STRUCTURAL-ANALYSIS OF SAPOSIN-C AND SAPOSIN-B - COMPLETE LOCALIZATION OF DISULFIDE BRIDGES

Saposins A, B, C, and D are a group of homologous glycoproteins derive d from a single precursor, prosaposin, and apparently involved in the stimulation of the enzymatic degradation of sphingolipids in lysosomes , Ah saposins have six cysteine residues at similar positions, In the present study we have investigated the disulfide structure of saposins B and C using advanced mass spectrometric procedures. Electrospray an alysis showed that deglycosylated saposins B and C are mainly present as 79- and 80-residue monomeric polypeptides, respectively. Fast atom bombardment mass analysis of peptide mixtures obtained by a combinatio n of chemical and enzymatic cleavages demonstrated that the pairings o f the three disulfide bridges present in each saposin are Cys(4)-Cys(7 7), Cys(7)-Cys(71), Cys(36)-Cys(47) for saposin B and Cys(5)-Cys(78), Cys(8)-Cys(72), Cys(36)-Cys(47) for saposin C. We have recently shown that saposin C interacts with phosphatidylserine-containing vesicles i nducing destabilization of the lipid surface (Vaccaro, A. M., Tatti, M ., Ciaffoni, F., Salvioli, R., Serafino, A., and Barca, A. (1994) FEBS Lett. 349, 181-186); this perturbation promotes the binding of the ly sosomal enzyme glucosylceramidase to the vesicles and the reconstituti on of its activity, It was presently found that the effects of saposin C on phosphatidylserine liposomes and on glucosylceramidase activity are markedly reduced when the three disulfide bonds are irreversibly d isrupted, These results stress the importance of the disulfide structu re for the functional properties of the saposin.