(Received for publication, October 13, 1994; and in revised form, November 29, 1994)
From the
A clone has been isolated from a human lymphoblastic cDNA expression library that complements a mutant Chinese hamster cell defective in the uptake of the folate analogue methotrexate. When transfected with this clone the mutant cells regain the ability to transport the drug and, as a consequence, become sensitive to its cytotoxic action.
The clone is 2863 base pairs long and has an open reading frame of 1770 base pairs that codes for a putative protein of 64 kDa. The putative protein has 51 and 50% identity at the amino acid level with the mouse and hamster functions, respectively, involved in the transport of reduced folates. Together these three proteins share 47% identity and have similar predicted structural features.
The data are consistent with this human clone encoding either the reduced folate transporter or an auxiliary function that interacts with this transporter.
Folates are essential components required for several metabolic pathways involving the biosynthesis of amino acids, purines, and pyrimidines. These compounds cannot be synthesized by eukaryotic cells and thus must be obtained from extracellular sources.
Studies in
various systems have identified two transport systems whereby folic
acid and its reduced forms including the chemotherapeutic agent
methotrexate (Mtx) ()enter cells. One system consists of a
membrane-bound folate receptor, termed the folate-binding protein,
which is a 40-kDa glycoprotein. It has a high affinity for folates,
including folic acid and a low affinity for Mtx (1, 2, 3, 4, 5, 6, 7, 8) .
It is present in both human and mouse cells and its cDNA has been
cloned from several
sources(9, 10, 11, 12, 13, 14) .
The second transport system, called the reduced folate carrier, has been identified in mouse(15, 16, 17) , human(18, 19, 20) , and hamster (21) cells. It has an apparent mass ranging from 36 to 78 kDa(16, 20, 22, 23) , is heavily glycosylated in human cells(23, 24) , and shows a preference for binding and transporting reduced folates and Mtx over folic acid.
In some cell systems, both transport systems are present and may interact in tandem to allow the transfer of folates across the membrane (25) . Other studies indicate that the two systems act independently (26, 27) .
In order to develop a better understanding of the folate transport process, we have previously isolated a series of Chinese hamster ovary (CHO) cell lines that are resistant to the cytotoxic action of Mtx because of an inability to take-up the drug(21, 28) . Such mutant cells require higher levels of reduced folates for growth as compared with wild-type cells(29) . This provided a selection scheme whereby we were able to obtain both a genomic DNA fragment (30) and a cDNA clone (pMtxT9) (31) which when introduced into the mutant cells could complement the phenotype. When the cDNA was transfected into mutant cells which did not express an mRNA for this gene, it conferred the ability to grow on low levels of folinic acid as a source of folates, to bind and take up Mtx, and as a consequence become sensitive to the drug's cytotoxic action(31) . The properties of the putative protein encoded by this cDNA are consistent with it being the reduced folate carrier. A function with a high degree of similarity to the hamster gene has also been cloned from mouse cells(32) .
In this report, we describe the isolation of a human cDNA homologous to the hamster gene using the latter as a molecular probe. The nucleotide sequence and predicted amino acid sequence of the putative encoded protein are similar to both the respective sequences from hamster and mouse, although the human protein is larger in its predicted size. When transfected into the mutant CHO cells, the human cDNA is able to complement the Mtx-resistant phenotype and restore wild-type phenotypic properties to the cells. This indicates that the hamster and human proteins are functionally similar.
For screening,
approximately 2 10
colony-forming units were
plated, transferred to Biotrans membranes, processed according to the
directions supplied by the manufacturer, and hybridized with a
radiolabeled 2.5-kb DNA fragment from the plasmid pMtxT9, which
contains the hamster gene for Mtx transport(31) , using the
hybridization conditions described previously(33) .
Sequence information was obtained from clones sequenced in both directions and/or from sequences of multiple clones covering the same regions such that the sequences obtained were unambiguously determined.
To identify cDNA clones, approximately 2
10
recombinant colony-forming units from the human
lymphoblast library were screened. Five clones giving strong
hybridization signals were isolated and designated as pHuMtxT1 to T5.
The inserts from these clones could be removed intact by digestion of
the DNA with BamHI and were shown to have approximate sizes of
4.3, 2.0, 2.5, 2.6, and 2.5 kb for the clones T1 to T5, respectively.
To determine whether any of these clones could complement mutant CHO cells, DNA from each plasmid clone was transfected into the mutant cells (MtxRII 5-3) which are Mtx-resistant because of defective drug uptake and lack the message for the gene involved in the transport of the drug(31) . The resulting transfectants were selected for growth in a low level of folinic acid. As shown in Table 1, DNA from plasmids pHuMtxT3, pHuMtxT4, and pHuMtxT5 were able to complement the mutant CHO phenotype to permit growth in 2 nM folinic acid. The frequency of colonies recovered with these three plasmid DNAs was similar to or higher than that obtained with the DNA from the plasmid containing the hamster gene (pMtxT9). Several transfectants tested were sensitive to Mtx. This indicates that these three plasmids contained full-length copies of a human cDNA capable of correcting the defect in hamster cells and consequently rendering them sensitive to Mtx.
Since plasmids pHuMtxT1 and pHuMtxT2 did not complement the mutant phenotype, no further analysis was carried out on these clones.
Figure 1:
Cellular uptake of
0.4 µM [H]Mtx by various cell lines.
Cells were incubated at 37 °C and [
H]Mtx
uptake was measured as described under ``Materials and
Methods.''
, wild-type CHO;
, mutant CHO MtxRII 5-3;
, MtxRII 5-3 transfected with pHuMtxT3 DNA;
, MtxRII 5-3
transfected with pHuMtxT4 DNA;
, MtxRII 5-3 transfected with
pHuMtxT5 DNA;
, MtxRII 5-3 transfected with pMtxT9
DNA.
Figure 2:
Autoradiogram of Northern hybridizations. A, approximately 5 µg of poly(A) RNA
isolated from human Hep G2 (lane 1), human fibroblasts (lane 2), CHO line MtxRII 5-3 (lane 3), CHO line
MtxRII 5-3 transfected with plasmid pHuMtxT3 DNA (lane 4), and
CHO line MtxRII 5-3 transfected with plasmid pHuMtxT4 DNA (lane
5) cells were separated on agarose gels, blotted, and hybridized
with the 2.5-kb BamHI fragment from pHuMtxT5 as described
under ``Materials and Methods.'' RNA molecular mass markers
(in kilobases) from Life Technologies, Inc. are shown. B, the
blot in A was stripped and rehybridized with radiolabeled
hamster dihydrofolate reductase cDNA.
As shown in B in Fig. 2, under these hybridization conditions, the hamster dihydrofolate reductase cDNA does not detect the human dihydrofolate reductase homologue.
Figure 3: Nucleotide and predicted amino acid sequence of pHuMtxT4. The nucleotide sequence is numbered on the left, and the amino acid sequence is numbered on the right. The putative Kozak sequence (38) at position 1 and the putative polyadenylation signal at position 2692 are underlined. Consensus site for N-linked glycosylation is marked with a +.
Sequence analysis of clones pHuMtxT3 and pHuMtxT5, which can also complement the mutant CHO phenotype, indicate that they possess identical sequences to pHuMtxT4 in the 5`-noncoding and coding region but are truncated in the 3`-noncoding region (data not shown).
The
deduced amino acid composition of the putative protein encoded by
pHuMtxT4 is shown in Fig. 3. The open reading frame contains 589
amino acids, the predicted molecular mass is 63,954 Da, and the pI is
9.12. A consensus site for N-linked glycosylation occurs at
amino acid residue 58. An amino acid hydropathy plot using the criteria
of Hopp and Woods (39) (Fig. 4) indicates that a large
part of the molecule is hydrophobic, with hydrophilic regions near both
the NH and COOH termini as well as in the middle of the
molecule. Secondary structure predictions according to the criteria of
Chou and Fasman (40) indicate that a majority of the molecule
conforms to
-sheets. These predicted indices for the putative
human protein are similar to those of the hamster protein(31) .
Figure 4: Hydropathy plot and predicted secondary structure of the putative protein encoded by pHuMtxT4. A, Hopp and Woods (39) analysis of the deduced amino acid sequences using a window size of 6 is shown. B, the secondary structure predictions using the criteria of Chou and Fasman (40) is indicated.
Figure 5: Comparison of the amino acid sequences for the mouse, hamster, and human cDNAs. The numbers refer to the amino acid sequences in the predicted coding region for the human gene as contained in pHuMtxT4. The dots indicate spaces in order to maximize the homology. The boxed areas indicate regions of identity.
Using a hamster cDNA clone as a molecular probe for a
function involved in the transport of reduced folates, we have been
able to isolate the corresponding human cDNA from a lymphoblastic cDNA
expression library. This human cDNA codes for a putative hydrophobic
protein with an apparent molecular mass of 64 kDa and with considerable
predicted -sheet structure.
The nucleotide sequence of this cDNA and the predicted amino acid sequence of the putative encoded protein show a high degree of similarity and identity to both the hamster and the mouse counterparts that have been shown to be involved in the uptake of reduced folates (31, 32) . The putative human protein, however, is larger than either the mouse or hamster proteins which both have predicted sizes of 52 kDa (31, 32) . The human protein appears to have a majority of its additional amino acid residues at the COOH-terminal end. Changes may occur post-translationally and it will be of interest to determine the sizes of the proteins present in the cells.
These proteins are also functionally similar. Transfection of mutant CHO cells, lacking the message for this function, with the human cDNA clones, restores wild-type properties to these cells. They take up the drug and thus become sensitive to its cytotoxic action. This is the same result obtained when these cells were transfected with the homologous hamster cDNA clone(31) . This implies that this function is conserved between hamster and human.
At present the exact role that similar proteins from hamster, mouse, and human cells play in folate transport is not clear. The data are consistent that these putative proteins are the reduced folate transporters from the three species. CHO cells do not appear to express the folate-binding protein component of folate transport (41, 42) and obtain folates via the reduced folate transporter. Mutant CHO cells, which are defective in this transport, when transfected with either the hamster cDNA (31) or the human cDNAs, regain the ability to transport Mtx. Similarly, the mouse cDNA can restore reduced folate carrier activity to human cells that are defective in this mode of folate transport(32) . It is also possible that these cloned functions are not the reduced folate carrier but rather an additional component that interacts with it to facilitate the transport of Mtx. Clearly, further biochemical studies are required to distinguish between these alternatives.
The reduced folate transporter from human cells is extensively glycosylated containing both N- and O-linked oligosaccharides(23, 24) . In its N-deglycosylated form in human K562 cells it has an apparent molecular mass of 68 kDa(23) . The similar protein from human CCRF-CEM cells when grown in the presence of tunicamycin and subsequently treated with N-glycanase had a molecular mass of 50 kDa(24) . The predicted size of the protein encoded by pHuMtxT4 is 64 kDa. These differences may be due in part to processing and additional post-translational modifications. It is of interest to note that the putative protein has a single consensus site for N-linked glycosylation at residue 58 which appears to be in an exposed region of the protein.
The hydropathy and secondary
structure prediction plots for both the hamster and human putative
proteins are similar. Both proteins contain stretches of hydrophobic
amino acids, suggesting that these regions are buried in the membrane.
At present it is too early to estimate the number and extent of the
transmembrane regions until more is know about the protein in its
mature, native form in the cell. A structural comparison has been made
between the presumed mouse reduced folate carrier and the human GLUT1
glucose transporter(32) . These two proteins showed remarkable
similarity and 12 potential transmembrane regions were identified in
the mouse reduced folate carrier. The structure description of the
GLUT1 transporter was based on the idea that the transmembrane regions
formed -helices (43) and recent work supports
this(44) . Other work, however, suggests that these regions may
form
-barrels(45) . This latter observation would be
consistent with the predicted structure of the hamster and human
putative reduced folate carrier proteins described here in which a
majority of the hydrophobic regions have a predicted
-sheet
structure.
The availability of a human cDNA for a function involved in the transport of Mtx will allow an investigation of the role that this gene product may have in the development of clinical resistance to this important chemotherapeutic agent.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17566[GenBank].