Crystal structures of saposins A and C

Abstract

Saposins Aa nd Ca re sphingolipid activator proteins requiredf or thel ysosomal breakdowno f galactosylceramide and glucosylceramide,r espectively.T he saposins interact with lipids, leading to an enhanced accessibility of thel ipid headgroups to their cognate hydrolases. We have determinedt he crystal structures of humans aposinsAandCt o2 .0 A˚and 2.4Å ,r espectively,a nd both reveal the compact, monomeric saposin fold. We confirmed that these twoproteins were monomeric in solution at pH 7.0b yanalytical centrifugation. However, at pH 4.8,inthe presence of thedetergent C 8 E 5 ,saposin Aa ssembled intod imers, while saposin Cf ormed trimers. Saposin Bw as dimeric under allc onditions tested.T he self-associationo ft he saposins is likelyt ob er elevantt oh ow theses mall proteins interact with lipids, membranes, andh ydrolase enzymes.Keywords: saposins;X -ray crystallography; analyticalu ltracentrifugation; protein-detergent interactions Saposins A, B, C, and Da re small, nonenzymatic proteins required for the breakdown of glycosphingolipids within the lysosome (Kolter and Sandhoff2005). They are derived from the proteolytic processing of the precursor protein prosaposin, producing the four individual saposins. The four saposin domains contained within prosaposin most likely arose from two tandem duplications of an ancestral gene into one single copy gene (Hazkani-Covoetal. 2002). Each saposin ''activates''the breakdown of particular lipid substrates by facilitating the access of the lipid headgroups to the active sites of cognate hydrolases. It is generally believed that in the absence of sphingolipid activator proteins, the oligosaccharide chains of the membranebound lipids do not extend far enough into the lysosomal lumen to be accessible to the active sites of the hydrolases. Mechanistically,t he saposins appear to activate lipid hydrolysis by solubilizing the lipid substrates or possibly by destabilizing the membrane structure (Vaccaro et al. 1993(Vaccaro et al. , 1995(Vaccaro et al. , 1997Wilkening et al. 1998;Salvioli et al. 2000). Article publishedonline aheado fp rint.A rticle and publicationd ate area th ttp://www.proteinscience.org/cgi

Saposin B is the dominant saposin that facilitates lipid binding to human CD1d molecules

“…Saposins A, C, and D have high homology to each other, with pairwise sequence identities ranging from 34% to 39%. Saposin B is more distantly related, with sequence identities of 22%, 15%, and 21% with saposins A, C, and D, respectively (35). The more important difference is in the lipid activation mechanism.…”

“…The affinity of saposin B for lipid bilayers is low at both neutral and acidic pH, consistent with its function as an extractor/solubilizer that has only transient interactions with the membrane. Furthermore, low pH affects the structure of saposin B differently from the way it affects saposin A or C (35). Saposins A and C, as well as most other saposin-like proteins, have a compact, monomeric fold that buries a small hydrophobic core, whereas saposin B, like GM2 activator protein, has a major conformational variant that forms a homodimer that has a large hydrophobic cavity (26).…”

“…Saposins A and C, as well as most other saposin-like proteins, have a compact, monomeric fold that buries a small hydrophobic core, whereas saposin B, like GM2 activator protein, has a major conformational variant that forms a homodimer that has a large hydrophobic cavity (26). For saposins A and C, low pH and the presence of detergent is required for dimer formation, whereas saposin B is dimeric at both neutral and low pH and in the presence or absence of detergent (35). These data again support the argument that the two categories of saposins interact differently with lipids in a manner relevant to the superior capacity of saposin B to facilitate lipid binding to CD1d.…”

Molecular imaging of membrane interfaces reveals mode of β-glucosidase activation by saposin C

“…sapC was labeled with the Alexa Fluor 546 (A546) fluorophore at residue 22. This position is in a short loop between helices ␣1 and ␣2 of the protein and is the site normally glycosylated in the natural protein (24). GCase was chemically labeled using an amine-reactive Alexa Fluor 488 (A488) succinimidyl conjugate.…”

Novel CDNF/MANF family of neurotrophic factors

“…Interestingly, the saposin-like domain of CDNF crystallized as a dimer at pH 4.6, whereas the full-length, mature MANF crystallized as a monomer at neutral pH (Parkash et al, 2009). Saposin C and Saposin A form oligomers in the presence of a detergent at low pH 4.8 (Ahn et al, 2006). Acidic pH also increases the ability of Saposin C and Saposin D to bind lipid vesicles and destabilize phospholipid-containing membranes (Vaccaro et al, 1995).…”