimated. Cystathionine ketimine, H-7, staurosporine, genistein, and herbimycin A used were 50 M, 20 M, 35 nM, 25 M and 20 M, respectively.
and obtained the results suggesting that TK participates in the fMLP-mediated O 2⫺ generation by CK or NAc-OCPC-treated HPPMN. 2. Effect of Cystathionine and Cystathionine Metabolites on the Phosphorylation of Tyrosine Residues in Human Neutrophils The stimulation of O 2⫺ generation by CK or NAcOCPC was inhibited by genistein, an inhibitor of TK, and was enhanced by H-7, an inhibitor of protein kinase C. The results suggested that the priming mechanism of HPPMN by CK or NAc-OCPC might involve TK (31). The contribution of TK to the priming of HPPMN has also been reported on several priming compounds (17). fMLP-induced phosphorylation of tyrosine residues of HPPMN protein has been detected by immunoblotting with anti-phosphotyrosine antibody. Using this method, we examined the effect of CK or NAc-OCPC on the phosphorylation of tyrosine residue of HPPMN protein. The phosphorylation of tyrosyl residues of 45 kDa protein was increased by preincubation with CK or NAc-OCPC, but was not by preincubation with cystathionine, NAc-cystathionine and CT, a reduced product of CK. This enhancement of tyrosyl phosphorylation consisted with that observed for the stimulus-specific effect of CK or NAc-OCPC on the fMLP-induced O 2⫺ generation by neutrophils. To determine whether TK or PKC participate in the CK or NAc-OCPC-stimulated phosphorylation of 45 kDa protein in HPPMN, the effect of protein kinase inhibitors on the phosphorylation of 45 kDa protein was examined. The phosphorylation process of tyrosine residue of 45 kDa protein was inhibited by genistein and herbimycin A, inhibitors of TK, but not by H-7 and staurosporine, inhibitors of PKC (Table 2).
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FIG. 3. The stimulus-specific enhancing effect of CS-I and CS-II on the O 2⫺ generation in human neutrophils. The cells were preincubated with CS-I or CS-II for 3 min prior to the assay of each stimulus-induced O 2⫺ generation. The final concentration of fMLP (A) and PMA (B) used were 12.5 and 1 nM, respectively. The O 2⫺ generation for the 100% was 0.163, 0.209, 0.184, and 0.174 nmol/10 6 cells for CS-I (A), CS-II (A), CS-I (B), and CS-II (B), respectively.
3. Different Effect of Diastereoisomers of L-Cystathionine Sulfoxide on the Stimulus-Coupled Responses of Human Neutrophils The synthetic L-cystathionine sulfoxide consists of two diastereoisomers, CS-I and CS-II. The presence of diastereoisomers was also reported on the L-cystathionine sulfoxides obtained from the urine samples of patients with cystathioninuria (21). The fMLP-induced O 2⫺ generation was enhanced by CS-II but not by CS-I (Fig. 3A). On the contrary, the PMA-induced O 2⫺ generation was enhanced by CS-I but not by CS-II (Fig. 3B). To determine whether PKC and TK participate in the priming mechanisms of CS-I and CS-II, the effect of protein kinase inhibitors on the O 2⫺ generation induced by PMA and fMLP was examined. The PMA-induced O 2⫺ generation by CS-I-treated cells was suppressed by H-7, an inhibitor of protein kinase C, showing a dependency on H-7 concentration, whereas genistein, an inhibitor of tyrosine kinase, rather enhanced the O 2⫺ generation. On the contrary, the fMLP-induced O 2⫺ generation by CS-II-treated cells was suppressed by genistein, showing a dependency on genistein concentration, whereas H-7 rather enhanced the O 2⫺ generation. The similar results were obtained by using other inhibitors; staurosporine for PKC and herbimycin A for TK. These results suggest that CS-I and CS-II enhance the O 2⫺ generation in neutrophils via activation of PKC and TK, respectively. DISCUSSION We have identified various sulfur-containing amino acids as cystathionine metabolites in the urine of the patients with cystathioninuria. However, the biochem-
ical and physiological relevance of these sulfur-containing amino acids has not yet been clarified. In the present study, we examined the effect of these sulfurcontaining amino acids on O 2⫺ generation by HPPMN. We found some cystathionine metabolites are new priming factors, for fMLP-induced O 2⫺ generation by HPPMN and obtained the results suggesting that TK participated in the fMLP-mediated O 2⫺ generation by CK or NAc-OCPC-treated HPPMN. The phosphorylation of tyrosine residues in 45 kDa protein in CK or NAc-OCPC-treated HPPMN markedly increased with time. The increase of O 2⫺ generation and the phosphorylation depended on the concentrations of these compounds. It has been widely appreciated that phosphorylation of proteins is involved in metabolic as well as morphological responses of the cell to external stimulation. There are several reports about phosphorylation of neutrophil protein with a molecular weight around 45 kDa. It was reported that 47 kDa protein was phosphorylated in human neutrophils after stimulation with fMLP or PMA on a bases of SDS-PAGE (23). Yamaguchi et al. reported that many stimuli, which cause respiratory burst in guinia pig neutrophils, commonly phosphorylated 46 kDa protein. They mentioned that 46 kDa protein is probably an equivalent to p47-phox (24), one of the cytosolic factors for activation of NADPH oxidase in human neutrophils. It is generally accepted, however, p47-phox is phosphorylated on serine/threonine by PKC-like enzymes. Furthermore, in our experiment, the 45 kDa protein noticed in our study was distinguishable from p47-phox (33). Stefano et al. reported that tyrosyl phosphorylation of 43 kDa protein was induced by fMLP and that of 45 kDa protein was induced by PMA (25). They men-
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tioned these two proteins are mitogen-activated protein kinase (MAPK) isoforms; p43 MAPK and p45 MAPK. At present, whether the 45 kDa protein found in our experiment is equivalent to p45 MAPK remains to be investigated. Enhancement of tyrosyl phosphorylation of 45 kDa protein in HPPMN by CK or NAcOCPC might seem to underlie the triggering mechanism for priming dependent respiratory burst. Priming of neutrophils results in the rapid mobilization of such cellular factors to the cell surface to increase the number of some receptor function (26). This change in function can alter the ligand binding properties of the receptor, and can allow the receptor become coupled to a new signalling pathway that leads the cells to activation. At present, the mechanism for activation of TK by CK or NAc-OCPC and whether CK or NAc-OCPC alters the property of the fMLP-receptor, by direct binding or indirectly by changing the membrane environment, are unknown. Comparing the structures of NAcOCPC, NAc-cysta, cystathionine sulfoxide, CK and cystathionine, we suggest it may be necessary for a primer to have one or more electronegative atoms and double bonds. Biological functions of the 45 kDa protein and the mechanism of TK activation in HPPMN priming should be studied further. As some patients with cystathioninuria have mental retardation, their mental retardation might be caused by the enhancement of O 2⫺ generation in neutrophils by cystathionine metabolites such as NAc-OCPC, cystathionine sulfoxide and CK. The studies on the mechanisms of O 2⫺ generation and tyrosyl phosphorylation of 45 kDa protein in HPPMN primed by NAc-OCPC, cystathionine sulfoxide and CK may make it possible to clarify the relation between the physiological roles and clinical symptom of NAc-OCPC, cystathionine sulfoxide and CK in the patients with cystathioninuria. REFERENCES 1. Stossal, T. P. (1974) N. Engl. J. Med. 290, 717–723. 2. Goldstein, I. M., Roos, D., Kaplan, H. B., and Weissmanm, G. (1975) J. Clin. Invest. 56, 1155–1163. 3. Rossi, F. (1986) Biochim. Biophys. Acta 853, 65– 89. 4. Takahashi, R., Edashige, K., Sato, E. F., Inoue, M., Matsuno, T., and Utsumi, K. (1991) Arch. Biochem. Biophys. 285, 325–330. 5. McCall, C. E., Bass, D. A., Dechatelt, L. R., Link, Jr., A. S., and Mann, M. (1979) J. Infect. Dis. 140, 277–286. 6. McPhail, L. C., Clayton, C. C., and Snyderman, R. (1983) J. Biol. Chem. 259, 5768 –5775. 7. Perianin, A., Snyderman, R., and Malfroy, B. (1989) Biochem. Biophys. Res. Commun. 161, 520 –524. 8. Combadiere, C., Hakin, J., Giroud, J. P., and Periain, A. (1990) Biochem. Biophys. Res. Commun. 168, 65–70.
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