J. Inher. Metab. Dis. 13 (1990) 247-249 © SSIEM and KluwerAcademicPublishers. Printed in the Netherlands
Short Communication
Diagnosis of a Novel Glycogen Storage Disease: Type laSP A. BURCHELL and I. D. WADDELL Department of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee DDI 9SY, UK
Classical type la glycogen storage disease is a severe metabolic disorder caused by a deficiency of the hepatic microsomal glucose-6-phosphatase enzyme. Glucose-6phosphatase is now known to be a multimeric complex of at least five potypeptides: the catalytic subunit of the glucose-6-phosphatase enzyme is a 36.5 kDa polypeptide (Burchell et al., 1988b; Countaway et al., 1988) and in vitro glucose-6-phosphatase activity is also dependent on the presence of a regulatory 21 kDa Ca 2+ binding polypeptide termed stabilizing protein (SP) (Burchell et al., 1985; Waddell and Burchell, 1987). Three transport proteins termed Tt, T2 and T3 respectively allow the substrates and products glucose-6-phosphate, phosphate (and pyrophosphate) and glucose to cross the endoplasmic reticulum membrane and their deficiencies are termed types lb, lc and ld glycogen storage disease.
METHODS
Assay ofglucose-6-phosphatase: All reagents were as previously described (Waddell and Burchell, 1988). Microsomes were isolated from fresh (unfrozen) human liver samples as described previously (Burchell et al., 1987). Glucose-6-phosphatase and mannose-6-phosphatase activities and microsomal intactness were measured and calculated as described in Burchell et al. (1988a) and expressed as/~mol Pi released min - t mg microsomal protein- 1. Non-specific hydrolysis of glucose-6-phosphate was assayed and corrected for as described in Burchell et al. (1988a). Immunoblot analysis: Immunoblot analysis of microsomal samples was carried out as previously described (Waddell and Burchell, 1988). Visualization was with monospecific antibodies to the glucose-6-phosphatase enzyme catalytic subunit (Burchell et al, 1988b; Countaway et al., 1988), T2, the pyrophosphate/phosphate translocase (Waddell et al., 1988), or SP, the stabilizing protein (Burchell et al., 1985) with a biotinylated anti-sheep second antibody and a streptavidin linked peroxidase complex as the detection system, using 4-chtoronaphthol as substrate. 247
248
BurchelI and Waddell
Samples: The control liver samples (n = 35) were small portions of hepatic needle or wedge biopsy samples which had been taken for the investigation of the original condition for which the patient was referred. Biochemical analysis of liver samples had previously shown that the catalytic subunit of the glucose-6-phosphatase protein was absent from the type la patient (BurcheU et al., 1988a,b) and that the T2 protein was absent from the type lc patient (Waddell et aI., 1988). Ethical approval was given by Tayside Health Board Ethical Committee.
RESULTS A liver biopsy sample was obtained from a 2-week-old boy with all the symptoms of type la glycogen storage disease. Biochemical analysis of the liver biopsy revealed elevated hepatic glycogen levels. Assay of glucose-6-phosphatase activity in fully disrupted microsomes revealed an abnormally low glucose-6-phosphatase enzyme activity, Vmax 0.04 #mol min- ~mg- ~ (normal age-matched value 0.44 + 0.06 #mol rain- ~mg- 1). In addition, the low activity was very unstable in a time and temperature dependent manner. The transport capacities of the proteins associated with glucose6-phosphatase were normal. Dot blotting of hepatic microsomes isolated from the patient with monospecific antibodies revealed the presence of normal levels of the 36.5kDa protein (Figure la) and normal levels of the T2 protein (Figure lb). In contrast, the 21 kDa stabilizing protein was completely absent (Figure lc).
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Figure 1 Dot blot analysis of human liver microsomes. Immunodetection was carried out using: (a) monospecific antibody to the catalytic subunit of the glucose-6-phosphatase enzyme; (b) monospecific antibody to T2; (c) monospecific antibody to the 21kDa Ca2+ binding stabilizing protein. Key: 1-5, control human hepatic microsomes (9-11 pg); 6, microsomes isolated from a patient with type la glycogen storage disease (6#g); 7, microsomes isolated from a patient with type lc glycogen storage disease (5/~g); 8, rat liver microsomes (2#g); centre well, microsomes isolated from the patient (5/~g). J. lnher. Metab. Dis. 13 (1990)
Type laSP Glycogen Storage Disease
249
DISCUSSION This is the first description of a deficiency of the 21 kDa regulatory stabilizing protein of hepatic microsomal glucose-6-phosphatase. We have termed the deficiency type laSP glycogen storage disease as the patient has exactly the same symptoms as patients with type ta glycogen storage disease, which has previously been shown to be caused by a deficiency of the 36.5kDa catalytic subunit of the glucose-6phosphatase enzyme (Burchell et al., 1988). The data presented here shows not only that the stabilizing protein is essential for glucose-6-phosphatase activity in vivo but also that it is probably a regulatory subunit of the glucose-6-phosphatase enzyme.
ACKNOWLEDGEMENTS This work was supported by a grant from the Scottish Hospital Endowments Research Trust to A.B.A.B. is a Lister Institute Research Fellow. We thank Miss L. Stewart for excellent technical assistance.
REFERENCES
Burchell, A., Burchell, B., Monaco, M., Walls, H. E. and Arion, W. J. Stabilization of glucose6-phosphatase activity by a 21000 dalton hepatic microsomal protein. Biochem. J. 230 (t985) 489-495 Burchell, A., Jung, R. T., Lang, C. C., Bennet, W. and Shepherd, A. N. Diagnosis of type la and type lc glycogen storage diseases in adults. Lancet 1 (1987) 1059-t062 Burchell, A., Hume, R. and Burchell, B. A new microteehnique for the analysis of the human hepatic rnicrosomal glucose-6-phosphatase system. Clin. Chim. Acta. 173 (1988a) 183-192 Burchell, A., Waddell, I. D., Countaway, J. L., Arion, W. J. and Hume, R. Identification of the human hepatic microsomal glucose-6-phosphatase enzyme. FEBS Lett. 242 (1988b) 153-156 Countaway, J. L., Waddell, I. D., Burchell, A. and Arion, W. J. The phosphohydrolase component of the hepatic microsomal glucose-6-phosphatase system is a 36.5-kilodalton polypeptide, d. Biol. Chem. 263' (1988) 2673-2678 Waddell, I. D. and Burchell, A. The hepatic microsomal glucose-6-phosphatase stabilizing protein is a calcium binding protein. Biochem. Soc. Trans. 15 (1987) 1t26-1127 Waddell, I. D. and Burchell, A. The microsomal glucose-6-phosphatase enzyme of pancreatic islets. Biochem. J. 255 (1988) 471-476 Waddell, I. D., Lindsay, J. G. and Burchell, A. The identification of T2, the phosphate/pyrophosphate transport protein of the hepatic microsomal glucose-6-phosphatase system. FEBS Lett. 229 (1988) 179-182
J. Inher. Metab. Dis. 13 (1990)
Short Communication
Diagnosis of a Novel Glycogen Storage Disease: Type laSP A. BURCHELL and I. D. WADDELL Department of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee DDI 9SY, UK
Classical type la glycogen storage disease is a severe metabolic disorder caused by a deficiency of the hepatic microsomal glucose-6-phosphatase enzyme. Glucose-6phosphatase is now known to be a multimeric complex of at least five potypeptides: the catalytic subunit of the glucose-6-phosphatase enzyme is a 36.5 kDa polypeptide (Burchell et al., 1988b; Countaway et al., 1988) and in vitro glucose-6-phosphatase activity is also dependent on the presence of a regulatory 21 kDa Ca 2+ binding polypeptide termed stabilizing protein (SP) (Burchell et al., 1985; Waddell and Burchell, 1987). Three transport proteins termed Tt, T2 and T3 respectively allow the substrates and products glucose-6-phosphate, phosphate (and pyrophosphate) and glucose to cross the endoplasmic reticulum membrane and their deficiencies are termed types lb, lc and ld glycogen storage disease.
METHODS
Assay ofglucose-6-phosphatase: All reagents were as previously described (Waddell and Burchell, 1988). Microsomes were isolated from fresh (unfrozen) human liver samples as described previously (Burchell et al., 1987). Glucose-6-phosphatase and mannose-6-phosphatase activities and microsomal intactness were measured and calculated as described in Burchell et al. (1988a) and expressed as/~mol Pi released min - t mg microsomal protein- 1. Non-specific hydrolysis of glucose-6-phosphate was assayed and corrected for as described in Burchell et al. (1988a). Immunoblot analysis: Immunoblot analysis of microsomal samples was carried out as previously described (Waddell and Burchell, 1988). Visualization was with monospecific antibodies to the glucose-6-phosphatase enzyme catalytic subunit (Burchell et al, 1988b; Countaway et al., 1988), T2, the pyrophosphate/phosphate translocase (Waddell et al., 1988), or SP, the stabilizing protein (Burchell et al., 1985) with a biotinylated anti-sheep second antibody and a streptavidin linked peroxidase complex as the detection system, using 4-chtoronaphthol as substrate. 247
248
BurchelI and Waddell
Samples: The control liver samples (n = 35) were small portions of hepatic needle or wedge biopsy samples which had been taken for the investigation of the original condition for which the patient was referred. Biochemical analysis of liver samples had previously shown that the catalytic subunit of the glucose-6-phosphatase protein was absent from the type la patient (BurcheU et al., 1988a,b) and that the T2 protein was absent from the type lc patient (Waddell et aI., 1988). Ethical approval was given by Tayside Health Board Ethical Committee.
RESULTS A liver biopsy sample was obtained from a 2-week-old boy with all the symptoms of type la glycogen storage disease. Biochemical analysis of the liver biopsy revealed elevated hepatic glycogen levels. Assay of glucose-6-phosphatase activity in fully disrupted microsomes revealed an abnormally low glucose-6-phosphatase enzyme activity, Vmax 0.04 #mol min- ~mg- ~ (normal age-matched value 0.44 + 0.06 #mol rain- ~mg- 1). In addition, the low activity was very unstable in a time and temperature dependent manner. The transport capacities of the proteins associated with glucose6-phosphatase were normal. Dot blotting of hepatic microsomes isolated from the patient with monospecific antibodies revealed the presence of normal levels of the 36.5kDa protein (Figure la) and normal levels of the T2 protein (Figure lb). In contrast, the 21 kDa stabilizing protein was completely absent (Figure lc).
I
2
3
1
2
3
1
2
3
7
6
5
O
7
6
5
a
7
6
b
5
c
Figure 1 Dot blot analysis of human liver microsomes. Immunodetection was carried out using: (a) monospecific antibody to the catalytic subunit of the glucose-6-phosphatase enzyme; (b) monospecific antibody to T2; (c) monospecific antibody to the 21kDa Ca2+ binding stabilizing protein. Key: 1-5, control human hepatic microsomes (9-11 pg); 6, microsomes isolated from a patient with type la glycogen storage disease (6#g); 7, microsomes isolated from a patient with type lc glycogen storage disease (5/~g); 8, rat liver microsomes (2#g); centre well, microsomes isolated from the patient (5/~g). J. lnher. Metab. Dis. 13 (1990)
Type laSP Glycogen Storage Disease
249
DISCUSSION This is the first description of a deficiency of the 21 kDa regulatory stabilizing protein of hepatic microsomal glucose-6-phosphatase. We have termed the deficiency type laSP glycogen storage disease as the patient has exactly the same symptoms as patients with type ta glycogen storage disease, which has previously been shown to be caused by a deficiency of the 36.5kDa catalytic subunit of the glucose-6phosphatase enzyme (Burchell et al., 1988). The data presented here shows not only that the stabilizing protein is essential for glucose-6-phosphatase activity in vivo but also that it is probably a regulatory subunit of the glucose-6-phosphatase enzyme.
ACKNOWLEDGEMENTS This work was supported by a grant from the Scottish Hospital Endowments Research Trust to A.B.A.B. is a Lister Institute Research Fellow. We thank Miss L. Stewart for excellent technical assistance.
REFERENCES
Burchell, A., Burchell, B., Monaco, M., Walls, H. E. and Arion, W. J. Stabilization of glucose6-phosphatase activity by a 21000 dalton hepatic microsomal protein. Biochem. J. 230 (t985) 489-495 Burchell, A., Jung, R. T., Lang, C. C., Bennet, W. and Shepherd, A. N. Diagnosis of type la and type lc glycogen storage diseases in adults. Lancet 1 (1987) 1059-t062 Burchell, A., Hume, R. and Burchell, B. A new microteehnique for the analysis of the human hepatic rnicrosomal glucose-6-phosphatase system. Clin. Chim. Acta. 173 (1988a) 183-192 Burchell, A., Waddell, I. D., Countaway, J. L., Arion, W. J. and Hume, R. Identification of the human hepatic microsomal glucose-6-phosphatase enzyme. FEBS Lett. 242 (1988b) 153-156 Countaway, J. L., Waddell, I. D., Burchell, A. and Arion, W. J. The phosphohydrolase component of the hepatic microsomal glucose-6-phosphatase system is a 36.5-kilodalton polypeptide, d. Biol. Chem. 263' (1988) 2673-2678 Waddell, I. D. and Burchell, A. The hepatic microsomal glucose-6-phosphatase stabilizing protein is a calcium binding protein. Biochem. Soc. Trans. 15 (1987) 1t26-1127 Waddell, I. D. and Burchell, A. The microsomal glucose-6-phosphatase enzyme of pancreatic islets. Biochem. J. 255 (1988) 471-476 Waddell, I. D., Lindsay, J. G. and Burchell, A. The identification of T2, the phosphate/pyrophosphate transport protein of the hepatic microsomal glucose-6-phosphatase system. FEBS Lett. 229 (1988) 179-182
J. Inher. Metab. Dis. 13 (1990)
