Abstract
The saposins are essential cofactors for the normal lysosomal degradation of complex glycosphingolipids by acid hydrolase enzymes; defects in either saposin or hydrolase function lead to severe metabolic diseases. Saposin A (SapA) activates the enzyme β-galactocerebrosidase (GALC), which catalyzes the breakdown of β-D-galactocerebroside, the principal lipid component of myelin. SapA is known to bind lipids and detergents in a pH-dependent manner; this is accompanied by a striking transition from a `closed' to an `open' conformation. However, previous structures were determined at non-lysosomal pH. This work describes a 1.8 Å resolution X-ray crystal structure determined at the physiologically relevant lysosomal pH 4.8. In the absence of lipid or detergent at pH 4.8, SapA is observeed to adopt a conformation closely resembling the previously determined `closed' conformation, showing that pH alone is not sufficient for the transition to the `open' conformation. Structural alignments reveal small conformational changes, highlighting regions of flexibility.
Highlights
The lysosomal breakdown of complex glycosphingolipids (GSLs) is essential for normal cellular homeostasis and recycling of essential macromolecular building blocks
Expression was induced for 4 h at 37C by the addition of 1.0 mM isopropyl -d-1-thiogalactopyranoside (IPTG) when the culture reached an optical density at 600 nm of 0.6
Human saposin A (SapA) crystals belong to the monoclinic space group P21 and have a relatively low solvent content of 33.2% as estimated by the Matthews method (Matthews, 1968; VM = 1.84 A 3 DaÀ1; Fig. 1b)
Summary
The lysosomal breakdown of complex glycosphingolipids (GSLs) is essential for normal cellular homeostasis and recycling of essential macromolecular building blocks. GSL substrates for degradation are embedded in membranes of lysosomal intralumenal vesicles, where complex glycan head groups are disassembled one residue at a time by the sequential action of lysosomal acid hydrolase enzymes (Sandhoff & Kolter, 1996; Kolter & Sandhoff, 2005, 2010). The active sites of these enzymes are typically shallow; in a membrane environment the steric crowding of head groups and lateral association of GSLs into clusters prevents enzymes from accessing the scissile bonds of their target substrates. Saposins A, B, C and D comprise a family of small, nonenzymatic lipid-transfer proteins that are essential for the activation of GSL catabolism; genetic defects in saposin function cause specific forms of lysosomal storage diseases (Spiegel et al, 2005; Sun et al, 2010; Sun & Grabowski, 2013). Each saposin shows functional specificity for the activation of certain catabolic reactions: saposin A (SapA) activates the hydrolysis of -d-galactocerebroside to ceramide and galactose by the enzyme -galactocerebrosidase (GALC; EC 3.2.1.46; Morimoto et al, 1989; Harzer et al, 1997)
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