The Light Chain of CD98 Is Identified as E16/TA1 Protein*

The 80/40-kDa CD98 protein complex was purified using an anti-CD98 heavy chain monoclonal antibody coupled to Sepharose beads. Eluted proteins were subjected to preparative SDS-polyacrylamide gel electrophoresis, and protein corresponding to the 40-kDa CD98 light chain was excised. Following proteolysis with trypsin, a peptide fragment was sequenced by mass spectrometry. The nine residues obtained were identical to established C-terminal sequences of the human E16 and rat TA1 proteins, suggesting that TA1/E16 protein is the CD98 light chain. Consistent with this, anti-TA1/E16 antibodies specifically immunoblotted the ; 35–40-kDa light chain present upon immunoprecipitation of the human CD98 complex. Furthermore, anti-CD98 heavy chain antibody specifically co-immunoprecipitated hemaggluti-nin-tagged light chain from cells transfected with hem-agglutinin-tagged E16 cDNA. In conclusion, the CD98 light chain is identical to the TA1/E16 protein, based on partial amino acid sequence identity, antibody cross-reactivity, genetic reconstitution evidence, similar molecular size, and comparable cell distribution. The CD98 (4F2, FRP-1) molecule is a heterodimer of approx-imately 80 and 40 kDa that was originally reported as a T cell activation (1). It is also expressed on normal prolifer-ating previously (30). Preparation of E16 Transfectants— The open reading frame of E16 cDNA was N-tagged with an HA tag by polymerase chain reaction and subcloned into pLXIN vector (CLONTECH) in the sense and antisense orientation. Transient transfection of f NX-Eco cells was performed by calcium phosphate transfection protocol as described (35). Briefly, 2 3 10 6 cells/60-mm plate were transfected with 10 mg of DNA. 48 h post-transfection the cells were lysed and used for immunoprecipitation experiments. Purification, Proteolysis, and Amino Acid Sequencing— Molt4 cells (2 3 10 10 ) from 24 roller bottles were washed with PBS and lysed for 90 min in 1% Triton X-100 in PBS in the presence of protease inhibitors (10 m g/ml ion trap mass spectrometer Microchemistry Immunoprecipitation and Immunoblotting— For immunoprecipitation, HT1080 or f NX-Eco cells were lysed in 1% Triton X-100 in 150 m M NaCl, 20 m M Hepes, pH 7.5, for 60 min at 4 °C. After preincubation with protein A-Sepharose 4CL, the cell extract was incubated with specific mAb directly conjugated to Sepharose 4B (or to protein A-Sepharose) for 60 min, washed 3 times with lysis buffer, and eluted with Laemmli sample buffer, and proteins were resolved on 10–12% SDS-PAGE under reducing conditions. For immunoblotting, proteins were transferred to a nitrocellulose membrane that was preblocked using 10% nonfat dry milk, 0.5% Tween 20 in PBS for 2 h. The membrane was then incubated with a mixture of anti-TA1 mAbs (P5B4, P5F5, and P17E4) in 5% nonfat dry milk, 1% Tween 20, 1 M D -glucose, 10% glycerol in PBS for ; 12 h. Blots were washed 5 times using 0.5% Tween 20 in PBS and then incubated with biotinylated goat anti-mouse heavy chain specific antibody, followed by ExtrAvidin-peroxidase (Sigma), and developed using Renaissance chemiluminescent reagent (NEN Life Science Products). In another experiment blots were incubated with anti-HA monoclonal antibody (HA.11) in 5% nonfat dry milk in Tris-buffered saline, washed with 5% nonfat dry milk in Tris-buffered saline, then incubated with goat anti- mouse heavy chain specific antibody coupled to peroxidase, and developed using Renaissance chemiluminescent reagent.

The CD98 (4F2, FRP-1) molecule is a heterodimer of approximately 80 and 40 kDa that was originally reported as a T cell activation antigen (1). It is also expressed on normal proliferating tissue such as the basal layer of squamous epithelia (2,3), all rapidly growing tumor cells (1,4,5), and on cells having secretion or transport functions (6). The CD98 protein may be involved in cell proliferation and activation (4,(7)(8)(9)(10), cell survival/death (11), metal ion transport (12,13), cell fusion (14 -17), and amino acid transport. In the latter case, the CD98 heavy chain in oocytes dramatically up-regulates amino acid transport activity resembling both system y ϩ L (18 -21) and system L (22). The former transports neutral and dibasic amino acids, whereas the latter transports branched chain and aromatic amino acids (23). Recently, the CD98 protein has also been implicated in the regulation of ligand binding functions of the integrin ␣ IIb ␤ 3 and cell adhesion functions mediated by ␤ 1 integrins (24). Furthermore, co-stimulation of T cells by an anti-CD98 antibody was blocked specifically by an anti-integrin ␤ 1 antibody, thus providing further evidence for a functional linkage between CD98 and integrins. 1 The CD98/4F2 protein was first characterized in 1981-1982 (1, 25), and the heavy chain was subsequently sequenced from human (26), mouse (11), rat (22), and hamster (24) sources. Because the CD98 heavy chain itself does not resemble a transport type of protein, it has been hypothesized that the light chain must contain the actual amino acid transport activity (23,27). However, the CD98 light chain has remained uncharacterized, despite substantial efforts in several different laboratories. To begin to better understand how CD98 might participate in so many diverse functions, we have now characterized the CD98 light chain. The molecular, immunologic, genetic, and biochemical results all strongly indicate that the CD98 light chain is identical to the E16/TA1 gene product (28,29). In parallel with the CD98/4F2 heavy chain gene, the E16/TA1 gene is also present on nearly all rapidly growing tumor cells and on activated T cells (28 -30). Furthermore, the E16/TA1 protein sequence predicts that it may have amino acid transport activity, as might be expected for a CD98 light chain.
Antibodies were selected based on reactivity with recombinant TA1 protein, as well as selective reactivity with rat and human carcinoma tissue. Recombinant TA1 was produced in Sf9 cells, using a baculovirus expression system (34). The baculovirus transfer vector (pVL1392) contained the entire TA1 coding region from an ApoI digest of pTA1 (28) and also coded for a string of six histidine residues. Recombinant TA1 protein (ϳ35 kDa) was purified to homogeneity using a nickel affinity column (Qiagen, Inc.).
Molt4, a human T leukemic cell line, was grown in RPMI media, supplemented with 10% fetal bovine serum and antibiotics. HT1080 and NX-Eco cells were grown in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum and antibiotics. Tissue extracts of human colon carcinoma and controls were obtained as described previously (30).
Preparation of E16 Transfectants-The open reading frame of E16 cDNA was N-tagged with an HA tag by polymerase chain reaction and subcloned into pLXIN vector (CLONTECH) in the sense and antisense orientation. Transient transfection of NX-Eco cells was performed by calcium phosphate transfection protocol as described (35). Briefly, 2 ϫ 10 6 cells/60-mm plate were transfected with 10 mg of DNA. 48 h post-transfection the cells were lysed and used for immunoprecipitation experiments.
Purification, Proteolysis, and Amino Acid Sequencing-Molt4 cells (2 ϫ 10 10 ) from 24 roller bottles were washed with PBS and lysed for 90 min in 1% Triton X-100 in PBS in the presence of protease inhibitors (10 g/ml leupeptin, 10 g/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride). All purification steps were carried out at 4°C. The lysate (ϳ500 ml) was incubated for 60 min with 7.5 ml of protein A-Sepharose beads and then for 60 min with 1.5 ml of control IgG-Sepharose to deplete nonspecific binding activity. Then, the lysate was incubated for 60 min with 1.2 ml of 6B12-Sepharose (coupled at ϳ3 mg of mAb/ml of packed beads) and washed with at least 6 column volumes of 0.01% Triton X-100 in PBS, followed by 5 column volumes of 0.01% Triton X-100 in 50 mM glycine, pH 4.1. Elution was achieved by adding 0.01% Triton X-100 in 100 mM NaCl, 100 mM glycine, pH 2.0. Fractions of ϳ700 l were collected, with purified CD98 proteins appearing in fractions 2 and 3. Purified protein was concentrated ϳ10-fold using a Microcon ultrafiltration device with a 20-kDa cutoff (Amicon Co.), SDS was added (to ϳ1%), and samples were stored at Ϫ70°C. Next, ϳ60 l of thawed CD98 protein sample was fractionated by non-reduced 10% SDS-PAGE, 0.75-mm thick, run at 9 mA. Staining with 0.2% Coomassie Blue in 50% methanol revealed the presence of a substantial amount of ϳ40-kDa light chain that had been spontaneously reduced. The visualized gel slice containing the light chain was excised, washed using 50% formamide in distilled H 2 O, dried under N 2 , and stored at Ϫ70°C. In-gel trypsin digestion was done as described (36), except that it was carried out at pH 7.0 on a gel slice that had not been reduced and alkylated. Tryptic fragments were analyzed by tandem mass spectrometry (MS/MS) as described (36), using a microcapillary HPLC-quadrupole ion trap mass spectrometer at the Harvard Microchemistry Facility, Cambridge, MA.
Immunoprecipitation and Immunoblotting-For immunoprecipitation, HT1080 or NX-Eco cells were lysed in 1% Triton X-100 in 150 mM NaCl, 20 mM Hepes, pH 7.5, for 60 min at 4°C. After preincubation with protein A-Sepharose 4CL, the cell extract was incubated with specific mAb directly conjugated to Sepharose 4B (or to protein A-Sepharose) for 60 min, washed 3 times with lysis buffer, and eluted with Laemmli sample buffer, and proteins were resolved on 10 -12% SDS-PAGE under reducing conditions. For immunoblotting, proteins were transferred to a nitrocellulose membrane that was preblocked using 10% nonfat dry milk, 0.5% Tween 20 in PBS for 2 h. The membrane was then incubated with a mixture of anti-TA1 mAbs (P5B4, P5F5, and P17E4) in 5% nonfat dry milk, 1% Tween 20, 1 M D-glucose, 10% glycerol in PBS for ϳ12 h. Blots were washed 5 times using 0.5% Tween 20 in PBS and then incubated with biotinylated goat anti-mouse heavy chain specific antibody, followed by ExtrAvidin-peroxidase (Sigma), and developed using Renaissance chemiluminescent reagent (NEN Life Science Products). In another experiment blots were incubated with anti-HA monoclonal antibody (HA.11) in 5% nonfat dry milk in Tris-buffered saline, washed with 5% nonfat dry milk in Tris-buffered saline, then incubated with goat antimouse heavy chain specific antibody coupled to peroxidase, and developed using Renaissance chemiluminescent reagent.

RESULTS AND DISCUSSION
Purification of CD98 -The T leukemic cell line Molt4 was chosen as a source for CD98 purification because it contains high levels of cell surface CD98, and it could be grown to high density in suspension culture (ϳ3 ϫ 10 6 cells/ml). Approximately 2 ϫ 10 10 Molt4 cells were lysed in 1% Triton X-100, and then background binding proteins were removed by preincubation with control Sepharose preparations. Next, the lysate was incubated with Sepharose conjugated to anti-CD98 mAb 6B12. Then after washing, purified CD98 protein was eluted at low pH (pH 2.0) and fractions were collected. As indicated (Fig. 1) the majority of silver stained protein appeared in fraction 2.
This material included a prominent protein of ϳ40 kDa, as expected for the CD98 light chain. Also prominent were proteins corresponding to the CD98 heavy chain (ϳ80 kDa) and possibly unreduced 80/40 complex (ϳ120 kDa).
Partial Sequencing of CD98 Light Chain-Numerous attempts to obtain the N-terminal sequence from up to 50 pmol of purified CD98 light chain were unsuccessful. Likewise, we failed in initial attempts to digest CD98 light chain with chymotrypsin, trypsin, thermolysin, or ArgC proteases, even though bovine serum albumin was digested under similar conditions. However, subsequent studies revealed that significant digestion with trypsin could be achieved at pH 7.0 (not shown). Consequently, purified CD98 light chain was digested with trypsin at pH 7.0, fragments were separated using HPLC, and from one of the purified fractions, an MS/MS ion sequence spectra was obtained that correlated with the sequence "LMQVVPQET" present in the C termini of the E16 (29) and TA1 (28) protein sequences. Consistent with it being the product of a trypsin digestion, the LMQVVPQET sequence is preceded by lysine in the published sequences. No other sequence identity was found in a GenBank search.
Confirmation That CD98 Light Chain Corresponds to E16/ TA1 Protein-To confirm the identity of the CD98 light chain, we first tested whether it was recognized by anti-TA1/E16 antibody in an immunoblotting experiment. The CD98 heterodimer was immunoprecipitated from human HT1080 cells, and proteins were resolved using 10% SDS-PAGE. Then, anti-E16/TA1 antibodies were shown to cross-react specifically with a protein of ϳ35-40 kDa, corresponding to the CD98 light chain (Fig. 2). No E16/TA1 cross-reactivity was observed in immunoprecipitates obtained using a negative control mAb or anti-major histocompatibility class I protein. In a positive control experiment, anti-E16/TA1 antibodies did show reactivity with whole cell lysate from a colon carcinoma but not from normal colon cells (Fig. 2, right panel). This agrees with previous evidence showing that E16/TA1 mRNA and protein were absent from similar specimens of normal colon but greatly up-regulated in colon carcinoma (30). In a separate experiment, immunoprecipitation of rat CD98 (using mAb B3), but not rat CD71, yielded a ϳ35-40-kDa protein that was again recognized by anti-E16/TA1 antibodies (not shown).
To further establish that E16/TA1 corresponds to the CD98 light chain, E16 cDNA was prepared to include an HA tag, and this cDNA was transiently expressed in human NX-Eco cells. As indicated (Fig. 3), immunoprecipitation of the CD98 heavy chain, followed by immunoblotting with an anti-HA tag, revealed the association of E16 protein with the heavy chain of CD98. E16 protein was not present in a control immunoprecipitation obtained using anti-␤ 1 integrin antibody A-1A5 (not shown).
The size and distribution of the E16/TA1 gene and/or protein are consistent with the expected size and distribution of the CD98 light chain. The E16/TA1 gene is predicted to code for a protein of 241 amino acids, with a size of 26.5 kDa (28, 29). Anti-TA1 polyclonal antibodies blotted proteins in the 30 -40-kDa range (30), which are thus comparable with the 35-40- FIG. 1. Purification of CD98. CD98 proteins were purified from Molt4 cell lysate as described under "Materials and Methods." From a column containing ϳ1 ml of 6B12-Sepharose beads, bound protein was eluted at pH 2.0, eluted fractions of 0.75 ml were collected, and 1% of each fraction was analyzed using 10% SDS-PAGE under reducing conditions. Proteins were visualized by silver staining.
CD98 Light Chain Is E16/TA1 Protein 33128 kDa size estimated for the CD98 light chain. In addition to being expressed on some normal cell types, the E16/TA1 protein is expressed on nearly all rapidly dividing cell lines, as well as on freshly activated peripheral blood lymphocytes (28 -30). Again, this distribution is generally consistent with the distribution of the CD98 heavy chain (5,6,8,10).
As previously noted (30) the E16/TA1 protein sequence shows a 51% identity to an amino acid permease from Schistosoma mansoni and a 30% identity to a methionine permease from Saccharomyces. Indeed, the presence of six or seven putative transmembrane domains in the E16/TA1 coding region is consistent with a possible transport function. In fact, recent work shows that E16/TA1 message levels are altered in response to amino acid availability in primary hepatocyte cultures, characteristic of some amino acid transporters (37). These properties of the E16/TA1 protein may help to explain the ability of the CD98 heavy chain to regulate system y ϩ L-like and system L-like amino acid transport activity (18 -22). The CD98 heavy chain shows ϳ29% identity to another type II transmembrane protein called D2/NBAT, which also may regulate amino acid transport (20,23,27,38). Like CD98, the D2/NBAT protein may also contain a disulfide-linked smaller subunit (39). However, the putative small subunit of D2/NBAT is ϳ50 kDa (39) and thus appears to be distinct from the E16/TA1 protein/CD98 light chain.
The predicted structure of the E16/TA1 protein includes relatively little extended extracellular regions, thus perhaps explaining the scarcity of monoclonal antibodies. Also, there are no potential glycosylation sites (28,29), as expected for CD98 light chain (25). The E16/TA1 protein was not previously known to be part of a heterodimer, mostly because suitable reagents were not available for protein biochemistry. Among the models proposed for the structure of E16/TA1, we now favor a model in which the membrane is spanned 7 times (28), such that the C-terminal segment would be on the extracellular side. This would allow at least one cysteine residue to be available for disulfide linkage with the CD98 heavy chain.
In conclusion, we have identified the CD98 light chain as being equivalent to the previously described E16/TA1 protein. This conclusion is based on peptide amino acid sequence identity, immunologic cross-reactivity, E16 cDNA encoding for a product that associates with CD98 heavy chain, and similarities in size, distribution, and association with amino acid transport. The new information presented here, together with the availability of E16/TA1 cDNA for expression studies, should greatly facilitate future studies of CD98 function. For example, it should be possible to determine whether the E16/TA1 protein contributes to the emerging connections between CD98 and functions mediated by members of the integrin family of cell adhesion receptors. Since this article was submitted, other reports have now provided functional evidence that E16/TA1related proteins dimerize with CD98/4F2 heavy chain and mediate L-type amino acid transport (40,41). It remains to be seen whether another light chain might be found that contributes to CD98-mediated y ϩ L transport.
FIG. 3. E16 cDNA encodes for a protein that associates with CD98 heavy chain. The CD98 complex was immunoprecipitated using anti-heavy chain mAb 4F2 directly coupled to protein A-Sepharose. Cell lysates were from FNX-Eco cells (2 ϫ 10 6 cell eq/sample) that had been transiently transfected with the vector (lane 1), antisense (lane 2), or sense E16-HA cDNA (lane 3). After proteins were resolved by 12% SDS-PAGE, immunoblotting with anti-HA monoclonal antibody HA.11 was performed.