Complexing of Glycolipids and Their Transfer between Membranes by the Activator Protein for Degradation of Lysosomal Ganglioside G_M2

Abstract

The lysosomal degradation of ganglioside G M Z by hexosaminidase A depends on the presence of the specific activator protein which mediates the interaction between micellar or membrane-bound ganglioside and water-soluble hydrolase. The mechanism and the glycolipid specificity of this activator were studied in more detail.1. It could be shown with three different techniques (isoelectric focusing, centrifugation and electrophoresis) that the activator protein extracts glycolipid monomers from micelles or liposomes to give water-soluble complexes with a stoichiometry of 1 mol of glycolipid/mol of activator protein. Liposome-bound ganglioside C,, is considerably more stable against extraction and degradation than micellar ganglioside.2. In the absence of enzyme the activator acts in vitro as glycolipid transfer protein, transporting glycolipids from donor to acceptor membranes.3. The activator protein is rathcr specific for ganglioside GM2. Other glycolipids (GM3, G M I , GDla and GA2) form less stable complexes with the activator and are transferred at a slower rate (except for ganglioside G, than ganglioside GM,The catabolism of glycolipids is accomplished by the sequential action of acid lysosomal glycosidases [I -51. An 'inherited deficiency of one of these enzymes results in the accumulation of all of its substrates, leading to the clinical picture of a lipidosis [2 -61. To study the pathogenesis of these diseases, a number of the enzymes involved have been purified and studied in vitro (cf. [4, 51). It was found that the watersoluble enzymes, such as the hexosaminidases, were active on water-soluble substrates, but had hardly any effect on their putative lipid substrates, owing to the latter's aggregation. In vitro, this difficulty can be overcome by the addition of suitable detergents [7-1 I]. In human tissues, proteins have been identified that facilitate the interaction between water-soluble enzymes and their lipid substrates [12, 131. The physiological importance of these 'activator' proteins is demonstrated by the finding that fatal glycolipid accumulation in variant AB of infantile GM2 gangliosidosis is caused by the deficiency of the activator protein for the degradation of ganglioside GMZ by hexosaminidase A [14]. The purification and partial characterization of this protein has been reported [15]. In the present paper the mechanism by which the activator stimulates ganDedicated to Professor Horst Jatzkewitz on the occasion of his 70th birthday.Ahhreviatiorm. Cer, ceramide or N-acylsphingoid; Gal, D-galactose; GalNAc, 2-acetamido-2-deoxy-~-galactopyranose; Glc, D-glucose; GlcNAc, 2-acetamido-2-deoxy-~-glucopyranose; Lac, lactose; NeuAc, N-acetylneuraniinic acid ; GAZ or glycosphingolipid GAZ, GgOseJCer or GalNAcp1 +4Galp1 +4Glc/j1-*1 Cer; G,,, or ganglioside G,,,, I13NeuAc,lV3NeuAc-GgOse4Cer or Gal(3t2ctNeuAc)fii +3GalNAcfi1 -4Ga1(3 t 2~N e u A c ) f i l --t 4Glc/31 4 1Cer; G,, or ganglioside G,,, I13NeuAc-GgOse,Cer or Galpl + 3GalNAcfl1+ 4Ga1(3 + 2xNeuAc)fil -4Glc/31 +ICer; GMZ or ganglioside GMZ, II3...

Degradation of Membrane-bound Ganglioside GM2 by β-Hexosaminidase A

“…Lipid bilayers containing GM3 or glucosylceramide showed a similar GM2AP binding curve as membranes devoid of ganglioside, whereas the GM2AP binding behavior is comparable for bilayers containing either GM1 or GM2, including the strong drop of the signal upon injection of GM2AP. This demonstrates that GM2AP shows some specificity but is not completely specific for GM2, which is in agreement with earlier glycolipid transfer studies using GM2AP (5,28). An interesting point is that studies on the enzymatic degradation of GM1 led to the conclusion that the GM2AP, similar to SAP-B, stimulates this degradation step in the presence of BMP (35).…”

“…5). These results indicate that GM2AP differentiates between different glycolipids but is not completely specific for GM2, which is in accordance with earlier results (5,28).…”

“…Since the binding of GM2AP to gangliosides had been reported not to be completely specific for GM2 (5,28), we decided to analyze whether a similar effect as that observed for GM2 was also observed for other glycolipids. To do this, we investigated the binding of GM2AP to lipid bilayers containing 10 mol % GM1, glucosylceramide, or GM3, all spiked with 10 mol % BMP, by surface plasmon resonance.…”

Biosynthesis, Processing, and Intracellular Transport of GM2 Activator Protein in Human Epidermal Keratinocytes

“…Since the function of G M2 AP is to serve as a lipid-binding protein (1), and since G M2 AP is able to transfer gangliosides from donor to acceptor liposomes in vitro (56), an intriguing option would be that it is transported and segregated in direct association to the lipid bilayer. Such an interaction has also been proposed for glucocerebrosidase, which does not bear M6P residues (57) and is possibly bound to acidic phospholipids (58).…”

The binding of glucosylceramidase to glucosylceramide is promoted by its activator protein

“…The best characterized mechanism by which activator proteins promote the hydrolysis of sphingolipids is that described for the GMz-ganglioside activator [17]. It extracts the ganglioside from the micelles to form a water-soluble activator-lipid complex which can interact with its hydrolysing water-soluble enzyme, ,&hexosaminidase A.…”