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 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 preparative scale synthesis of GalNAc-threonine was successfully standardized. The glycosylation of Fmoc-Thr-OBzl with protected sugar followed by its reductive acetylation afforded the anomeric mixture of N^a-Fmoc-Thr(Ac₃-a-D-GalNAc)-OBzl. The separation of anomers were tried by using different grades of silica gel for column chromatography and finally the separation was achieved by using prepacked silica gel column on HPLC.

Formaecin I has sequence similarity with apidaecin class of peptides which belong to nonglycosylated proline rich family. The apidaecins peptides show the comparable antibacterial activity to glycosylated formaecin I. Further designing of nonglycosylated analoges of formaecin I was done by incorporating the conserved residues of apidaecins in formaecin sequentially to delineate the important residues important for the activity. The designed peptides were synthesized and assayed for their antibacterial activities against various gram negative strains. The comparison of the antibacterial activities of the designed analogs suggested that incorporation of conserved residues from apidaecins in nonglycosylated formaecin I can compensate for the loss of sugar. The hemolytic activities of these analogs were also assayed on freshly isolated rat erythrocytes. All the analogs were found to be ineffective in lysing the erythrocytes.

To investigate a possible specific interaction of formaecin I with the target molecule, the nonglycosylated D-enantiomer was synthesized and compared its antibacterial activity against E. coli with the L-nonglycosylated analog. The D-enantiomer was found to be completely inactive. It suggests that the three dimensional structure of formaecin I is important for its mode of action and the peptide may act on the gram negative bacteria through a stereospecific target.

The conformational studies of these analogs were done by circular dichroism and their comparison was done with the glycosylated peptide. In addition to the random structure, CD spectrum of the native formaecin I shows a hump at 206 nm corresponding to PPII conformation which is the only major feature of the designed active nonglycosylated analog.

Similar studies involving another glycosylated peptide, drosocin, were undertaken. Drosocin is a 19-residue proline rich disaccharide containing antibacterial peptide which was isolated from Drosophila. The monoglycosylated form of drosocin was synthesized. Several nonglycosylated analogs of drosocin were designed on the basis of apidaecins. These peptides were synthesized and subjected to their antibacterial activities against E. coli and Salmonella typhimurium. It was found that the introduction of the conserved residues of apidaecin in nonglycosylated drosocin enhance the antibacterial activity but not to the level of native drosocin.

To study the mechanism of action of glycosylated and nonglycosylated antibacterial peptides, the binding of these peptides with DNA and RNA was examined. The gel retardation experiment showed that formaecin and its nonglycosylated analogs could not suppress the migration of DNA even at the 200 weight ratio of peptide to DNA whereas the drosocin analogs inhibited the migration at the weight ratio of 50.

Publications

Original peer-reviewed articles

1.     Nair DT, Kaur KJ, Singh K, Mukherjee P, Rajagopal D, George A, Bal V, Rath S, Rao KV and Salunke DM (2003) Mimicry of native peptide antigens by the corresponding retro-inverso analogs is dependent on their intrinsic structure and interaction properties J Immunol 170:1362-1373.

2.     Nagpal S, Kaur KJ, Jain D and Salunke DM (2002) Plasticity in structure and interactions is critical for the action of indolicidin, an antibacterial peptide of innate immune origin. Protein Sci 11:2158-2167.