Conformational and Amino Acid Residue Requirements for the Saposin C Neuritogenic Effect

Qi, Xiaoyang; Kondoh, Keiji; Krusling, David; Kelso, Gregory J.; Leonova, Tatyana; Grabowski, Gregory A.

doi:10.1021/bi990079o

Cited by 23 publications

(16 citation statements)

References 37 publications

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“…The sapC-induced increase in GCase activity may also be related to a higher intrinsic activity of the enzyme within an activator complex, possibly involving a conformational change in the enzyme (44), as seen in the case of pancreatic lipase. This is supported by the fact that sapC can increase the hydrolysis of soluble substrate analogs by up to 17-fold in the presence of large unilamellar vesicles (12,33).…”

Section: Discussionmentioning

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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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%

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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“…Serine was chosen as an isosteric substitution for cysteines 4,16,18,23,126, and 248. The first four cysteines participate in disulfide bonds as follows: Cys 4 -Cys 16 and Cys 18 -Cys 23 . Cys 126 , Cys 248 , and Cys 342 are free (see "Results").…”

Section: Methodsmentioning

confidence: 99%

“…Additional Mutations in GCase and Their Effect on GCase Properties-The disulfide bonds in wild-type GCase are between residues Cys 4 and Cys 16 and residues Cys 18 and Cys 23 . These are located in an isolated loop structure (domain 1) in the crystal (33) (Fig.…”

Section: Volume 281 • Number 7 • February 17 2006mentioning

confidence: 99%

Analyses of Variant Acid β-Glucosidases

Liou

Kazimierczuk

Zhang

et al. 2006

Journal of Biological Chemistry

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132

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Acid ␤-glucosidase (GCase) is a 497-amino acid, membrane-associated lysosomal exo-␤-glucosidase whose defective activity leads to the Gaucher disease phenotypes. To move toward a structure/ function map for disease mutations, 52 selected single amino acid substitutions were introduced into GCase, expressed in an insect cell system, purified, and characterized for basic kinetic, stability, and activator response properties. The variant GCases from Gaucher disease patients and selected variant GCases from the mouse had decreased relative k cat and differential effects on active site binding and/or attachment of mechanism-based covalent (conduritol B epoxide) or reversible (deoxynojirimycin derivatives) inhibitors. A defect in negatively charged phospholipid activation was present in the majority of variant GCases but was increased in two, N370S and V394L. Deficits in saposin C enhancement of k cat were present in variant GCases involving residues 48 -122, whereas ϳ2-fold increases were obtained with the L264I GCase. About 50% of variant GCases each had wild-type or increased sensitivity to in vitro cathepsin D digestion. Mapping of these properties onto the crystal structures of GCase indicated wide dispersion of functional properties that can affect catalytic function and stability. Site-directed mutagenesis of cysteine residues showed that the disulfide bonds, Cys 4 -Cys 16 and Cys 18 -Cys 23 , and a free Cys 342 were essential for activity; the free Cys 126 and Cys 248 were not. Relative k cat was highly sensitive to a His substitution at Arg 496 but not at Arg 495 . These studies and high phylogenetic conservation indicate localized and general structural effects of Gaucher disease mutations that were not obvious from the nature of the amino acid substitution, including those predicted to be nondisruptive (e.g. Val 3 Leu). These results provide initial studies for the engineering of variant GCases and, potentially, molecular chaperones for therapeutic use.

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“…The reaction mixtures were in 0.005 M sodium citrate, 0.01 M sodium phosphate, pH 4.7, to reduce noise at wavelengths below 200 nm. Data acquisition and deconvolution calculations were done as described (31,41). The deconvolution programs were from Jasco with reference to an average of seven x-ray structures of known proteins, i.e.…”

Section: Methodsmentioning

confidence: 99%

Differential Membrane Interactions of Saposins A and C

Grabowski

2001

Journal of Biological Chemistry

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Saposins are small, heat-stable glycoprotein activators of lysosomal glycosphingolipid hydrolases that derive from a single precursor, prosaposin, by proteolytic cleavage. Three of these saposins (B, C, and D) share common structural features including a lack of tryptophan, a single glycosylation sequence, the presence of three conserved disulfide bonds, and a common multiamphipathic helical bundle motif. Saposin A contains an additional glycosylation site and a single tryptophan. The oligosaccharides on saposins are not required for in vitro activation functions. Saposins A and C were produced in Escherichia coli to contain single tryptophans at various locations to serve as intrinsic fluorescence reporters, i.e. as topological probes, for interaction with phospholipid membranes. Maximum emission shifts, aqueous and solid quenching, and resonance energy transfer were quantified by fluorescence spectroscopy. Amphipathic helices at the amino-and carboxyl termini of saposins A and C were shown to insert into the lipid bilayer to about five carbon bond lengths. In comparison, the middle region of saposins A or C were either embedded in the bilayer or solvent-exposed, respectively. Conformational changes of saposin C induced by phosphatidylserine interaction suggested the reorientation of functional helical domains. Differential interaction models are proposed for the membrane-bound saposins A and C. By site-directed mutagenesis of saposin A and C, their membrane topological structures were correlated with their activation effects on acid ␤-glucosidase. These findings show that proper orientation of the middle segment of saposin C to the outside of the membrane surface is critical for its specific and multivalent interaction with acid ␤-glucosidase. Such membrane interactions and orientations of the saposins determine the proximity of their activation and/or binding sites to lysosomal hydrolases or lipoid substrates.Saposins, a family of small (ϳ80 amino acids) heat-stable glycoproteins, are essential for the in vivo hydrolytic activity of several lysosomal enzymes in the catabolic pathway of glycosphingolipids (1-3). Four members of saposins, A, B, C, and D, are proteolytically derived from a single precursor protein, termed prosaposin (4 -8). The primary sequences of saposins are highly homologous with ϳ60% amino acid similarity. In addition, each of the saposins has six conserved cysteines that form three intradomain disulfide bridges whose placements are identical (9). In a native state, five consensus N-glycosylation sequences in prosaposin are occupied by oligosaccharide chains (10 -12). Each of saposins B, C, and D has one such sequence, whereas saposin A has two. Nonglycosylated saposins retain their respective activation effects using in vitro assays (12-15).A multiple ␣-helical bundle motif, characterized by a threeconserved-disulfide structure and several amphipathic peptides, is found in the saposins and also in saposin-like proteins and domains, i.e. NK-lysin, surfactant-associated protein B (SP-B), 1 a...

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Conformational and Amino Acid Residue Requirements for the Saposin C Neuritogenic Effect

Cited by 23 publications

References 37 publications

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

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

Analyses of Variant Acid β-Glucosidases

Differential Membrane Interactions of Saposins A and C

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