The objective of the proposed studies is to determine the in vivo effects of prosaposin (PS)-derived sphingolipid activator protein (saposins A, B, C and D) deficiencies on the control of glycosphingolipid (GSL) metabolism. The hypothesis is that GSL catabolism is modulated by the interactions of these saposins in the metabolic pathway, and that this represents an example of intragenic epistasis. Furthermore, we propose that saposin B is the key modulator that controls flow through this pathway. These studies are based on the observations that the rare, not fully detailed, human (B and C) and mouse (A) isolated saposin deficiencies have significantly different phenotypes (both clinically and biochemically), than simultaneous deficiency (PS-/-) of all four PS-derived saposins. Similarly, the PS-/- genotype leads to new phenotypes by masking the GSL storage of isolated B, C or A deficiency. We postulate that these altered phenotypes result from the interactions and redundancies of the saposins at specific steps in the GSL catabolic pathway, suggested by in vitro/ex vivo analyses. For these studies, mice with mono- or di- saposin deficiencies will be created by targeted introduction of specific point mutations that, based on human and mouse models, have produced functionally intact prosaposin and """"""""non-mutated saposins,"""""""" and isolated deficiencies of the saposins. The mutations will disrupt the conserved disulfide structure of the saposins by substitution of Phe or Ser for Cys at selected residues: ex vivo expression analyses will evaluate the overall effects of these mutations on PS and saposin stability prior to ES cell targeting. Mono-saposin B, C or D deficiencies will be created first and the resultant phenotypes characterized at the clinical, histologic and biochemical [in vivo and in vitro (e.g., cultured fibroblasts, neurons, hepatocytes)] levels: Saposin A deficient mice are available to us. Disaposin deficiencies of saposins A and B, and B and C will be created to evaluate the interactions at specific steps in GSL catabolism: e.g., the proposed need of saposins B and C for lactosylceramide degradation and the """"""""rescue"""""""" of the saposin A deficient phenotype by inclusion of saposin B deficiency. These studies have implications for GSL metabolism, and lysosomal storage disease phenotypic expression and therapy.
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