A synthetic membrane-anchored antigen efficiently promotes uptake of antifluorescein antibodies and associated protein A by mammalian cells

Full text of DOI:10.1021/ja017087o

12712
J. Am. Chem. Soc. 2001, 123, 12712-12713
Scheme 1
A Synthetic Membrane-Anchored Antigen Efficiently
Promotes Uptake of Antifluorescein Antibodies and
Associated Protein A by Mammalian Cells
Stephen L. Hussey, Enfei He, and Blake R. Peterson*
Department of Chemistry
The PennsylVania State UniVersity
UniVersity Park, PennsylVania 16802
a 2-Nitrobenzenesulfonyl chloride, DIEA, 0 °C, 1 h. b BrCH2CO2t-
Bu, K2CO3, DMF, 85 °C, 12 h. c HCO2H/Et2O (1:1), 65 °C, 3 h. d SOCl2,
CH2Cl2, reflux, 2 h. e 5-Aminofluorescein (NH2-Fl), THF, 25 °C, 3 h.
f PhSH, K2CO3, DMF, 25 °C, 12 h.
ReceiVed September 14, 2001
The efficient delivery of small molecules, proteins, and DNA
to living cells is critical for the effectiveness of therapeutics and
molecular probes. Although most small-molecule drugs enter cells
though passive diffusion across low-polarity cell membranes,
macromolecules are typically not cell-permeable and require
specific active transport mechanisms to gain access to intracellular
receptors.1 The cellular uptake of both natural and nonnatural
molecules can be enhanced by numerous methods including
modification with cationic peptides,2 proteins,3 peptoids,4 and
lipids,5 encapsulation with liposomes,6 dendrimers,7 and sidero-
phores,8 and complexation with cationic polymers.9 The efficiency
of these methods varies substantially, however, and improved
methods that enhance the cellular uptake of macromolecules are
needed in diverse areas from basic cell biology to drug delivery
and genetic therapy.
membranes.11 5-Aminofluorescein was incorporated to provide a
fluorescent tag bearing high affinity for antifluorescein immu-
noglobulins (IgG) (e.g. Kd ) 0.7 nM for monoclonal antifluo-
rescein 4-4-20 binding to fluorescein-biotin). This system was
chosen for investigation because molecular recognition between
fluorescein and antifluorescein IgG proteins has been extensively
characterized by structural,12 computational,13 spectroscopic,14
kinetic,13,14 thermodynamic,15 and mutagenic16 methods. Further-
more, studies of antifluorescein IgG proteins bound to fluorescein-
conjugated lipids on model biomembrane monolayers, bilayers,
and vesicles have been described.17
Compound 1 was prepared as shown in Scheme 1 from 3â-
cholesterylamine (2), which was synthesized following a previ-
ously reported method.18 To determine whether 1 interacts with
cellular plasma membranes, compound-treated Jurkat lymphocytes
were examined by epifluorescence microscopy. Treatment of cells
with 1 (10 µM) revealed intense green fluorescence at the
periphery of 100% of living cells (Figure 1B). This staining pattern
was analyzed by comparison with cells bearing red fluorescent
plasma membranes from treatment with sulfosuccinimidyl biotin,
which acylates amino groups on the cell surface, followed by
addition of cell-impermeable texas red-conjugated streptavidin.19
Comparison of red and green fluorescence confirmed that 1 is
persistently and nearly exclusively localized at the cellular plasma
membrane (compare Figure 1, parts B and D).
The avidity of antifluorescein IgG for 1 in cellular plasma
membranes was assessed by the addition to cells of nonfluorescent
IgG complexed with red fluorescent conjugates of commercially
available Protein A (PrA) from Staphlococcus aureus. PrA
comprises a 57 kDa protein that binds to the invariant Fc fragment
of rabbit-derived IgG proteins with a dissociation constant of ca.
60 nM.20 Pretreatment of cells with 1 followed by addition of
rabbit polyclonal antifluorescein IgG and Alexa Fluor-594-
conjugated PrA afforded red fluorescence at the plasma membrane
of 100% of living cells within 10 min of addition of the IgG-PrA
complex (Figure 1G).
We describe here a novel synthetic ligand (1), termed a
“memtigen” (membrane-anchored antigen), that enables dose-
dependent uptake of proteins by mammalian cells as a conse-
quence of non-covalent interactions at cellular plasma membranes.
Compound 1 is a derivative of cholesterol designed to function
as a fluorescent membrane anchor with high affinity for both
cellular plasma membranes and commercially available antifluo-
rescein antibodies. 3â-Cholesterylamine was employed as the
membrane anchor component because cholesterol is an abundant
membrane-associated steroid,10 and protonated amines such as
the headgroup of 1 favorably interact with anionic phospholipids
under physiological conditions to increase affinity for plasma
(1) Smith, D. A.; van de Waterbeemd, H. Curr. Opin. Chem. Biol. 1999,
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10.1021/ja017087o CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/22/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 50, 2001 12713
Figure 2. Analysis of protein uptake in Jurkat lymphocytes by flow
cytometry. (A) Dose-response from treatment with 1 (1 h) followed by
antifluorescein IgG (0.05 mg/mL) and Alexa Fluor 633-conjugated Protein
A (0.01 mg/mL) for 4 h at 37 °C. (B) Inhibition of endocytosis in cells
treated with proteins and 1 (1 µM). (C) Competition and omission control
experiments. Each bar represents the median fluorescence of 10 000 living
Jurkat lymphocytes.
Figure 1. Epifluorescence and differential interference contrast (DIC)
microscopy of compound-treated Jurkat lymphocytes. (A, B) Treatment
with 1 (10 µM) for 1 h. (C, D) Control cells bearing plasma membranes
labeled with biotin and Texas Red-conjugated streptavidin at 4 °C. (E-
G) Cells treated with 1 (10 µM) for 1 h followed by antifluorescein IgG
(0.05 mg/mL) and Alexa Fluor 594-conjugated Protein A (0.01 mg/mL)
for 10 min at 37 °C. (H-J) Cells treated as shown in E-G for 4 h.
Fluorescence excitation (Ex.) and emission (Em.) wavelengths (nm) are
explicitly shown.
Remarkably, treatment of cells with 1 and the IgG-PrA complex
for 4 h engendered both red and green fluorescence in the interior
of >99% of the cells examined, indicating that the proteins and
1 were internalized (Figures 1I, 1J, and 2). No intracellular or
membrane-associated fluorescence was observed when the anti-
body complex and 1 were premixed prior to addition to cells,
possibly due to aggregation of the amphipathic complex formed.
Flow cytometry was employed to quantitate uptake of anti-
fluorescein IgG bound to Alexa Fluor-633-conjugated PrA by
Jurkat lymphocytes (Figure 2). Since this technique does not
distinguish membrane-associated from intracellular fluorescence,
cells were washed twice with 5-aminofluorescein (10 µM) prior
to analysis to competitively displace any noninternalized protein.
Flow cytometry revealed that 1 effects dose-dependent uptake of
fixed concentrations of the IgG-PrA complex at ligand concentra-
tions as low as 100 nM. This effect was blocked by competition
with 5-aminofluorescein, confirming that the IgG interacts specif-
ically with 1 (Figure 2C). Omission of 1, the IgG, or the PrA
conjugate yielded no significant cellular red fluorescence (Figure
2C).
Mammalian cells employ both receptor-mediated and nonre-
ceptor-mediated endocytosis to internalize specific small mol-
ecules, macromolecules, and particles. In addition, viruses, toxins,
and symbiotic microorganisms exploit endocytic pathways to gain
entry into cells.21 This process is dependent on time, temperature,
pH, and energy.21 We hypothesized that protein uptake mediated
by 1 might involve endocytosis promoted by the high effective
concentration of proteins immobilized at the plasma membrane.
Hence, conditions that block endocytosis were examined. Protein
uptake was suppressed by addition to the media of sodium azide,22
an inhibitor of energy-dependent cellular processes, decreasing
Figure 3. Schematic of compound-mediated protein uptake by mam-
malian cells. Compound 1 is first incorporated into the plasma membrane.
Addition of antifluorescein IgG and PrA results in protein plasma
membrane association and subsequent internalization.
the pH of the media to 5.5 with acetic acid, or decreasing the
temperature to 4 °C (Figure 2B). Thus, the mechanism of protein
internalization promoted by 1 meets the basic criteria for an
endocytic process.21
Figure 3 illustrates this novel strategy for the delivery of IgG
and PrA conjugates to >99% of viable mammalian cells, which
includes adherent cell lines such as Chinese Hamster Ovary
(CHO) cells. In summary, a novel plasma membrane-associated
synthetic ligand promotes dose-dependent uptake of antifluores-
cein IgG and associated PrA in mammalian cells by an endocytic
mechanism. This strategy may enable regulated intracellular
delivery of numerous cell-impermeable molecules conjugated or
noncovalently linked to antibodies or Protein A.
Acknowledgment. We thank the NIH (CA83831) for financial
support. S.L.H. thanks the DOD for a predoctoral fellowship. We thank
Ms. Elaine Kunze for assistance with flow cytometry.
Supporting Information Available: Experimental procedures, char-
acterization data for new compounds, micrographs of uptake of Texas
Red-conjugated antifluorescein IgG, and micrographs of protein uptake
in adherent CHO cells (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
JA017087O
(21) Mukherjee, S.; Ghosh, R. N.; Maxfield, F. R. Physiol. ReV. 1997, 77,
759-803.
(22) Sandvig, K.; Olsnes, S. J. Biol. Chem. 1982, 257, 7504-7513.