Design of therapeutically improved analogue of the antimicrobial peptide, indolicidin, using a glycosylation strategy
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Indolicidin is a member of cathelicidin family which displays broad spectrum antimicrobial activity. Severe toxicity and aggregation propensity associated with indolicidin pose a huge limitation to its probable therapeutic application. We are reporting the use of glycosylation strategy to design an analogue of indolicidin and subsequently explore structural and functional effects of sugar on it. Our study led to the design of a potent antibacterial glycosylated peptide, [βGlc-T9,K7]indolicidin, which showed decreased toxicity against erythrocytes and macrophage cells and thus a higher therapeutic selectivity. The incorporation of sugar also increased the solubility of the peptide. The mode of bacterial killing, functional stability, LPS binding, and cytokine inhibitory potential of the peptide, however, seemed unaffected upon glycosylation. Absence of significant changes in structure upon glycosylation accounts for the possibly retained functions and mode of action of the peptide. Our report thus presents the designing of an indolicidin analogue with improved therapeutic potential by substituting aromatic amino acid with glycosylated amino acid as a promising strategy for the first time.
KeywordsAntimicrobial peptides Indolicidin Glycosylation Toxicity Therapeutic selectivity Aggregation
Computer-aided resonance assignment
Dulbecco’s modified Eagle’s medium
4-(2-Hydroxyethyl)-1-piperazine ethanesulfonic acid
High-performance liquid chromatography
Lysogeny broth (Luria–Bertani broth)
Matrix-assisted laser desorption and ionization
Minimum haemolytic concentration
Scanning electron microscopy
Total correlation spectroscopy
Tumor necrosis factor alpha
We thank Department of Science and Technology (Project no.: SR/S1/OC-63/2012), India for financial support. We thank Department of Biotechnology (DBT), Government of India and ICGEB New Delhi for the NMR facility. We thank Ms. Shanta Sen for help with HRMS data. We also thank Ms. Rekha Rani for help with SEM studies and Dr. Sharad Vashisht for discussions regarding structural studies of glycopeptide.
KK conceived the idea of present work. KK and RD designed the experiments. RD performed the experiments. KK and RD analyzed the data and wrote the manuscript. NSB and PA performed the NMR studies.
Compliance with ethical standards
Conflict of interest
Authors declare that they have no conflicts of interest.
Research involving human participants and/or animals
This article does not contain any studies with human participants or animals performed by any of the authors.
- Case DA, Ben-Shalom IY, Brozell SR, Cerutti DS, Cheatham TE III, Cruzeiro VWD, Darden TA, Duke RE, Ghoreishi D, Gilson MK, Gohlke H, Goetz AW, Greene D, Harris R, Homeyer N, Izadi S, Kovalenko A, Kurtzman T, Lee TS, LeGrand S, Li P, Lin C, Liu J, Luchko T, Luo R, Mermelstein DJ, Merz KM, Miao Y, Monard G, Nguyen C, Nguyen H, Omelyan I, Onufriev A, Pan F, Qi R, Roe DR, Roitberg A, Sagui C, Schott-Verdugo S, Shen J, Simmerling CL, Smith J, Salomon-Ferrer R, Swails J, Walker RC, Wang J, Wei H, Wolf RM, Wu X, Xiao L, York DM, Kollman PA (2014) Amber. University of California, San FranciscoGoogle Scholar
- Coltart DM, Royyuru AK, Williams LJ, Glunz PW, Sames D, Kuduk SD, Schwarz JB, Chen XT, Danishefsky SJ, Live DH (2002) Principles of mucin architecture: structural studies on synthetic glycopeptides bearing clustered mono-, di-, tri-, and hexasaccharide glycodomains. J Am Chem Soc 124:9833–9844CrossRefPubMedGoogle Scholar
- Hartmann M, Berditsch M, Hawecker J, Ardakani MF, Gerthsen D, Ulrich AS (2010) Damage of the bacterial cell envelope by antimicrobial peptides gramicidin S and PGLa as revealed by transmission and scanning electron microscopy. Antimicrob Agents Chemother 54:3132–3142CrossRefPubMedPubMedCentralGoogle Scholar
- Hsu CH, Chen C, Jou ML, Lee AYL, Lin YC, Yu YP, Huang WT, Wu SH (2005) Structural and DNA-binding studies on the bovine antimicrobial peptide, indolicidin: evidence for multiple conformations involved in binding to membranes and DNA. Nucleic Acids Res 33:4053–4064CrossRefPubMedPubMedCentralGoogle Scholar
- Keller R (2004) The computer aided resonance assignment tutorial, 1st edn. Verlag, CANTINAGoogle Scholar
- Kustanovich I, Shalev DE, Mikhlin M, Gaidukov L, Mor A (2002) Structural requirements for potent versus selective cytotoxicity for antimicrobial dermaseptin S4 derivatives. Biochemistry 277:16941–16951Google Scholar
- Lee HS, Qi Y, Im W (2015) Effects of N-glycosylation on protein conformation and dynamics: protein Data Bank analysis and molecular dynamics simulation study. Sci Rep 5:1–7Google Scholar
- Lingwood C, Mylvaganam M, Minhas F, Binnington B, Branch DR, Pomès R (2005) The sulfogalactose moiety of sulfoglycosphingolipids serves as a mimic of tyrosine phosphate in many recognition processes: prediction and demonstration of Src homology 2 domain/sulfogalactose binding. J Biol Chem 280:12542–12547CrossRefPubMedGoogle Scholar
- Nan YH, Park KH, Park Y, Jeon YJ, Kim Y, Park IS, Hahm KS, Shin SY (2009a) Investigating the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin. FEMS Microbiol Lett 292:134–140CrossRefPubMedGoogle Scholar
- Roth BL, Poot M, Yue ST, Millard PJ, Roth BL, Poot M, Yue ST, Millard PJ (1997) Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain. Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain. Appl Environ Microbiol 63:2421–2431PubMedPubMedCentralGoogle Scholar
- Savjani KT, Gajjar AK, Savjani JK (2012) Drug solubility: importance and enhancement techniques. ISRN Pharm 2012:1–10Google Scholar
- Ueda T, Tomita K, Notsu Y, Ito T, Fumoto M, Takakura T, Nagatome H, Takimoto A, Mihara S, Togame H et al (2009) Chemoenzymatic synthesis of glycosylated glucagon-like peptide 1: effect of glycosylation on proteolytic resistance and in vivo blood glucose-lowering activity. J Am Chem Soc 14:6237–6245CrossRefGoogle Scholar
- Wu WG, Pasternack L, Huang DH, Koeller KM, Lin CC, Seitz O, Wong CH (1999) Structural study on O-glycopeptides: glycosylation-induced conformational changes of O-GlcNAc, O-LacNAc, O-Sialyl-LacNAc, and O-Sialyl- Lewis-X peptides of the mucin domain of MAdCAM-1. J Am Chem Soc 121:2409–2417CrossRefGoogle Scholar