Principal Investigator :    Kanwaljeet Kaur

The project is aimed at 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 include synthesis and structural characterization of glycoconjugates and structure-function analysis of the synthetic glycoconjugates in the context of host-parasite interactions

The synthesis of mono-O-glycosylated peptide, formaecin at preparative scale was started. The synthesis of GalNAc-threonine could be accomplished by following ten steps scheme. The glycosylation of Fmoc-Thr-OBzl with protected sugar followed by its reductive acetylation afforded the anomeric mixture of Na-Fmoc-Thr(Ac3-a-D-GalNAc)-OBzl. The seperation of anomers is being tried by using different types of silica gel for column chromatography.

The antibacterial activity of formaecin and its nonglycosylated form against various strains of gram negative bacteria showed the higher activity for glycosylated peptide as compared to nonglycosylated one, although a differential pattern of activity was observed for different strains. However, the extent of contribution of the sugar was comparable in each case. To check their toxicity, the hemolytic activity of these two peptides was assayed on freshly isolated rat erythrocytes. It was found that both the peptides were ineffective in lysing erythrocytes. The conformational studies of these two peptides were done by circular dicroism. CD spectra revealed no evidence of regular structure in aqueous solution for both the peptides.

It was found that formaecin has homology with nonglycosylated antibacterial peptides of proline rich class. These nonglycosylated peptides show antibacterial activity comparable to glycosylated ones. In an effort to obtain the structural insight in the functional context, it is of interest to systematically design analogs of formaecin containing no sugar and test for their activity in order to delineate the residues that may be responsible for the antibacterial activity. We have designed a nonglycosylated analog of formaecin by making two changes in it. First was the removal of glycosylated threonine and second, a proline residue was introduced at 9th position resulting the sequence as GRPNPVNNPKPPHPRL. The peptide was synthesized and subjected to its antibacterial activity assay against E. coli and Salmonella typhimurium. Antibacterial activity of formaecin and its nonglycosylated analog was determined by radial diffusion assay. It was observed that both the peptides showed comparable antibacterial activity. Probably the introduction of proline is providing the conformation to peptide similar as formaecin. Further an analog in which Pro-9 residue was substituted by Ala was analyzed to characterize the role of proline in the antibacterial activity. The homology modeling of the formaecin may provide the structural basis of the activity differences between different analogs.

The initial event common to all the cationic peptides is the binding of positively charged residues to the negatively charged molecules exposed at the target cell surface. In order to examine the importance of the presence of positively charged argenine residues at C and N terminals in modulating the biological activity, an analog was designed in which C terminal arginine was substituted with alanine. The peptide was synthesized and assayed for its antibacterial activity against Gram negative strains.

Publications

Original peer-reviewed articles

1.     Jain D, Kaur KJ and Salunke DM (2001) Enhanced binding of a rationally designed peptide ligand of concanavalin A arises from improved geometrical complementarity. Biochemistry 40:12059-12066.

2.     Goel M, Jain D, Kaur KJ, Kenoth R, Maiya BG, Swamy MJ and Salunke DM (2001) Functional equality in the absence of structural similarity, an added dimension to molecular mimicry.
J Biol Chem 276:39277-39281.

3.     *Kaur KJ, Jain D, Goel M and Salunke DM (2001) Immunological implications of structural mimicry between a dodecapeptide and a carbohydrate moiety. Vaccine 19:3124-3130 (*in press last year, since published).

4.     *Jain D, Kaur KJ and Salunke DM (2001) Plasticity in protein-peptide recognition: crystal structures of two different peptides bound to concanavalin A. Biophysical J 80:2912-2921 (*in press last year, since published).