(Received for publication, September 20, 1995)
From the
Almost complete purification (>95%) of the 46-kDa murine,
one-carbon, reduced folate transporter (RFT) at a recovery of 20% was
obtained by ligand-directed immunoaffinity fractionation from
transporter overproducing L1210/R83 cells. These cells were labeled
with the N-hydroxysuccinimide ester of
[H]aminopterin (AMT), the isolated plasma
membrane alkaline washed to remove nonintegral membrane proteins,
detergent-solubilized, and RFT-separated on an anti-AMT
antibody-protein G-Sepharose column followed by preparative
SDS-polyacrylamide gel electrophoresis. Anti-RFT antibody, subsequently
derived, differentially blotted (L1210/R83
L1210/0) a 46-kDa
protein during SDS-polyacrylamide gel electrophoresis of plasma
membrane from L1210/R83 and L1210 cells and in L1210/R83 cells after
trichloroacetic acid precipitation.
In contrast to that reported for
human tumor cells, glycosidase treatment of RFT revealed no common N- or O-linked core oligosaccharides associated with
this protein. The same 46-kDa protein at different relative levels was
revealed in a Western blot of plasma membrane from other murine tumors.
Blotting of plasma membrane from methotrexate resistant, transport
defective L1210 cell variants exhibited wild-type levels of a less
electrophoretically mobile RFT or greater levels of the same 46-kDa RFT
which could not be affinity labeled with N-hydroxysuccinimide-[H]AMT. The same
antibody differentially blotted a 83-kDa plasma membrane protein from
human HL-60 and CCRF-CEM cells with different levels of reduced folate
transport and affinity labeling of RFT, verifying the conserved nature
of this protein consistent with earlier functional studies.
The preferred route for mediated entry of reduced folates and
folate analogues through the plasma membrane of tumor cells (reviewed
in (1) and (2) ) is the one-carbon, reduced folate
transport (RFT) ()system. This transport system is a major
determinant of cytotoxicity (2, 3) and of therapeutic
responsiveness of tumors(3) , and genetic alteration of its
biochemical properties has been shown (4) to be a common form
of acquired resistance to classical folate analogues in many animal and
human tumors. More recent studies utilizing affinity- or photoaffinity
labeling have characterized the transporter (RFT) for the one-carbon,
reduced folate transport system as an integral membrane protein of
molecular mass = 43-46 kDa in murine L1210
cells(5, 6, 7) , but exhibiting a much higher
mass in human CCRF-CEM, K562, and HL-60 cells (8, 9, 10) . The larger mass of the human
transporter appears to relate to its content of core
oligosaccharides(8, 9, 11) . Further studies
on the biochemical properties of this transporter and the regulation of
its gene expression have been hampered by the relatively low level of
its expression (1) in all of the tumor cells studied and the
unavailability of facile methods for its purification. Progress in
regard to the former limitation was first documented (12) in
our studies describing the isolation by one of two methods of variants
of the L1210 cell expressing high levels of the transporter. The
isolation of similar variants of human CCRF-CEM and K562 leukemia cells
by one of these methods was subsequently reported (8, 13) by others. More recently (10) , we
applied the same methodology to the isolation of a similar variant of
HL-60 cells.
In a preliminary report(14) , a method for purification of the RFT from L1210 cells was recently described. These workers utilized affinity purification by streptavidin ``capture'' of the transporter labeled with a biotinylated folate analogue to obtain microgram amounts of the transporter. More recently, a second approach was described (15) using lectin-affinity chromatography which appeared to be successful when applied to a highly glycosylated reduced folate transporter(8, 11) from an ``overproducing'' variant of K562 cells. These are extremely interesting approaches with apparent potential for widespread use. However, the application of these approaches in the authors' laboratory for the purification of transporter from L1210 cells ``overproducing'' the transporter was consistently unsuccessful. As an alternative approach, we now report on the purification of this transporter from an ``overproducing'' L1210 cell variant by ligand-directed immunoaffinity chromatography. This methodological approach is now described in detail along with data obtained with anti-RFT polyclonal antibodies on the native properties of the transporter and its relative expression and properties in parental and transport altered variants of murine and human tumor cells.
Figure 1:
SDS-PAGE of plasma membrane protein
from L1210/R83 and L1210/R25 cells after affinity labeling with
NHS-[H]AMT. Cells were affinity labeled with 1
µM of NHS-[
H]AMT, plasma membrane
prepared, alkaline washed and 50 µg dissolved in sample buffer
prior to SDS-PAGE on a 10% polyacrylamide case gel. Fractions were
collected by means of a gel slicer. See text for additional details.
Protein standards employed were phosphorylase b (94 kDa),
bovine serum albumin (67 kDa), ovalbumin (43 kDa),
glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase
(30 kDa), and soybean trypsin inhibitor (20 kDA). The results of
typical separation are shown.
Data pertaining to the purification of
[H]AMT-RFT from alkaline washed L1210/R83 plasma
membrane are given in Fig. 2and 3 and Table 1. Binding of
solubilized NHS-[
H]AMT-labeled proteins to
antibody-protein G-Sepharose after preclearing occurred to the extent
of 70-80% and approximately 85% of the bound material could be
readily removed from the antibody by elution with glycine HCl buffer.
Approximately 75% of the eluted material was recovered in the peak
fractions for a total recovery of tritium labeled protein in the range
of 50% (Table 1). Analytical SDS-PAGE analysis of this material
is shown in Fig. 2. This migration profile predicted that the
majority of the eluted material would be recovered in the major peak
fractions at 46 kDa following preparative electrophoresis. However, the
actual recovery of [
H]AMT-RFT in the peak
fractions from the Bio-Rad 491 cell usually selected (see Table 1and Fig. 3) was considerably less (40%) due to
aggregation and proteolysis.
Figure 2: Analytical SDS-PAGE of immunoaffinity purified RFT from L1210/R83 cells. Aliquots of 10 µl of eluate from the anti-AMT antibody-protein G-Sepharose column were solubilized in sample buffer and electrophoresed on a 10% polyacrylamide disc gel. See text and legend of Fig. 1for additional details. The results of a typical separation are shown.
Figure 3: Preparative SDS-PAGE of immunoaffinity purified RFT from L1210/R83 cells. Peak fractions of eluate from the anti-AMT antibody-protein G-Sepharose Column were solubilized in sample buffer and electrophoresed on 10.5% polyacrylamide with the aid of a Bio-Rad 491 cell. Elution of fractions was in electrophoreses buffer. Additional details are provided in the text and the legend to Fig. 1. The results of a typical separation are shown.
In Table 1, we compare the total protein recovered at each purification stage. We also compare the disintegratons/min of radioactivity per mg of protein at each stage. From these values, we estimate that approximately 500-fold purification was required to obtain highly purified RFT from the alkaline washed plasma membrane of these cells and that final recovery was in the range of 20%. Although the data suggest that complete purity was achieved, in actual practice, the purity is probably only in the range of 96-98%. Following SDS-PAGE (slab gel) of this purified material, duplicate transblots were stained with Coomassie Blue or immunoblotted with anti-AMT monoclonal antibody using ECL detection. In each case a single protein band (approximately 46 kDa) was delineated (Fig. 4) on the blot.
Figure 4: Detection of purified RFT following SDS-PAGE by direct staining and Western blotting with anti-AMT antibody. Aliquots of two different preparations of purified RFT (RFT#1 and RFT#2) were subjected to SDS-PAGE (10% gel), transblotted to nylon (polyvinylidene difluoride), and either stained with Coomassie Blue (A) or immunoblotted (B). Additional experimental details are given in the legend to Fig. 1and in the text.
Figure 5: Relative amount and electrophoretic properties of RFT in plasma membrane of L1210 and L1210/R83 cell detected by Western blotting with anti-RFT polyclonal antibody. Following SDS-PAGE, purified RFT, RFT in crude or plasma membrane, or trichloroacetic acid precipitate of parental or variant (L1210/R83) L1210 cells was transblotted to nitrocellulose and immunoblotted with anti-RFT antibody. A, 2-µg sample of plasma membrane from L1210 and L1210/R83 cells. B, 7-µg sample of trichloroacetic acid precipitate of L1210 and L1012/R83 cells. C, 5-µg sample of crude membranes from L1210/R83 cells or 8 ng of purified RFT. Additional details are given in the text or legend to Fig. 1.
Figure 6: The lack of an effect of N- and O-glycanase on the electrophoretic properties of RFT in L1210/R83 cells. 50 ng of RFT were solubilized in 2% SDS and diluted 10-fold in 10 mM Tris-HCl (pH 7.2), 1% N-octylglucoside, and proteinase inhibitors(6) . Following incubation for 18 h at 37 °C with and without 10 units/ml of either N-glycanase or O-glycanase with neuraminidase, aliquots of 5 µg of the sample were added to a 10% SDS-PAGE gel. The various glycoprotein standards (100 µg) were treated with N-glycanase in the same manner. Detection of RFT by Western blotting following SDS-PAGE was by ECL.
Figure 7: Relative amount and electrophoretic properties of RFT in plasma membrane of murine tumors and their MTX transport altered variants. 2-µg samples of purified plasma membrane from different murine tumor cells (A), L1210, L1210/R24, and L1210/R25 cells (B), or L1210 and L1210/R26A-D cells (C). Additional details are given in the text or legends to Fig. 4and Fig. 5. The results shown are of separate blots (one of three) run on different days.
The ECL data shown in Fig. 7, B and C, also delineate a single
protein band in the vicinity of 46 kDa following SDS-PAGE of plasma
membrane from several independently isolated transport defective L1210
cell variants. All of these variants have markedly lower levels (Table 2) of one-carbon, reduced folate transport in comparison
to parental L1210 cells and in contrast to the latter,
trans-stimulation (2) by internalized L-L5CHO-folateH of [
H]MTX
influx could not be demonstrated. Also, variants L1210/R26A-D
have reduced influx V
, while L1210/R24 and R25
have reduced V
in addition to increased K
for MTX influx. On the basis of affinity
labeling and Western blotting, these variants fall into two distinct
classes as shown in Fig. 7, B and C, and Table 2. The variants L1210/R26A-D exhibit levels of RFT
somewhat higher than parental L1210 cells. However, RFT in all of these
variants migrates during SDS-PAGE at a rate slightly reduced relative
to parental cell RFT, so that an aberrant molecular size was obtained
for each which was approximately 10% higher than that determined for
parental cell RFT (46 kDa). Variants L1210/R24 and 25 were even more
unusual. RFT in these variants was not affinity labeled by
NHS-[
H]AMT (Fig. 2, Table 2) but was
found in the plasma membrane, based upon the ECL data, at levels 4-
(L1210/R24) to 8-fold (L1210/R25) higher than in parental L1210 cells.
Figure 8: Relative amount and properties of RFT in the plasma membrane of human leukemia cells and their variants as detected by Western blotting. The indicated amounts of purified plasma membrane from CCRF-CEM cells and its transport elevated (CEM/7A) or transport reduced (CEM/T) variants and HL-60 cells were run on a 7.5% polyacrylamide gel prior to transblotting. Additional details pertaining to this typical blot are (one of two) provided in the text.
The results demonstrate that ligand-directed immunoaffinity fractionation with preparative SDS-PAGE is a reasonable approach to the purification of RFT from L1210 cells. Also, the availability of an L1210 cell variant overproducing RFT to a very substantial extent (6) contributed greatly to our realizing adequate amounts of purified RFT for further studies. Although this method of purification has inherent limitations with regard to the maximum degree of purification attainable, it seems likely that the RFT consistently recovered will continue to be greater than 95% in purity. No indication of more substantial amounts of impurity, i.e. manifested as multiple protein bands in the size selected region, was obtained either by direct staining of protein or immunoblotting following SDS-PAGE. Also, the results in Fig. 7, A and B, showing differences in the level of RFT among various tumors and a small but perceptible shift in electrophoretic migration of RFT from four transport-defective variants, could not be obtained if the antiserum to purified RFT contained significant amounts of antibody to contaminating proteins similar in size to RFT. Conversely, since most integral plasma membrane proteins are glycosylated, significant amounts of such proteins as contaminants would have been visible after migration away from glycanase-treated RFT during SDS-PAGE.
The amount of RFT generated by the above methodology was very adequate for eliciting polyclonal antibody formation in rabbits. Western blotting with this immunoabsorbed antibody preparation of plasma membrane from L1210 and L1210/R83 cells detected the 46-kDa RFT in relative amounts commensurate with higher one-carbon, reduced folate transport and NHS-AMT affinity labeling of RFT in L1210/R83 cells(6) . Of interest as well were results showing that these antibodies also blotted RFT in plasma membrane of other tumors of different histologic origin and an 83-kDa RFT in plasma membrane from HL-60 and CCRF-CEM cells. Again, the relative extent of blotting was similar to the relative level of one-carbon, reduced folate transport and specific NHS-AMT labeling found (9, 27, 28) in parental and variant CCRF-CEM cells. These results on relative tissue content and interspecies cross-immunoreactivity of RFT lend considerable support to the notion we have expressed earlier(1) , that one-carbon, reduced folate transport is a conserved property among tumor cells of different species.
These and earlier studies from the authors laboratory (6) and others (5, 7, 29) utilizing alternative methods of size estimation suggested that L1210 cell RFT exists in the plasma membrane as a 43-46-kDa protein in a form with little or no core oligosaccharides attached (14) . In the latter case, this was concluded from an experiment examining (14) the effect of N-glycanase on the migration of stained purified RFT during SDS-PAGE. We have extended these studies in greater detail to show that immunologically delineated RFT has no detectable N- or O-linked oligosaccharides attached. These are in sharp contrast to results of very extensive prior studies (11, 15, 23) by others revealing N- and O-linked oligosaccharides on RFT in plasma membrane of human leukemia cells (K562 and CCRF-CEM). Most importantly, these studies showed that N-glycanase treatment of plasma membrane from cells grown in tunicamycin markedly reduced the molecular size of RFT to approximately 50 kDa, a size similar to that delineated for murine tumor RFT.
The availability of antibody to RFT has made it possible (15) (this study) to examine in more detail the properties of RFT in tumor cell variants with defective one-carbon, reduced folate transport that are resistant to classical folate analogues. The variants examined here revealed interesting differences in the amount and physical properties of RFT in addition to the functional modifications documented in Table 2. One group of independently isolated L1210 cell variants (L1210/R26A-D) exhibited an isoform of RFT with a modest, but consistent, decrease in electrophoretic mobility compared to wild-type RFT. Two other variants (L1210/R24 and R25) exhibited markedly increased amounts of RFT despite the fact that these 46-kDa proteins could not be affinity labeled with NHS-AMT. Prior studies (7, 29) of similar transport defective variants of the L1210 cell detected no differences in the amount or physical properties of RFT, while a transport-defective variant of CCRF-CEM cells exhibited slightly elevated amounts of a more rapidly migrating isoform of RFT during SDS-PAGE. In contrast, the transport-defective, variant of CCRF-CEM cells examined here and the variants of K562 cells examined by immunoblotting by others (15) showed only lower levels of RFT in the plasma membrane. Thus, it would appear that impaired function of one-carbon, reduced folate transport in tumor cell variants with acquired resistance to folate analogues can result from a variety of alterations affecting both the structure and amount of RFT.
The recent isolation of cDNAs
from L1210 (30) and hamster ovary (31) cell
recombinant cDNA libraries, which restore MTX transport in
transport-defective cells, has potentially important implications for
one-carbon, reduced folate transport in tumor cells. It is of interest,
therefore, that our results further verify that the molecular size of
native RFT expressed in L1210 cells is in the neighborhood of 46 kDa,
while pRFC-1 derived from the L1210 cell cDNA library and a related
hamster ovary cell cDNA appear to code for a protein of 58 kDa. An
NHS-[H]AMT affinity-labeled protein of 46 kDa was
previously delineated (6) among the plasma membrane proteins
from L1210/R83 cells during molecular sieve chromatography, most likely
ruling out that the 46-kDa RFT reflects an aberrancy in electrophoretic
mobility. This discrepancy with regard to the molecular size of RFT
remains unexplained at this time, but it clearly has implications for
the identity of the protein coding for pRFC-1. Therefore, further work
will be required before the exact relationship between pRFC-1 and its
hamster and human homologue and RFT is understood, and we present these
comments and our findings in this regard with no bias intended.