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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS034071-08
Application #
6627665
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Tagle, Danilo A
Project Start
1995-05-01
Project End
2004-12-31
Budget Start
2003-01-01
Budget End
2003-12-31
Support Year
8
Fiscal Year
2003
Total Cost
$242,319
Indirect Cost
Name
Cincinnati Children's Hospital Medical Center
Department
Type
DUNS #
071284913
City
Cincinnati
State
OH
Country
United States
Zip Code
45229
Sun, Ying; Qi, Xiaoyang; Grabowski, Gregory A (2003) Saposin C is required for normal resistance of acid beta-glucosidase to proteolytic degradation. J Biol Chem 278:31918-23
You, Hong Xing; Qi, Xiaoyang; Grabowski, Gregory A et al. (2003) Phospholipid membrane interactions of saposin C: in situ atomic force microscopic study. Biophys J 84:2043-57
Sun, Ying; Witte, David P; Jin, Peng et al. (2003) Analyses of temporal regulatory elements of the prosaposin gene in transgenic mice. Biochem J 370:557-66
Wang, Ying; Grabowski, Gregory A; Qi, Xiaoyang (2003) Phospholipid vesicle fusion induced by saposin C. Arch Biochem Biophys 415:43-53
Qi, X; Grabowski, G A (2001) Molecular and cell biology of acid beta-glucosidase and prosaposin. Prog Nucleic Acid Res Mol Biol 66:203-39
Jin, P; Sun, Y; Grabowski, G A (2001) In vivo roles of RORalpha and Sp4 in the regulation of murine prosaposin gene. DNA Cell Biol 20:781-9
Qi, X; Grabowski, G A (2001) Differential membrane interactions of saposins A and C: implications for the functional specificity. J Biol Chem 276:27010-7
Sun, Y; Jin, P; Witte, D P et al. (2000) Prosaposin: promoter analysis and central-nervous-system-preferential elements for expression in vivo. Biochem J 352 Pt 2:549-56
Chang, M H; Bindloss, C A; Grabowski, G A et al. (2000) Saposins A, B, C, and D in plasma of patients with lysosomal storage disorders. Clin Chem 46:167-74
Qi, X; Kondoh, K; Krusling, D et al. (1999) Conformational and amino acid residue requirements for the saposin C neuritogenic effect. Biochemistry 38:6284-91

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