We have described a hereditary, lysosomal storage disease in humans which results in accumulation of glutamyl ribose-5-phosphate, the linkage region of poly-(ADP-ribosylated) histones. We propose to characterize the molecular basis of glutamyl ribose-5-phosphate storage disease by investigating ADP-ribosyl protein lyase deficiency and its effects on poly(ADP-ribosylated) protein metabolism. Both the synthetic and catabolic enzymes of poly(ADP-ribose) metabolism have been measured in cultured fibroblasts from the patient, his parents and a control. These results indicate a deficiency of ADP-ribose protein lyase inherited as an X-linked condition. The data also suggest that a deficiency of this enzyme has a feedback inhibitory effect on poly(ADP-ribose) synthetase. These studies will be extended to characterize in detail the enzymology in cultured fibroblasts and the patient's tissues using enzyme kinetics, immunologic staining of enzyme protein and product, and pulse-chase studies to determine the effects on synthetase protein and activity. Further studies will utilize NAD labeled in the adenine, ribose and phosphate moieties in permeabilized fibroblasts to label ADP-ribosylated proteins. These proteins and their degradation products will be analyzed for molecular weight and degree of labeling by SDS-PAGE and HPLC. The quantity and chain length of poly(ADP-ribose) in these proteins/peptides will be analyzed by phosphodiesterase digestion and chromatography. Non-ester bound material will be proteolytically degraded and sequenced to assess any non-glutamic acid linkages. ADP-ribosyl protein lyase will be purified by an established method in order to generate polyclonal antibodies. Cultured cells labeled with 35S-methionine will be precipated with anti-lyase antibody to study the synthesis, post-translational modification and degradation of ADP-ribosyl protein lyase in the normal and deficient state. These studies will characterize the enzyme deficiency in glutamyl ribose-5-phosphate storage disease and the effect of ADP-ribosyl protein lyase deficiency on the metabolism of poly(ADP-ribosylated) proteins. As this post-translational modification has a putative role in DNA expression and the control of cell growth, analysis of a human abnormality of poly(ADP-ribose) metabolism may shed light on these basic processes.
| Souba, W W; Roughneen, P T; Goldwater, D L et al. (1987) Postoperative alterations in interorgan glutamine exchange in enterectomized dogs. J Surg Res 42:117-25 |
