PH-DEPENDENT CONFORMATIONAL PROPERTIES OF SAPOSINS AND THEIR INTERACTIONS WITH PHOSPHOLIPID-MEMBRANES

Saposins A, B, C, and D are small lysosomal glycoproteins released by proteolysis from a single precursor polypeptide, prosaposin. We have p resently investigated the conformational states of saposins and their interaction with membranes at acidic pH values similar to those presen t in lysosomes. With the use of phase partitioning in Triton X-114, ex perimental evidence was provided that, upon acidification, saposins (S ap) A, C, and D acquire hydrophobic properties, while the hydrophilici ty of Sap B is apparently unchanged. The pH-dependent exposure of hydr ophobic domains of Sap C and D paralleled their pH-dependent binding t o large unilamellar vesicles composed of phosphatidyl-choline, phospha tidylserine, and cholesterol. In contrast, the binding of Sap A to the vesicles was very restricted, in spite of its increased hydrophobicit y at low pH. A low affinity for the vesicles was also shown by Sap B, a finding consistent with its apparent hydrophilicity both at neutral and acidic pH. At the acidic pH values needed for binding, Sap C and D powerfully destabilized the phospholipid membranes, while Sap A and B minimally affected the bilayer integrity. In the absence of the acidi c phospholipid phosphatidylserine, the induced destabilization markedl y decreased. Of the four saposins, only Sap C was able to promote the binding of glucosylceramidase to phosphatidylserine containing membran es. This result is consistent with the notion that Sap C is specifical ly required by glucosylceramidase to exert its activity. Our finding t hat an acidic environment induces an increased hydrophobicity in Sap A , C, and D, making the last two saposins able to interact and perturb phospholipid membranes, suggests that this mechanism might be relevant to the mode of action of saposins in lysosomes.