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Biotechnology and Applied Biochemistry (2001) 34, (151–159) (Printed in Great Britain)

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Identification of an import signal for, and the nuclear localization of, human lactoferrin

Silvana Penco*, Sonia Scarfi†, Marco Giovine‡, Gianluca Damonte†, Enrico Millo†, Barbara Villaggio§, Mario Passalacqua†, Marina Pozzolini†, Cecilia Garrè* and Umberto Benatti†¹

*Department of Oncology, Biology and Genetics, University of Genoa, Viale Benedetto XV, 6 Genoa, Italy, †Department of Experimental Medicine, Section Biochemistry, University of Genoa, Viale Benedetto XV, 1 Genoa, Italy, ‡CNR-Biotechnology Target Project, Via Leon Battista Alberti 4, Genoa, Italy, and §Department of Internal Medicine, University of Genoa, Viale Benedetto XV, 6 Genoa, Italy

Key words: nuclear localization signal, peptide nucleic acids.

Abbreviations used: NLS, nuclear localization signal; NES, nuclear export signal; (h)Lf, (human) lactoferrin; ES-MS, electrospray MS; PNA, peptide nucleic acid; RS, reverse phase; TFA, trifluoroacetic acid; Rhod-Lf-peptide, rhodaminated Lf-derived peptide; GRRRR, one-letter code for the pentapeptide Gly-Arg-Arg-Arg-Arg; Fmoc, fluoren-9-ylmethoxycarbonyl; EGFP, enhanced green fluorescent protein; HTLV, human T-cell leukaemia virus.

¹ To whom correspondence should be addressed (e-mail benatti@unige.it).

Many different unique functions have been attributed to lactoferrin (Lf), including DNA and RNA binding, and transport into the nucleus, where Lf binds to specific sequences and activates transcription. A pentapeptide, Gly-Arg-Arg-Arg-Arg, corresponding to a region of the N-terminal portion of human Lf rich in basic amino acids, was synthesized and its intracellular localization was investigated. Peptide internalization was assayed using the rhodaminated form of the same molecule. This N-terminal peptide sequence is able to be internalized within less than 10 min at concentration as low as 1 µM, and its intracellular localization is nuclear, mainly nucleolar. Similar behaviour was observed using peptides composed of either all L or D amino acids, the last one being a retro-inverse peptide. The internalization process does not involve an endocytotic pathway, since no inhibition of the uptake was observed at 4 °C. The kinetics of peptide internalization was also evaluated. The internalization properties of such a short Lf pentapeptide have been assayed for its ability to transport peptide nucleic acids (PNAs) inside cells in order to improve their efficacy. The abundant transmembrane transport and nuclear localization of the proposed peptide, deriving from hLf and, for the first time, identified as a nuclear localization signal, could be used as an alternative strategy to tackle the unsolved problem of intracellular accumulation of antisense and antigene drugs and for the development of new pharmacological tools.

Received 1 June 2001; accepted 21 July 2001

Portland Press Ltd © 2001