Lingual antimicrobial peptide

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 LAP(23-55), LAP(42-64), LAP(21-64), LAP(1-26) and LAP(1-64) were synthesized using solid phase fluorenylmethoxycarbonyl (Fmoc) chemistry. Circular Dichroism spectroscopy of synthesized peptides revealed predominantly beta-structure for LAP(23-55,) LAP(42-64) and LAP(21-64) with less alpha-helix in different solutions. Quantitation of secondary structure indicated the highest beta-structure for all these three peptides in membrane mimetic SDS solution. The helicogenic solvent TFE could not induce helix in LAP(23-55) however TFE induced helical propensity was observed in LAP(42-64) and LAP(21-64). The quantitation of secondary structure indicated the highest ordered structure for LAP(23-55) followed by LAP(42-64) and LAP(21-64). The antibacterial activity of LAP(23-55) was found to be more potent against Staphylococcus aureus, Listeria monocytogens, Escherichia coli and Salmonella typhimurium followed by LAP(42-64) and LAP(21-64). Minimum inhibitory concentration (MIC) also showed similar trend with lowest value for LAP(23-55) followed by LAP(42-64) and LAP(21-64). Haemolysis and cytotoxicity was observed above 3 fold for LAP(21-64,) above six fold for LAP(23-55) and LAP(42-64) of their MIC. The LAP(1-26) and LAP(1-64) 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.

The present study was undertaken to examine whether potential levels of innate immune factors (lingual antimicrobial peptide (LAP), lactoferrin (LF) and lactoperoxidase (LPO)) in colostrum are associated with subsequent milk somatic cell count (SCC) in dairy cows. Quarter milk samples were collected daily for 1 week postpartum to measure LAP and LF concentrations and LPO activity. SCC in milk was determined weekly for 2 months postpartum and its correlations to concentrations of LAP and LF and LPO activity were examined. Only small variations of all immune factors were found among four udders in each individual cow, whereas there were great differences in these factors among cows. Negative correlation was detected only between LPO activity and mean and maximum SCC, whereas its relationship was not significant. LAP and LF concentrations were significantly correlated positively to mean, maximum and minimum SCC. These results suggest that the great difference in innate immune factors among animals and high LAP and LF concentrations in colostrum may be associated with subsequent high incidence of SCC increase.

Mammalian beta-defensins are innate host defense peptides with potential as a therapeutic agent. Present study reports expression of beta-defensin mRNA transcript in teat mucosal layer of Bubalus bubalis, which is homologous to lingual antimicrobial peptide (LAP) of cattle. The PCR-amplified and cloned full-length cDNA encodes for a putative peptide of 64 residues ( approximately 7 kDa) with pI of 11.324 and possess characteristic features of beta-defensin family including presence of six evolutionary conserved cysteine residues. The first 21 amino acids constitute a putative signal peptide, suggesting that it is a secretory protein. The peptide shares 85%-100% sequence identity with beta-defensins of cattle (Bos taurus). Nonetheless, the maximum identity at nucleotide (100%) and amino acid (98.5%) level was with cattle LAP, which is in consistent with phylogeny analysis report. Hence, the cloned beta-defensin has been designated as buffalo LAP. This study envisages role of beta-defensin peptides in innate immunity of mammary gland.