Role of carbohydrates in host-parasite interactions

Principal Investigator : Kanwal J Kaur

The project is aimed for understanding the differential roles of carbohydrate domains in host-parasite interactions by using synthetic glycoconjugates involving model systems such as antimicrobial glycopeptides of innate immune origin and Entamoeba histolytica lectin. The main objectives are i) synthesis and structural characterization of glycoconjugates and ii) structure-function analysis of the synthetic glycoconjugates in the context of host-parasite interactions

The synthesis of a proline rich 16-residue mono-O-glycosylated peptide, formaecin, is initiated for assessing the role of glycosylation in its antibacterial activity. The key challenge in the chemical synthesis of formaecin is installation of a GalNAc residue into the peptide sequence. A glycosylated threonine was incorporated into the peptide sequence via solid phase peptide synthesis. The synthesis of the critical building block, GalNAc-threonine, was accomplished by following ten-step scheme. In brief, the tetraacetyl galactosyl bromide, which was readily prepared from free galactose in two quantitative steps, was used crude in a zinc-mediated reduction to form galactal. The triphasic reaction conditions (Zn dust/AcOH/H2O/ether) afforded 90% yield for this reaction. The purified galactal was converted to azidonitrate by using ceric-ammonium nitrate/NaN3/CH3CN. The conversion of azidonitrate to azidobromide is accomplished by LiBr, which yielded a coupling partner that was used after its purification, with Fmoc-benzyl-protected threonine. Fmoc-Thr-OBzl was synthesized from threonine in two steps. The glycosylation of azidobromide with Fmoc-threonine benzyl ester afforded the desired a-glycoside along with the trace amount of b-anomer, which was removed following reductive acetylation by column chromatography. Deprotection of the benzyl ester by hydrogenolysis yielded the final product, Na-Fmoc-Thr(Ac3-a-D-GalNAc)-OH in small quantity for subsequent use. All the intermediate compounds and the final product were characterized by 1H NMR.

The small amount of glycosylated amino acid which was obtained during the standardization of the synthesis of glycosylated threonine, was utilized to prepare and standardize the synthesis of the glycosylated peptide, formaecin. The peptide was synthesized on HMP resin using Na-Fmoc-protected amino acids and DCC-mediated HOBt ester activation in NMP (ABI 431A Synthesizer). The glycosylated amino acid Na -Fmoc-Thr(Ac3-a-D-GalNAc)-OH was coupled using 2.5 eq of amino acid and activation with HBTU (2 eq) in the presence of HOBt and N,N-diisopropylethylamine (2 eq each); the reaction time was 1.5h. The peptide was cleaved from the resin by treatment with trifluoroacetic acid/thioanisole/phenol/water/1,2-ethanedithiol. The crude peptide was purified using RP-HPLC. Purified glycopeptide was treated with 5% hydrazine hydrate in water to deacetylate the sugar moiety; reaction was monitored by RP-HPLC and was complete in 40min. The completely deprotected glycopeptide was again purified by C18 column. The purity was verified using C18 analytical column. Elution of the peptide was accomplished with a linear gradient from 15 to 80% acetonitrile containing 0.1% trifluoroacetic acid over 30min. Characterization was performed by molecular mass determination.

The nonglycosylated form of formaecin was also synthesized for comparison of its biological and structural properties with formaecin. The antibacterial activity of formaecin and its nonglycosylated form against Salmonella typhimurium and E.coli was determined by radial diffusion assay. It was observed that formaecin was about 15 times more active than its nonglycosylated form, when the comparative activities of these peptides inferred on the basis of inhibition zone area at 0.25nmol. The LPS binding assays were also carried out using affinity sensor; the formaecin and its nonglycosylated analog show the affinity constants as 570mM and 580mM, respectively.

Publications

Original peer-reviewed articles

1.   Kaur KJ, Jain D, Goel M and Salunke DM (2001) Immunological implications of structural mimicry between a dodecapeptide and a carbohydrate moiety. Vaccine (in press).

2.   *Jain D, Kaur KJ, Sundaravadivel B and Salunke DM (2000) Structural and functional consequences of peptide-carbohydrate mimicry: crystal structure of a carbohydrate-mimicking peptide bound to concanavalin A. J Biol Chem 275: 16098-16102 (*in press last year, since published).

3.   Jain D, Kaur KJ, Goel M and Salunke DM (2000) Structural basis of functional mimicry between carbohydrate and peptide ligands of Con A. Biochem Biophys Res Commun 272:843-849.

4.    Jain D, Kaur KJ and Salunke DM (2001) Plasticity in protein-peptide recognition: crystal structures of two different peptides bound to concanavalin A. Biophys J (in press).