Key Concepts:

Figure 1. Dysmorphology of TSD neurons. A ‘membranous cytoplasmic body’ (MCB), as seen in an electron micrograph of a neuronal cell from a GM2 gangliosidosis patient. MCBs are enlarged lysosomes containing stored GM2 ganglioside.

Figure 2. (a) Structure of GM2 ganglioside. Individual carbohydrates are colour coded according to the representation of GM2 ganglioside shown in the lower panel: N-acetylgalactosamine (GalNAc, red hexagon); sialic acid (NANA, green hexagon), its carboxylate (–COO^–) contained in a small red circle; Gal (galactose, bule circle) and glucose (Glc, grey circle). (b) Overview of the roles of the two subunits of Hex A and the GM2 activator protein in the hydrolysis of GM2 ganglioside. C-terminal ends of the - (pink) and - (light blue) subunits are denoted by the single letter ‘C’. Shown are the positions of Arg424 (denoted by +) in the active site (in grey) of the -subunit interacting with the carboxylate of the NANA residue of GM2 ganglioside and the equivalently positioned Asp452 (–) in the active site (grey) of the -subunit.

Figure 3. Comparison and alignment of the amino acid sequences of the - and -subunits of human Hex. Identical amino acids at aligned positions in the two subunits are denoted by asterisks (^*). Sequences bearing the consensus sequon NXT/S, corresponding to sites of N-linked glycosylation, are shown underlined. Signal sequences, removed co-translationally from the N-terminus of both subunits, are highlighted in blue above a dashed line. Sequences comprising the mature, lysosomal _p and _p chains are in square brackets [ ], those making up the _m and _b chains are in braces { } and those associated with the _a chain are in parentheses ( ). Disulfide bridges between Cys residues are indicated by connecting black lines beneath the secondary structural elements. Conserved motif (HXgGDE), found at the active site of Hex, is boxed outlined in red. The basic sequence (N-R424) in the -subunit, involved in pairing with the carboxylate group of NANA, and its aligned acidic sequence (D-L453) in the -subunit are shown boxed and outlined in blue. These are adjacent to Y427 and Y456 which are required for the stabilisation of their companion subunit's active site (see Figure 4). Note that an indicated Y456S substitution has been associated with acute Sandhoff disease. The boundary between Domains I and II derived from the 3D structures of Hex B is demarcated by vertical lines labelled with the corresponding domain. Secondary structural elements found in the 3D structure of Hex B corresponding to helices and -strands are denoted as green rectangular boxes and blue ribbons terminated with an arrowhead, respectively. Residues found at the interface between the - and -subunits are highlighted in yellow. The figure was derived from Mark et al. (2003).

Figure 4. GM2 ganglioside computationally docked into the active site of Hex A. For clarity only selected residues in the active site interacting with GalNAc and parts of sialic acid (NANA) are shown. Residue Arg424 (highlighted in blue) in the active of the -subunit is highlighted, showing the bidentate hydrogen bonds formed with the negatively charged carboxylate group (arrow) from NANA. A negatively charged aspartic acid (Asp452) residue in the equivalent position in the active site of the -subunit would repel the similarly charged carboxylate group found in GM2. The stereogram was adapted, with permission, from Lemieux et al. (2006).

Figure 5. Proposed catalytic mechanism for Hex A/B and other members of glucoside hydrolase family GH20. Representations of the conserved catalytic acid/base residues in the active site of - and -subunits are shown highlighted in pink and blue, respectively. A defining feature of the GH20 family members that retain the stereochemistry at the anomeric centre is that they use a substrate-assisted catalysis mechanism to hydrolyse the glycosidic -linkage of the nonreducing N-acetylhexosamide at the end of oligosaccharides, polysaccharides and glycolipids. The 2-acetamido group on the substrate, along with the Asp (322; 354) and Glu (355;323) residues (part of the conserved sequon, HXgGDE, see Figure 3) located in the active site, act together to bring about hydrolysis of the substrate (Michaelis complex). According to this model, Glu acts as a general acid–base catalyst. Acting as an acid, it initially protonates the oxygen atom in the glycosidic linkage (first transition), thereby inducing a positive charge on the anomeric centre to form the first transition intermediate (oxazolinium ion intermediate) bearing a partial carbenium ion (positively charged carbon atom). Contemporaneously, the carboxylate anion in the Asp polarises the nitrogen atom on the acetamido group of the substrate, whereas at the same time orienting and partially ionising the carbonyl atom for nucleophillic attack of the carbon atom at the anomeric centre. These coordinated movements result in the formation of an oxazolinium intermediate (a stable thiazoline analogue of the intermediate, N-acetylglucosamine thiazoline (NGT), which also functions as a nanomolar competitive inhibitor (K_i=200 nM) of lysosomal Hex A/B, provided initial experiment support for the existence of this intermediate) and release of the hydrolysed glycoconjugate (ROH). To complete the reaction, the ionised Glu now acts as a base and deprotonates an incoming water molecule producing a hydroxyl ion that attacks the anomeric carbon atom in the oxazolinium intermediate regenerating the 2-acetamido group and re-inverting the anomeric carbon atom to the original configuration (Product complex). Adapted, with permission, from Mark et al. (2003).

Figure 6. Three-dimentional model of the quaternary Hex A:GM2 gaglioside:GM2 Activator complex. Two orientations of the complex (A and B) are related by 180° rotation about the z-axis. Surface rendering of a model of Hex A showing the -subunit (dark grey) and a homology-based model of -subunit (residues differing between - and -subunits highlighted in light brown whereas shared identical residues are coloured in blue). Three-dimensional ribbon representation of GM2 Activator (magenta) containing a bound molecule of GM2 ganglioside (yellow spheres). The hydrophobic tail of the ganglioside is docked into the cavity formed by the -cup fold of the Activator, and the GalNAc and NANA residues of GM2 are docked into the active site of the -subunit, the latter through its carboxylate group. The corresponding active site in the -subunit is highlighted in orange. Surface loops in the -subunit, capable of interacting with GM2 activator, are coloured green (IleProVal:396–398) and magenta (GlySerGluPro:280–283), respectively. Adapted, with permission, from Mark et al. (2003).

Figure 7. Flow chart depicting the biosynthesis, assembly and intracellular transport of the Hex isozymes. [Pro/]UF indicates the concentration of unfolded - or -propolypeptides, whereas [Pro/]F indicates the concentration of either folded subunit. Transport from the cis-Golgi to the trans-Golgi network is through bulk flow, with secretion being the cell's default pathway. ER, endoplasmic reticulum; MPR, mannose phosphate receptor and ERGIC, ER Golgi intermediate compartment. Adapted, with permission, from Mahuran (1999).