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Cited by 68

The DOI matches its Crossref record; 34 of the 34 references it cites were found in a registry or an index.

Guanidinylated Neomycin Delivers Large, Bioactive Cargo into Cells through a Heparan Sulfate-dependent Pathway

Elson-Schwab, Lev, Garner, Omai B., Schuksz, Manuela, Crawford, Brett E., Esko, Jeffrey D., Tor, Yitzhak·2007·Journal of Biological Chemistry
DOI 10.1074/jbc.m700463200
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Abstract

Facilitating the uptake of molecules into living cells is of substantial interest for basic research and drug delivery applications. Arginine-rich peptides have been shown to facilitate uptake of high molecular mass cargos into cells, but the mechanism of uptake is complex and may involve multiple receptors. In this report, we show that a derivative of the aminoglycoside antibiotic neomycin, in which all of the ammonium groups have been converted into guanidinium groups, can carry large (>300 kDa) bioactive molecules across cell membranes. Delivery occurs at nanomolar transporter concentrations and under these conditions depends entirely on cell surface heparan sulfate proteoglycans. Conjugation of guanidinoneomycin to the plant toxin saporin, a ribosome-inactivating agent, results in proteoglycan-dependent cell toxicity. In contrast, an arginine-rich peptide shows both heparan sulfate-dependent and -independent cellular uptake. The high selectivity of guanidinoneomycin for heparan sulfate suggests the possibility of exploiting differences in proteoglycan compositions to target delivery to different cell types.

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Crossref · 10.1074/jbc.m700463200

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Figures

Image 1
CC-BY-NC-ND · From the PDF
FIGURE2 Uptakeoffluorescentcarriers in CHO cells. a , biotinylated guanidinoneomycin ( G-Neo ), biotinylated neomycin ( Neo ), and biotinylated Arg 9 (Arg 9 ) were conjugated to streptavidin-PE-Cy5 ( ∼ 300 kDa). Wild-type ( blue ) and heparan/chondroitin sulfate-deficient pgsA cells ( green ) were incubated with the different conjugates at concentrations from 10 to 1000 nM for 1 h at 37 °C. After washing the cells, they were released with EDTA and analyzed by flow cytometry. Cells incubated with streptavidin-PE-Cy5 alone are shown in red . b , mean fluorescence values show that both guanidinoneomycin and neomycin display glycosaminoglycan-dependent uptake; however, guanidinoneomycin has a considerably higher uptake efficiency than neomycin. The uptake of Arg 9 does not appear to depend exclusively on heparan/chondroitin sulfate glycosaminoglycans.
FIGURE2 Uptakeoffluorescentcarriers in CHO cells. a , biotinylated guanidinoneomycin ( G-Neo ), biotinylated neomycin ( Neo ), and biotinylated Arg 9 (Arg 9 ) were conjugated to streptavidin-PE-Cy5 ( ∼ 300 kDa). Wild-type ( blue ) and heparan/chondroitin sulfate-deficient pgsA cells ( green ) were incubated with the different conjugates at concentrations from 10 to 1000 nM for 1 h at 37 °C. After washing the cells, they were released with EDTA and analyzed by flow cytometry. Cells incubated with streptavidin-PE-Cy5 alone are shown in red . b , mean fluorescence values show that both guanidinoneomycin and neomycin display glycosaminoglycan-dependent uptake; however, guanidinoneomycin has a considerably higher uptake efficiency than neomycin. The uptake of Arg 9 does not appear to depend exclusively on heparan/chondroitin sulfate glycosaminoglycans.CC-BY-NC-ND · From the PDF
FIGURE 3 Binding and uptake of guanidinoneomycin by CHO cells and mutants. Guanidinoneomycin-biotin streptavidin-PE-Cy5 conjugates were incubated with wild-type and mutant CHO cells at 60 nM for 1 h at 37 °C under the indicated conditions. Binding and uptake was analyzed by flow cytometry, and the mean fluorescence values were determined. These data show that the cellular uptake of guanidinoneomycin depends on the presence of cell surface heparan sulfate.
FIGURE 3 Binding and uptake of guanidinoneomycin by CHO cells and mutants. Guanidinoneomycin-biotin streptavidin-PE-Cy5 conjugates were incubated with wild-type and mutant CHO cells at 60 nM for 1 h at 37 °C under the indicated conditions. Binding and uptake was analyzed by flow cytometry, and the mean fluorescence values were determined. These data show that the cellular uptake of guanidinoneomycin depends on the presence of cell surface heparan sulfate.CC-BY-NC-ND · From the PDF
FIGURE 4 CHOcell binding of guanidinoneomycin at 4 °C. Guanidinoneomycin-biotin streptavidin-PE-Cy5 conjugates were incubated with wild-type ( blue ) and pgsA ( pink ) cells at concentrations from 10 nM to 30 𝛍 M at 4 °C. Binding was analyzed by flow cytometry, and the mean fluorescence values were determined. The difference between mean values ( green ) shows that proteoglycan-dependent binding sites for guanidinoneomycin start to become saturated at low to mid micromolar concentrations. The inset shows relative mean fluorescence values for untreated wild-type cells and wild-type cells treated with guanidinoneomycin at 37 and 4 °C.
FIGURE 4 CHOcell binding of guanidinoneomycin at 4 °C. Guanidinoneomycin-biotin streptavidin-PE-Cy5 conjugates were incubated with wild-type ( blue ) and pgsA ( pink ) cells at concentrations from 10 nM to 30 𝛍 M at 4 °C. Binding was analyzed by flow cytometry, and the mean fluorescence values were determined. The difference between mean values ( green ) shows that proteoglycan-dependent binding sites for guanidinoneomycin start to become saturated at low to mid micromolar concentrations. The inset shows relative mean fluorescence values for untreated wild-type cells and wild-type cells treated with guanidinoneomycin at 37 and 4 °C.CC-BY-NC-ND · From the PDF
FIGURE 5 Inhibition of FGF and Arg 9 binding by guanidinoneomycin. a , wild-type CHO cells were incubated with biotinylated FGF-2 (10 ng/ml) for 1hinthepresenceofincreasing concentrations of guanidinoneomycin. Cells were then stained with streptavidin-phycoerythrin-cychrome and analyzed by flow cytometry. Guanidinoneomycin inhibited FGF-2 binding to cells with an IC 50 of ∼ 20 𝛍 M. b , wild-type and pgsA cells were incubated with 0.5 mM Arg9-BODIPY and increasing concentrations of guanidinoneomycin. After 1 h the cells were analyzed by flow cytometry. Guanidinoneomycin is able to partially block Arg 9 binding to the surface of cells. The signal saturates once it reaches that of pgsA cells incubated with the fluorescent peptide. Inset , relative fluorescence of untreated wild-type cells ( a ), wild-type cells treated with fluorescent Arg 9 ( b ), pgsA cells treated with Arg 9 ( c ), and wild-type cells treated with Arg 9 and 1 𝛍 M ( d ) or 300 𝛍 M guanidinoneomycin ( e ).
FIGURE 5 Inhibition of FGF and Arg 9 binding by guanidinoneomycin. a , wild-type CHO cells were incubated with biotinylated FGF-2 (10 ng/ml) for 1hinthepresenceofincreasing concentrations of guanidinoneomycin. Cells were then stained with streptavidin-phycoerythrin-cychrome and analyzed by flow cytometry. Guanidinoneomycin inhibited FGF-2 binding to cells with an IC 50 of ∼ 20 𝛍 M. b , wild-type and pgsA cells were incubated with 0.5 mM Arg9-BODIPY and increasing concentrations of guanidinoneomycin. After 1 h the cells were analyzed by flow cytometry. Guanidinoneomycin is able to partially block Arg 9 binding to the surface of cells. The signal saturates once it reaches that of pgsA cells incubated with the fluorescent peptide. Inset , relative fluorescence of untreated wild-type cells ( a ), wild-type cells treated with fluorescent Arg 9 ( b ), pgsA cells treated with Arg 9 ( c ), and wild-type cells treated with Arg 9 and 1 𝛍 M ( d ) or 300 𝛍 M guanidinoneomycin ( e ).CC-BY-NC-ND · From the PDF
Image 6
CC-BY-NC-ND · From the PDF
FIGURE 6 Visualization of guanidinoneomycin uptake in CHO cells. a , wild-type cells were incubated with aconjugateofbiotinylatedguanidinoneomycinandstreptavidinylatedAlexa-488for1hat37 °C.Anoverlayof the 4 ′ ,6-diamidino-2-phenylindole nuclear stain ( blue ) and fluorescent guanidinoneomycin conjugate ( green ) shows internalization in punctate vesicular structures. When the same experiment was performed with heparan/chondroitin sulfate-deficient pgsA cells ( b ), no cell-associated guanidinoglycoside fluorescence was observed. c , incubation of wild-type cells with 0.4 M sucrose inhibited uptake, whereas incubation with 5 mM amiloride had no effect.
FIGURE 6 Visualization of guanidinoneomycin uptake in CHO cells. a , wild-type cells were incubated with aconjugateofbiotinylatedguanidinoneomycinandstreptavidinylatedAlexa-488for1hat37 °C.Anoverlayof the 4 ′ ,6-diamidino-2-phenylindole nuclear stain ( blue ) and fluorescent guanidinoneomycin conjugate ( green ) shows internalization in punctate vesicular structures. When the same experiment was performed with heparan/chondroitin sulfate-deficient pgsA cells ( b ), no cell-associated guanidinoglycoside fluorescence was observed. c , incubation of wild-type cells with 0.4 M sucrose inhibited uptake, whereas incubation with 5 mM amiloride had no effect.CC-BY-NC-ND · From the PDF
Image 8
CC-BY-NC-ND · From the PDF

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  1. Flavell, 1998, Curr. Top Microbiol. Immunol., 234, 57

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Metadata

Authors
Elson-Schwab, L., Garner, O. B., Schuksz, M., Crawford, B. E., Esko, J. D., & Tor, Y.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2007-05
Volume
282
Issue
18
Pages
13585-13591
ISSN
0021-9258
DOI
10.1074/jbc.m700463200
Cited by
68
References
34
Language
en
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