AaeAP1 and AaeAP2: Novel Antimicrobial Peptides from the Venom of the Scorpion, Androctonus aeneas: Structural Characterisation, Molecular Cloning of Biosynthetic Precursor-Encoding cDNAs and Engineering of Analogues with Enhanced Antimicrobial and Anticancer Activities
Abstract
:1. Introduction
2. Results
2.1. Identification and Structural Analysis of Mature Novel AMPs from Venom
2.2. Cloning of AMP Precursor-Encoding cDNAs
#1 | b(1+) | b(2+) | Sequence | y(1+) | y(2+) | #2 |
---|---|---|---|---|---|---|
1 | 148.07570 | 74.54149 | F | - | - | 19 |
2 | 261.15977 | 131.08352 | L | 1869.13213 | 935.06970 | 18 |
3 | 408.22819 | 204.61773 | F | 1756.04806 | 878.52767 | 17 |
4 | 495.26022 | 248.13375 | S | 1608.97964 | 804.99346 | 16 |
5 | 608.34429 | 304.67578 | L | 1521.94761 | 761.47744 | 15 |
6 | 721.42836 | 361.21782 | I | 1408.86354 | 704.93541 | 14 |
7 | 818.48113 | 409.74420 | P | 1295.77947 | 648.39337 | 13 |
8 | 905.51316 | 453.26022 | S | 1198.72670 | 599.86699 | 12 |
9 | 1004.58158 | 502.79443 | V | 1111.69467 | 556.35097 | 11 |
10 | 1117.66565 | 559.33646 | I | 1012.62625 | 506.81676 | 10 |
11 | 1188.70277 | 594.85502 | A | 899.54218 | 450.27473 | 9 |
12 | 1245.72424 | 623.36576 | G | 828.50506 | 414.75617 | 8 |
13 | 1358.80831 | 679.90779 | L | 771.48359 | 386.24543 | 7 |
14 | 1457.87673 | 729.44200 | V | 658.39952 | 329.70340 | 6 |
15 | 1544.90876 | 772.95802 | S | 559.33110 | 280.16919 | 5 |
16 | 1615.94588 | 808.47658 | A | 472.29907 | 236.65317 | 4 |
17 | 1729.02995 | 865.01861 | I | 401.26195 | 201.13461 | 3 |
18 | 1885.13107 | 943.06917 | R | 288.17788 | 144.59258 | 2 |
19 | - | - | C-Amidated | 132.07676 | 66.54202 | 1 |
#1 | b(1+) | b(2+) | Sequence | y(1+) | y(2+) | #2 |
---|---|---|---|---|---|---|
1 | 148.07570 | 74.54149 | F | - | - | 19 |
2 | 261.15977 | 131.08352 | L | 1841.10083 | 921.05405 | 18 |
3 | 408.22819 | 204.61773 | F | 1728.01676 | 864.51202 | 17 |
4 | 495.26022 | 248.13375 | S | 1580.94834 | 790.97781 | 16 |
5 | 608.34429 | 304.67578 | L | 1493.91631 | 747.46179 | 15 |
6 | 721.42836 | 361.21782 | I | 1380.83224 | 690.91976 | 14 |
7 | 818.48113 | 409.74420 | P | 1267.74817 | 634.37772 | 13 |
8 | 905.51316 | 453.26022 | S | 1170.69540 | 585.85134 | 12 |
9 | 976.55028 | 488.77878 | A | 1083.66337 | 542.33532 | 11 |
10 | 1089.63435 | 545.32081 | I | 1012.62625 | 506.81676 | 10 |
11 | 1160.67147 | 580.83937 | A | 899.54218 | 450.27473 | 9 |
12 | 1217.69294 | 609.35011 | G | 828.50506 | 414.75617 | 8 |
13 | 1330.77701 | 665.89214 | L | 771.48359 | 386.24543 | 7 |
14 | 1429.84543 | 715.42635 | V | 658.39952 | 329.70340 | 6 |
15 | 1516.87746 | 758.94237 | S | 559.33110 | 280.16919 | 5 |
16 | 1587.91458 | 794.46093 | A | 472.29907 | 236.65317 | 4 |
17 | 1700.99865 | 851.00296 | I | 401.26195 | 201.13461 | 3 |
18 | 1857.09977 | 929.05352 | R | 288.17788 | 144.59258 | 2 |
19 | - | - | C-Amidated | 132.07676 | 66.54202 | 1 |
2.3. Secondary Structure Prediction and Physiochemical Properties of AaeAP1 and -2 and Their Cationicity-/Amphipathicity-Enhanced Analogues, AaeAP1a and -2a
Species | Peptide | Primary structure | Nucleotide sequence database |
---|---|---|---|
Androctonus aeneas | AaeAP1 | FLFSLIPSVIAGLVSAIRNa | (Accession No. HG792997) |
Androctonus aeneas | AaeAP2 | --------A----------a | (Accession No. HG792998) |
Mesobuthus martensii | Kb1 | --------A-S--I--FKa | (Accession No. AF159979) |
Mesobuthus eupeus | caerin-like AMP | --------A-S--I--FKa | (Accession No. KC108907) |
Androctonus amoreuxi | AamAP1 | -------HA-G--I--FKa | (Accession No. FR821613) |
Isometrus maculatus | imcroporin | -F---L--L-G------Ka | (Accession No. FJ750949) |
Lychas mucronatus | mucroporin | --G----L-G-----FKa | (Accession No. EU669864) |
Mesobuthus martensii | BmKb2 | --S-----A-S--I--FKa | (Accession No. AF543048) |
Tityus costatus | AMP clone 5 | -F------L-G---F--Ka | (Accession No. AY740687) |
Androctonus amoreuxi | AamAP2 | -P-----HA-GG-ISAIKa | (Accession No. FR821614) |
Peptides | Secondary structure | α-Helix (%) | Hydrophobicity (H) | Hydrophobic moment (μH) | Net charge |
---|---|---|---|---|---|
AaeAP1 | FLFSLIPSVIAGLVSAIRN cccccccchhhheeeeeec | 21.05 | 0.849 | 0.455 | +2 |
AaeAP2 | FLFSLIPSAIAGLVSAIRN cccccccchhhheeeeeec | 21.05 | 0.801 | 0.428 | +2 |
AaeAP1a | FLFKLIPKAIKGLVKAIRK ccceecchhhhhheeeeec | 31.58 | 0.610 | 0.669 | +7 |
AaeAP2a | FLFKLIPKVIKGLVKAIRK ccceecchhhhhheeeeec | 31.58 | 0.562 | 0.641 | +7 |
2.4. Antimicrobial/Haemolytic Activities of AaeAP1 and -2 and Their Cationicity-/Amphipathicity-Enhanced Analogues, AaeAP1a and -2a
Peptides | MIC (mg/L) | MBC (mg/L) 100% hemolysis (mg/L) | |||||
---|---|---|---|---|---|---|---|
S. aureus | E. coli | C. albicans | S. aureus | E. coli | C. albicans | Horse red cells | |
AaeAP1 | 16 | >512 | 32 | 32 | NT | 64 | 16 |
AaeAP2 | 16 | >512 | 32 | 16 | NT | 64 | 64 |
AaeAP1a | 4 | 16 | 4 | 32 | 32 | 16 | 32 |
AaeAP2a | 4 | 16 | 4 | 32 | 32 | 16 | 64 |
Peptide | Primary structure | Mass (Da) |
---|---|---|
AaeAP1 | FLFSLIPSVIAGLVSAIRNamide | 2016.18 |
AaeAP2 | FLFSLIPSAIAGLVSAIRNamide | 1986.15 |
AaeAP1a | FLFKLIPKVIKGLVKAIRKamide | 2209.48 |
AaeAP2a | FLFKLIPKAIKGLVKAIRKamide | 2181.45 |
* * * * * |
2.5. The Antiproliferative Effects of AaeAP1 and -2 and Their Cationicity-/Amphipathicity-Enhanced Analogues, AaeAP1a and -2a, against a Panel of Human Cancer Cell Lines
3. Discussion
4. Experimental Section
4.1. Acquisition of Scorpion Venom
4.2. Isolation and Structural Characterisation of Antimicrobial Activity from Lyophilised Venom
4.3. Antimicrobial Screening Assays
4.4. Molecular Cloning of Antimicrobial Peptide Biosynthetic Precursor-Encoding cDNAs from the Venom-Derived cDNA Library
4.5. Prediction of Putative AMP Secondary Structures and Physicochemical Properties
4.6. Peptide Synthesis and Purification
4.7. Antimicrobial Minimal Inhibitory Concentration and Minimum Bactericidal Assays
4.8. Haemolysis Assay
4.9. Culture and Maintenance of Human Cancer Cell Lines
4.10. Assessment of Anti-Proliferative Effects of AMPs on Human Cancer Cells Using the MTT Cell Viability Assay
5. Conclusions
Author Contributions
Conflicts of Interest
References
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Du, Q.; Hou, X.; Wang, L.; Zhang, Y.; Xi, X.; Wang, H.; Zhou, M.; Duan, J.; Wei, M.; Chen, T.; et al. AaeAP1 and AaeAP2: Novel Antimicrobial Peptides from the Venom of the Scorpion, Androctonus aeneas: Structural Characterisation, Molecular Cloning of Biosynthetic Precursor-Encoding cDNAs and Engineering of Analogues with Enhanced Antimicrobial and Anticancer Activities. Toxins 2015, 7, 219-237. https://doi.org/10.3390/toxins7020219
Du Q, Hou X, Wang L, Zhang Y, Xi X, Wang H, Zhou M, Duan J, Wei M, Chen T, et al. AaeAP1 and AaeAP2: Novel Antimicrobial Peptides from the Venom of the Scorpion, Androctonus aeneas: Structural Characterisation, Molecular Cloning of Biosynthetic Precursor-Encoding cDNAs and Engineering of Analogues with Enhanced Antimicrobial and Anticancer Activities. Toxins. 2015; 7(2):219-237. https://doi.org/10.3390/toxins7020219
Chicago/Turabian StyleDu, Qiang, Xiaojuan Hou, Lei Wang, Yingqi Zhang, Xinping Xi, Hui Wang, Mei Zhou, Jinao Duan, Minjie Wei, Tianbao Chen, and et al. 2015. "AaeAP1 and AaeAP2: Novel Antimicrobial Peptides from the Venom of the Scorpion, Androctonus aeneas: Structural Characterisation, Molecular Cloning of Biosynthetic Precursor-Encoding cDNAs and Engineering of Analogues with Enhanced Antimicrobial and Anticancer Activities" Toxins 7, no. 2: 219-237. https://doi.org/10.3390/toxins7020219