D. J. Kalita

Structure-function studies of Bubalus bubalis lingual antimicrobial peptide analogs.

Antimicrobial peptides expressed on different epithelial lining are major components of the innate immune system. Based on the deduced amino acid sequence of Bubalus bubalis lingual antimicrobial peptide (LAP) cDNA (Accession No. DQ458768), five overlapping peptides LAP23–55, LAP42–64, LAP21–64, LAP1–26 and LAP1–64 were synthesized using solid phase fluorenylmethoxycarbonyl (Fmoc) chemistry. Circular Dichroism spectroscopy of synthesized peptides revealed predominantly β-structure for LAP23–55, LAP42−64 and LAP21–64 with less α-helix in different solutions. Quantitation of secondary structure indicated the highest β-structure for all these three peptides in membrane mimetic SDS solution. The helicogenic solvent TFE could not induce helix in LAP23–55 however TFE induced helical propensity was observed in LAP42–64 and LAP21–64. The quantitation of secondary structure indicated the highest ordered structure for LAP23–55 followed by LAP42–64 and LAP21–64. The antibacterial activity of LAP23–55 was found to be more potent against Staphylococcus aureus, Listeria monocytogens, Escherichia coli and Salmonella typhimurium followed by LAP42–64 and LAP21–64. Minimum inhibitory concentration (MIC) also showed similar trend with lowest value for LAP23–55 followed by LAP42–64 and LAP21–64. Haemolysis and cytotoxicity was observed above 3 fold for LAP21–64, above six fold for LAP23–55 and LAP42–64 of their MIC. The LAP1–26 and LAP1–64 could not produce any characteristic CD spectra and did not show any antimicrobial activity, indicating that N- terminal of the peptide negates the antimicrobial activity.

Molecular cloning and characterization of lingual antimicrobial peptide cDNA of Bubalus bubalis.

Antimicrobial peptides form a crucial component of innate immune system, making it a highly effective first line of defense in animals. In the study, lingual antimicrobial peptide cDNA of Bubalus bubalis has been characterized. The characterized cDNA has complete ORF of 195 bases. The signal sequence of buffalo LAP comprised of N-terminal 1-20 amino acids and mature peptide from 23-64 amino acids. The percentage of similarity of buffalo LAP and buffalo EBD at nucleotide and amino acid level was 96.4% and 92.3% respectively. The identity of buffalo LAP with cattle LAP and TAP at nucleotide level was 92.8% and 90.3%. Both at nucleotide and amino acid level buffalo LAP is closer to buffalo EBD followed by cattle LAP and TAP. Phylogenetic tree at nucleotide and amino acid level also showed close relationship of buffalo LAP with buffalo EBD, cattle LAP and TAP. The synthesized LAP fragment had antibacterial activity.