FUNCTIONAL-ORGANIZATION OF SAPOSIN-C - DEFINITION OF THE NEUROTROPHICAND ACID BETA-GLUCOSIDASE ACTIVATION REGIONS

Saposin C is an essential co-factor for the hydrolysis of glucosylcera mide by acid beta-glucosidase in mammals, In addition, prosaposin prom otes neurite outgrowth in vitro via sequences in saposin C. The region al organization of these neurotrophic and activation properties of sap osin C was elucidated using recombinant or chemically synthesized sapo sin Cs from various regions of the molecule. Unreduced and reduced pro teins were analyzed by electrospray-mass spectrometry to establish the complement of disulfide bonds in selected saposin Cs. Using saposin B as a unreactive backbone, chimeric saposins containing various length segments of saposin B and C localized the neurotrophic and acid beta- glucosidase activation properties to the carboxyl- and NH2-terminal 50 % of saposin C, respectively. The peptide spanning residues 22-31 had neurotrophic effects. Molecular modeling and site-directed mutagenesis localized the activation properties of saposin C to the region spanni ng residues 47-62. Secondary structure was needed for retention of thi s property. Single substitutions of R and S at the conserved cysteines at 47 or 78 diminished but did not obliterate the activation properti es. These results indicate the segregation of neurotrophic and activat ion properties of saposin C to two different faces of the molecule and suggest a topographic sequestration of the activation region of prosa posin for protection of the cell from adverse hydrolytic activity of a cid beta-glucosidase.