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

Abstract

According to a recent hypothesis, glycosphingolipids originating from the plasma membrane are degraded in the acidic compartments of the cell as components of intraendosomal and intralysosomal vesicles and structures. Since most previous in vitro investigations used micellar ganglioside GM2 as substrate, we studied the degradation of membrane-bound ganglioside GM2 by water-soluble ␤-hexosaminidase A in the presence of the GM2 activator protein in a detergent-free, liposomal assay system. Our results show that anionic lipids such as the lysosomal components bis(monoacylglycero)phosphate or phosphatidylinositol stimulate the degradation of GM2 by ␤-hexosaminidase A up to 180-fold in the presence of GM2 activator protein. In contrast, the degradation rate of GM2 incorporated into liposomes composed of neutral lysosomal lipids such as dolichol, cholesterol, or phosphatidylcholine was significantly lower than in negatively charged liposomes. This demonstrates that both, the GM2 activator protein and anionic lysosomal phospholipids, are needed to achieve a significant degradation of membrane-bound GM2 under physiological conditions. The interaction of GM2 activator protein with immobilized membranes was studied with surface plasmon resonance spectroscopy at an acidic pH value as it occurs in the lysosomes. Increasing the concentration of bis(monoacylglycero)phosphate in immobilized liposomes led to a significant drop of the resonance signal in the presence of GM2 activator protein. This suggests that in the presence of bis(monoacylglycero)phosphate, which has been shown to occur in inner membranes of the acidic compartment, GM2 activator protein is able to solubilize lipids from the surface of immobilized membrane structures.The degradation of glycosphingolipids (GSLs) 1 endocytosed from the plasma membrane takes place in the acidic compartments of the cell. According to a recently proposed hypothesis of the topology of lysosomal digestion (1), GSLs reach the lysosomal compartments as membrane-bound components of intraendosomal and intralysosomal vesicles and structures. The degradation of GSLs with short oligosaccharide head groups and ceramide requires two proteins: a water-soluble lysosomal sphingolipid hydrolase and a sphingolipid activator protein (SAP) (2, 3). Recent studies suggest that SAPs mediate the interaction of water-soluble sphingolipid hydrolases with their respective membrane-bound substrates (4). The enzymatic, lysosomal degradation of the ganglioside GM2 is catalyzed by ␤-hexosaminidase A (HexA) and requires the GM2 activator protein (GM2AP) as cofactor (5).Human lysosomal ␤-hexosaminidases (EC 3.2.1.52) cleave off terminal ␤-glycosidically-bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates, including glycoproteins, oligosaccharides, and GSLs such as GM2, its asialo derivative GA2, and globoside (6). A total of three different ␤-hexosaminidase isoenzymes have been identified. These are composed of different combinations of two noncovalently linked, structur...

Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion

“…It has been shown previously that the degradation of (glyco)sphingolipids by lysosomal exohydrolases is stimulated by BMP in the presence of sphingolipid activator proteins (39, 54, 58, 59). It has also been shown to play a crucial role in the transfer of cholesterol in the lysosomal compartment (22, 29, 30).…”

“…BMP (C18:1) has been reported to enhance degradation of sphingolipids and glycosphingolipids by lysosomal exohydrolases in the presence of sphingolipid activator proteins (39, 54, 58, 59). Its ability to stimulate cholesterol transfer at lysosomal pH has been previously reported (29).…”

Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2

“…The lysosomal catabolism of membrane bound (glyco)sphingolipids such as GM1 (51), GM2 (52), sulfatide (53), sulfated gangliotriaosylceramide (54), glucosylceramide (55), and Cer (56) is strongly stimulated by anionic phospholipids. Dysfunction in the lysosomal sphingolipid digestion leads to lipid accumulation resulting in fatal lipid storage diseases (1).…”

Molecular and Biochemical Characterization of a Novel β-N-Acetyl-D-Hexosaminidase with Broad Substrate-Spectrum from the Aisan Corn Borer, Ostrinia Furnacalis

“…The R312-Q313-N314-K315 in Segment I in pro-Hsβ is removed by specific proteolytic processing after its delivery to lysosome 26. However in Hsα, the G280-S281-E282-P283 in Segment I constitutes a flexible loop that binds a specific lipid binding protein named GM2 activator protein, which significantly stimulates the hydrolysis rate of HsHexA toward the negatively-charged ganglioside GM2 27, 30. Moreover, the residue pair N423-R424 (Hsα) / D452-L453 (Hsβ), which determines the substrate specificity for charged/ non-charged substrates of HsHexA/ HsHexB 25-28, are not conserved in OfHex2 and other IBS-Hexs (Fig.…”