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Lymphocyte γ-glutamyltranspeptidase activity in ataxia-telangiectasia
BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
36,
377-381
(1986)
SHORT COMMUNICATION Lymphocyte r-Glutamyltranspeptidase
Activity in Ataxia-Telangiectasia
y-Glutamyltranspeptidase (GGTP, EC 2.3.2.2) catalyzes the transfer of the glutamyl moiety of glutathione and other y-glutamyl compounds to a variety of acceptors such as amino acids, peptides, and water (1,2). This membrane-bound enzyme is known to be localized in epithelial cells of many organs (3-6). It has been shown that this enzyme is also present on the lymphoid cell surface (7,8). The surface localization of GGTP makes this enzyme useful as a surface marker for normal and neoplastic cells in various stages of differentiation and maturation (7-10). Ataxia-telangiectasia (A-T) is an autosomal recessive human genetic disease characterized by neurological disorders, immune deficiencies, a high incidence of cancer, and sensitivity to ionizing radiation (11). Extensive clinical, genetic, immunologic and biochemical studies have been conducted to elucidate the pathogenesis of A-T. The biochemical studies were mainly centered on DNA synthesis and repair mechanisms, on hypersensitivity to ionizing radiation (ll-15), and on the activities of several enzymes, including superoxide dismutase and catalase (16). The activities of some membrane-bound enzymes (alkaline phosphatase, total ATPase, Mg*+ATPase, and 5’-nucleotidase) were studied in A-T patients’ lymphocytes and all of them, except 5’-nucleotidase, were found to be significantly increased (17). The purpose of this study was to investigate the membrane-bound GGTP activity in peripheral blood lymphocytes in patients with A-T. MATERIALS
AND METHODS
Studies were performed on six A-T patients (age 7 to 14 years) in the Immunology Unit of the Department of Pediatrics, Hacettepe University, Ankara, Turkey. Fourteen controls (age 5 to 14 years) were selected from patients hospitalized for minor surgery; none of them had systemic disease or immunodeficiency. The determination of serum immunoglobulins (IgG, IgA, and IgM levels) and the E-rosette test were performed using previously described methods (18). Peripheral blood lymphocytes were obtained from control subjects and patients by the method of Boyum (19). Six milliliters of venous blood drawn in heparinized syringes was mixed with 3 ml of phosphate-buffered saline (PBS; 10 mM sodium phosphate buffer containing 150 mM NaCl, pH 7.2) and layered over Ficoll377 0885-4505/86
$3.00
Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
378
SHORT
COMMUNICATION
Hypaque (Sigma, St. Louis, MO.). After centrifugation at SOOg, for 30 min at 4°C the lymphocytes from the interface were collected and transferred to another tube and treated with 10 ml of 0.87% NH4C1 to lyse erythrocytes. The cells were washed twice with PBS and suspended in 10 mM Tris-HCl (pH 8.0) containing 1.50 mM NaCl to give a final protein concentration of 1-2 mg/ml. The lymphocytes were immediately homogenized in a Teflon-glass homogenizer at 4°C for 10 min (7). The protein concentration in the homogenate was determined by the method of Lowry et al. (20). y-Glutamyltranspeptidase activity was measured in a total volume of 1 ml in the following test system (final concentrations): 50 mM Tris-HCl (pH 8.0), 75 mM NaCl, 2.5 mM L-y-glutamyl-p-nitroanilide, 30 mM glycylglycine, and 0.2 mg of protein (homogenate) (7,21). After 10 min of incubation at 37°C the reaction was stopped by addition of 1 ml cold 1.4 N trichloroacetic acid. The absorbance of liberated p-nitroaniline (PNA) was measured at 410 nm. The activity was expressed as nanomoles PNA/60 min/mg protein. A control was run in parallel with each case. The Mann-Whitney U test was used for statistical analysis (22). RESULTS AND DISCUSSION
Characteristics of patients, including immunologic features, are shown in Table 1. E-Rosette percentage was low in two of these patients. IgA was not detectable in four out of six patients and one patient had a lower level than normal. Sinusitis, chronic fibrotic changes in the lungs, and horizontal nystagmus were the prominant physical and radiological findings in patients with A-T. Figure 1 shows the comparison of GGTP activities measured in lymphocytes from A-T patients and controls. In the cells of patients, mean activity for GGTP (349.0 5 24.1) was significantly lower than in the cells of controls (523.4 + 12.8), (P < 0.001). A-T patients demonstrate genetic and therefore clinical heterogeneity. Some hypotheses that have been proposed for the pathogenesis of the disease are defects in the DNA synthesis and repair mechanisms, in tissue differentiation, and in chromosomal translocations and dysfunction of the immune system (12,13,2325). The reduction in lymphocyte counts, the unresponsiveness to mitogens and antigens, lack of delayed hypersensitivity responses (skin test), and the presence of low or undetectable serum IgA levels are indicative of defects in cellular and humoral immune systems. The reduced levels of serum IgA in A-T patients may indicate a defect in transformation of B cells to IgA-secreting plasma cells. It is also suggested that the helper (Tn) cells which are necessary for the maturation of B cells may not be functioning properly (26-30). &er et al. (17) showed that the lymphocytes of patients with A-T had higher activities of membrane-bound enzymes, alkaline phosphatase, total ATPase, and Mg*+ATPase than corresponding normal cells. They suggested abnormality in the plasma membranes of the patients’ lymphocytes. The measurements of GGTP activity are used as a membrane marker in several studies. The specific activity of GGTP was found to be lower than normal in the peripheral blood lymphocytes of B-cell disorders and in the peripheral blood blast cells in the acute leukemias. But, in myelomatosis and in chronic T-cell
M M M F M M Normal values
Sex
14 I4 8 8 7 14
Age (years)
Note. ND = not determined.
1 2 3 4 5 6
Patient
4 I.5 2 3 I 2 ____
3 3 4 5 I 4 2100 2120 1408 2200 3780 1100 2000 ._.
68 ND 39 60 71 51 67 t 7
TABLE 1 Clinical Description of Ataxia-Telangiectasia Patients -_. Peripheral Age at Age at blood onset of onset of lymphocyte E-Rosettes ataxia telangiectasia counts/$ (%) 1100 I080 II70 880 1080 1510 1200 2 30 ~~~.
w
103 35 0 0 0 0 138 2 37
&A
bM 115 157 129 173 215 193 120 -c 35 ___-
~___
Serum immunoglobulins tmg/dl) ~~~___
E 2 E 5
8
CA g z
SHORT COMMUNICATION
380
0’
CONTROLS
PATIENTS
FIG. 1. y-Glutamyltranspeptidase activities in the lymphocytes of A-T patients and control subjects. Enzyme activities are expressed along the ordinate. Experimental conditions are as described under Materials and Methods.
disorders, the specific activity of this enzyme was found to be higher than that of normal peripheral blood lymphocytes. They suggested that such variations may be related to the age of the cell, the stage it has reached in differentiation and maturation, and its metabolic activity (9,lO). In another study, lymphocyte GGTP activity was reported to be lower in systemic lupus erythematosus (SLE) than in healthy individuals. Their data indicate that the altered activity may be due to impaired surface membranes and changes in activation of these cells in SLE patients (31). The present study showed that lymphocyte membrane-bound GGTP activity was significantly decreased in patients with A-T. This result supports the view of abnormality in the plasma membrane of lymphocytes from patients with AT. Therefore, lymphocyte membranes of these patients deserve attention and require further investigation. SUMMARY
y-Glutamyltranspeptidase (GGTP) is a membrane-bound enzyme, that catalyzes y-glutamyl transfer from y-glutamyl compounds to amino acid and peptide acceptors. One of the most important clinical findings about ataxia-telangiectasia (A-T), a multisystemic and autosomal-recessive disease, is dysfunction of the immune system. In this study, the activity of GGTP was determined in the lymphocytes from patients with A-T. Lymphocyte GGTP activity in A-T patients was found to be significantly lower than that of control lymphocytes (P < 0.001). This change may be due to the abnormality in the membrane of lymphocytes of AT patients.
SHORT COMMUNICATION
381
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
Meister, A., Life Sci. 15, 177 (1974). Meister, A., Ann. Intern. Med. 81, 247 (1974). Marathe, G. V., Nash, B., Haschemeyer, R. H., and Tate, S. S., FEBS Lett. 107, 436 (1976). Mamelak, R. D., Groth, D. F., and Prusiner, S. B., Amer. Chem. Sot. 19, 2367 (1980). Rosalki, S. B., in “Advances in Clinical Chemistry” (0. Bodansky and A. L. Latner, Eds.), Vol. 17, p. 53. Academic Press, New York, 1975. Meister, A., Tate, S. S., and Griffith, 0. W., in “Methods in Enzymology” (W. B. Jakoby, Ed.), Vol. 77, p. 237. Academic Press, New York, 1981. Novogrodsky, A., and Tate, S. S., Proc. Natl. Acad. Sci. USA 73, 2414 (1976). Marathe, G. V., Damle, N. S., Haschemeyer, R. H., and Tate, S. S., FESS Let?. 115, 273 (1980). Hultberg, B., and Sjogren, U., Acta Haematol. 63, 132 (1980). Kramers, M., and Catovsky, D., Brit. J. Haemato/. 38, 453 (1978). Paterson, C. M., and Smith, P. J., Annu. Rev. Gene?. 13, 291 (1979). Painter, C. M., and Young, B. R., Proc. Natl. Acad. Sci. USA 77, 7315 (1980). Murnane, J. P., and Painter, R. B., Biochemistry 22, 1217 (1983). Taylor, A. M. R., Metcalfe, J. A., Oxford, J. M., and Hamden, D. G., Nature (London) 260, 441 (1976). Natarajan, A. T., and Meyers, M., Hum. Genet. 52, 127 (1979). Sheridan, R. B., and Huang, P. C., Mutat. Res. 61, 381 (1979). Gzer, N. K., Ciliv, G., Berkel, A. I., Sanal, 0.. Yegin, 0.. and Ersoy, F., C/in. Exp. Immunol. 61, 118 (1985). Ersoy, F., and Berkel, A. i., Turk. J. Pediutr. 16, 145 (1974). Boyum, A., J. Lab. Invest. 21 (Suppl. 27). 77 (1968). Lowry, 0. H., Rosebrough, N. J., Farr, A. L.. and Randall, R. J.. J. Biol. Chem. 193, 265 (1951). Losa, G., Morell, A., and Barandun, S., Amer. J. Pathol. 107, 191 (1982). Rohlf, F. J., and Sokal, R. R., “Statistical Tables.” Freeman, San Francisco, 1969. Setlow, R. B., Nature (London) 271, 713 (1978). Al Saadi, A., Palutke, M., and Kumar. G. K., Hum. Gener. 55, 23 (1980). Tamura, T., Chiba, S., Abo, W., Chiba, Y., and Nakao, T., Infect. Immun. 28, 49 (1980). Hecht, F., McCaw, B. K., and Koler, R. D., N. Et@. J. Med. 289, 286 (1973). Saxon A., Stevens, R. H., and Golde, D. W., N. Engl. J. Med. 300, 700 (1979). O’Connor, R. D., and Linthicum, D. S., C/in. Immunol. Immunopathol. 15, 66 (1980). Bridges, B. A., and Hamden, D. G., Nature (London) 289, 222 (1981). Waldman, T. A., Broder, S., Goldman. C. K., Frost, K., Krosmeyer. S. J., and Medici, M. A., J. Clin. Invest. 71, 282 (1983). Rovensky, J., Stancikova, M., Tovarek, J., and Jaroskova, L., Arch. Zmmunol. Ther. Exp. 30, 111 (1982).
A. NE$E COKUGRA$*,’ AYSEN KARAN* NESR~N KARTAL ~ZER* A. ~ZZET BERKEL? *Department of Biochemistry Faculty of Pharmacy and fZnstitute of Child Health Hacettepe University Ankara, Turkey Received September 3, 1985 ’ To whom correspondence
should be addressed.
MEDICINE
AND
METABOLIC
BIOLOGY
36,
377-381
(1986)
SHORT COMMUNICATION Lymphocyte r-Glutamyltranspeptidase
Activity in Ataxia-Telangiectasia
y-Glutamyltranspeptidase (GGTP, EC 2.3.2.2) catalyzes the transfer of the glutamyl moiety of glutathione and other y-glutamyl compounds to a variety of acceptors such as amino acids, peptides, and water (1,2). This membrane-bound enzyme is known to be localized in epithelial cells of many organs (3-6). It has been shown that this enzyme is also present on the lymphoid cell surface (7,8). The surface localization of GGTP makes this enzyme useful as a surface marker for normal and neoplastic cells in various stages of differentiation and maturation (7-10). Ataxia-telangiectasia (A-T) is an autosomal recessive human genetic disease characterized by neurological disorders, immune deficiencies, a high incidence of cancer, and sensitivity to ionizing radiation (11). Extensive clinical, genetic, immunologic and biochemical studies have been conducted to elucidate the pathogenesis of A-T. The biochemical studies were mainly centered on DNA synthesis and repair mechanisms, on hypersensitivity to ionizing radiation (ll-15), and on the activities of several enzymes, including superoxide dismutase and catalase (16). The activities of some membrane-bound enzymes (alkaline phosphatase, total ATPase, Mg*+ATPase, and 5’-nucleotidase) were studied in A-T patients’ lymphocytes and all of them, except 5’-nucleotidase, were found to be significantly increased (17). The purpose of this study was to investigate the membrane-bound GGTP activity in peripheral blood lymphocytes in patients with A-T. MATERIALS
AND METHODS
Studies were performed on six A-T patients (age 7 to 14 years) in the Immunology Unit of the Department of Pediatrics, Hacettepe University, Ankara, Turkey. Fourteen controls (age 5 to 14 years) were selected from patients hospitalized for minor surgery; none of them had systemic disease or immunodeficiency. The determination of serum immunoglobulins (IgG, IgA, and IgM levels) and the E-rosette test were performed using previously described methods (18). Peripheral blood lymphocytes were obtained from control subjects and patients by the method of Boyum (19). Six milliliters of venous blood drawn in heparinized syringes was mixed with 3 ml of phosphate-buffered saline (PBS; 10 mM sodium phosphate buffer containing 150 mM NaCl, pH 7.2) and layered over Ficoll377 0885-4505/86
$3.00
Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
378
SHORT
COMMUNICATION
Hypaque (Sigma, St. Louis, MO.). After centrifugation at SOOg, for 30 min at 4°C the lymphocytes from the interface were collected and transferred to another tube and treated with 10 ml of 0.87% NH4C1 to lyse erythrocytes. The cells were washed twice with PBS and suspended in 10 mM Tris-HCl (pH 8.0) containing 1.50 mM NaCl to give a final protein concentration of 1-2 mg/ml. The lymphocytes were immediately homogenized in a Teflon-glass homogenizer at 4°C for 10 min (7). The protein concentration in the homogenate was determined by the method of Lowry et al. (20). y-Glutamyltranspeptidase activity was measured in a total volume of 1 ml in the following test system (final concentrations): 50 mM Tris-HCl (pH 8.0), 75 mM NaCl, 2.5 mM L-y-glutamyl-p-nitroanilide, 30 mM glycylglycine, and 0.2 mg of protein (homogenate) (7,21). After 10 min of incubation at 37°C the reaction was stopped by addition of 1 ml cold 1.4 N trichloroacetic acid. The absorbance of liberated p-nitroaniline (PNA) was measured at 410 nm. The activity was expressed as nanomoles PNA/60 min/mg protein. A control was run in parallel with each case. The Mann-Whitney U test was used for statistical analysis (22). RESULTS AND DISCUSSION
Characteristics of patients, including immunologic features, are shown in Table 1. E-Rosette percentage was low in two of these patients. IgA was not detectable in four out of six patients and one patient had a lower level than normal. Sinusitis, chronic fibrotic changes in the lungs, and horizontal nystagmus were the prominant physical and radiological findings in patients with A-T. Figure 1 shows the comparison of GGTP activities measured in lymphocytes from A-T patients and controls. In the cells of patients, mean activity for GGTP (349.0 5 24.1) was significantly lower than in the cells of controls (523.4 + 12.8), (P < 0.001). A-T patients demonstrate genetic and therefore clinical heterogeneity. Some hypotheses that have been proposed for the pathogenesis of the disease are defects in the DNA synthesis and repair mechanisms, in tissue differentiation, and in chromosomal translocations and dysfunction of the immune system (12,13,2325). The reduction in lymphocyte counts, the unresponsiveness to mitogens and antigens, lack of delayed hypersensitivity responses (skin test), and the presence of low or undetectable serum IgA levels are indicative of defects in cellular and humoral immune systems. The reduced levels of serum IgA in A-T patients may indicate a defect in transformation of B cells to IgA-secreting plasma cells. It is also suggested that the helper (Tn) cells which are necessary for the maturation of B cells may not be functioning properly (26-30). &er et al. (17) showed that the lymphocytes of patients with A-T had higher activities of membrane-bound enzymes, alkaline phosphatase, total ATPase, and Mg*+ATPase than corresponding normal cells. They suggested abnormality in the plasma membranes of the patients’ lymphocytes. The measurements of GGTP activity are used as a membrane marker in several studies. The specific activity of GGTP was found to be lower than normal in the peripheral blood lymphocytes of B-cell disorders and in the peripheral blood blast cells in the acute leukemias. But, in myelomatosis and in chronic T-cell
M M M F M M Normal values
Sex
14 I4 8 8 7 14
Age (years)
Note. ND = not determined.
1 2 3 4 5 6
Patient
4 I.5 2 3 I 2 ____
3 3 4 5 I 4 2100 2120 1408 2200 3780 1100 2000 ._.
68 ND 39 60 71 51 67 t 7
TABLE 1 Clinical Description of Ataxia-Telangiectasia Patients -_. Peripheral Age at Age at blood onset of onset of lymphocyte E-Rosettes ataxia telangiectasia counts/$ (%) 1100 I080 II70 880 1080 1510 1200 2 30 ~~~.
w
103 35 0 0 0 0 138 2 37
&A
bM 115 157 129 173 215 193 120 -c 35 ___-
~___
Serum immunoglobulins tmg/dl) ~~~___
E 2 E 5
8
CA g z
SHORT COMMUNICATION
380
0’
CONTROLS
PATIENTS
FIG. 1. y-Glutamyltranspeptidase activities in the lymphocytes of A-T patients and control subjects. Enzyme activities are expressed along the ordinate. Experimental conditions are as described under Materials and Methods.
disorders, the specific activity of this enzyme was found to be higher than that of normal peripheral blood lymphocytes. They suggested that such variations may be related to the age of the cell, the stage it has reached in differentiation and maturation, and its metabolic activity (9,lO). In another study, lymphocyte GGTP activity was reported to be lower in systemic lupus erythematosus (SLE) than in healthy individuals. Their data indicate that the altered activity may be due to impaired surface membranes and changes in activation of these cells in SLE patients (31). The present study showed that lymphocyte membrane-bound GGTP activity was significantly decreased in patients with A-T. This result supports the view of abnormality in the plasma membrane of lymphocytes from patients with AT. Therefore, lymphocyte membranes of these patients deserve attention and require further investigation. SUMMARY
y-Glutamyltranspeptidase (GGTP) is a membrane-bound enzyme, that catalyzes y-glutamyl transfer from y-glutamyl compounds to amino acid and peptide acceptors. One of the most important clinical findings about ataxia-telangiectasia (A-T), a multisystemic and autosomal-recessive disease, is dysfunction of the immune system. In this study, the activity of GGTP was determined in the lymphocytes from patients with A-T. Lymphocyte GGTP activity in A-T patients was found to be significantly lower than that of control lymphocytes (P < 0.001). This change may be due to the abnormality in the membrane of lymphocytes of AT patients.
SHORT COMMUNICATION
381
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
Meister, A., Life Sci. 15, 177 (1974). Meister, A., Ann. Intern. Med. 81, 247 (1974). Marathe, G. V., Nash, B., Haschemeyer, R. H., and Tate, S. S., FEBS Lett. 107, 436 (1976). Mamelak, R. D., Groth, D. F., and Prusiner, S. B., Amer. Chem. Sot. 19, 2367 (1980). Rosalki, S. B., in “Advances in Clinical Chemistry” (0. Bodansky and A. L. Latner, Eds.), Vol. 17, p. 53. Academic Press, New York, 1975. Meister, A., Tate, S. S., and Griffith, 0. W., in “Methods in Enzymology” (W. B. Jakoby, Ed.), Vol. 77, p. 237. Academic Press, New York, 1981. Novogrodsky, A., and Tate, S. S., Proc. Natl. Acad. Sci. USA 73, 2414 (1976). Marathe, G. V., Damle, N. S., Haschemeyer, R. H., and Tate, S. S., FESS Let?. 115, 273 (1980). Hultberg, B., and Sjogren, U., Acta Haematol. 63, 132 (1980). Kramers, M., and Catovsky, D., Brit. J. Haemato/. 38, 453 (1978). Paterson, C. M., and Smith, P. J., Annu. Rev. Gene?. 13, 291 (1979). Painter, C. M., and Young, B. R., Proc. Natl. Acad. Sci. USA 77, 7315 (1980). Murnane, J. P., and Painter, R. B., Biochemistry 22, 1217 (1983). Taylor, A. M. R., Metcalfe, J. A., Oxford, J. M., and Hamden, D. G., Nature (London) 260, 441 (1976). Natarajan, A. T., and Meyers, M., Hum. Genet. 52, 127 (1979). Sheridan, R. B., and Huang, P. C., Mutat. Res. 61, 381 (1979). Gzer, N. K., Ciliv, G., Berkel, A. I., Sanal, 0.. Yegin, 0.. and Ersoy, F., C/in. Exp. Immunol. 61, 118 (1985). Ersoy, F., and Berkel, A. i., Turk. J. Pediutr. 16, 145 (1974). Boyum, A., J. Lab. Invest. 21 (Suppl. 27). 77 (1968). Lowry, 0. H., Rosebrough, N. J., Farr, A. L.. and Randall, R. J.. J. Biol. Chem. 193, 265 (1951). Losa, G., Morell, A., and Barandun, S., Amer. J. Pathol. 107, 191 (1982). Rohlf, F. J., and Sokal, R. R., “Statistical Tables.” Freeman, San Francisco, 1969. Setlow, R. B., Nature (London) 271, 713 (1978). Al Saadi, A., Palutke, M., and Kumar. G. K., Hum. Gener. 55, 23 (1980). Tamura, T., Chiba, S., Abo, W., Chiba, Y., and Nakao, T., Infect. Immun. 28, 49 (1980). Hecht, F., McCaw, B. K., and Koler, R. D., N. Et@. J. Med. 289, 286 (1973). Saxon A., Stevens, R. H., and Golde, D. W., N. Engl. J. Med. 300, 700 (1979). O’Connor, R. D., and Linthicum, D. S., C/in. Immunol. Immunopathol. 15, 66 (1980). Bridges, B. A., and Hamden, D. G., Nature (London) 289, 222 (1981). Waldman, T. A., Broder, S., Goldman. C. K., Frost, K., Krosmeyer. S. J., and Medici, M. A., J. Clin. Invest. 71, 282 (1983). Rovensky, J., Stancikova, M., Tovarek, J., and Jaroskova, L., Arch. Zmmunol. Ther. Exp. 30, 111 (1982).
A. NE$E COKUGRA$*,’ AYSEN KARAN* NESR~N KARTAL ~ZER* A. ~ZZET BERKEL? *Department of Biochemistry Faculty of Pharmacy and fZnstitute of Child Health Hacettepe University Ankara, Turkey Received September 3, 1985 ’ To whom correspondence
should be addressed.