An extracellular Pseudomonas cepacia lipase, LipA, is inactive when expressed in the absence of the product of the limA gene. Evidence has been presented that LimA is a molecular chaperone. The lipA and limA genes have been cloned in separate and independently inducible expression systems in Escherichia coli. These systems were used to test the molecular chaperone hypothesis by investigating whether LimA could activate presynthesized prelipase and whether presynthesized LimA could activate newly synthesized prelipase. The results show that LimA cannot activate presynthesized prelipase and that presynthesized LimA can activate only a limited number of de novo synthesized prelipase molecules. Co-immunoprecipitation of prelipase/lipase with LimA generated a 1:1 complex of prelipase/lipase and LimA. The results suggest that a 1:1 complex of LipA and LimA is required for prelipase processing and secretion of active lipase.
Mice carrying a targeted disruption of the rhodopsin gene develop a severe degenerative retinopathy, failing to elaborate rod photoreceptor outer segments (ROS), having no recordable rod electroretinogram (ERG) and losing all of their rod cells over a period of approximately 12 weeks. Murine and human rhodopsins differ in their amino acid sequences. Whether, or to what extent, such variability might influence the ability of human rhodopsin to serve as an adequate structural and functional substitute for the endogenous protein in mouse rod cells bears direct relevance to exploiting the full utility of Rho-/-animals as a model of degenerative retinal disease in man. We crossed Rho-/-mice with mice expressing a wild-type human rhodopsin transgene at levels approximating to those of the endogenous protein. Immunohistological examination of retinal selections from such animals demonstrated ROS of normal number and length and temporal expression of rhodopsin similar to that observed in wild-type animals; that is, immunoreactivity to an anti-rhodopsin antibody became clearly evident by day 3 post-partum. Whereas Rho-/-mice never display a rod ERG response, and even lose cone responses by 12 weeks of age, rescued mice showed 75% normal maximum amplitudes and had ERG b-wave thresholds (based on a 50 microV criterion) within 0.1 log unit of normal wild-type at 20 weeks, and cone amplitudes remained normal at this age. These data demonstrate very substantial structural and functional rescue of the rod photoreceptors of Rho-/-mice and long-term preservation by the human rhodopsin transgene.
The lipA gene of Pseudomonas cepacia DSM3959 requires a downstream gene, limA, in oder to express lipase activity. The product of the lim gene, LimA, is a molecular chaperone required during the folding of lipase in oder for the lipase to adopt an active conformation. The lipase and LimA proteins have been shown to form a complex precipitable with either an anti-lipase or anti-LimA antibody. LimA has been shown to form a 1:1 complex with with prelipase and lipase isolated from "natural" P. cepacia system. The mature lipase (lacking its signal peptide) has been expressed in the presence and absence of LimA in Escherichia coli. LimA can activate mature lipase during a urea denaturation-renaturation experiment, indicating that the signal peptide is not required for the lipase to be activated by LimA. The effects of various reagents on the renaturation of lipase from 8 M urea have been examined. We propose a mechanism for the function of the LimA chaperone during the production of active extracellular lipase.
To explore the possible influence of defined genetic backgrounds on photoreceptor viability and function in mice carrying a targeted disruption of the rhodopsin gene, the severities of retinopathies in Rho-/- mice on C57BL/6J and 129Sv congenic backgrounds were compared by light microscopy and electroretinography and qualitatively by in situ end labeling of DNA in apoptotic photoreceptor nuclei of retinal sections. Cone photoreceptor viability and function were shown to deteriorate more slowly on the C57BL/6J background in comparison to that of the 129Sv, with significantly greater numbers of outer nuclear layer nuclei in the retinas of C57BL/6J mice at 3 and 4 months of age. Both amplitude and waveform features of the ERG were shown to be remarkably different in the two strains, indicating an approximately 6-fold difference in C57BL/6J Rho-/- mice compared to 129Sv Rho-/- mice at 80 days. Thus, in comparison with the 129Sv strain, genetic modifiers appear to constitute a component of the C57BL/6J background, the expression of which significantly protects cone photoreceptors from apoptotic death in a mutation-induced murine retinopathy. The differences in phenotype revealed in this study are sufficient in principle to provide a basis for comparisons to be made between QTLs in light-induced and mutation-induced systems.
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