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3 Transportans Margus Pooga, Mattias Hällbrink, and Ülo Langel CONTENTS 3.1 Introduction ....................................................................................................53 3.2 The Discovery of Transportan .......................................................................53 3.3 Cell Penetration of Transportan.....................................................................55 3.4 Properties of Transportan...............................................................................57 3.5 Structure–Activity Relationship of Transportan............................................58 3.6 Transportan as a Delivery Vector...................................................................59 3.6.1 Delivery of Peptides...........................................................................59 3.6.2 Delivery of PNA ................................................................................60 3.6.3 Delivery of Proteins ...........................................................................61 3.7 Methodological Considerations .....................................................................66 3.7.1 Cargo–CPP Conjugation Strategies ...................................................66 3.7.2 Disulfide Heterodimers ......................................................................66 3.7.3 Conjugation of Transportan to Proteins ............................................66 3.8 Structural Organization of Transportan .........................................................67 Acknowledgments....................................................................................................69 References................................................................................................................69 3.1 INTRODUCTION The first discovered cell-penetrating peptides (CPPs) were derived from naturally occurring proteins like the Antennapedia homeodomain of Drosophila, 1 Tat of HIV- 1, 2 and VP22 of HSV, 3 followed soon by designed chimeric peptides like MTS- NLS, 4 transportan, 5 and model peptides. 6 Numerous analogues of these CPPs have now been designed and synthesized, giving rise to families of respective peptides. Both classes of CPPs, protein-derived and designed or combined, have been vastly expanded by new peptides and derivatives. In this chapter we summarize the discovery, properties, and application of the cell-penetrating peptide transportan and its analogues. 3.2 THE DISCOVERY OF TRANSPORTAN The predecessor of transportan, galparan, comprises a fragment of the neuropeptide galanin (1–13) and the wasp venom peptide mastoparan. 7 (Table 3.1) It was designed under a program aimed at creating galanin receptor ligands by using a chimeric 0-8493-1141-1/02/$0.00+$1.50 © 2002 by CRC Press LLC 53
54 Cell-Penetrating Peptides: Processes and Applications TABLE 3.1 Sequences and Uptake Parameters of Mastoparan (MP), Transportan (TP), and Transportan Analogues (TP2 to TP16) Length a Cellular uptake b Speed of uptake, τ0.5 Sequence (a.a.) 0°C 37°C (min) Galparan GWTLNSAGYLLGPINLKALAALAKKIL 27 nd nd nd TP GWTLNSAGYLLGK c INLKALAALAKKIL 27 +++ +++ 3.4 TP2 GWTLNSAGYLLGK c INLKAKAALAKKLL 27 ++ ++ TP3 C6H5CH2CO(D-Y(Me))FQNRPRYK c -(Ahx) -INLKALAALAKKIL ++ ++ nd TP4 GWTLNSAGYLLGK c FLPLILRKIVTAL 26 — — — TP5 GWTLNPAGYLLGK c INLKALAALAKKIL 27 + + nd TP6 GWTLNPPGYLLGK c INLKALAALAKKIL 27 + + nd TP7 ...LNSAGYLLGK c INLKALAALAKKIL 24 +++ +++ 4.3 TP8 .........LLGK c INLKALAALAKKIL 18 — +/– — TP9 GWTLNSAGYLLGK c ..LKALAALAKKIL 25 +++ +++ 6.5 TP10 ......AGYLLGK c INLKALAALAKKIL 21 +++ +++ 8.6 TP11 GWTLNS......K c INLKALAALAKKIL 21 + + nd TP12 ...LNSAGYLLGK c ..LKALAALAKKIL 22 ++ ++ 10.7 TP13 ...LNSAGYLLGK c ....ALAALAKKIL 20 — +/– — TP14 ......AGYLLGK c ..LKALAALAKKIL 19 ++ ++ nd TP15 ...LNSAGYLLGK c ..LKALAALAK… 19 — +/– — TP16 GWTLNSAGYLLGK c INLKAPAALAKKIL 27 — +/– — MP d INLKALAALAKKIL 14 + + nd a Number of amino acid residues. b Detection of biotin by indirect immunofluorescence: +++ denotes comparable, ++ or + decreased, and +/– weak uptake in relation to that of transportan. c Biotinylated at ε-amino group of Lys. d Biotinylated at the N terminus. strategy. The combination of these two peptides was motivated by the following observations: 1. Galanin (1–13) had been shown to be the smallest fragment of this neuropeptide that exhibited a reasonably high affinity toward the galanin receptor type 1. 8 2. Many chimeric galanin receptor ligands were shown to possess very high affinity to galanin receptors. They consisted of galanin (1–13) in N terminus and a fragment of other neuropeptide or just a stretch of hydrophobic amino acids in C terminus. 9 3. The C-terminal part of the chimeras was suspected to interact primarily with cellular membranes rather than with the receptor protein. Therefore, mastoparan, known to interact with lipid membranes, was used to Cterminally extend galanin (1–13) in order to increase the affinity toward membranes. 7
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CELL- PENETRATING PEPTIDES Processe
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Pharmacology and Toxicology: Basic
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Library of Congress Cataloging-in-P
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to the handbook are prominent resea
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REFERENCES 1. Green, M. and Loewens
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Contributors Mats Andersson Microbi
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Erin T. Pelkey Department of Chemis
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Contents Section I Classes of Cell-
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Chapter 16 Cell-Penetrating Peptide
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Page 26 and 27: The Tat-Derived Cell-Penetrating Pe
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Page 45 and 46: 24 Cell-Penetrating Peptides: Proce
Page 76 and 77: Transportans 55 Indeed, galparan is
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Page 80 and 81: Transportans 59 In the penetration
Page 82 and 83: Transportans 61 A 21-mer antisense
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Page 86 and 87: Transportans 65 antibiotin antibodi
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Page 94 and 95: Model Amphipathic Peptides 73 its D
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Signal Sequence-Based Cell-Penetrat
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116 Cell-Penetrating Peptides: Proc
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Interactions of Cell-Penetrating Pe
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Structure Prediction of CPPs and It
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FIGURE 9.7 (Color Figure 9.7 follow
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FIGURE 9.8 The center of the figure
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Biophysical Studies of Cell-Penetra
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Toxicity and Side Effects of Cell-P
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Kinetics of Uptake of Cell-Penetrat
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Cell-Penetrating Peptide Conjugatio
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Cell-Penetrating Peptides as Vector
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TABLE 16.1 Examples of Transport of
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CPP 2 Cargo or mRNA CAP Antisense A
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Microbial Membrane-Permeating Pepti
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Index A Abaecin, 129 Abz radiolabel
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Index 399 Diffraction, 168 Disulphi
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Index 401 structure prediction, 187
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Index 403 pRB proteins, Tat-E1A bin
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Index 405 lipid perturbation (secon
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