Function of saposin C in the reconstitution of glucosylceramidase by phosphatidylserine liposomes

Abstract

The function of saposin C (Sap C), a glucosylceramidase activator protein, in the enzyme stimulation by phosphatidylserine (PS) liposomes has been investigated. Using gel filtration experiments evidence was obtained for Sap C binding to PS large unilamellar vesicles (LUV) but not to glucosylceramidase. PS LUV, which by themselves are unable to tightly bind and stimulate the enzyme, acquire the capacity to also bind the enzyme after interaction with Sap C, making it express its full activity. Our results indicate that the primary step in the Sap C mode of action resides in its association with PS membranes; in turn, this association promotes the interaction between the membranes and glucosylceramidase.

Molecular imaging of membrane interfaces reveals mode of β-glucosidase activation by saposin C

“…Although sapC has the potential to solubilize membrane lipids (12,15), solution studies with liposomes have led to conflicting conclusions in assigning the GCase/sapC pair to either the ''solubilizer'' or the ''liftase'' mode of action (12,33). Our direct visualization of the events occurring at the membrane interface suggests that GCase hydrolyzes its substrate at the bilayer level with the help of sapC within a complex at the membrane surface.…”

“…This is supported by the fact that sapC can increase the hydrolysis of soluble substrate analogs by up to 17-fold in the presence of large unilamellar vesicles (12,33). However, this activation cannot be explained by an increase in the accessibility of catalytic pocket, because the crystal structure of isolated GCase showed that the active site is easily accessible to solution compounds (23).…”

“…Purified GCase was previously shown to interact only weakly with liposomes and to exhibit a low level of liposomal GlcCer hydrolysis (29). Unlike the pancreatic lipase/colipase system (22), there is no indication of an interaction between sapC and purified delipidated GCase in solution in the absence of membrane lipids (30)(31)(32)(33). The association of the proteins and hydrolase activation is recovered when lipids, especially negatively charged lipids, are added (30)(31)(32)(33).…”

“…Unlike the pancreatic lipase/colipase system (22), there is no indication of an interaction between sapC and purified delipidated GCase in solution in the absence of membrane lipids (30)(31)(32)(33). The association of the proteins and hydrolase activation is recovered when lipids, especially negatively charged lipids, are added (30)(31)(32)(33). [Earlier GCase preparations were shown to bind Sepharose-coupled sapC (34,35), and sapC induced the lowering of the GCase K m value for 4-methylumbelliferyl-␤-D-glucopyranoside (MUG) hydrolysis (35).…”

“…Close proximity of sapC and GCase molecules is confirmed by our results showing colocalization of labeled sapC and GCase fluorescences in the bilayer, as well as sapC/GCase FRET. However, because purified and delipidated GCase does not seem to interact with sapC in solution (30)(31)(32)(33), GCase binding may depend on the specific recognition of membrane-bound conformations of sapC.…”