Role of carbohydrates in host-parasite interactions

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 specific objectives of the project 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 GalNAc-threonine was synthesized at preparative scale for which standardization was done last year. The glycosylated amino acid was utilized to prepare the formaecin I. The comparison of antibacterial activity of formaecin I and its nonglycosylated analog against ATCC strain of E.coli which is used as the standard for checking drug resistance, was determined by radial diffusion assay. It was observed that glycosylated peptide, formaecin I, was more active than its nonglycosylated form. Formaecin I was found to be 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 0.25nmole. The other designed nonglycosylated analogs of formaecin I which were synthesized by incorporating the conserved residues of apidaecins were also assayed for their antibacterial activities against E.coli ATCC 25922. The pattern of activity for these designed nonglycosylated analogs of formaecin I was similar as we had shown earlier for other gram-negative strains.

The comparative conformational studies of formaecin I and its nonglycosylated analog were done by CD spectroscopy in water, 2,2,2-trifluoroethanol (TFE), and sodium lauryl sulphate (SDS). The CD spectrum of nonglycosylated peptide in water exhibited a negative band at 200nm, which indicated a peptide without conformational preferences. The glycosylated peptide (formaecin I) resulted in a slightly decreased band intensity without any change in the shape of the CD curve. In the structure promoting solvent such as TFE, the peptide without sugar showed a CD pattern qualitatively similar to type C spectrum, characterized by two negative bands at 225nm and 203nm, respectively. In case of formaecin I, the broad band at 200nm in water was somewhat red shifted to 205nm and became sharper in TFE. Micellar SDS has been used as a model of the negatively charged bacterial lipid membranes, with which cationic peptides first interact. In this environment, CD spectrum of both of these peptides was similar to the pattern obtained in TFE.

To identify the interacting bacterial macromolecular targets, the nonglycosylated analog of formaecin I was tested for its binding to the bacterial heat shock protein DnaK. The binding experiments were carried out by using affinity sensor. The biotin labeled nonglycosylated analog was synthesized for its immobilization at streptavidin activated cuvette. The 33-residue long stretch which is the part of multihelical lid over the peptide binding pocket in heat shock protein DnaK, was synthesized and used for its binding to the labeled nonglycosylated formaecin I. It was observed that DnaK can bind to the nonglycosylated peptide.

An example of disaccharide containing antibacterial peptide, drosocin, was undertaken for assaying the role of glycosylation in its antibiotic activity. Drosocin has Galb(1®3)GalNAca1®OThr in its sequence. The synthesis of this disaccharide containing threonine has been initiated.

Publications

Original peer-reviewed articles

1.     Goel M, Anuradha P, Kaur KJ, Maiya BG, Swamy MJ and Salunke DM (2004) Porphyrin binding to jacalin is facilitated by the inherent plasticity of the carbohydrate binding site: novel mode of lectin-ligand interaction. Acta Crystallogr D Biol Crystallogr 60:281-288.

2.     Chakrabarty P, Sethi DK, Padhan N, Kaur KJ, Salunke DM, Bhattacharya S and Bhattacharya A (2004) Identification and characterization of EhCaBP2: a second member of the calcium-binding protein family of the protozoan parasite Entamoeba histolytica. J Biol Chem (in press).