Further studies on the reconstitution of glucosylceramidase activity by Sap C and anionic phospholipids.
The reconstitution of the activity of the lysosomal enzyme glucosylceramidase requires anionic phospholipids and, at least, a protein factor, saposin C (Sap C). We have previously proposed a mechanism for the glucosylceramidase activation [Vaccaro et al. (1993) FEBS Lett. 336, 159-162] which implies that Sap C promotes the association of the enzyme with anionic phospholipid-containing membranes, thus favoring the contact between the enzyme and its lipid substrate, glucosylceramide. We have further investigated the properties of Sap C using a fluorescent hydrophobic probe such as 4, 4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS). The binding between bis-ANS and Sap C was pH-dependent, indicating that protonation leads to increased exposure of hydrophobic surfaces of Sap C. The interaction of Sap C with membranes, triggered by the development of hydrophobic properties at low pH values, was affected by the content of anionic phospholipids, such as phosphatidylserine or phosphatidylinositol, suggesting that anionic phospholipids have the potential to modulate the insertion of Sap C in the hydrophobic environment of lysosomal membranes. We previously showed that Sap C and anionic phospholipids are both required for the binding of glucosylceramidase to large vesicles. We have presently observed that Sap C is able to promote the association of glucosylceramidase with the lipid surface only when anionic phospholipids exceed a concentration of 5-10%. This level can be reached by summing lower amounts of individual anionic phospholipids, since they have additive effects. The present data extend and refine our model of the mechanism of glucosylceramidase activation and stress the key role of pH, Sap C and anionic phospholipids in promoting the interaction of the enzyme with membranes.
Salvioli
R
R
Department of Metabolism and Pathological Biochemistry, Istituto Superiore Sanita, Viale Regina Elena 299, 00161, Rome, Italy.
Tatti
M
M
Ciaffoni
F
F
Vaccaro
A M
AM
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D000838:Anions; D002417:Cattle; D004530:Egg Yolk; D005456:Fluorescent Dyes; D005962:Glucosylceramidase / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000737:chemistry; D008247:Lysosomes / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry*; D049231:Saposins; D013050:Spectrometry, Fluorescence; D049128:Sphingolipid Activator Proteins
D000817:Anilino Naphthalenesulfonates; D000838:Anions; D005456:Fluorescent Dyes; D006023:Glycoproteins; C483345:PSAP protein, human; D010743:Phospholipids; D049231:Saposins; D049128:Sphingolipid Activator Proteins; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D005962:Glucosylceramidase