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The stoichiometry of peptide-heparan sulfate binding as a determinant of uptake efficiency of cell-penetrating peptides

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Abstract

Binding to negatively charged heparan sulfates (HS) at the cell surface is considered the first step in the internalization of cationic cell-penetrating peptides (CPPs). However, little is known about the relation of the characteristics of the HS-CPP interaction such as affinity, stoichiometry, and clustering with uptake. In this study, we investigated a collection of mutants of a cyclic CPP derived from human lactoferrin with respect to HS binding and uptake. The thermodynamic parameters of HS binding were determined by isothermal titration calorimetry, clustering of HS was investigated by dynamic light scattering, and cellular uptake by flow cytometry and confocal microscopy. Whereas mutations of non-arginine amino acids that are conserved across lactoferrins of different mammalia only had a minor effect on uptake efficiency, changes in the number of arginine residues influenced the uptake significantly. In general, introduction of arginine residues and cyclization improved the HS affinity and the ability to cluster HS. In particular, there was a strong negative correlation between stoichiometry and uptake, indicating that crosslinking of HS is the driving force for the uptake of arginine-rich CPPs. Using glycan microarrays presenting a collection of synthetic HS, we show that a minimal chain length of HS is required for peptide binding.

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Abbreviations

CPP:

Cell-penetrating peptide

DLS:

Dynamic light scattering

GAG:

Glycosaminoglycan

HS:

Heparan sulfate

hLF:

Human lactoferrin CPP

ITC:

Isothermal titration calorimetry

NZ:

Nucleation zones

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Acknowledgments

We would like to thank the Max Planck Society and the Studienstiftung des Deutschen Volkes for their generous support of this work (scholarship to AR).

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Authors and Affiliations

  1. Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Medical Centre, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands

    Rike Wallbrecher, Wouter P. R. Verdurmen, Samuel Schmidt, Petra H. Bovee-Geurts, Toin H. van Kuppevelt & Roland Brock

  2. Department of Biomolecular Systems, Max-Planck-Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476, Potsdam, Germany

    Felix Broecker, Anika Reinhardt & Peter H. Seeberger

  3. Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195, Berlin, Germany

    Felix Broecker, Anika Reinhardt & Peter H. Seeberger

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  1. Rike Wallbrecher
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  2. Wouter P. R. Verdurmen
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  3. Samuel Schmidt
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  7. Toin H. van Kuppevelt
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  9. Roland Brock
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Correspondence to Roland Brock.

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Wallbrecher, R., Verdurmen, W.P.R., Schmidt, S. et al. The stoichiometry of peptide-heparan sulfate binding as a determinant of uptake efficiency of cell-penetrating peptides. Cell. Mol. Life Sci. 71, 2717–2729 (2014). https://doi.org/10.1007/s00018-013-1517-8

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