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.
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Children's Hospital Research Foundation, Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-3039, USA.
Qin
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Sun
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Kondoh
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Grabowski
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GA
D000595:Amino Acid Sequence; D004789:Enzyme Activation; D005962:Glucosylceramidase / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016501:Neurites / Q000378:metabolism*; D049231:Saposins
D006023:Glycoproteins; D049231:Saposins; D005962:Glucosylceramidase