Objectives
To characterise, at the biochemical and molecular level, schistosome molecules (specifically glycoconjugates) that stimulate the host’s innate immune response by:
- Mass spectroscopic characterisation of glycoproteins and glycolipids from cercariae and eggs.
- Fractionation of parasite extracts by affinity, or size, chromatography.
- Characterisation of glycoproteins and glycolipids using specific anti-carbohydrate monoclonal antibodies.
- Isolatation of fractions of parasite extracts that have immunoreactive properties.
- Synthesising, or obtaining, defined molecular structures that have potential immunoregulatory function.
Description of work
Parasite material released by cercariae and eggs (ES products) are known to have immune regulatory activity and will be biochemically characterised, fractionated and then purified. Throughout WP4, partly characterised material will be tested for immunological activity as an iterative process between partners 1 and 2.
Initially, enzymatic (e.g. glycosidases) and chemical (e.g. periodate) treatment of the ES products will determine whether N-glycans or O-glycans, or specific monosaccharides are important. The identity of glycans will be determined following release of N- and O-glycans by enzymatic (PNGaseF/A) and chemical (reductive β-elimination) procedures prior to profiling by MALDI-TOF-MS and nano-LC.NS to provide an overview of glycan composition and abundance.
ES products, before or after glycan release, will be fractionated (e.g. gel filtration, ion-exchange, lectin affinity chromatography, normal phase, reversed phase, graphitized carbon) and the resulting fractions tested in assays of immunoreactivity (WP3), characterised by mass spectrometry, and further fractionated as necessary. Anti-helminth glycans mAbs (e.g. LDN, LDN-F, F-LDN, core-fucose) on affinity columns will facilitate glycan/ gycoconjugate fractionation and purification.
Ultimately, we seek to synthesise glycoconjugates with the same immunological activity as the identified glycan elements but since this can be a substantial process, we will also select from an available set of synthetic tri- and tetrasaccharides those with structurally similarity to our immunoreactive fractions, or use appropriate glycoconjugates from commercial and collaborative sources. The isolated and synthesised structures will be directly tested for immunoreactivity using in vitro assays as detailed in WP5.