(Received for publication, July 31, 1995; and in revised form, September 11, 1995)
From the
Intracellular polyamine pools are partially maintained by an
active transport apparatus that is specific for and regulated by
polyamines. Although mammalian transport activity has been
characterized by kinetic studies, the actual protein itself has yet to
be identified, purified, or cloned. As one approach to this problem, we
attempted photoaffinity labeling of plasma membrane proteins using two
specifically designed and synthesized polyamine conjugates as
photoprobes. The first is a spermidine conjugate bearing the
photoreactive moiety 4-azidosalicylic acid at the N position via an alkyl linkage, and the second is a norspermine
conjugate with 4-azidosalicylic acid at the N
position via an acyl linkage. Labeling of murine L1210
lymphocytic leukemia cells was carried out at 4 °C to promote
selective alkylation of cell surface proteins. Separation of plasma
membrane proteins from cells cross-linked with the N
-spermidine conjugate by SDS-polyacrylamide gel
electrophoresis revealed two heavily labeled proteins at
118 and
50 kDa (designated p118 and p50, respectively). Band p118 was more
well defined and much more intensely labeled. Analogous proteins were
also observed in human U937 lymphoma cells. Specificity of labeling was
strongly suggested by competition with polyamines and analogs during
labeling and further indicated by the nearly identical labeling of the
same protein by the N
-norspermine photoprobe but
not by the unconjugated photoreagent. Neuraminidase pretreatment of
L1210 cells increased mobility of the p118, suggesting that it was
glycosylated and, thus, of plasma membrane origin. In
transport-deficient L1210 cells, p118 and p50 were found to have a
slightly higher molecular mass and were accompanied by a less distinct
protein band (
100 kDa). These findings indicate the presence of a
polyamine binding protein at the surface of murine and human leukemia
cells, which could be directly or indirectly related to the polyamine
transport apparatus.
Cell proliferation is critically dependent on a constant supply
of the intracellular polyamines: putrescine, spermidine (Spd), ()and spermine (Spm). Under in vivo conditions,
this supply is thought to be maintained by sensitively regulated
processes involving polyamine biosynthesis, catabolism, and transport.
While the key enzymes responsible for biosynthesis and catabolism of
polyamines have been biochemically purified, characterized, and cloned,
the putative proteins responsible for polyamine transport in mammalian
systems have, thus far, only been studied at the functional level.
From kinetic studies, polyamine transport is known to be an active process that is energy- and temperature-dependent and adheres to Michaelis-Menten kinetics(1, 2, 3) . Although the transporter is highly specific for polyamines, various synthetic molecules such as polyamine analogs, methylglyoxal-bis(guanylhydrazone) (MGBG), and paraquat utilize the carrier as a primary means for gaining entry into cells(4) . By contributing to their intracellular accumulation, the transporter undoubtedly determines their in vitro and in vivo pharmacological behavior and hence their therapeutic potential.
Like polyamine biosynthesis, polyamine transport is sensitively and negatively regulated by intracellular pools. Polyamine depletion results in increases in uptake, while polyamine excess causes decreases in transport(5, 6, 7) . In the absence of appropriate molecular probes, the basis for regulation of polyamine transport remains uncertain. Recent findings suggest that, like the polyamine biosynthetic enzyme ornithine decarboxylase, transport may be at least partially regulated by antizyme, which apparently contributes to the regulated degradation of these proteins(8, 9) . In order to investigate such possibilities more directly, the mammalian transporter needs to be purified and/or cloned.
Due to the relatively weak ionic interactions between polyamines and their receptors, standard means of isolation and purification such as affinity chromatography have not proved useful in such systems. Efforts to clone the protein have included cross-species transfection of DNA from transport-positive cells into transport-negative cells as has been done with the proteins involved in folate transport(10) . Efforts by Byers et al. (11) led to the transferal of polyamine transport activity by this means but not to the isolation of gene sequences encoding the transporter. In contrast to studies in mammalian systems, Igarashi and co-workers (12, 13, 14) have used this approach in bacterial systems to isolate the genes encoding multiple polyamine transporters of Escherichia coli.
In this study,
photoaffinity labeling was attempted in an effort to identify and
characterize cell surface proteins in mammalian systems that bind
polyamines and may be involved in polyamine transport. The approach
allows for the covalent modification of specific protein sites by the
activation of a light-labile moiety affixed to a particular ligand.
First proposed by Ji(15) , photolabeling has proved useful in
the identification of various plasma membrane transporters and surface
receptors(16, 17) . Under conditions designed to
optimize selective interaction with plasma membrane proteins,
custom-synthesized polyamine photoprobes (Fig. 1) were used to
modify covalently putative polyamine binding sites. This approach led
to the identification of a plasma membrane polyamine binding protein
having a molecular mass of 118 kDa and demonstrating high specific
activity of labeling with the polyamine photoprobes; its relevance to
polyamine transport is uncertain. These studies have been previously
presented in abstract form(18) .
Figure 1:
Structure of photoprobes N-ASA-[
I]Spd and N
-ASA-[
I]nSpm.
The synthesis of N-ASA-nSpm
began with the commercially available intermediate 3-bromopropylamine
hydrochloride, which was converted to the corresponding N-(p-NO
-Cbz) derivative (21) by
treating it with 4-nitrobenzylchloroformate. The resulting
bromopropylcarbamate was then condensed with the previously described
intermediate
1-phthalimido-4-(N-benzyl)-7-{[(2-mesitylene)sulfonyl]amino}-4-azaheptane (22) in the presence of sodium hydride to afford a
heterogenously tetraprotected intermediate possessing the requisite
norspermine backbone. In order to preserve the amide linkage in the
immediate precursor to the target photoprobe, it was necessary to
manipulate the nitrogen-protecting groups early in the synthesis to
produce an intermediate that could be deprotected under mild
conditions. Thus, the mesityl-protecting group was removed by treatment
with 30% HBr in acetic acid(23) , and the resulting secondary
nitrogen was reprotected as the corresponding N-(p-NO
-Cbz) derivative (21) .
The phthalimide was then removed by hydrazinolysis(24) , and
the resulting primary amine was converted to the corresponding N-Boc derivative(25) . The N-benzyl- and N-(p-NO
-Cbz)-protecting groups were
simultaneously removed by hydrogenolysis (26) to afford a
norspermine derivative in which one of the two primary nitrogens was
protected. The primary nitrogen in this intermediate was then reacted
regiospecifically with the commercially available reagent
NHS-ASA(27) , yielding the penultimate precursor to the target
photoprobe. This intermediate, as well as the target molecule, were
protected from light at all times to avoid premature photolysis.
Removal of the N-Boc-protecting group under mild conditions
(trifluoroacetic acid, room temperature, 1 h) then afforded the target
molecule N
-ASA-nSpm as the trifluoroacetate salt.
All intermediates in the pathway including the target photoprobe were
fully characterized by NMR and IR spectroscopy, and all spectra were
consistent with the assigned structures. Purity was assessed to be
95%. Because the ASA moiety was affixed to a terminal amine via an
acyl linkage, the recognizable polyamine moiety based on charge was
that of norspermine (i.e. having three charged nitrogens).
The I-iodinated probe
was cleaned of any free radionuclide by reverse phase column
chromatography. Octadecyl columns obtained from J. T. Baker
(Phillipsburg, NJ) were prepared as per the manufacturer's
recommendations. The radioactive solution was added to the column by
vacuum pressure, and the void volume was collected. The C18 column was
rinsed twice with sodium phosphate buffer, and each wash was collected.
The sample was eluted from the column by a methanol, 0.1 N HCl
wash. Each wash and sample eluate was tested for the presence of the
photoprobe by spotting 2 µl on fluorescent thin layer
chromatography paper and viewing with a short wavelength lamp. The
MeOH/HCl eluate was the only positively fluorescent spot. The sample
was then dried under a gentle stream of nitrogen and redissolved in
sodium phosphate buffer. Each of the eluates and the final redissolved
sample was counted in a Beckman
counter, model Gamma 5500. The
concentration of the iodinated and the noniodinated photoprobe was
calculated by using a previously determined extinction coefficient
(data not shown) at the wavelength of 267 nm. The specific activity of
the polyamine photoprobes generally ranged from 0.7 to 2
10
cpm/µmol, while the specific activity of the
unconjugated photoprobe NHS-[
I]ASA was 7
10
cpm/µmol.
Earlier structure function studies involving polyamine uptake (32, 33) have shown that derivatization of Spd via an N-alkyl linkage is better tolerated than
derivatization at the N
position. Thus, a Spd
photoprobe was designed and synthesized in which the photoreactive
group ASA was affixed to the N
position of Spd via
a 2-carbon alkyl bridge (Fig. 1). The purity of the compound was
assessed via elemental analysis and thin layer chromatography and found
to be 89.7%. The ability of N
-ASA-Spd photoprobe
to compete with Spd for transport into murine L1210 leukemia cells was
examined by Michaelis-Menten kinetic parameters for uptake. With an
apparent K
of 52 µM, the photoprobe
was found to be within the range of other compounds that utilize the
polyamine transport system including MGBG, which competes in these
cells with an apparent K
of 71 µM (Table 1). Since photoaffinity probes with binding constants
in the low micromolar range have been used successfully in the labeling
of plasma membrane proteins such as a folate binding
protein(28) , we presumed that the N
-Spd
conjugate could be similarly useful in photocross-linking of polyamine
binding proteins at the cell surface.
Previous plasma membrane
labeling protocols (34) have established that membrane
cross-linking should be carried out at 4 °C in order to minimize
probe internalization and restrict labeling to the cell surface. To
confirm this strategy, accumulation studies using
[H]Spd were performed to demonstrate that while
large amounts of radioactivity were taken up by intact cells at 37
°C, virtually none was accumulated at 4 °C (Fig. 2). In
accordance with previous protocols, all photoaffinity labeling was done
at 4 °C. In preliminary studies, concentrations of photoprobe at
100 µM were found to be saturating (data not shown). In
order to conserve photoprobe, labeling was conducted at 35
µM unless otherwise indicated.
Figure 2:
Temperature dependence of Spd
accumulation in L1210 cells. L1210 cells were incubated for varying
times in the presence of [H]Spd at 37 °C
(+) or 4 °C (
).
Separation of proteins contained in plasma membrane preparations of photolabeled cells was achieved by SDS-polyacrylamide gel electrophoresis. Labeled proteins were then identified by autoradiography following exposures of 5-14 days. By Coomassie Blue staining, >60 separate protein bands were visible (Fig. 3). Of these, several were found by autoradiography to be diffusely labeled to varying degrees of intensity. Two, however, demonstrated a high specific activity of labeling, and both were found by Coomassie staining to represent relatively minor membrane proteins. The first was a prominent well-defined band at 118 kDa, and the second was a broader, more diffuse band at 50 kDa (Fig. 3). We have designated the former of these proteins p118 and the latter p50.
Figure 3:
SDS-Polyacrylamide gel electrophoresis
separation of L1210 plasma membrane proteins (lane 2) and the
identification of photoaffinity-labeled proteins by autoradiography (lanes 3 and 4). Note the high affinity of the Spd
photoprobe for a protein at 118 kDa and a protein at
50
kDa.
To examine the possible
relevance of p118 to polyamine transport activity, the plasma membrane
protein labeling pattern in polyamine transport-deficient L1210/MG
cells (19) was compared with that of parental L1210 cells. In
agreement with the original studies by Persson et
al.(19) , L1210/MG cells were found to be 1000-fold
resistant to MGBG and to accumulate much less radiolabeled Spd than the
parental cells (data not shown). By Coomassie Blue staining, no obvious
difference in stained protein pattern was apparent between L1210 and
L1210/MG cells (data not shown). Following photoaffinity labeling with
the radioiodinated N
-Spd photoprobe (Fig. 4, lanes 1 and 3), it was observed that
the primary labeled protein, p118, consistently exhibited a slightly
higher molecular mass form in the L1210/MG cells, and the labeling of a
minor band (100 kDa) was more apparent. Even following prolonged
autoradiographic exposure (data not shown), no other differences in
labeled protein bands were apparent between the parent and
transport-deficient lines.
Figure 4:
Comparison of plasma membrane protein
labeling patterns of L1210 and L1210/MG cells and the effects of
neuraminidase pretreatment. Cells were labeled with 50 µM
N-ASA-[
I]Spd. Note that in
comparing labeling patterns of L1210 cells (lane 1) with those
of L1210/MG cells (lane 3), there is a slight upward shift of
p118 and p50 and the appearance of another band at
100 kDa. Cells
represented in lanes 2 and 4 were pretreated with
neuraminidase for 30 min at 37 °C using 1 unit of enzyme activity/5
10
cells/ml. Following such treatment, there is a
downward shift of p118 and the appearance of a diffuse band at
100
kDa.
Neuraminidase pretreatment was used to examine whether negatively charged sialic acid residues contributed to p118 labeling and to compare the glycosylation status of labeled proteins in L1210 and L1210/MG cells (Fig. 4, lanes 2 and 4). This treatment failed to diminish labeling intensity, suggesting that sialic acid was not responsible for photoprobe binding. It did, however, produce a clear increase in mobility of p118 in both L1210 and L1210/MG cells, indicating that the protein was similarly glycosylated in both cell types.
The
specificity of p118 as a possible polyamine binding protein was
evaluated by first comparing labeling with the unconjugated
photoaffinity reagent NHS-ASA. In uptake studies, NHS-ASA was
ineffective in competing with [H]Spd for uptake
into L1210 cells (K
>100 µM). When
the unconjugated probe NHS-ASA-
I was used to photolabel
cells, the protein banding patterns were significantly different from
those obtained with N
-ASA-[
I]Spd (Fig. 5). In particular, neither p118 nor p50 were significantly
labeled, thus suggesting that the polyamine ligand of the photoprobe
strongly influenced targeting specificity to sites on the plasma
membrane.
Figure 5:
Comparison of photolabeling with polyamine
conjugated N-Spd photoprobe (lanes 1 and 2) and an unconjugated photoprobe (lane 3). L1210
cells were labeled with 100 and 200 µMN
-ASA-[
I]Spd or with 100
µM NHS-ASA-
I.
The labeling pattern was also compared with another
photoaffinity reagent, N-ASA-[
I]nSpm, which was
synthesized in order to further evaluate the binding specificity of
p118. With this conjugate, the ASA moiety is affixed via an acyl bond
to the N
of the polyamine so that the remaining three
charged nitrogens of nSpd presumably are available to act as the
binding determinants (Fig. 1). In uptake studies, the N
-nSpm conjugate competed with
[
H]Spd transport more effectively than N
-ASA-Spd, with an apparent K
value of 23 µM (Table 1). Thus, N
-ASA-nSpm was a potentially more specific
photoaffinity reagent than the N
-conjugate.
Although we demonstrated that N
-ASA-Spd and
especially N
-ASA-nSpm competed with
[
H]Spd for polyamine transport, these data do not
indicate the ability of the conjugate to actually utilize the
transporter to enter cells. To test this possibility, L1210 and
L1210/MG cells were incubated with either of the iodinated conjugates
in the dark for 30 min, washed, and then processed for radioactivity
content. A clear difference in uptake of the two polyamine photoprobes
was seen between the two cell lines, where normal L1210 cells
accumulated much more of either photoprobes than the
transport-deficient cells (Fig. 6). Accumulation of
radioactivity by transport-deficient cells suggests that in addition to
utilizing the polyamine transporter, the photoprobes may, to a lesser
degree, enter cells via other means, such as passive diffusion.
Figure 6:
Photoprobe accumulation in L1210 versus L1210/MG cells. L1210 and L1210/MG were incubated for
30 min at 37 °C in phosphate-buffered saline containing 2 g/liter
glucose + .1 g/liter CaCl and 2.5 µM photoprobe. N
-ASA-[
I]nSpm accumulates
to a greater amount, and as indicated by the differential between the
two cells types, it is more specific for the polyamine transport
system.
Photoaffinity tagging of L1210 cells with N-ASA-[
I]nSpm resulted in
labeling of p118 that was identical to that seen with N
-ASA-[
I]Spd (Fig. 7). Thus, a photoprobe that exhibits a
2-fold higher
affinity for the transporter also intensely labels p118. The more
potent binding affinity of the N
-nSpm photoprobe
seemed to be reflected in higher intensity of protein labeling, which
was apparent in the shorter exposure times required for
autoradiography. Photolabeling of cells with the N
-nSpm probe also resulted in a band at >200
kDa. A similar but more faintly labeled protein was occasionally seen
with the N
-Spd probe. Additional studies using a
7.5% gel led to the conclusion that the apparent 200-kDa band
represented labeled protein that had precipitated at the
stack:separating gel interface (data not shown). Comparison of labeling
patterns for L1210 and L1210/MG cells tagged with the N
-nSpm probe failed to yield any differences
beyond those seen with the N
-Spd probe.
Figure 7:
Comparison of photoaffinity labeling with
the photoprobes N-ASA-Spd (lanes 1 and 2) or N
-ASA-nSpm (lanes 3 and 4). Cells were labeled with 35 µM of either N
-ASA-[
I]Spd or the N
-ASA-[
I]nSpm. The two
probes produce nearly identical protein labeling patterns. Additional
studies using a 7.5% gel suggested that the apparent 200-kDa band was
labeled protein that had precipitated at the stack:separating gel
interface.
Specificity of p118 labeling was also tested by competition assays
whereby excess amounts of polyamines or polyamine analogs were present
during the cross-linking reaction. In a series of such assays, cells
were incubated with competing Spd, Spm, DESpm, or the noniodinated
probe for 10 min before and during exposure to N-ASA-[
I]Spd. A
dose-dependent decline in labeling was apparent (Fig. 8). At
100-1000-fold concentrations, both the natural polyamines and the
polyamine analog, DESpm, competed effectively with p118 labeling,
reducing the covalent cross-linking by as much as 75%. As per
expectations, N
-ASA-Spd was the most potent
competitor, reducing labeling at only 10-fold excess. It should be
noted that during uv exposure, N
-ASA-Spd binding
is covalent and irreversible, and therefore not subject to reversal by
competing agonists.
Figure 8:
Competition of polyamines or analogs
during photoaffinity labeling. Nonradioactive polyamines or analogs
were added to L1210 cells 10 min prior to and during labeling in 35
µMN-Spd photoprobe. Concentrations
were 100, 300, and 1000-fold greater than those of the photoprobe or 1,
3, and 10 times that of photoprobe. At 1000-fold excess, Spd, Spm, and
DESpm all competed effectively in photoprobe labeling of p118. At a
10-fold concentration, the noniodinated photoprobe
N
-ASA-Spd competed successfully for p118
labeling.
To test the generality of p118 labeling, human
U937 lymphoma cells were photoaffinity-labeled with the N-ASA-[
I]Spd photoprobe.
Prior dose-response curves comparing the U937 cells with the murine
lines showed that the human lymphoma cells were sensitive to MGBG (data
not shown), indicating that they expressed an analogous polyamine
transport apparatus. A labeling pattern was observed similar to that
seen in murine cells (Fig. 9). An intensely labeled protein
appeared at
112 kDa and may represent the human analog to murine
p118. The slight molecular mass difference could be related to a
different degree of glycosylation.
Figure 9:
Comparison of photoaffinity-labeled plasma
membrane proteins from murine L1210 lymphocytic leukemia cells (lane 1) and human U937 lymphoma cells (lanes 2 and 3). Cells were labeled with 50 µMN-ASA-[
I]-Spd. A similar
protein labeling pattern is observed in both cell lines. In U937 cells,
the apparent molecular mass of the labeled protein analogous to p118 is
112 kDa.
To our knowledge, this use of photoaffinity labeling
demonstrates for the first time, the presence of polyamine binding
proteins at the surface of mammalian cells. Previously, Morgan et
al. (36) used an azidonitrobenzoyl-Spm conjugate to
describe and map discrete binding sites on nucleosome core particles by
photoaffinity labeling. The reagent was similar to N-ASA-nSpm used here by having the photoprobe
moiety affixed via an N
acyl linkage. It differed
in that the probe was radiolabeled as
C on the Spm moiety
as opposed to
I on the photoprobe moiety as used here.
The present findings suggest that despite low affinity binding of
polyamine receptors, photoaffinity labeling of such proteins is
possible due to the irreversible bond formed during binding and
photoactivation. A similar approach might also prove useful in probing
small ion channels such as the N-methyl-D-aspartate
receptor and inward rectifying potassium channels where polyamines have
recently been shown to play a potent modulatory
role(37, 38, 39, 40) .
Of the two
photoprobes synthesized and used in this study, it was unexpected to
find that N-ASA-nSpm was a much more effective
competitor for Spd uptake than N
-ASA-Spd based on
our previous findings with Spd
analogs(32, 33, 41) . In this regard, it
should be noted that although ASA is affixed to nSpm, the attachment is
via an acyl linkage so that the charge on the derivatized N
is lost. Thus, with only three of the four
nitrogens charged, the recognizable polyamine ligand is that of nSpd.
Consistent with its ability to compete more effectively for uptake, N
-ASA-nSpm labeled p118 more intensely, as
indicated by the shorter exposure times required for gel
autoradiography.
Of the various L1210 cell proteins cross-linked with the polyamine-conjugated photoprobes, p118 was most intensely labeled even though by Coomassie Blue staining, it represented a relatively minor membrane protein species. An analogous protein 112 kDa in size was also found on human leukemia cells. Characterization of p118 as a plasma membrane protein was based on both experimental design and findings. Labeling of intact cells at 4 °C minimizes probe internalization (42) and thus favors interaction with cell surface proteins. This bias is further enhanced by the preparation and study of plasma membrane preparations from labeled cells. Differences in p118 mobility from cells treated with neuraminidase strongly suggest that p118 contains sialic acid residues and is therefore of plasma membrane origin. Persistence of labeling following neuraminidase treatment also indicates that negatively charged sialic acid residues are probably not involved in binding photoprobes.
The specificity of
p118 as a polyamine binding protein is strongly suggested by (a) similar labeling of p118 with two structurally different
polyamine conjugates, (b) the absence of similar protein
labeling with the unconjugated photoprobe NHS-ASA, and (c) the
ability of exogenous polyamines to effectively compete in labeling of
p118. The high concentration of polyamines or the analog, DESpm,
required to compete with photoprobe labeling is consistent with the
irreversible nature of photoprobe binding in the presence of uv light.
Competitors would only be expected to antagonize binding during the
recognition phase but not once photocross-linking is established. This
deduction was confirmed in a very recent study by Leroy et
al.(35) , who used a polyamine photoconjugate to map the
polyamine activating site the -subunit of casein kinase 2. In that
study, competition experiments required similarly high levels of
polyamines to those reported here to achieve labeling decreases of 50%.
Those authors offer a similar interpretation of their findings.
The evidence that p118 may be part of the polyamine transport apparatus is somewhat more tenuous. Comparison of plasma membrane protein labeling patterns for parental L1210 cells with polyamine transport-deficient L1210/MG cells failed to indicate a loss of p118 protein or a decrease in its labeling. Likewise, no differences were observed in any other labeled or unlabeled protein distinguishable by autoradiography or protein staining, respectively. It is possible, however, that transport deficiency is due to a functional change in the transporter protein complex (12, 13, 14, 43) and/or one of its regulatory proteins. In this regard, it is interesting that on the basis of gel mobility, p118 from L1210/MG cells consistently displayed a slightly higher molecular weight than parental line protein. This may be due to changes in primary protein structure and/or to differences in post-translational modification of p118. Since the shift in p118 mobility persisted following neuraminidase treatment, post-translational differences involving sialic acid residues were excluded.
If not directly involved in polyamine transport, p118 may play a collateral role such as the folate-binding proteins play in folate transport (44, 45, 46) or function as a regulatory protein. Finally, the possibility that the polyamine binding function of p118 may be completely unrelated to polyamine transport must be considered. Polyamines, for example, have been shown to interact with the N-methyl-D-aspartate receptor (37) and inward rectifying potassium channels(38, 39, 40) , both of which are on the plasma membrane. It is expected that the nature of this interesting protein will become more apparent as current purification and characterization studies progress.