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Chemical synthesis, structure–activity relationship, and properties of shepherin I: a fungicidal peptide enriched in glycine-glycine-histidine motifs

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Abstract

Although glycine-rich antimicrobial peptides (AMPs) are found in animals and plants, very little has been reported on their chemistry, structure activity-relationship, and properties. We investigated those topics for Shepherin I (Shep I), a glycine-rich AMP with the unique amino acid sequence G1YGGHGGHGGHGGHGGHGGHGHGGGGHG28. Shep I and analogues were synthesized by the solid-phase method at 60 °C using conventional heating. Purification followed by chemical characterization confirmed the products' identities and high purity. Amino acid analysis provided their peptide contents. All peptides were active against the clinically important Candida species, but ineffective against bacteria and mycelia fungi. Truncation of the N- or C-terminal portion reduced Shep I antifungal activity, the latter being more pronounced. Carboxyamidation of Shep I did not affect the activity against C. albicans or C. tropicalis, but increased activity against S. cerevisiae. Carboxyamidated analogues Shep I (3-28)a and Shep I (6-28)a were equipotent to Shep I and Shep Ia against Candida species. As with most cationic AMPs, all peptides had their activity significantly reduced in high-salt concentrations, a disadvantage that is defeated if 10 µM ZnCl2 is present. At 100 µM, the peptides were practically not hemolytic. Shep Ia also killed C. albicans MDM8 and ATCC 90028 cells. Fluo-Shep Ia, an analogue labeled with 5(6)-carboxyfluorescein, was rapidly internalized by C. albicans MDM8 cells, a salt-sensitive process dependent on metabolic energy and temperature. Altogether, such results shed light on the chemistry, structural requirements for activity, and other properties of candidacidal glycine-rich peptides. Furthermore, they show that Shep Ia may have strong potential for use in topical application.

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Abbreviations

Shep I :

Shepherin I

SAR:

Structure–activity relationship

AMPs:

Antimicrobial peptides

MIC:

Minimal inhibitory concentration

Fluo :

5(6)-Carboxyfluorescein

GRP:

Glycine-rich peptide or protein

Boc:

Tert-butyloxycarbonyl

Fmoc:

9-Fluorenylmethyloxycarbonyl

Tos:

Tosyl

PAM:

4-Hydroxymethyl-phenylacetamidomethyl

NMP:

N-methyl-2-pyrrolidone

DMSO:

Dimethyl sulfoxide

PBS:

Phosphate-buffered saline

IGP:

Isotonic glucose phosphate

PBC:

Phosphate-borate-citrate

TFE:

2,2,2-Trifluoroethanol

SDS:

Sodium dodecyl sulfate

PDB:

Potato dextrose broth

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Acknowledgments

This work was supported by FAPESP (grants 04/14376-7 and 08/11695-1 to MTM) and CNPq (142022/2003-9, a doctoral fellowship for CR). We thank Dr. Nilton Lincopan (for providing C. albicans ATCC 90028 and HU 168 strains), Dr. Cleber W. Liria (for amino acid analyses), Adriana Y. Matzukuma and Roberto C. Modia (for assistance with FACS and confocal microscopy, respectively), and Gustavo P.B. Carretero (for theoretical structure prediction).

Conflict of interest

The authors declare that they have no conflicts of interest.

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Author notes

  1. César Remuzgo

    Present address: Special Laboratory of Pain and Signaling, Butantan Institute, São Paulo, Brazil

Authors and Affiliations

  1. Laboratory of Peptide Chemistry, Department of Biochemistry, Institute of Chemistry, University of São Paulo, P.O. Box 26077, 05513-970, São Paulo, SP, Brazil

    César Remuzgo, Thaís S. Oewel, Thiago R. S. Lopes & M. Teresa Machini

  2. Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

    Sirlei Daffre

  3. Laboratory of Structural Biology, Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil

    Fabio H. Dyszy & Shirley Schreier

  4. Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

    Gláucia M. Machado-Santelli

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  1. César Remuzgo
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  2. Thaís S. Oewel
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Correspondence to M. Teresa Machini.

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Remuzgo, C., Oewel, T.S., Daffre, S. et al. Chemical synthesis, structure–activity relationship, and properties of shepherin I: a fungicidal peptide enriched in glycine-glycine-histidine motifs. Amino Acids 46, 2573–2586 (2014). https://doi.org/10.1007/s00726-014-1811-2

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