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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Glycine and Histidine-rich Antifungal Peptides: On the Way tothe Mode of Action of Shepherin ICésar Remuzgo 1* , Thiago R. S. Lopes 1 , Thaís S. Oewel 1 ,Gláucia M. Machado-Santelli 2 , Sirlei Daffre 3 , andM. Terêsa Machini Miranda 11 Department of Biochemistry, Institute of Chemistry; Departments of 2 Cell andDevelopmental Biology and of 3 Parasitology, Institute of Biomedical Sciences;05508-000, University of São Paulo, São Paulo, Brazil*Present address: Special Laboratory of Pain and Signalling,Butantan Institute, São Paulo, BrazilIntroductionThe emergence of microorganisms resistant to the commercial antibiotics has become aworldwide problem due to the lack of new drugs for the treatment of infectious diseases.Thus, the investigation of novel antimicrobial molecules has been considered critical forthe development of a new generation of antibiotics [1].Shepherin I (Shep I) is a 28-mer peptide isolated from the roots of the Capsellabursa-pastoris plant that is characterized by its almost exclusive glycine (67.9%) andhistidine (28.6%) contents, presence of six tandem repeats of the motif Gly-Gly-His andexpression of antimicrobial activity against yeast phase-fungi and mycelial fungi [2].With the aim of studying this glycine- and histidine-rich antimicrobial peptide, wesynthesized truncated, amidated, fluorescently labeled and/or tryptophan-containinganalogues, tested them against some Candida strains and Saccharomyces cerevisiae at lowand high salt concentration in absence or presence of Zn 2+ ions and evaluated its toxicity onhuman erythrocytes. Also, we determined the killing kinetics of Shep Ia against C. albicansMDM8 and investigated its internalization into these cells.Results and DiscussionStepwise solid-phase peptide syntheses were performed manually at 60°C usingconventional heating and customized protocols [3,4]. The crude peptides werecharacterized by LC-ESI/MS and purified by RP-HPLC. The overall purities of finalpeptides, evaluated by RP-HPLC and confirmed by LC-ESI/MS, were higher than 95%.Their peptide contents were obtained by full hydrolysis followed by amino acid analysis ofthe hydrolyzates. Their anticandidal, anticandidacidal and hemolytic activity weremeasured as earlier described [5,6]. The internalization of the fluorescently labeledanalogues into the yeast cells was also verified by confocal microscopy and FACSanalysis.Shep Ia, Shep I (3-28)a and Shep I (6-28)a were as active as Shep I against C. albicansstrains, C. tropicalis Squibb 1600, C.parapsilosis ATCC 22019 and S.cerevisiae ATCC 2601, indicating thatShep I (6-28)a may be the minimal fullyactive portion of Shep Ia. Shep Ia wasmore active (4-8 fold) than Shep I againstC. krusei ATCC 6258. The effect of saltconcentration on the anticandidal activitywas less pronounced for Shep Ia, Shep I(3-28)a and Shep I (6-28)a than for thecorresponding carboxyl-free analogues.On the other hand, the activity of Shep Iand amidated analogues weresignificantly enhanced in the presence ofthe Zn 2+ ion (Table 1). Shep Ia killed C.Fig. 1. Killing kinetics of Shep Ia againstC. albicans MDM8.albicans MDM8 cells at 62.5 μM in 30min (Figure 1), caused low hemolysis inhuman erythrocytes in isotonic glucose414