Effect of Basic Amino Acids on the Biological Activity of Helical Antimicrobial Peptide†

doi:10.7503/cjcu20170596

Abstract

Abstract:

In order to investigate the effect of basic amino acids on the biological activity of antimicrobial peptides, an α helical antimicrobial peptides of HPRP-A1 was used to design several peptide derivatives with different K/R ratio by substitution lysine(K) with arginine(R). The relationship between the structure and activity of helical antimicrobial peptides was studied by combining the biophysical properties such as helicity, hydrophobicity and peptide association parameter of antimicrobial peptides. The interaction between helical antimicrobial peptides and different types of cell membranes was further studied by liposome mimic membrane and the cell permeation ability of peptide was examined by 1-N-pbenyl-naphtylamine(NPN) and o-nitrophenyl-β-D-galactopyranoside(ONPG). The results confirmed that the increasing numbers of arginine substitution increased the hydrophobicity and helicity of the antimicrobial peptides, resulting in increasing toxicity of the helical antimicrobial peptides against eukaryotic cell membrane. However, the increasing of arginine was accompanied by decreasing in the ability of peptide association and the permeability of the antimicrobial peptide against the prokaryotic cell membrane, resulting in decreasing of the antibacterial activity of the antimicrobial peptide.

Key words: Antimicrobial peptide, Arginin, Lysine, Liposome mimic membrane

CLC Number:

O629.7

TrendMD:

ZHAO Wencai, HAN Lili, PENG Yingjun, WANG Xiaojing, LIU Shengyu, LI Pengfei, HUANG Yibing, CHEN Yuxin. Effect of Basic Amino Acids on the Biological Activity of Helical Antimicrobial Peptide^†[J]. Chem. J. Chinese Universities, 2018, 39(4): 681.

Figures/Tables 6

Table 1 Biophysical properties of HPRP-A1 and its derivatives*

Peptide	Amino acid sequence	M_w	Hydrophobicity t_R0/min	P_A
HPRP-A1	Ac-FKKLKKLFSKLWNWK-amide	2036.54	46.93	2.342
HPRP-A1-2R	Ac-FRKLKKLFSKLWNWR-amide	2092.56	47.28	2.205
HPRP-A1-4R	Ac-FRKLKRLFSRLWNWR-amide	2148.59	47.65	1.229
HPRP-A1-6R	Ac-FRRLRRLFSRLWNWR-amide	2204.62	48.45	1.092

Fig.1 CD spectra of HPRP-A1 and its derivatives(A) In the presence of PBS buffer and 2,2,2-Trifluoroethanol(TFE)(volume ratio 1:1);(B) in PBS buffer(50 mmol/L KH2PO4/K2HPO4+100 mmol/L KCl, pH=7.4) at pH=7.4, 25 ℃(B).

Table 2 Biological activities of HPRP-A1 and its derivatives*

Peptide	MIC/(μmol·L^-1)				MHC/(μmol·L^-1)	Therapeutic index
Peptide	P. aeruginosa	S. aereus	E. coil	B. subtilis	MHC/(μmol·L^-1)	Therapeutic index
HPRP-A1	16	16	8	8	64	5.33
HPRP-A1-2R	32	32	8	4	64	3.37
HPRP-A1-4R	64	16	16	4	32	1.28
HPRP-A1-6R	64	32	32	8	32	0.94

Fig.2 Outer membrane permeabilization assay of HPRP-A1 and its derivativesNPN uptake in P. aeruginosa ATCC 27853 was continuously measured by fluorescence spectrophotometer for 12 min at 25 ℃ endued by 4 μmol/L HPRP-A1 and its derivatives(A) and 2, 4 and 8 μmol/L HPRP-A1(B).

Fig.3 Inner membrane permeabilization assay of HPRP-A1 and its derivativesRelease of cytoplasmic β-galactosidase activity were measured by UV-visible spectrophotometer at 420 nm after adding ONPG(30 mmol/L) and various concentrations peptides into E. coli ML-35 bacterial suspension.

Fig.4 Fluorescence emission spectra(A, B) and Stern-Volmer plot(C, D) of peptides with various liposome models at 25 ℃Stern-Volmer plots were obtained by the sequential addition of the fluorescence quencher KI. PC/PG liposomes(mass fraction 7:3) were used to mimic prokaryotic membrane in (A, C) and PC/cholesterol(mass fraction 8:1) liposomes were used to mimic eukaryotic membrane in (B, D), respectively.

References 27

[1]	Zhang Y. H., Li L. C., Yuan W. C., Zhang X. M., Chem. Res. Chinese Universities, 2015, 31(3), 381-387
[2]	Feng H. Y., Gao L., Ye X. H., Wang L., Xue Z. C., Kong J. M., Li L. Z., Chem. Res. Chinese Universities, 2017, 33(1), 155-159
[3]	Cheng S. Q., Liang G. D., Jiang X. F., Wang C., Liu K. L., Chem. J. Chinese Universities, 2016, 37(7), 1287-1292
	(程思绮, 梁国栋, 姜喜凤, 王潮, 刘克良. 高等学校化学学报, 2016, 37(7), 1287-1292)
[4]	Andersson D. I., Hughes D., Kubicek-Sutherland J. Z., Drug Resist. Updates, 2016, 26, 43-57
[5]	Hancock R. E. W., Sahl H. G., Nat. Biotechnol., 2006, 24, 1551-1557
[6]	Huang Y. B., Zhai N. C., Gao G., Chen Y. X., Chem. J. Chinese Universities, 2012, 33(6), 1252-1258
	(黄宜兵, 翟乃翠, 高贵, 陈育新. 高等学校化学学报, 2012, 33(6), 1252-1258)
[7]	Huang Y. B., He L. Y., Li G. R., Zhai N. C., Jiang H. Y., Chen Y. X., Protein & Cell, 2014, 5, 631-642
[8]	Huang Y. B., Huang J. F., Chen Y. X., Protein & Cell, 2010, 1, 143-152
[9]	Bechara C., Sagan S., Febs Lett., 2013, 587, 1693-1702
[10]	Vasconcelos L., Parn K., Langel U., Ther. Deliv., 2013, 4, 573-591
[11]	Hao X. Y., Yan Q. Y., Zhao J., Wang W. R., Huang Y. B., Chen Y. X., PLoS One, 2015, 10, e0138911
[12]	Schmidt N. W., Tai K. P., Kamdar K., Mishra A., Lai G. H., Zhao K., Ouellette A. J., Wong G. C., J. Biol. Chem., 2012, 287, 21866-21872
[13]	Zou G. Z., de Leeuw E., Li C., Pazgier M., Li C. Q., Zeng P., Lu W. Y., Lubkowski J., Lu W., J. Biol. Chem., 2007, 282, 19653-19665
[14]	Huang Y. B., Wang X. F., Wang H. Y., Liu Y., Chen Y. X., Mol. Cancer Ther., 2011, 10, 416-426
[15]	Mant C. T., Chen Y., Hodges R. S., J. Chromatogr. A, 2003, 1009, 29-43
[16]	Chen Y. X., Guarnieri M. T., Vasil A. I., Vasil M. L., Mant C. T., Hodges R. S., Antimicrob. Agents Chemother., 2007, 51, 1398-1406
[17]	Yi T. H., Huang Y. B., Chen Y. X., Journal of Peptide Science, 2015, 21, 46-52
[18]	Sun S. Y., Zhao G. X., Huang Y. B, Cai M. J., Shan Y. P., Wang H. D., Chen Y. X., Sci. Rep., 2016, 6, e29145
[19]	Chen Y. X., Mant C. T., Farmer S. W., Hancock R. E. W., Vasil M. L., Hodges R. S., J. Biol. Chem., 2005, 280, 12316-12329
[20]	Huang H. W., Biochemistry, 2000, 39, 8347-8352
[21]	Yi T. H., Huang Y. B., Chen Y. X., Chem. Bio. Drug Des., 2015, 85, 598-607
[22]	Eriksson M., Nielsen P. E., Good L., J. Biol. Chem., 2002, 277, 7144-7147
[23]	Li L. B., Vorobyov I., Allen T. W., J. Phys. Chem. B, 2013, 117, 11906-11920
[24]	Harris F., Dennison S. R., Singh J., Phoenix D. A., Med. Res. Rev., 2013, 33, 190-234
[25]	Lopez-Exposito I., Amigo L., Recio I., BBA-Biomembranes,2008, 1778, 2444-2449
[26]	Mant C. T., Kovacs J. M., Kim H. M., Pollock D. D., Hodges R. S., Biopolymers,2009, 92, 573-595
[27]	Qiao Y., Gao Z. H., Liu Y., Cheng Y., Yu M. X., Duan Y., Liu Y., Biomed. Res. Int., 2014, 2014, e869186