Mechanism of activation of glucocerebrosidase by CO-β-glucosidase (glucosidase activator protein)

Abstract

The nature of the stimulatory action of the protein 'coglucosidase' on glucocerebrosidase was investigated with the use of highly purified cofactor from bovine spleen, radioactive glucosyl ceramide and methylumbelliferyl-beta-glucoside. A complex between glucosidase and either substrate could not be detected under equilibrium and non-equilibrium binding conditions. Complex formation between stimulating protein and the enzyme could be shown by the binding of the enzyme to an affinity column containing coglucosidase. This binding could be blocked by adding phosphatidylserine to the enzyme. The lipid also stimulated the enzyme. Additional evidence for binding of the enzyme to the two kinds of stimulators was the finding that they protected the enzyme against inactivation by N-ethylmaleimide and chloromercuriphenylsulfonate. A role for lipids in the stimulatory action of coglucosidase was shown by extracting lipids from the enzyme; this resulted in a loss of basal enzyme activity and of sensitivity to activation by the protein. Adding back to the lipids or phosphatidylserine increased the sensitivity of the delipidated enzyme to coglucosidase. Using the crude, unextracted enzyme we could show that low concentrations of phosphatidylserine augmented the effectiveness of coglucosidase but high concentrations of the lipid blocked the effect of the protein. It is proposed that lipids, particularly acidic ones, act on solubilized glucocerebrosidase to produce an enzyme conformation which allows binding and stimulation by coglucosidase. At higher lipid concentrations, the acidic lipids bind, in competition with coglucosidase, to the latter's binding site on the enzyme.

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

“…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). However, the same preparations were shown to contain considerable amounts of lipids (35).]…”

“…[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). However, the same preparations were shown to contain considerable amounts of lipids (35).] Acidic pH and the presence of the negatively charged lipids 1,2-diacyl-sn-3-phosphoinositol (PI) or bis(monoacylglycero)phosphate (BMP), which are abundant in intralysosomal compartment (36), are required for the activation of GCase activity by sapC (12,27).…”

Functional Organization of Saposin C

“…Saposin C binds to and may transfer gangliosides between artificial membranes (12), but it does not bind glucosylceramide, the acid ␤-glucosidase substrate (13). Saposin C binds directly to acid ␤-glucosidase (13) and may alter the structure of negatively charged phospholipid, particularly phosphatidylserine, interfaces (10).…”

“…Saposin C binds directly to acid ␤-glucosidase (13) and may alter the structure of negatively charged phospholipid, particularly phosphatidylserine, interfaces (10). These function to conform acid ␤-glucosidase and possibly alter binding at the lipid interface to effect an enhancement of substrate hydrolytic rates (13,14). Interaction of saposin C with acid ␤-glucosidase is complex and involves high (specific) and low affinity sites (15).…”

Sneak peak at galactocerebrosidase, Krabbe disease's lysosomal hydrolase

“…Alternatively, ceramide might not need to bind to the enzyme directly. Instead, substrate presentation from the lysosomal membrane could be assisted by saposins, small anionic proteins required for maximal activity of both GALC (13) and GCase (14). Although the exact mode of lysosomal hydrolase activation for these proteins is not fully understood and may be enzyme-specific (15,16), GALC displays an enticing positively charged patch near the active site for a potential complexation.…”