Kalita Dhruba Jyoti, Kumar Ashok, Kumar Satish
Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India.
Vet Res Commun. 2009 Feb;33(2):149-61. doi: 10.1007/s11259-008-9081-7. Epub 2008 Jul 24.
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.
在不同上皮衬里表达的抗菌肽是固有免疫系统的主要组成部分。基于水牛舌抗菌肽（LAP）cDNA（登录号DQ458768）推导的氨基酸序列，使用固相芴甲氧羰基（Fmoc）化学合成了五个重叠肽LAP(23 - 55)、LAP(42 - 64)、LAP(21 - 64)、LAP(1 - 26)和LAP(1 - 64)。合成肽的圆二色光谱显示，LAP(23 - 55)、LAP(42 - 64)和LAP(21 - 64)在不同溶液中主要为β结构，α螺旋较少。二级结构定量表明，在模拟膜的SDS溶液中，这三种肽的β结构含量最高。螺旋诱导溶剂TFE不能在LAP(23 - 55)中诱导螺旋，但在LAP(42 - 64)和LAP(21 - 64)中观察到TFE诱导的螺旋倾向。二级结构定量表明，LAP(23 - 55)的有序结构含量最高，其次是LAP(42 - 64)和LAP(21 - 64)。发现LAP(23 - 55)对金黄色葡萄球菌、单核细胞增生李斯特菌、大肠杆菌和鼠伤寒沙门氏菌的抗菌活性更强，其次是LAP(42 - 64)和LAP(21 - 64)。最低抑菌浓度（MIC）也呈现类似趋势，LAP(23 - 55)的值最低，其次是LAP(42 - 64)和LAP(21 - 64)。LAP(21 - 64)在其MIC的3倍以上观察到溶血和细胞毒性，LAP(23 - 55)和LAP(42 - 64)在其MIC的6倍以上观察到溶血和细胞毒性。LAP(1 - 26)和LAP(1 - 64)不能产生任何特征性的CD光谱，也没有显示出任何抗菌活性，表明该肽的N端消除了抗菌活性。
