It is now established that CD1 molecules present lipid antigens to T cells, although it is not clear how the exchange of lipids between membrane compartments and the CD1 binding groove is assisted. We report that mice deficient in prosaposin, the precursor to a family of endosomal lipid transfer proteins (LTP), exhibit specific defects in CD1d-mediated antigen presentation and lack Vα14 NKT cells. In vitro, saposins extracted monomeric lipids from membranes and from CD1, thereby promoting the loading as well as the editing of lipids on CD1. Transient complexes between CD1, lipid, and LTP suggested a "tug-of-war" model in which lipid exchange between CD1 and LTP is on the basis of their respective affinities for lipids. LTPs constitute a previously unknown link between lipid metabolism and immunity and are likely to exert a profound influence on the repertoire of self, tumor, and microbial lipid antigens.CD1 molecules have evolved a unique hydrophobic binding groove that binds lipid antigens in both the secretory and endosomal compartments for presentation to T lymphocytes (1). In mice, the main population of CD1-restricted T cells, called Vα14 NKT cells, express a † These authors contributed equally to this work. To whom correspondence should be addressed. abendela@bsd.uchicago.edu (A.B.); lteyton@scripps.edu (L.T.). * These authors contributed equally to this work. CIHR Author Manuscript CIHR Author Manuscript CIHR Author Manuscriptsemi-invariant Vα14-Jα18/Vβ8 T cell receptor (TCR). These cells exhibit reactivity against CD1d in combination with endogenous ligands (2) that can be mimicked by α-galactosylceramide (αGC) (3). This population is conserved among mammalian species and regulates immune responses (4, 5). Like human CD1b-restricted T cells specific for mycobacterial glycolipids (6), Vα14 NKT cells are dependent on endosomal trafficking of CD1d for natural antigen recognition (7-10). Other endogenous or exogenous antigens do not require endosomal trafficking, however, suggesting that loading may be achieved in distinct cellular compartments depending on the nature of the antigen (9,11,12). CD1 endosomal trafficking is tightly controlled by cytoplasmic tail-encoded tyrosine-containing motifs binding adaptor protein 2 and 3 (AP-2 and AP-3) complexes, as well as by association with the invariant chain (Ii) or Ii/major histocompatibility (MHC) class II complexes (13-16).Because lipids are integral membrane components that might require lipid transfer proteins (LTP) (17) for extraction, we investigated whether various families of LTP might assist antigen presentation. We focused on the Vα14 NKT cell endosomal-dependent system to test the possible involvement of LTPs localized in the endocytic pathway, such as saposins (18) (Fig. 1D). These findings demonstrate a selective defect in the development of Vα14 NKT cells in the absence of prosaposin.Many NKT cell hybridomas can be activated in vitro to release interleukin-2 (IL-2) by coculture with fresh CD1d-expressing cells, such as thymocytes, which are...

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.

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)

Surfactant protein B (SP-B) is a hydrophobic, 79 amino acid peptide that regulates the structure and function of surfactant phospholipid membranes in the airspaces of the lung. Addition of SP-B to liposomes composed of DPPC/PG (7:3) leads to membrane binding, destabilization, and fusion, ultimately resulting in rearrangement of membrane structure. The goal of this study was to map the fusogenic and lytic domains of SP-B and assess the effects of altered fusion and lysis on surface activity. Synthetic peptides were generated to predicted helices and/or interhelical loops of SP-B and tested for fusion, lytic, and surface activities. The N-terminal half of SP-B (residues 1-37), which includes the nonhelical N-terminal amino acids in addition to helices 1 and 2, promoted rapid liposome fusion whereas shorter peptides were significantly less effective. The requirements for optimal surface tension reduction were similar to those for fusion; in contrast, helix 1 (residues 7-22) alone was sufficient for liposome lysis. The C-terminal half of SP-B (residues 43-79), which includes helices 3, 4, and 5, exhibited significantly lower levels of fusogenic, lytic, and surface tension reducing activities compared to the N-terminal region. These results indicate that SP-B fusion, lytic and surface activities map predominantly to the N-terminal half of SP-B. Amino acid substitutions in synthetic peptides corresponding to the N-terminal half of SP-B indicated that, in general, decreased fusion or lytic activities were associated with altered surface tension reducing properties of the peptide. However, the presence of fusion and lytic activities alone could not account for the surface tension reducing property of SP-B. We propose a model in which association of helix 1 with lipids leads to membrane permeabilization but not aggregation; helix 2 mediates membrane cross-linking (aggregation), which, in turn, facilitates lipid mixing, membrane fusion, and interfacial adsorption/surface tension reduction.

of the Hyp. A significant proportion of rhamnogalacturonan-associated sugars co-solubilized and co-purified along with the extensin fragments following the trypsinization. By sodium dodecyl sulfate gel electrophoresis and gel filtration, the glycopeptides fel1 into two classes. One class contained distinctly sized molecules with relative molecular weights in the range of 4,000 to 24,000. The other class did not enter the resolving gel and was hetero-disperse. After complete deglycosylation by a 0°C anhydrous hydrogen fluoride treatment, the first class was little affected i n i t s electrophoretic mobility, whereas the larger heterogeneous material mostly entered the separating gel. After further trypsinization of the deglycosylated peptides and analysis by capillary zone electrophoresis, the peptides i n both size classes were shown to contain the sequences described above. From our observations we suggest that cotton extensin becomes insolubilized into cell walls in part by pectinprotein cross-links in addition to the protein-protein (or proteinphenolic-protein) cross-links that have been repeatedly suggested.

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.

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.

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.