Saposin B mobilizes lipids from cholesterol‐poor and bis(monoacylglycero)phosphate‐rich membranes at acidic pH

Abstract

Sphingolipid activator proteins (SAPs), GM2 activator protein (GM2AP) and saposins (Saps) A-D are small, enzymatically inactive glycoproteins of the lysosome. Despite of their sequence homology, these lipid-binding and -transfer proteins show different specificities and varying modes of action. Water-soluble SAPs facilitate the degradation of membrane-bound glycosphingolipids with short oligosaccharide chains by exohydrolases at the membrane-water interface. There is strong evidence that degradation of endocytosed components of the cell membrane takes place at intraendosomal and intralysosomal membranes. The inner membranes of the lysosome differ from the limiting membrane of the organelle in some typical ways: the inner vesicular membranes lack a protecting glycocalix, and they are almost free of cholesterol, but rich in bis(monoacylglycero)phosphate (BMP), the anionic marker lipid of lysosomes. In this study, we prepared glycosylated Sap-B free of other Saps by taking advantage of the Pichia pastoris expression system. We used immobilized liposomes as a model for intralysosomal vesicular membranes to probe their interaction with recombinantly expressed Sap-B. We monitored this interaction using SPR spectroscopy and an independent method based on the release of radioactively labelled lipids from liposomal membranes. We show that, after initial binding, Sap-B disturbs the membrane structure and mobilizes the lipids from it. Lipid mobilization is dependent on an acidic pH and the presence of anionic lipids, whereas cholesterol is able to stabilize the liposomes. We also show for the first time that glycosylation of Sap-B is essential to achieve its full lipid-extraction activity. Removal of the carbohydrate moiety of Sap-B reduces its membrane-destabilizing quality. An unglycosylated Sap-B variant, Asn215His, which causes a fatal sphingolipid storage disease, lost the ability to extract membrane lipids at acidic pH in the presence of BMP.Abbreviations BMP, bis(monoacylglycero)phosphate; GM2AP, ganglioside GM2 activator protein; PtdCho, phosphatidylcholine; pSap, Sap precursor; RU, response unit; SAP, sphingolipid activator protein; Sap, saposin.

Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation

“…To investigate the role of saposins as lipid transfer proteins independently of any storage phenotype, we designed cell-free assays and measured binding of Escherichia coli-purified recombinant saposins to synthetic lipids and subsequent loading of CD1d molecules. Although there is evidence that glycosylation influences the ability of recombinant saposins to mobilize lipids from membranes and liposomes (51,52), nonglycosylated proteins have been shown to retain comparable lipid binding and hydrolase-activating capacity. Despite high homology, the four saposins have different lipid specificities, as demonstrated by the phenotype of patients with selective deficiency of individual molecules (53).…”

Saposin B-dependent Reconstitution of Arylsulfatase A Activity in Vitro and in Cell Culture Models of Metachromatic Leukodystrophy

“…Native Sap B was purified from pig kidney and human urine, respectively (11). Recombinant human Saps A, B, C, and D were produced using the Pichia pastoris expression system (12,13). Prosaposin was purified as described (14).…”

“…(12). After reacting under standard conditions, the assay was terminated by the addition of 0.5 ml of chloroform/methanol (1/1, v/v).…”

“…The radioactivity of the suspensions was determined in triplicate. Metabolic studies with prosaposindeficient fibroblasts were performed as described (12). For metabolic studies with murine kidney cells, different combinations of rhASA, Sap B, and prosaposin were added.…”

“…5A). In the cell culture assay the ability of externally added Sap B (25 g/ml) to compensate the defective sulfatide catabolism of prosaposin-deficient human fibroblasts was exploited (12). Feeding of prosaposin-deficient cells revealed no difference in the corrective capacity of unmodified Sap B and […”