Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans.
Cell surface proteoglycans (PGs) appear to promote uptake of arginine-rich cell-penetrating peptides (CPPs), but their exact functions are unclear. To address if there is specificity in the interactions of arginines and PGs leading to improved internalization, we used flow cytometry to examine uptake in relation to cell surface binding for penetratin and two arginine/lysine substituted variants (PenArg and PenLys) in wildtype CHO-K1 and PG-deficient A745 cells. All peptides were more efficiently internalized into CHO-K1 than into A745, but their cell surface binding was independent of cell type. Thus, PGs promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Uptake of each peptide was linearly dependent on its cell surface binding, and affinity is thus important for efficiency. However, the gradients of these linear dependencies varied significantly. Thus each peptide's ability to stimulate uptake once bound to the cell surface is reliant on formation of specific uptake-promoting interactions. Heparin affinity chromatography and clustering experiments showed that penetratin and PenArg binding to sulfated sugars is stabilized by hydrophobic interactions and result in clustering, whereas PenLys only interacts through electrostatic attraction. This may have implications for the molecular mechanisms behind arginine-specific uptake stimulation as penetratin and PenArg are more efficiently internalized than PenLys upon interaction with PGs. However, PenArg is also least affected by removal of PGs. This indicates that an increased arginine content not only improve PG-dependent uptake but also that PenArg is more adaptable as it can use several portals of entry into the cell.
Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
Helene L
HL
Amand
Hanna A
HA
Rydberg
Louise H
LH
Fornander
Per
P
Lincoln
Bengt
B
Nordén
Elin K
EK
Esbjörner
D000818:Animals; D001120:Arginine / Q000737:chemistry*; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D057846:Cell-Penetrating Peptides; D006224:Cricetinae; D003412:Cricetulus; D005453:Fluorescence; D006493:Heparin / Q000378:metabolism; D008239:Lysine / Q000737:chemistry*; D011485:Protein Binding; D011509:Proteoglycans / Q000378:metabolism*
D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D011509:Proteoglycans; D006493:Heparin; D001120:Arginine; C414409:penetratin; D008239:Lysine