Functional organization of saposin C - Definition of the neurotrophic and acid beta-glucosidase activation regions

Saposin C is an essential co-factor for the hydrolysis of glucosylceramide by acid beta-glucosidase in mammals, In addition, prosaposin promotes neurite outgrowth in vitro via sequences in saposin C. The regional organization of these neurotrophic and activation properties of saposin C was elucidated using recombinant or chemically synthesized saposin Cs from various regions of the molecule. Unreduced and reduced proteins 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 spanning residues 47-62. Secondary structure was needed for retention of this property. Single substitutions of R and S at the conserved cysteines at 47 or 78 diminished but did not obliterate the activation properties. These results indicate the segregation of neurotrophic and activation properties of saposin C to two different faces of the molecule and suggest a topographic sequestration of the activation region of prosaposin for protection of the cell from adverse hydrolytic activity of acid beta-glucosidase.