Characterization of the cell penetrating properties of a human salivary proline-rich peptide.
Saliva contains hundreds of small proline-rich peptides most of which derive from the post-translational and post-secretory processing of the acidic and basic salivary proline-rich proteins. Among these peptides we found that a 20 residue proline-rich peptide (p1932), commonly present in human saliva and patented for its antiviral activity, was internalized within cells of the oral mucosa. The cell-penetrating properties of p1932 have been studied in a primary gingival fibroblast cell line and in a squamous cancer cell line, and compared to its retro-inverso form. We observed by mass-spectrometry, flow cytometry and confocal microscopy that both peptides were internalized in the two cell lines on a time scale of minutes, being the natural form more efficient than the retro-inverso one. The cytosolic localization was dependent on the cell type: both peptide forms were able to localize within nuclei of tumoral cells, but not in the nuclei of gingival fibroblasts. The uptake was shown to be dependent on the culture conditions used: peptide internalization was indeed effective in a complete medium than in a serum-free one allowing the hypothesis that the internalization could be dependent on the cell cycle. Both peptides were internalized likely by a lipid raft-mediated endocytosis mechanism as suggested by the reduced uptake in the presence of methyl-ß-cyclodextrin. These results suggest that the natural peptide may play a role within the cells of the oral mucosa after its secretion and subsequent internalization. Furthermore, lack of cytotoxicity of both peptide forms highlights their possible application as novel drug delivery agents.
Istituto di Biochimica e Biochimica Clinica, Facoltà di Medicina, Catholic University, L.go F. Vito, 1, 00168 Rome, Italy. Electronic address: gradicioni@yahoo.it.
Giorgia
G
Radicioni
Dipartimento di Tecnologie e Salute, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy. Electronic address: annarita.stringaro@iss.it.
Annarita
A
Stringaro
Dipartimento di Tecnologie e Salute, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy. Electronic address: agnese.molinari@iss.it.
Agnese
A
Molinari
Istituto di Biochimica e Biochimica Clinica, Facoltà di Medicina, Catholic University, L.go F. Vito, 1, 00168 Rome, Italy. Electronic address: g.nocca@rm.unicatt.it.
Giuseppina
G
Nocca
Istituto per la Chimica del Riconoscimento Molecolare, Italian National Research Council, Via Mario Bianco, 9, 20100 Milan, Italy. Electronic address: renato.longhi@icrm.cnr.it.
Renato
R
Longhi
Istituto di Biochimica e Biochimica Clinica, Facoltà di Medicina, Catholic University, L.go F. Vito, 1, 00168 Rome, Italy. Electronic address: davide.pirolli@rm.unicatt.it.
Davide
D
Pirolli
Dipartimento di Otorinolaringoiatria, Facoltà di Medicina, Catholic University, Largo A. Gemelli, 8, 00168 Rome, Italy. Electronic address: escarano@rm.unicatt.it.
Emanuele
E
Scarano
Istituto di Biochimica e Biochimica Clinica, Facoltà di Medicina, Catholic University, L.go F. Vito, 1, 00168 Rome, Italy. Electronic address: federica.iavarone@rm.unicatt.it.
Federica
F
Iavarone
Dipartimento di Scienze Applicate ai Biosistemi, University of Cagliari, Cittadella Universitaria, Monserrato, 09042 Cagliari, Italy. Electronic address: bmanconi@unica.it.
Barbara
B
Manconi
Dipartimento di Scienze Applicate ai Biosistemi, University of Cagliari, Cittadella Universitaria, Monserrato, 09042 Cagliari, Italy. Electronic address: tcabras@unica.it.
Tiziana
T
Cabras
Dipartimento di Scienze Applicate ai Biosistemi, University of Cagliari, Cittadella Universitaria, Monserrato, 09042 Cagliari, Italy. Electronic address: imessana@unica.it.
Irene
I
Messana
Istituto di Biochimica e Biochimica Clinica, Facoltà di Medicina, Catholic University, L.go F. Vito, 1, 00168 Rome, Italy. Electronic address: massimo.castagnola@icrm.cnr.it.
Massimo
M
Castagnola
Istituto per la Chimica del Riconoscimento Molecolare, Italian National Research Council, Rome, L. go F. Vito, 1, 00168 Rome, Italy. Electronic address: alberto.vitali@icrm.cnr.it.
Alberto
A
Vitali
D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D057846:Cell-Penetrating Peptides / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D005881:Gingiva / Q000166:cytology; D006801:Humans; D018613:Microscopy, Confocal; D010455:Peptides / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D055273:Salivary Proline-Rich Proteins / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D047392:beta-Cyclodextrins / Q000494:pharmacology
D057846:Cell-Penetrating Peptides; D003470:Culture Media; D016895:Culture Media, Serum-Free; D010455:Peptides; D055273:Salivary Proline-Rich Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin
Cell internalization; Flow cytometry; Laser scanning confocal microscopy; Proline-rich peptide; Saliva