Differential Membrane Interactions of Saposins A and C: IMPLICATIONS FOR THE FUNCTIONAL SPECIFICITY

Xiaoyang Qi,Gregory A Grabowski

doi:10.1074/jbc.m101075200

Xiaoyang Qi, Gregory A Grabowski

Open Access

https://doi.org/10.1074/jbc.m101075200

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Abstract

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 beta-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 beta-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.

Highlights

Saposins are small, heat-stable glycoprotein activators of lysosomal glycosphingolipid hydrolases that derive from a single precursor, prosaposin, by proteolytic cleavage
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
Together with spectral shifting experiments, the results indicate that the middle region of saposin C around Ser-37 was exposed to the aqueous phase, whereas the aminoand carboxyl-terminals regions were associated with membrane lipids

Summary

IMPLICATIONS FOR THE FUNCTIONAL SPECIFICITY*

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. A multiple ␣-helical bundle motif, characterized by a threeconserved-disulfide structure and several amphipathic peptides, is found in the saposins and in saposin-like proteins and domains, i.e. NK-lysin, surfactant-associated protein B (SP-B), acid sphingomyelinase, acyloxylacyl hydrolase, plant aspartic proteases and, pore-forming peptides (amoebapores) (for review, see Ref. 16). Negatively charged phospholipid bilayers are required for the interactions of saposin A and/or C with acid ␤-glucosidase in vitro. Mutant saposins provided insight into the molecular basis for the conformational requirement of membrane-bound saposins to the specific activation functions

EXPERIMENTAL PROCEDURES

RESULTS

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DISCUSSION

Deconvolution calculations