Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin.

Typical plant aspartic protease zymogens comprise a characteristic and plant-specific insert (PSI). PSI domains can interact with membranes, and a role as a defensive weapon against pathogens has been proposed. However, the potential of PSIs as antimicrobial agents has not been fully investigated and explored yet due to problems in producing sufficient amounts of these domains in bacteria. Here, we report the development of an expression platform for the production of the PSI domain of cirsin in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. We successfully generated K. lactis transformants expressing and secreting significant amounts of correctly processed and glycosylated PSI, as well as its nonglycosylated mutant. A purification protocol with protein yields of ∼4.0 mg/liter was established for both wild-type and nonglycosylated PSIs, which represents the highest reported yield for a nontagged PSI domain. Subsequent bioactivity assays targeting phytopathogenic fungi indicated that the PSI of cirsin is produced in a biologically active form in K. lactis and provided clear evidence for its antifungal activity. This yeast expression system thereby emerges as a promising production platform for further exploring the biotechnological potential of these plant saposin-like proteins.

Efficient one-step chromatographic purification and functional characterization of recombinant human Saposin C.

Saposin (Sap) C is a small lysosomal disulfide bridge-containing glycoprotein required for glucosylceramide (GC) hydrolysis by glucosylceramidase (GCase). Sap C deficiency causes a variant form of Gaucher disease (GD), a rare genetic disorder characterized by GC accumulation in lysosomes of monocyte/macrophage lineage. Efforts to develop fast and efficient methodologies to express and purify Sap C have been made in the last years. Here, human Sap C was expressed in a bacterial strain that greatly enhances disulfide bond formation, and the recombinant protein was purified in a single chromatographic step using an affinity tag-based protein purification system. Mass spectrometry analysis demonstrated that disulfide bridges required for Sap C stability and functionality were retained. Consistently, the recombinant protein was shown to interact with anionic phospholipids-containing vesicles, and reconstitute GCase activity in vitro. Recombinant Sap C was efficiently endocytosed by Sap C-deficient fibroblasts, and targeted to lysosomes. These findings document that the bacterially purified Sap C exerts biological properties functionally equivalent to those observed for the native protein, indicating its potential use in the development of therapeutic intervention.

Characterization and localization of saposin-like protein-2 (SAP-2) in Fasciola gigantica.

Fasciola gigantica saposin-like protein-2 (FgSAP-2) belongs to a family of lipid-interacting proteins that are involved in the cytolysis of target cells. In this study, we have cloned and expressed FgSAP-2 and produced the antibody against this recombinant protein. Rabbit antiserum against rFgSAP-2 reacted with a similar native protein in the whole body extracts of the 4-week-old juvenile and adult stage, as well as a protein in their excretion-secretion, but not in the tegument. In situ hybridization and immunofluorescence detection revealed the presence of SAP-2 mRNA transcripts and proteins in the cecal epithelial cells of 4-week-old juvenile and adult parasites, but not in the metacercariae and newly excysted juveniles. Moreover, SAP-2 is present only in the cecal epithelial cells lining the distal part of the digestive tract, but not in the tegumental-type epithelium lining the proximal part of the digestive tract. The rFgSAP-2 reacted with antisera from rabbits infected with F. gigantica metacercariae collected at 5 weeks, but not at 2 weeks after infection. Anti-rFgSAP-2 did not exhibit any cross-reactivity with the other parasites' antigens, including Opisthorchis viverrini, Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gigantocotyle explanatum, Paramphistomum cervi, Setaria labiato-papillosa, and Haemonchus placei. This finding indicated that SAP-2 is a unique protein that is expressed only in late juvenile and adult F. gigantica, and it could be considered for immunodiagnostic and as a vaccine candidate for fasciolosis.

Crystal structures of human saposins C andD: implications for lipid recognition and membrane interactions.

Human saposins are essential proteins required for degradation of sphingolipids and lipid antigen presentation. Despite the conserved structural organization of saposins, their distinct modes of interaction with biological membranes are not fully understood. We describe two crystal structures of human saposin C in an "open" configuration with unusual domain swapped homodimers. This form of SapC dimer supports the "clip-on" model for SapC-induced vesicle fusion. In addition, we present the crystal structure of SapD in two crystal forms. They reveal the monomer-monomer interface for the SapD dimer, which was confirmed in solution by analytical ultracentrifugation. The crystal structure of SapD suggests that side chains of Lys10 and Arg17 are involved in initial association with the preferred anionic biological membranes by forming salt bridges with sulfate or phosphate lipid headgroups.

A new triterpenoid saponin from the roots of Silene viscidula.

A new triterpenoid saponin, silenoviscoside F (1), was isolated from the roots of Silene viscidula, together with three known saponins, dianchinenoside D (2), sinocrassuloside I (3) and sinocrassuloside II (4). The structure of compound 1 was elucidated by spectroscopic data, GC-MS and chemical methods. The structures of compounds 2-4 were determined on the basis of spectroscopic data comparison with the literature values, which were isolated from the genus Silene for the first time.

Saposin A mobilizes lipids from low cholesterol and high bis(monoacylglycerol)phosphate-containing membranes: patient variant Saposin A lacks lipid extraction capacity.

Saposin A (Sap-A) is one of five known sphingolipid activator proteins required for the lysosomal degradation of sphingolipids and for the loading of lipid antigens onto antigen-presenting molecules of the CD1 type. Sap-A assists in the degradation of galactosylceramide by galactosylceramide-beta-galactosidase in vivo, which takes place at the surface of intraendosomal/intralysosomal vesicles. Sap-A is believed to mediate the interaction between the enzyme and its membrane-bound substrate. Its dysfunction causes a variant form of Krabbe disease. In the present study we prepared glycosylated Sap-A free of other Saps, taking advantage of the Pichia pastoris expression system. Using liposomes and surface plasmon resonance spectroscopy, we tested the binding and lipid mobilization capacity of Sap-A under different conditions. Along the endocytic pathway, the pH value decreases, and the lipid composition of intraendosomal and intralysosomal membranes changes drastically. In the inner membranes the cholesterol concentration decreases, and that of the anionic phospholipid bis(monoacylglycero)phosphate increases. Here, we show that Sap-A is able to bind to liposomes and to mobilize lipids out of them at acidic pH values below pH 4.7. Low cholesterol levels and increasing concentrations of bis(monoacylglycero)phosphate favor lipid extraction significantly. Galactosylceramide as a bilayer component is not essential for lipid mobilization by Sap-A, which requires intact disulfide bridges for activity. We also show for the first time that glycosylation of Sap-A is essential for its lipid extraction activity. Variant Sap-A proteins, which cause storage of galactosylceramide in humans (Krabbe disease, Spiegel, R., Bach, G., Sury, V., Mengistu, G., Meidan, B., Shalev, S., Shneor, Y., Mandel, H., and Zeigler, M. (2005) Mol. Genet. Metab. 84, 160-166) and in mutant mice (Matsuda, J., Vanier, M. T., Saito, Y., Tohyama, J., and Suzuki, K. (2001) Hum. Mol. Genet. 10, 1191-1199) are deficient in lipid extraction capacity.