Myo-inositol oxygenase molecular enzymology and tissue specific expression /

by Arner, Ryan.

myo-Inositol (MI) and its various biochemical derivatives are very widely distributed in mammalian tissues, higher plants, fungi and some bacteria where they play an important role in many aspects of cellular regulation including membrane structure, signal transduction and osmoregulation (Holub, 1986;Majerus, 1992;Loewus and Loewus, 1983). The first committed step in the metabolism of MI occurs predominantly in the kidney and involves the oxidative cleavage of the ring to give D-glucuronic acid (Howard and Anderson, 1967;Charalampous and Lyras, 1957). This reaction is catalyzed by the enzyme myo-inositol oxygenase (EC 1.13.99.1, MIOX). In order to generate sufficient pure enzyme for mechanistic study, as well as to clarify the discrepancies of past research, a porcine MIOX clone was generated and expressed in a bacterial system. A full-length cDNA was isolated from a porcine kidney library with an open reading frame of 849 bp and a corresponding protein subunit molecular mass of 32.7 kDa. The cDNA was expressed in a bacterial pET expression system and an active recombinant MIOX was purified from bacterial lysates to electrophoretic homogeneity. The purified enzyme displayed the same catalytic properties as the native enzyme with Km and kcat values of 5.9 mM and 11 min-1, respectively. The pI value was estimated to be 4.5. Preincubation with 1 mM Fe2+ and 2 mM cysteine was essential for the enzyme activity. D-chiro-inositol, a myo-inositol isomer, is a substrate for the rMIOX with an estimated Km of 33.5 mM. Both myo-inositol and D-chiro-inositol have been implicated in the pathogenesis of diabetes. Previously the native MIOX enzyme was reported to be likely found in a complex with the enzyme responsible for the second step of MI catabolism, i.e. glucuronate iii reductase (Reddy et al., 1981a), which is also known as aldehyde reductase or ALR1 (EC 1.1.1.2) (De Jongh et al., 1987). The MIOX:ALR1 complex was partially purified and the activity examined. MIOX activity was present without reactivation with Fe/Cys, as is required with pure MIOX. When inositol is supplied to the complex as substrate, activity can be detected by the consumption of NADPH by the reductase. Adding similar concentrations of free glucuronate as that produced by the MIOX activity resulted in no detectable activity, indicating the ALR1 was trapping acyclic glucuronate from MIOX. However, no activity was detected with inositol as substrate when the aldose reductase inhibitor Sorbinil was added at 10µM. The same concentration inhibited pure ALR1 by 90% when glucuronate was the substrate. No inhibition of recombinant MIOX by Sorbinil was observed. These results suggest the possibility that aldose reductase inhibitor treatment for diabetes complications may have an impact on the inositol catabolic pathway. In order to study the physiological relevance of the MIOX:ALR1 complex, the expression pattern of MIOX must be established. The expression pattern of MIOX in hog tissues was examined by Western blot, Northern blot, and RT-PCR methods. The predominant source of protein and mRNA was found in kidney. In situ hybridization further localized the MIOX to the kidney proximal tubule epithelial cells. However, protein was also detected by Western blot in retina tissue. To examine the lens, a human lens epithelial cell model, HLE-B3, was employed for protein and mRNA detection. Both MIOX protein and mRNA were detected in these cells. In human and mouse, in situ hybridization detected MIOX in the kidney. Sections of other human organs failed to detect MIOX. LLC-PK1 were tested as a model for the study of MIOX expression in iv kidney. These cells expressed low levels of MIOX compared to kidney tissue, and the MIOX was unresponsive to inducers and hyperglycemia. It was concluded that an animal model would be necessary for future studies of MIOX in vivo. In conclusion, this work represents a foundation for the future study of MIOX and its physiological relevance. The expression of MIOX is not confined to the kidney, but was also detected in retina and human lens epithelial cells. All of these tissues are subject to complications brought on by Diabetes Mellitus. Since MI metabolism is deranged in diabetic tissues, MIOX may play a major role in the pathogenesis of diabetic complications. v