The mechanism of glycosphingolipid degradation revealed by a GALC-SapA complex structure

Abstract

Sphingolipids are essential components of cellular membranes and defects in their synthesis or degradation cause severe human diseases. The efficient degradation of sphingolipids in the lysosome requires lipid-binding saposin proteins and hydrolytic enzymes. The glycosphingolipid galactocerebroside is the primary lipid component of the myelin sheath and is degraded by the hydrolase β-galactocerebrosidase (GALC). This enzyme requires the saposin SapA for lipid processing and defects in either of these proteins causes a severe neurodegenerative disorder, Krabbe disease. Here we present the structure of a glycosphingolipid-processing complex, revealing how SapA and GALC form a heterotetramer with an open channel connecting the enzyme active site to the SapA hydrophobic cavity. This structure defines how a soluble hydrolase can cleave the polar glycosyl headgroups of these essential lipids from their hydrophobic ceramide tails. Furthermore, the molecular details of this interaction provide an illustration for how specificity of saposin binding to hydrolases is encoded.

Erratum

“…SAPLIPS were first shown to be monomers such as the antimicrobial peptides NK lysin 32 and caenopore-5, 35 both with membrane pore-forming properties. More recently, however, very different structures were obtained for two other SAPLIP-family members, galactocerebrosidase 36 and acid sphingomyelinase, 37 which show dimeric species. Through a conformational change between helices H1 and H2, these structures can open up, thereby exposing large hydrophobic surfaces.…”

“…The two proteins form dimers in different ways that both involve packing of the exposed hydrophobic surfaces. Whereas the C and D chains of galactocerebrosidase pack in parallel fashion, 36 the two sphingomyelinase proteins pack in an antiparallel way. 37 Based on the highly conserved amphipathic helices and disulfide-bonding patterns, we propose that similar conformational changes occur in Canopy proteins leading to dimerization.…”

The lipid transfer protein Saposin B does not directly bind CD1d for lipid antigen loading

“…There are two proposed mechanisms for how saposins assist in lipid presentation to hydrolases: the "solubiliser" model, whereby saposins encapsulate lipids within a hydrophobic oligomeric complex for presentation to soluble enzymes, and the "liftase" model, where saposins bind directly to membranes destabilising them allowing membrane-associated hydrolases access to lipid substrates. Structural and cell-based evidence exists to support both these models 19,[26][27][28][29] . These two models are also relevant for the proposed mechanisms by which saposins load lipids onto CD1 molecules 30 .…”

“…SapA, SapB, SapC and SapD are the products of the proteolytic cleavage of a prosaposin (PSAP) precursor and are each small, nonenzymatic proteins possessing three disulphide bonds that stabilise an extremely heat-resistant helical structure. The saposins can exist in a "closed" monomeric, globular conformation or, at low pH, adopt a more "open" conformation, forming higher-order oligomers enclosing a hydrophobic cavity into which lipid acyl chains can be buried [14][15][16][17][18][19] . Each saposin functions in conjunction with specific hydrolases to facilitate the degradation of different glycosphingolipids and the loss of saposin function phenotypically resembles the loss of the associated hydrolases [20][21][22][23][24][25] .…”

Molecular models should not be published without the corresponding atomic coordinates

“…A clearer picture of how saposins transfer hydrophobic lipids to soluble hydrolases is essential to the molecular understanding of sphingolipidoses, a family of devastating human diseases. Recent atomicresolution crystal structures illustrate how saposins or saposin-like domains interact with lysosomal sphingolipid processing enzymes: the complex between β-galactocerebrosidase and saposin A (7), and the structures of acid sphingomyelinase (8)(9)(10). The atomic coordinates for these are publicly available.…”