M. G. Finn et al.
before the addition of diethyl ether (25 mL). This mixture was
stirred at RT for 45 min. The white precipitate was filtered off and
ethereal HCl (18 mL, 2n) was added to the filtrate with continuous
stirring. The yellow-white precipitate was filtered and dried under
vacuum to give 2 (2.1 g, 78%), which was characterized by H NMR
spectroscopy and matched the data previously reported.[25]
Qb-Tfn uptake was effectively inhibited by approximately
equivalent concentrations of free Tfn as were presented by the
particles, the Tfn-conjugated VLP ligands did not appear to
benefit from their polyvalency with respect to affinity or avidi-
ty.
1
Preparation of transferrin-alkyne conjugate (3): Transferrin
(2 mgmLÀ1) was incubated with sodium meta-periodate (1 mm) in
sodium acetate buffer (0.1m, pH 5.5; 30 mL) on ice in the dark for
30 min. The mixture was concentrated to less than 1 mL by using
centrifugal filter tubes (Millipore), and then dialyzed against HEPES
buffer (0.1m, pH 7.2) using Slide-A-Lyzer Dialysis Cassette Kit
(Pierce). The resulting oxidized transferrin was incubated with 5
(8.2 mm, 350-fold excess) in HEPES buffer with DMSO (20%, total
volume 25 mL) for 5 h at RT by gentle tumbling. Concentration
and dialysis as above provided 3 as a pink solution in HEPES
buffer; the protein concentration was estimated by using the Brad-
ford protein assay.
Conclusions
The oxidation and derivatization of the sialic acid residues of
transferrin is a convenient and effective method for introduc-
ing a connecting linkage that can provide a consistent display
geometry while retaining binding affinity to transferrin recep-
tors. Conjugation of transferrin-alkyne thus prepared to the
azide-functional groups on the Qb virus capsid was accom-
plished using the powerful CuAAC click reaction in a recently
reported optimized protocol.[16] These conjugates were specifi-
cally internalized by cells expressing transferrin receptors
through clathrin-mediated endocytosis.
Preparation of Qb-transferrin conjugate (5) by CuAAC reaction:
Two identical reaction mixtures were prepared, each containing
Qb-azide 1 (1.7 mgmLÀ1, 0.11 mm in protein subunits) and transfer-
rin-alkyne 3 (10.2 mgmLÀ1, 0.12 mm) in HEPES buffer (0.1m, pH 7.3,
1 mL), containing sodium ascorbate (5 mm), copper sulfate
(0.25 mm) and the ligand 4 (1.25 mm). CuSO4 was mixed with 4 in
a separate microtube prior to addition to each reaction mixture.
The reaction mixtures were allowed to stand at RT for 2 h and 5 h,
respectively. The resulting conjugates (5a, 5b) were purified by
size-exclusion FPLC on a Superose 6 column.
Our studies represent the first test of transferrin polyvalency
on receptor-mediated cell entry in which the protein ligands
are arranged in a well-defined platform-based manner. On a
per-unit basis, the Qb-Tfn conjugates have greater affinities for
TfnR-bearing cells than free Tfn, and the rates of uptake of Tfn-
bearing particles were strongly improved by the attachment of
greater numbers of Tfn ligands to each particle (Figure 6C).
These findings suggest that polyvalent transferrin conjugates
can enhance the targeting of specific cell populations in com-
plex mixtures. However, on a per-Tfn basis, the VLPs did not
exhibit significantly increased affinity relative to the free
ligand. This interesting disconnection of affinity (or avidity)[22]
and internalization efficiency remains unexplained at present,
but changes in recycling[23] and intracellular trafficking path-
ways exhibited by multivalent constructs may be at least par-
tially responsible. These findings make transferrin assemblies
potentially useful in the delivery of drugs for therapeutic pur-
poses.[24]
For all of the above steps, we used diferric Tfn under conditions
designed to minimize the loss of iron. After conjugation, the pro-
tein absorbance ratio (A465/A280) was found to be 0.043; this was
within the range (0.042–0.046) and indicated the presence of Fe in
the protein.[26] If Fe is lost, the ability of the attached Tfn to bind
its receptor would be somewhat diminished, as the affinity for
TfnR for apo-Tfn is approximately tenfold less than for Fe2Tfn.[21,27]
SDS-PAGE and Western blot analysis of Qb-Tfn conjugates: Qb
VLP samples 1 and 5a,b were analyzed on denaturing 4–12%
NuPage protein gels using 1ꢃ MES buffer (Invitrogen). The gel was
observed under UV illumination to detect fluorescent dye-labeled
bands before staining with Coomassie SimplyBlueTM SafeStain (Invi-
trogen). For Western blot analysis, after electrophoretic separation
on the gel, the proteins were transferred to a nitrocellulose mem-
brane (Millipore) by electrophoretic blotting. After blocking with
dry milk (5% (w/v)) in TBS-T for 1 h, transferrin conjugation to Qb
was detected with HRP-conjugated mouse monoclonal anti-trans-
ferrin antibody (Abcam), diluted 1:5000 in TBS-T buffer. HRP detec-
tion of peroxide was performed with SuperSignal chemiluminis-
cence substrate (Pierce) and exposure to X-ray film.
Experimental Section
Details of instrumentation and the purchase or preparation of all
reagents, including the Qb VLPs, are given in the Supporting Infor-
mation.
Preparation of AlexaFluor 568-labeled Qb-azide (1): A solution of
wild-type Qb VLPs (5 mgmLÀ1 in 0.1m phosphate buffer, pH 7) was
treated with a premixed DMSO solution of NHS-linker-azide (final
concentration 12 mm, 35-fold excess per Qb subunit) and the NHS
ester of the dye (final concentration 0.35 mm), such that the final
reaction mixture contained 20% DMSO. The solution was allowed
to stand for 12 h at RT, and the derivatized VLP was purified away
from excess reagents on a sucrose gradient (10–40%) and concen-
trated by ultrapelleting. The virus pellet was resuspended in HEPES
buffer (0.1m, pH 7.3). FPLC analysis of 1 indicated that >95% of
the virus consisted of intact particles. Protein concentration was
analyzed by using the Coomassie Plus (Bradford) Protein Assay
(Pierce).
Cell culture and uptake studies: BSC1 monkey kidney epithelial
cells stably expressing rat brain EGFP-clathrin light chain (EGFP-
LCa) were provided by Dr. T. Kirchhausen, Harvard Medical School
and cultured in DMEM supplemented with fetal bovine serum
(FBS; 10%) and G418 (500 mgmLÀ1). For microscopy studies, cells
were plated on glass coverslip at a density of 8.3ꢃ103 cellscmÀ2
overnight. Cells were washed twice in PBS, and VLPs diluted in
PBS4+ (1 mm CaCI2, 1 mm MgCI2, 0.2% BSA (w/v), and 5 mm glu-
cose) at a concentration of 0.6 mgmLÀ1. The cells were either kept
at 08C for binding or shifted to 378C for the desired amount of
time. The cells were washed again twice in PBS, and then fixed in
paraformaldehyde (4%) for 30 min at RT. The cells were visualized
at 60ꢃ magnifications using an Olympus X71 epifluorescence
Synthesis of O-(prop-2-ynyl)hydroxylamine (2): Phthalimide-pro-
tected O-(prop-2-ynyl)hydroxylamine (5.0 g, 24.9 mmol) was stirred
with hydrazine monohydrate (1.4 g, 27.8 mmol) for a few minutes
1278
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 1273 – 1279