Xiaoyang Qi

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

CD1d molecules bind lipid antigens in the endocytic pathway, and access to the pathway is important for the development of CD1d-restricted natural killer T (NKT) cells. Saposins, derived from a common precursor, prosaposin, are small, heat-stable lysosomal glycoproteins required for lysosomal degradation of sphingolipids. Expression of prosaposin is required for efficient lipid binding and recognition of human CD1d molecules by NKT cells. Despite high sequence homology among the four saposins, they have different specificities for lipid substrates and different mechanisms of action. To determine the saposins involved in promoting lipid binding to CD1d, we expressed prosaposin deletion mutants lacking individual saposins in prosaposin-negative, CD1d-positive cells. No individual saposin proved to be absolutely essential, but the absence of saposin B resulted in the lowest recognition of α-galactosylceramide by NKT cells. When recombinant exogenous saposins were added to the prosaposin-negative cells, saposin B was the most efficient in restoring CD1d recognition. Saposin B was also the most efficient in mediating α-galactosylceramide binding to recombinant plate-bound CD1d and facilitating NKT cell activation. Saposin B could also mediate lipid binding to soluble CD1d molecules in a T cell-independent assay. The optimal pH for saposin B-mediated lipid binding to CD1d, pH 6, is higher than that of lysosomes, suggesting that saposin B may facilitate lipid binding to CD1d molecules throughout the endocytic pathway.

Proteolytic processing patterns of prosaposin in insect and mammalian cells.

Prosaposin is a multifunctional protein encoded at a single locus in humans and mice. The precursor contains, in tandem, four glycoprotein activators or saposins, termed A, B, C, and D, that are essential for specific glycosphingolipid hydrolase activities. Prosaposin appears to be a potent neurotrophic factor. To explore the proteolytic processing from prosaposin to mature activator proteins, metabolic labeling was done with human prosaposin expressed in insect cells, human fibroblasts, neuronal stem cells (NT2) and retinoic acid-differentiated NT2 neurons. In all cell types, the major processing pathway was through a tetrasaposin, A-B-C-D, from which saposin A was then removed. In mammalian cells monosaposins were derived from the trisaposin B-C-D by cleavage to the disaposins, B-C and C-D, that were processed to monosaposins. In insect cells the major end products were the disaposins, with A-B and C-D derived from the tetrasaposin, A-B-C-D, or with B-C and C-D derived from the trisaposin, B-C-D. In insect and mammalian cells, the nonsignal NH2-terminal peptide preceding saposin A (termed Nter) was usually removed prior to saposin A cleavage. In NT2-derived differentiated neurons, precursor tetrasaposins containing A-B-C-D were secreted with and without Nter. Immunofluorescence studies using prosaposin-specific antisera showed large steady state amounts of uncleaved prosaposin in Purkinje cells, cortical neurons, and other specific cell types in adult mice. These studies indicate that prosaposin processing is highly regulated at a proteolytic level to produce prosaposin, tetrasaposins, or mature monosaposins in specific mammalian cells.

Acid beta-glucosidase: intrinsic fluorescence and conformational changes induced by phospholipids and saposin C.

Acid beta-glucosidase is a lysosomal membrane protein that cleaves the O-beta-D-glucosidic linkage of glucosylceramide and aryl-beta-glucosides. Full activity reconstitution of the pure enzyme requires phospholipids and saposin C, an 80 aa activator protein. The deficiency of the enzyme or activator leads to Gaucher disease. A conformational change of acid beta-glucosidase is shown to accompany activity reconstitution by selected phospholipids or, particularly, phospholipid/saposin C complexes by intrinsic fluorescence spectral shifts, fluorescence quenching, and circular dichroism (CD). Negatively charged phospholipid (NCP) interfaces with unsaturated fatty acid acyl chains (UFAC) induced concordant blue-shifts in tryptophanyl fluorescence spectra and a loss of beta-strand structure by CD. The enzyme required an unsaturated fatty acid acyl chain in proximity (10-11 A) within liposomal membranes for activation, fluorescence blue-shifts, and changes in CD spectra. Activity enhancements were greatest when UFAC and the negatively charged headgroup were present on the same phospholipid. NCPs with UFAC protected the enzyme from fluorescence quenching by aqueous agents (I-, Cs+, acrylamide, TEMPO). Phosphatidylcholine with doxyl spin-labeled fatty acid acyl chains at carbons 7, 10, or 16 quenched enzyme fluorescence only when in NCP/PC liposomes. Saposin C (Trp-free) induced additional activity and fluorescence spectral changes in the enzyme only in the presence of NCP liposomes containing UFA. CD spectral changes indicated saposin C and acid beta-glucosidase interaction only in the presence of NCPs with UFA. These studies show that acid beta-glucosidase requires interfaces composed of NCPs, containing UFAC, for penetration into the outer leaflet of membranes. Furthermore, this interaction induces essential conformational changes for saposin C binding and further enhancement of acid beta-glucosidase catalytic activity.

Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

Prosaposin directly interacts with progranulin and facilitates progranulin lysosomal trafficking via the trafficking receptors M6PR and LRP1, independent of the previously identified progranulin trafficking pathway mediated by sortilin.

Cancer-Selective Targeting and Cytotoxicity by Liposomal-Coupled Lysosomal Saposin C Protein

Purpose: Saposin C is a multifunctional protein known to activate lysosomal enzymes and induce membrane fusion in an acidic environment. Excessive accumulation of lipid-coupled saposin C in lysosomes is cytotoxic. Because neoplasms generate an acidic microenvironment, caused by leakage of lysosomal enzymes and hypoxia, we hypothesized that saposin C may be an effective anticancer agent. We investigated the antitumor efficacy and systemic biodistribution of nanovesicles comprised of saposin C coupled with dioleoylphosphatidylserine in preclinical cancer models. Experimental Design: Neuroblastoma, malignant peripheral nerve sheath tumor and, breast cancer cells were treated with saposin C–dioleoylphosphatidylserine nanovesicles and assessed for cell viability, ceramide elevation, caspase activation, and apoptosis. Fluorescently labeled saposin C–dioleoylphosphatidylserine was i.v. injected to determine in vivo tumor-targeting specificity. Antitumor activity and toxicity profile of saposin C–dioleoylphosphatidylserine were evaluated in xenograft models. Results: Saposin C–dioleoylphosphatidylserine nanovesicles, with a mean diameter of ∼190 nm, showed specific tumor-targeting activity shown through in vivo imaging. Following i.v. administration, saposin C–dioleoylphosphatidylserine nanovesicles preferentially accumulated in tumor vessels and cells in tumor-bearing mice. Saposin C–dioleoylphosphatidylserine induced apoptosis in multiple cancer cell types while sparing normal cells and tissues. The mechanism of saposin C–dioleoylphosphatidylserine induction of apoptosis was determined to be in part through elevation of intracellular ceramides, followed by caspase activation. In in vivo models, saposin C–dioleoylphosphatidylserine nanovesicles significantly inhibited growth of preclinical xenografts of neuroblastoma and malignant peripheral nerve sheath tumor. I.v. dosing of saposin C–dioleoylphosphatidylserine showed no toxic effects in nontumor tissues. Conclusions: Saposin C–dioleoylphosphatidylserine nanovesicles offer promise as a novel, nontoxic, cancer-targeted, antitumor agent for treating a broad range of cancers. (Clin Cancer Res 2009;15(18):5840–51)

Phospholipid vesicle fusion induced by saposin C.

Saposin C is a small Trp-free, multifunctional glycoprotein that enhances the hydrolytic activity of acid beta-glucosidase in lysosomes. Saposin Cs functions have been shown to include neuritogenic/neuroprotection effects and membrane fusion induction. Here, the mechanism and kinetics of saposin Cs fusogenic activity were evaluated by fluorescence spectroscopic methods including dequenching, fluorescence resonance energy transfer, and stopped-flow analyses. Trp or dansyl groups were introduced as fluorescence reporters into selected sites of saposin C to serve as topological probes for protein-protein and protein-membrane interactions. Saposin C induction of liposomal vesicle enlargement was dependent upon anionic phospholipids and acidic pH. The initial fusion burst was completed in the timeframe of a few seconds to minutes and was dependent upon the unsaturated anionic phospholipid content. Two events were associated with saposin C-membrane interaction: membrane insertion of the saposin C terminal helices and reorientation of its central helical region. The latter conformational change likely exposed a binding site for saposins anchored on vesicles. Addition of selected saposin C peptides prior to intact saposin C in reaction mixtures abolished the liposomal fusion. These results indicated that saposin-membrane and saposin-saposin interactions are needed for the fusion process.

Molecular and cell biology of acid β-glucosidase and prosaposin

Publisher Summary The chapter discusses the molecular biologic, biochemical, and cell biology approaches to evaluate the disordered metabolism of the proteins and lipids that have resulted in Gaucher disease. Following is a review heavily focused on some of the effects, with reference to other seminal contributions, to the understanding of Gaucher disease, the acid β-glucosidase locus, and the sphingolipid activator locus, termed “prosaposin.” What is the evident from the work and others, is the enormous amount yet to be fully understood about the biological function of relatively simple genetic loci and the resultant gene products that result in the lysosomal disease termed “Gaucher disease.” Many issues still require resolution: (1) the relevance of the scheme to the natural environment in the cell, (2) the identification of natural acute promyelocytic leukemia (APL) activators, probably bi phosphate, (3) the native form (monomeric vs dimeric) for the interaction of the enzyme and saposin C, and (4) the relationship of the transcriptional and translational control, and the stoichiometry and activity of the enzyme and saposin C. Each of these issues has relevance to the biology of glucosylceramide and the therapy of acid β-glucosidase deficiency states. Continued elucidation of this enzymes role, of the need and functions of its cofactors (the saposins), and of the molecular enzymology, involved with its catalytic activity, should provide a rich and fertile resource for the general mechanisms of the reactions for these enzyme groups.

Systemic Delivery of SapC-DOPS Has Antiangiogenic and Antitumor Effects Against Glioblastoma

Saposin C-dioleoylphosphatidylserine (SapC-DOPS) nanovesicles are a nanotherapeutic which effectively target and destroy cancer cells. Here, we explore the systemic use of SapC-DOPS in several models of brain cancer, including glioblastoma multiforme (GBM), and the molecular mechanism behind its tumor-selective targeting specificity. Using two validated spontaneous brain tumor models, we demonstrate the ability of SapC-DOPS to selectively and effectively cross the blood-brain tumor barrier (BBTB) to target brain tumors in vivo and reveal the targeting to be contingent on the exposure of the anionic phospholipid phosphatidylserine (PtdSer). Increased cell surface expression of PtdSer levels was found to correlate with SapC-DOPS-induced killing efficacy, and tumor targeting in vivo was inhibited by blocking PtdSer exposed on cells. Apart from cancer cell killing, SapC-DOPS also exerted a strong antiangiogenic activity in vitro and in vivo. Interestingly, unlike traditional chemotherapy, hypoxic cells were sensitized to SapC-DOPS-mediated killing. This study emphasizes the importance of PtdSer exposure for SapC-DOPS targeting and supports the further development of SapC-DOPS as a novel antitumor and antiangiogenic agent for brain tumors.

Targeting and Cytotoxicity of SapC-DOPS Nanovesicles in Pancreatic Cancer

Only a small number of promising drugs target pancreatic cancer, which is the fourth leading cause of cancer deaths with a 5-year survival of less than 5%. Our goal is to develop a new biotherapeutic agent in which a lysosomal protein (saposin C, SapC) and a phospholipid (dioleoylphosphatidylserine, DOPS) are assembled into nanovesicles (SapC-DOPS) for treating pancreatic cancer. A distinguishing feature of SapC-DOPS nanovesicles is their high affinity for phosphatidylserine (PS) rich microdomains, which are abnormally exposed on the membrane surface of human pancreatic tumor cells. To evaluate the role of external cell PS, in vitro assays were used to correlate PS exposure and the cytotoxic effect of SapC-DOPS in human tumor and nontumorigenic pancreatic cells. Next, pancreatic tumor xenografts (orthotopic and subcutaneous models) were used for tumor targeting and therapeutic efficacy studies with systemic SapC-DOPS treatment. We observed that the nanovesicles selectively killed human pancreatic cancer cells in vitro by inducing apoptotic death, whereas untransformed cells remained unaffected. This in vitro cytotoxic effect correlated to the surface exposure level of PS on the tumor cells. Using xenografts, animals treated with SapC-DOPS showed clear survival benefits and their tumors shrank or disappeared. Furthermore, using a double-tracking method in live mice, we showed that the nanovesicles were specifically targeted to orthotopically-implanted, bioluminescent pancreatic tumors. These data suggest that the acidic phospholipid PS is a biomarker for pancreatic cancer that can be effectively targeted for therapy utilizing cancer-selective SapC-DOPS nanovesicles. This study provides convincing evidence in support of developing a new therapeutic approach to pancreatic cancer.

Saposin C Coupled Lipid Nanovesicles Enable Cancer-Selective Optical and Magnetic Resonance Imaging

PurposeNanovesicles composed of the phospholipid dioleylphosphatidylserine (DOPS) and a fusogenic protein, saposin C (SapC), selectively target and induce apoptotic cell death in a variety of human cancer cells in vitro and in vivo. We tested whether such tumor-homing nanovesicles are capable of delivering fluorescent probes and magnetic resonance (MR) contrast agents to cancerous tissue to aid in earlier detection and improve visualization.ProceduresSapC–DOPS nanovesicles labeled with either a far-red fluorescent probe (CellVue® Maroon, CVM) or conjugated with a dextran coated MR contrast agent, ultrasmall superparamagnetic iron oxide (USPIO), were systemically administrated into xenografts for tumor detection using optical and MR imaging systems.ResultsSapC–DOPS nanovesicles were effectively detected in vivo in tumor-bearing animals using both optical and MR imaging techniques, thereby demonstrating the cancer-selective properties of these nanovesicles.ConclusionsSapC–DOPS nanovesicles offer promise as a new and robust theranostic agent for broad cancer-selective detection, visualization, and potential therapy.