The objectives of the proposed studies are to elucidate the molecular control mechanisms of a unique multifunctional locus, prosaposin, in glycosphingolipid (GSL) metabolism. The discovery of a single locus, prosaposin, encoding in tandem, four small acidic activator proteins (saposins) of GSL hydrolase activity has intriguing and important implications for the control of these degradative pathways. Preliminary promoter studies, in vitro and in vivo, localized CNS and visceral activities to the immediate approximately 2400 by 5' to the coding sequence with the most 3' 310 bp needed for CNS expression. The proposed studies will define """"""""enhancer"""""""" elements for CNS expression in the 310-2400 bp region and determine the essential promoter elements for specific CNS cellular and developmental transcription of prosaposin. Efficient transgenic mice with """"""""natural promoter fragment""""""""/prosaposin/IRES/IacZ constructs will be used to facilitate co-localization expression analyses. The extent and patterns of phenotypic, histologic and biochemical """"""""rescue"""""""" of prosaposin knock-out mice (PSKO) will establish the physiologic functions of promoter fragments, and prosaposins with and without the alternatively spliced exon 8. This exon encodes QDQ that is thought to be critical to prosaposin secretion and extracellular functions, and for specific GSL binding to saposin B. Continuing studies will characterize the structural correlates of saposins C and B functional mechanisms for enhancing GSL hydrolase activities. Systematic site-directed mutagenesis, chimeric saposins, fluorescence energy transfer and enzyme activation will be used to identify such essential residues and regions for saposin C or B. The proposed studies build upon our previous results that outline the regional expression patterns and gross segregation of prosaposin and saposin C functions, respectively. These studies should provide insights into the modulation of this unique """"""""lysosomal"""""""" locus and of GSL flux during growth and development, as well as into the broader field of saposin-like protein function throughout phylogeny.
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