Functional human saposins expressed in Escherichia coli. Evidence for binding and activation properties of saposins C with acid beta-glucosidase.
Small (80-amino acid) glycoproteins or saposins are important for the in vivo function of several lysosomal hydrolases. Four saposins, A, B, C, and D, are encoded by a single locus termed prosaposin. Saposins C and A are thought to function in vivo as activators of acid beta-glucosidase. The physiologic role of saposin C has been confirmed, whereas that of saposin A role has not. To investigate the effects of saposins C and A on acid beta-glucosidase activity, the coding sequence for the individual saposins was expressed in Escherichia coli and the recombinant proteins purified to homogeneity. Recombinant and natural saposins A and C activated acid beta-glucosidase similarly only in micromolar amounts. Saposin C had specific activation of acid beta-glucosidase activity at < 200 nM. A second phase of activation was achieved at > 1 microM. In comparison, saposin A consistently activated acid beta-glucosidase only at > 1 microM. Two mutant saposins C (Cys382-->Phe and Cys382--Gly) were created and shown to compete with saposin C for a site on acid beta-glucosidase. The mutant saposins did not activate the enzyme. Recombinant saposin A (< 200 nM) competed with saposin C for a site on the enzyme but without activating effects. These studies show that saposin A is not an in vitro activator of acid beta-glucosidase at physiologic concentrations, although binding occurs without activating acid beta-glucosidase. The studies with mutant saposins C indicate that the binding and activation effects of saposins C are distinct events. These results indicate that the saposin C-induced conformational change in the enzyme occurs via highly specific, probably multivalent, interactions between acid beta-glucosidase and saposin C.
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Division of Human Genetics, Children's Hospital Research Foundation, Cincinnati, Ohio.
Leonova
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Grabowski
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005962:Glucosylceramidase / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010718:Phosphatidylserines / Q000494:pharmacology; D011498:Protein Precursors / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D049231:Saposins; D017386:Sequence Homology, Amino Acid; D049128:Sphingolipid Activator Proteins; D014158:Transcription, Genetic
D017931:DNA Primers; D006023:Glycoproteins; C483345:PSAP protein, human; D010718:Phosphatidylserines; D011498:Protein Precursors; D011994:Recombinant Proteins; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D005962:Glucosylceramidase