Angewandte
Chemie
tions of 2 (Figure 2B lanes 2–6)) showed concentration
dependent labeling. Pre-incubation of BM-DC lysate with
DCG-04 (lanes 7,8) or AS44 (lane 9; Supporting Informa-
tion), a truncated DCG-04 derivative, abolished labeling.
Western blot analysis with streptavidin-HRP against the
biotin tag of DCG04 showed an inversely proportional
relationship to the fluorescence intensity of 2 (lane 7,8
Figure 2B lower panel). Probe 7, lacking the oligomannose
cluster, gave a similar labeling profile as 2, except that the
bands run lower on the gel owing to the lower molecular
weight of 7 (Figure 2C). These results demonstrate that 2, 7,
and AS44 bind to the same cysteine cathepsins as DCG-04
and, importantly, that the poly-lysine mannoside moiety does
not interfere with cathepsin binding.
To determine the magnitude and the mechanism of
fluorescent ABP uptake, we performed pulse-chase experi-
ments monitored by flow cytometry analysis. Figure 3A
shows normalized flow cytometry results from immature
BM-DCs and bone-marrow derived macrophages (BM-MF),
both expressing the mannose receptor. Cells pulsed for 1 h
with 10 mm of 2 at 48C, washed and chased at 48C (Figure 3A,
lane 1) reveal no fluorescence uptake whereas cells treated at
378C do (Figure 3B, lane 2). The 48C pulse, 378C chase
experiment (Figure 3A, lane 3) shows an intermediate uptake
indicating that 2 endocytosis proceeds by an active and
temperature-dependent mechanism. Apparently, at 48C, the
extracellular 2-mannose receptor complex is stable enough to
withstand the washing step and is endocytosed at 378C but
not at 48C. The fact that ligand binding to the mannose
receptor is Ca2+ ion dependent[13] provides an alternative
means to investigate the uptake mechanism of mannose
clustered 2. Thus, when we used a phosphate buffered saline
(PBS) buffer containing 4 mm EDTA (ethylenediaminete-
traacetate) to harvest cells prior to flow cytometry analysis,
we found lowered fluorescence signal in cells chased at 48C
but not in cells chased at 378C. At 48C the EDTA in the
buffer sequesters the Ca2+ ions from the mannose receptor
upon which mannose clustered 2 is released, prohibiting
endocytosis at elevated temperatures. Adding EDTA to cells
treated with 2 at 378C has, as expected, less effect on the
fluorescence levels, as at this temperature mannose receptors
clustered to 2 are already internalized.
We found that uptake of 2 is blocked by the established
MR ligand, mannan (poly-b-1-6-mannose, Figure 3A lane 5)
as well as hexalysine mannoside 3 (Figure 3A, lane 4). This
result again strongly points towards the endocytosis of 2 in a
MR-dependent manner. Preincubation of cells with the
cathepsin blocker AS44 on the other hand did not inhibit 2
uptake (Figure 3A, lane 6) and demonstrates that MR-
mediated uptake is, again as expected, independent of
cathepsin activity. Additionally, internalization of 7 is not
temperature (Figure 3A, lanes 7,8,9) or AS44-inhibition
(Figure 3A, lane 10) dependent. This result indicates, in
agreement with the microscopy data, that 7 crosses the
plasma membrane by passive diffusion.
In parallel with the flow cytometry analysis, we lyzed
some of the cells at pH 7.5 (to inactivate cathepsins and
prevent post-lysis tagging) and analyzed cathepsin labeling by
SDS-PAGE and in-gel fluorescence (Figure 3B). In agree-
ment with the flow cytometry results, the absence of cathepsin
labeling at 48C (Figure 3B, lane 1) and in the presence of MR
blockers (Figure 3B, lane 4 and 5) demonstrates that 2 is not
cell permeable and is taken up through a MR-mediated
process. Moreover, a lipophilic compound, such as 7, readily
labels cathepsins at low temperature, strengthening the point
above. Cathepsin labeling with 2 was observed at 378C
(Figure 3B, lane 2) and was reduced to 25% in the 48C to
378C pulse-chase experiment (Figure 3B lane 3) confirming
the flow cytometry data. Pre-incubation with AS44 (Fig-
ure 3B, lane 7) blocked more than 50% of the fluorescent
labeling of cysteine cathepsins, but it did not inhibit the
cellular uptake of 2 as seen by flow cytometry.
In conclusion, we designed and synthesized a fluores-
cently tagged clustered mannoside DCG-04 analogue and
established that its uptake by antigen presenting cells (APCs)
and subsequent cathepsin labeling proceeds in a mannose-
receptor dependent manner. Compound 2 may thus find use
in the study of cysteine protease activities in the relevant
subcellular compartments of APCs and in the context of
antigen processing and (cross)presentation. The modular
approach to the assembly of 2 is flexible and in principle
allows variation in both the mannose cluster and the electro-
philic trap. Thus high-end fluorescent probes targeting other
receptors (for instance, members of the Toll-like receptor
family) and intracellular targets (for instance, any of the
broad family of lysosomal hydrolase activities) may be
considered and we feel that our modular strategy may find
application in areas of physiological relevance quite unrelated
to the subject at hand.
Figure 3. Cellular uptake mechanism of 2 and 7 in BM-DCs and BM-
MF. Reagents and conditions: A) Live cells (DCs and MF) normal-
ized flow cytometry analysis of 2 and 7 pulse-chase experiments:
lane 1–3 and 7–9 temperature dependent, lanes 4–5 MR blockers,
lanes 7 and 10 cathepsin inhibition; NC=non-treated cells; B) SDS-
PAGE (12.5%) in-gel fluorescence analysis of BM-DC lysates at pH 7.5
from lanes 1–6 of Figure 3A (10 mg protein/lane).
Received: November 12, 2008
Published online: January 20, 2009
Angew. Chem. Int. Ed. 2009, 48, 1629 –1632
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1631
Hillaert, Ulrik
