Cell-free expression, purification and immunoreactivity assessment of recombinant Fasciola hepatica saposin-like protein-2.

Cell free protein synthesis has become a powerful method for the high-throughput production of proteins that are difficult to express in living cells. The protein SAP2 of Fasciola hepatica (FhSAP2), which has demonstrated to be both, an excellent vaccine candidate against experimental fascioliasis and a good antigen for serodiagnosis of human chronic fascioliasis, is a typical example of a molecule that is difficult to produce. This is mainly due to its tendency to get over-expressed in inclusion bodies by prokaryotes. FhSAP2 expressed in an Escherichia coli-based expression system is poorly glycosylated, insoluble and often undergoes improper folding leading it to reduced immunogenicity. In this work, FhSAP2 was expressed in vitro using the eukaryote cell free system, TNT T7 Quick coupled transcription/translation, that has been designed for the expression of PCR-generated DNA templates. FhSAP2 was expressed in micro-volumes and purified by an affinity chromatography method, which gave a protein yield of 500 µg/ml as determined by bicinchoninic acid assay method. Circular dichroism, Western blotting and enzyme-linked immunosorbent assay analysis were used to confirm the secondary structure, purity and integrity of protein. Results demonstrate that FhSAP2 can be expressed in a cell-free system retaining its main conformational and antigenic properties. The protein purified could be used in immunization experiments and immunodiagnostic techniques.

Synthetic Glycoforms Reveal Carbohydrate-Dependent Bioactivity of Human Saposin†D.

The main glycoforms of the hydrophobic lysosomal glycoprotein saposin D (SapD) were synthesized by native chemical ligation. An approach for the challenging solid-phase synthesis of the fragments was developed. Three SapD glycoforms were obtained following a general and robust refolding and purification protocol. A crystal structure of one glycoform confirmed its native structure and disulfide pattern. Functional assays revealed that the lipid-binding properties of three SapD glycoforms are highly affected by the single sugar moiety of SapD showing a dependency of the size and the type of N-glycan.

Optical and nuclear imaging of glioblastoma with phosphatidylserine-targeted nanovesicles.

Multimodal tumor imaging with targeted nanoparticles potentially offers both enhanced specificity and sensitivity, leading to more precise cancer diagnosis and monitoring. We describe the synthesis and characterization of phenol-substituted, lipophilic orange and far-red fluorescent dyes and a simple radioiodination procedure to generate a dual (optical and nuclear) imaging probe. MALDI-ToF analyses revealed high iodination efficiency of the lipophilic reporters, achieved by electrophilic aromatic substitution using the chloramide 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen) as the oxidizing agent in an organic/aqueous co-solvent mixture. Upon conjugation of iodine-127 or iodine-124-labeled reporters to tumor-targeting SapC-DOPS nanovesicles, optical (fluorescent) and PET imaging was performed in mice bearing intracranial glioblastomas. In addition, tumor vs non-tumor (normal brain) uptake was compared using iodine-125. These data provide proof-of-principle for the potential value of SapC-DOPS for multimodal imaging of glioblastoma, the most aggressive primary brain tumor.

The effects of chemically synthesized saposin C on glucosylceramide-ß-glucosidase.

OBJECTIVE: Saposin C (SAP-C) is an essential activator of glucosylceramide (GlcCer)-ß-glucosidase (GCase), the enzyme deficient in Gaucher's disease. In this study, we investigated the effects of chemically synthesized SAP-Cs (synthetic SAP-Cs) on GCase. METHODS: Enzymatic assays and western blot analyses were carried out to evaluate the effects of two kinds of synthetic SAP-Cs, a non-glycosylated form and a N-glycosylated form bearing a complex type nonasaccharide, on GCase with respect to its activation, stabilization, and protection. Imiglucerase (Cerezyme) was used as the GCase. To mimic physiological conditions, GCase activity was assayed in the presence of 4-nitrobenzo-2-oxa-1,3-diazole-labeled GlcCer-containing liposomes composed of bis(monoacylglycero)phosphate, l-α-phosphatidylcholine, and cholesterol. RESULTS: GCase activities increased depending on the concentration of synthetic SAP-Cs. SAP-Cs at a concentration of 1µM increased GCase activities significantly, by 14- to 22-fold (non-glycosylated SAP-C: 22.9±0.16; nona-glycosylated SAP-C: 14.9±0.19; without SAP-C: 1.05±0.035pmol/h/ng GCase). These values equaled or surpassed previously published values obtained using recombinant non-glycosylated SAP-C. Both synthetic SAP-Cs were as effective as bovine serum albumin in stabilizing GCase at 37°C. Western blot analysis revealed that synthetic SAP-Cs specifically protected GCase from cathepsin D digestion. CONCLUSIONS: The results demonstrate that these novel, chemically synthesized SAP-Cs function as activators, stabilizers, and protectors of GCase, suggesting their utility in enzyme replacement therapy in patients with Gaucher's disease.

Prosaposin-derived peptide alleviates ischaemia-induced hearing loss.

CONCLUSION: An 18-mer peptide derived from the neurotrophic region of prosaposin (PS-pep) prevents hearing loss and cochlear damage due to transient cochlear ischaemia by activating an anti-apoptotic pathway. PS-pep is a potent candidate molecule for alleviating ischaemia-induced hearing loss. OBJECTIVE: PS-pep was investigated for its protective effects against ischaemia-induced hearing loss and cochlear damage. METHODS: Ischaemia was induced in both cochleae in Mongolian gerbils by pulling the ligatures around both vertebral arteries in an anterior direction using 5 g weights for 15 min. PS-pep was synthesized artificially and administered subcutaneously four times after the induction of transient cochlear ischaemia. RESULTS: An increase in the auditory brainstem response threshold was alleviated in animals treated with 2.0 mg/kg PS-pep. Histological examinations conducted on day 7 showed that the loss of inner hair cells (IHCs) was more prominent than that of outer hair cells. Higher doses of PS-pep significantly alleviated IHC loss. An increase in the anti-apoptotic factor bcl-2 was also noted in the IHCs treated with PS-pep.

Chemoenzymatic synthesis of hydrophobic glycoprotein: synthesis of saposin C carrying complex-type carbohydrate.

The complex-type N-linked octasaccharide oxazoline having LacNAc as the nonreducing end sugar was efficiently synthesized using the benzyl-protected LacNAc, mannose, and ß-mannosyl GlcNAc units as key building blocks. To achieve a highly ß-selective glycosylation with the LacNAc unit, the N-trichloroacetyl group was used for the protection of the amino group in the LacNAc unit. After complete assembly of these units and deprotection, the obtained free sugar was successfully derivatized into the corresponding sugar oxazoline. On the other hand, the N-acetylglucosaminylated saposin C, a hydrophobic lipid-binding protein, was chemically synthesized by the native chemical ligation reaction. On the basis of the previous results related to the synthesis of the nonglycosylated saposin C, the O-acyl isopeptide structure was introduced to the N-terminal peptide thioester carrying GlcNAc to improve its solubility toward aqueous organic solvents. The ligation reaction efficiently proceeded with the simultaneous O- to N-acyl shift at the O-acyl isopeptide moiety. After the removal of the cysteine-protecting group and folding, saposin C carrying GlcNAc was successfully obtained. The synthetic sugar oxazoline was then transferred to this glycoprotein using the mutant of endo-ß-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) (glycosynthase), and the saposin C carrying the complex-type nonasaccharide was successfully obtained.