Expression, purification, crystallization, and preliminary X-ray analysis of recombinant human saposin B

Ahn, Victoria E.; Faull, Kym F.; Whitelegge, Julian P.; Higginson, Jason; Fluharty, Arvan L.; Privé, Gilbert G.

doi:10.1016/s1046-5928(02)00597-1

Cited by 19 publications

(26 citation statements)

References 33 publications

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“…However, earlier reports have shown that nonglycosylated saposins retain their lipid-binding and enzyme activation effects (39), and recombinant sapC expressed in Escherichia coli was shown to have similar properties to sapC purified from natural tissues (40). Notably, E. coli-expressed saposin B (nonglycosylated) was as active as glycosylated human saposin B purified from urine in an in vitro activity assay (10) and could functionally complement saposin B-deficient human fibroblast cells (41). We have shown in a previous report (15) that both unglycosylated sapC and Alexalabeled N22C sapC have similar lipid extraction activities on supported bilayers.…”

Section: Discussionmentioning

confidence: 99%

“…Different saposins and enzymes are hypothesized to fall into a particular category of enzyme activation. For example, saposin B is thought to be a detergent-like lipid solubilizer (9,10), whereas sapC may act as a liftase at the bilayer surface (2,11,12). Strong binding of sapC to lipid bilayers was shown by using coprecipitation assays (13) and NMR spectroscopic titrations (14).…”

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confidence: 99%

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

Alattia

Shaw²,

Yip³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Acid ␤-glucosidase (GCase) is a soluble lysosomal enzyme responsible for the hydrolysis of glucose from glucosylceramide and requires activation by the small nonenzymatic protein saposin C (sapC) to gain access to the membrane-embedded glycosphingolipid substrate. We have used in situ atomic force microscopy (AFM) with simultaneous confocal and epifluorescence microscopies to investigate the interactions of GCase and sapC with lipid bilayers. GCase binds to sites on membranes transformed by sapC, and enzyme activity occurs at loci containing both GCase and sapC. Using FRET, we establish the presence of GCase/sapC and GCase/ product contacts in the bilayer. These data support a mechanism in which sapC locally alters regions of bilayer for subsequent attack by the enzyme in stably bound protein complexes.atomic force microscopy ͉ confocal microscopy ͉ FRET ͉ interfacial catalysis ͉ lipid storage disease

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Alattia

Shaw²,

Yip³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…The expression, purification, and crystallization of the selenomethionine-substituted protein has been described (15). Briefly, human saposin B was expressed in Escherichia coli strain AD494(DE3) and purified with heat treatment, followed by anion exchange, size-exclusion, and hydrophobic interaction chromatographies.…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of saposin B reveals a dimeric shell for lipid binding

Ahn

Faull²,

Whitelegge³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Saposin B is a small, nonenzymatic glycosphingolipid activator protein required for the breakdown of cerebroside sulfates (sulfatides) within the lysosome. The protein can extract target lipids from membranes, forming soluble protein-lipid complexes that are recognized by arylsulfatase A. The crystal structure of human saposin B reveals an unusual shell-like dimer consisting of a monolayer of ␣-helices enclosing a large hydrophobic cavity. Although the secondary structure of saposin B is similar to that of the known monomeric members of the saposin-like superfamily, the helices are repacked into a different tertiary arrangement to form the homodimer. A comparison of the two forms of the saposin B dimer suggests that extraction of target lipids from membranes involves a conformational change that facilitates access to the inner cavity.T he saposins are a group of four structurally related activator proteins that function in conjunction with hydrolase enzymes for the stepwise breakdown of glycosphingolipids within the lysosome (1, 2). These proteins act by modifying the local environment of target lipids, and ''activate'' the breakdown of their substrates by presenting them in a form in which the lipid scissile bonds are accessible to the active sites of the specific enzymes. Presumably, the hydrolases cannot form a catalytic complex with the glycosphingolipids when these are in unmodified membrane bilayers.Each of the four saposins activates one or more lipid exohydrolases. For example, saposin B (also known as the cerebroside sulfate activator, or CS-Act) facilitates the hydrolysis of the sulfate group from cerebroside sulfate by arylsulfatase A, resulting in the formation of galactosylceramide (3). This glycolipid is then catabolized to ceramide by ␤-galactosylceramidase in a reaction activated by saposin C. There seems to be more than one mechanism of activation by the saposins, and this may further depend on the particular target lipid. Thus, saposin B is able to extract and solubilize cerebroside sulfates from membranes, allowing arylsulfatase A to act on the small, diffusible protein-lipid complexes. Saposin B may also have a physiological role in activating the hydrolysis of the ganglioside GM1 to GM2 by lysosomal ␤-galactosidase, and in this case, the activator may act by modifying the local lipid structure at the membrane surface to allow catalysis to proceed (4).The saposins belong to a large and diverse family of small, cysteine-rich proteins that share a common ability to interact with membranes but act in a wide variety of functions. Other members of the ''saposin-like'' superfamily of proteins include the lung surfactant-associated protein B (SP-B), the tumorolytic protein NK-lysin, granulysin, the pore-forming amoebapores, and the membrane-targeting domain of some enzymes (5, 6). The saposin-like proteins are Ϸ80 residues in length and have a characteristic pattern of conserved cysteines ( Fig. 1A; refs. 7 and 8). To date, the three-dimensional modeling of the saposins and other members of the sapo...

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“…15,38 For fluorescence labeling, the saposin C glycosylation site residue Asn22 was mutated to a cysteine using the QuikChange sitedirected mutagenesis kit (Stratagene). Protein purification was carried out as with the native protein with the presence of 1 mM DL-dithiothreitol in all buffers, which was sufficient to maintain a reduced state for the N22C residue, but not enough to reduce the three native intrachain disulfide bonds.…”

Section: Cloning Expression and Purificationmentioning

confidence: 99%