(Received for publication, June 19, 1996)
From the Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 64978, Israel
In primary embryonal fibroblasts from transgenic
mice expressing H-2 genes and a miniature swine class I transgene
(PD1), transformation with the highly oncogenic Ad12 results in a
reduction in peptide transporter and proteasome-associated (LMP2 and
LMP7) gene expression, and suppression in transport and cell surface expression of all class I antigens. The selective suppression in
transport of H-2 (but not of PD1) molecules in cells reconstituted for
the expression of peptide transporter and LMP genes implied that an
additional factor(s) is involved in the assembly of class I complexes.
Here we show that the 2m, H-2Db, and
H-2Kb genes are transcribed and translated in
Ad12-transformed cells. However, unlike normal and E1Ad5-transformed
cells, in which
2m is either secreted unbound or bound
to class I heavy chains, in Ad12-transformed cells significant amounts
of
2m are retained in the cell bound to the membrane,
but free of class I heavy chains. This abnormal turnover of
2m in the Ad12-transformed cells suggests the existence
of a novel
2m-binding molecule(s) that sequesters
2m, and this process may provide a mechanism by which
transformation with Ad12 may subvert class I complex formation.
MHC1 class I molecules are polymorphic, integral membrane proteins that bind a diverse group of peptides derived from endogenous antigens and display these peptides for recognition by cytotoxic T-lymphocytes (1). This mechanism enables the immune system to control infectious diseases and the growth of tumor cells (2, 3). Indeed, cells infected by a variety of viruses that interfere with cell surface expression of class I antigens, as well as tumors of various origins that demonstrate suppressed levels of class I antigens, can escape immune surveillance (4).
The biochemistry and cell biology of antigen processing and
presentation by class I MHC molecules has been analyzed in detail in
recent years (5, 6). It is well established that the efficient
transport of class I molecules to the cell surface depends on the
assembly of the heavy chain/2m dimer with peptides that are usually 8-9 amino acids long. Such peptides, generated by cleavage
of proteins in the cytosol, are actively transported by a heterodimeric
complex, known as transporter associated with antigen presentation
(TAP), into the endoplasmic reticulum (ER), where assembly with class I
molecules takes place. The trimeric complex is then transported through
the Golgi apparatus to the cell surface. Mutant cell lines (5, 6, 7, 8),
"knockout" mice (9, 10), and tumor cells (4, 11, 12), which do not
express TAP genes, are generally devoid of cell surface MHC class I
molecules. Cell lines and knockout mice lacking
2m
expression also fail to display normal cell surface class I MHC
molecules (13, 14, 15). The crucial role of
2m is evident
from data demonstrating that it stabilizes cell surface expression of
class I heavy chains, facilitates the binding of purified class I
molecules to antigenic peptides both on cells and plastic surfaces, and generates additional high affinity peptide-binding sites in
preparations of soluble purified class I molecules (16, 17, 18, 19, 20). These
data, as well as the finding that
2m determines the fate
of class I heavy chains (21), suggest that
2m may play a
predominant role in maintaining the class I heavy chain in a
non-denatured conformation suitable for terminal folding upon binding
of peptide. Such a model predicts a role for extracellular
2m in providing a microenvironment that supports the
existence of cell surface
2m/heavy chain dimers capable
of binding peptides. Indeed, both constitutive and peptide-induced
class I surface expression can be augmented markedly, in some cases, by
the presence of exogenous
2m (21, 22). The existence of
a substantial pool of inactive heavy chains on the cell surface, which
are able to bind added
2m at 37 °C, has been
demonstrated by Rock et al. (23), implying a physiological
role for exogenous
2m in maintaining cell surface class
I heavy chains in a state suitable for subsequent binding of exogenous
peptides.
Increased levels of 2m can be detected both in the
peripheral circulation and locally, in many abnormal in vivo
conditions (24, 25, 26, 27, 28). Additionally, lymphoid and hepatoma cells have
been shown to produce and secrete
2m when stimulated with various cytokines (29, 30). If these augmented levels facilitate
the accumulation of class I-bound peptides, then the regulation of
2m secretion may have functional consequences. Supportive of such a role are data demonstrating the strong in vivo priming of cytotoxic T-lymphocytes to class I-binding
peptides if the latter are combined with
2m when
injected into mice (31).
We have shown previously that, in primary embryonal fibroblasts from
transgenic mice expressing both endogenous H-2 genes and a miniature
swine class I transgene (PD1), transformation with the highly oncogenic
Ad12 results in inhibition of the transport of newly synthesized class
I molecules via the Golgi apparatus, a significant reduction in peptide
transporter (TAP1 and TAP2) and in proteasome-associated (LMP2 and
LMP7) gene expression, and in suppression of cell surface expression of
all class I antigens (12, 32, 33, 34). Expression of these class I genes is either normal or up-regulated in cells transformed by the non-oncogenic virus-Ad5. Re-expression of TAP and LMP in an Ad12-transformed cell
line completely reconstituted the cell surface expression and transport
of PD1 and induced the transport of 20% of the H-2Db
molecules, but did not affect the transport or expression of H-2Kb molecules (34). These data, as well as the fact that
in Ad12-transformed cells, H-2 molecules were not recognized by
conformation-independent antibodies (33, 34), raised the possibility
that either the synthesis of 2m in these cells is
defective and the class I heavy chains compete for a limited amount of
2m molecules (PD1 being more competitive), or that
Ad12-transformed cells are deficient in a factor(s) that facilitate the
assembly of
2m with a particular set of class I heavy
chains (H-2). We now present data substantiating that
2m, as well as H-2Db and H-2Kb,
are transcribed and translated in Ad12-transformed cells. However, the
results show an abnormal turnover of
2m molecules and
suggest the existence of a novel
2m-binding molecule(s)
that acts to retain
2m in the transformed cells.
The Ad12-transformed (VAD12.79, VAD12.42, VAD12.20, VAD12.25, VAD12.36, VAD12.54, and VAD12.43), E1Ad5-transformed (A5O1 and A5O5), and TAP-transfected cell lines and the normal cell line M1 have been described previously (32, 33, 34). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 10% fetal calf serum, penicillin, streptomycin, gentamycin, and amphotericin B at the recommended concentrations (35). Media and supplements were purchased from Biological Industries (Bet Ha'emek, Israel).
Cell lines were treated with 600 units/ml /
interferons (Lee
Biomolecular Research Inc., San Diego, CA) for 16 h, or 100 units/ml
-interferon (Boehringer, Mannheim, Germany) for 48 h before harvesting.
TAP2-transfected Ad12-transformed
cells (VAD12.79 Cl1.1, Ref. 34) were transfected by the calcium
phosphate-DNA coprecipitation method (34). The transfection mixture
contained 10 µg of plasmid DNA (the 2m gene expressed
from an actin promoter in PBr was constructed in Dr. D. Margulies'
laboratory (NIAID, NIH, Bethesda, MD)), 1 µg of plasmid DNA
containing the hygromycin gene, and 5 µg of carrier DNA (sheared
salmon sperm DNA, Sigma). Twenty-four hours after
transfection, the cells were washed with PBS, fresh media was added and
after an additional 24 h the medium was supplemented with 110 units/ml hygromycin B (Calbiochem). Following selection of
hygromycin-resistant cells, individual cell colonies were isolated and
expanded in culture.
The following antibodies were used for
immunoprecipitation: 20.8.4S (recognizes the
1/
2 epitope on H-2Kb; Ref.
36); B22.249 (recognizes an
1 epitope on
H-2Db; Ref. 36); PT85A (recognizes a public determinant on
swine lymphocyte antigens (SLA); Ref. 37). Rabbit antibodies directed against
2m were a kind gift from Dr. J. E. Coligan
(NIAID, NIH, Bethesda, MD).
Cells were grown to 80% confluence and starved for 15 min in methionine-free medium. They were then labeled in methionine-free medium containing 250 mCi/ml [35S]methionine (Amersham International, Little Chalfort, United Kingdom (UK)) for 15 min unless otherwise indicated, washed with PBS, and chased for the indicated intervals. Cells that were labeled for 15 h were incubated in methionine-free medium containing 16% Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 mCi/ml [35S]methionine. The cells were lysed with buffer containing 0.5% Triton X-100, 50 mM Tris (pH 7.5), and 150 mM NaCl. The immunoprecipitates were washed with buffer containing 0.1% Triton X-100, 50 mM Tris (pH 7.5), and 150 mM NaCl. For Endo H treatment, immunoprecipitates were eluted by adding 25 µl containing 50 mM Tris (pH 8), 1% SDS, and boiled for 5 min. The samples were centrifuged and the supernatant was added to 25 µl of 50 mM citrate buffer (pH 5.5) containing 500 units of Endo H (New England Biolabs). The samples were incubated at 37 °C for 18 h, followed by the addition of sample buffer. All the buffers contained the following protease inhibitors; 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin (Sigma). The immunoprecipitates were fractionated on 10 or 15% SDS-polyacrylamide gels, and X-Omat AR x-ray films (Eastman Kodak Co.) were exposed to the dried gels.
Preparation of MembranesMembranes were prepared as described (38). Briefly, cell lysates were separated into two fractions: one containing cell membranes and the other containing soluble proteins. Cell pellets (5 × 107/ml) were resuspended in ice-cold Dounce buffer (10 mM Tris (pH 7.6), 0.5 mM MgCl) with protein inhibitors and incubated on ice for 30 min. The cells were lysed with a Dounce homogenizer, and the tonicity of the solution was restored by adding NaCl to a final concentration of 0.15 M. The suspension was spun at 500 × g to remove the nuclei, and the supernatant was further centrifuged for 45 min at 100,000 × g at 4 °C. The supernatant, which contained soluble proteins, was transferred to a separate tube, and the pellet was resuspended in lysis buffer.
Probes and PlasmidsThe following probes have been
described previously (32, 33, 34); the actin probe was a
PstI-PstI fragment from chicken actin
cDNA, the PD1-specific probe was a SacI-BamHI
genomic fragment containing exons 2-7 of the PD1 gene, the H-2 probe
was an EcoRI-HindIII fragment derived from
pH-2d33 (H-2Kd), the
2m probe
was a PstI-PstI fragment from
2m
cDNA, and the histone probe was a BamHI genomic
fragment. Plasmids containing H-2Kb cDNA and
H-2Db genomic (pMo/Db) fragments (40) were used
as templates for PCR reactions and were a kind gift from Dr. L. Eisenbach (The Weizmann Institute of Science, Rehovot, Israel) and from
Dr. D. Pardoll (Johns Hopkins University, Baltimore, MD),
respectively.
The hybridization solution contained 4 × SSC, 50% formamide, 0.2% SDS, 0.1% polyvinylpyrrolidone, and 100 µg/ml sheared salmon sperm DNA. Hybridizations were carried out at 42 °C, followed by washes with 2 × SSC, 0.1% SDS at room temperature, and 0.2 × SSC at temperatures ranging between 55 and 65 °C. After stripping with a boiling solution of 0.1% SDS, the blots were used for additional hybridizations.
RNA AnalysisCytoplasmic RNA was prepared using a
modification of the White and Bancroft method (41), as described
previously (34). RNA was denatured and fractionated on a 1.2%
formaldehyde/formamide agarose gel, blotted onto a Hybond-N membrane
(Amersham International, Little Chalfort, UK) and hybridized with the
appropriate probe, which had been labeled with
[-32P]dCTP (Rotem Industries, Dimona, Israel), using a
Random Priming labeling kit (U. S. Biochemical Corp.).
Polysomes were fractionated as
described previously (42). Cells were grown to 80% confluence and
harvested by trypsinization (0.25 M trypsin EDTA;
Biological Industries, Israel). A quantity of 6 × 107
cells was lysed per fractionation. The cells were washed with PBS, and
cell pellets were kept at 70 °C until used. Trypsin, PBS, and
media contained 100 µg/ml cycloheximide (Sigma).
Cells were thawed on ice and suspended in 450 µl of polysomal buffer containing 10 mM NaCl, 10 mM Tris (pH 7.4), 1.5 mM MgCl2, and 50 µl of ribonucleoside vanadyl
complexes (200 mM) (New England Biolabs). Cells were lysed
following the addition of 60 µl of polysomal buffer containing 10%
Triton X-100 and 10% deoxycholate by brief mixing before and after 3 min of incubation on ice. Nuclei were pelleted by centrifugation for 3 min at 4 °C, and the postnuclear supernatant was diluted with an
equal volume of 25 mM Tris (pH 7.5), 10 mM
MgCl2, 25 mM NaCl, 0.14 M sucrose,
500 µg/ml heparin, 0.05% Triton X-100. The suspension was layered
over 35 ml of 15-45% (w/w) sucrose gradient with a 2-ml cushion of
45% sucrose. The sucrose solution contained 25 mM Tris (pH
7.5), 25 ml of NaCl, 2 mM MgCl2. A volume of 36 µl of heparin (from a stock solution of 200 µg/ml) was added to
each gradient. The gradients were centrifuged at 26,000 rpm for 4 h at 4 °C in a Beckman SW27 rotor. After centrifugation, 38 fractions of 1 ml each were collected into tubes containing 10 µl of
10% SDS. The A260 was monitored, the polysome profile was determined,
and each 2-3 tubes were pooled to give a total of 8-10 tubes. RNA was
extracted from polysomal and subpolysomal fractions as described
before. Only gradients with identical polysomal profiles were
compared.
Reverse transcription of RNA was carried out for
1 h at 42 °C, in 20 µl of PCR buffer II (Perkin Elmer)
containing 1 µg of total cellular RNA, 2.5 µM
oligo(dT)18 primer (New England Biolabs), 1 mM
nucleotide mixture, 5 mM MgCl2, 35 units of
ribonuclease inhibitor (MBI Fermentas, Vilnius, Lithuania), and 7 units
of avian myeloblastosis virus reverse trascriptase (Promega, Madison, WI). The cDNA was stored at 20 °C until used.
PCR was carried out with 1 µl of the cDNA, and 50 pmol of the
following primers (40): a 5 consensus primer for H-Db and
H-2Kb (5
-CGC GAC GCT GCT GCG CAC AG-3
), and 3
primers
specific for H-2Db (5
-TAC AAT CTC GGA GAG ACA TT-3
) or
H-2Kb (5
-TAC AAT CTG GGA GAG ACA GA-3
), 40 µM nucleotide mixture, and 0.375 units of Taq
DNA polymerase (Promega). Each PCR cycle included 1 min of denaturation
at 94 °C, 1 min of annealing at 65 °C, and 1 min of
extension/synthesis at 72 °C. After the appropriate number of PCR
cycles (MiniCycler, MJ Research Inc., Watertown, MA), 10 µl or less
of the reaction mix were fractionated on a 1% agarose gel. The gel was
soaked twice for 20 min in 1.5 M NaCl, 0.5 M
NaOH at room temperature and blotted onto a Hybond-N membrane (Amersham
International, Little Chalfort, UK) with transfer buffer containing 1.5 M NaCl, 0.25 M NaOH. The membrane was baked for 10 min at 80 °C, and the DNA was cross-linked to the membrane by
exposure to UV.
Since both genes yield an RT-PCR product that is identical in size, two positive and two negative controls were included in each set of reactions. A plasmid containing a genomic fragment from H-2Db was amplified with H-2Db primers and with H-2Kb primers, and a plasmid containing H-2Kb cDNA was amplified with H-2Kb primers and with H-2Db primers. Since the yield of the PCR product is proportional to the starting amount of the template, only under conditions in which PCR amplification proceeds exponentially at a constant efficiency (43, 44) is the PCR reaction quantitative. A titration curve for amplified cDNA from each cell line and for each gene was plotted to ensure that the number of amplification cycles was below the plateau level. Detailed analysis of the results is presented under "Results."
Quantitation of Radioactive SignalsDried radioactive gels or blots were exposed to a phosphoimager screen and analyzed with a phosphoimager (Fuji BAS1000, Tokyo, Japan). The data are presented as phosphostimulated luminescence (PSL) units. In some cases, following scanning densitometric analysis of the x-ray films was performed.
Due to post-transcriptional interference
with class I assembly and transport (32, 33), most of the
Ad12-transformed cell lines express very low levels of cell surface
class I antigens despite having near normal levels of class I mRNA.
Fig. 1 (A and B) substantiates
previous data from our laboratory (12, 32), demonstrating that nearly
all the Ad12-transformed cell lines express normal or elevated levels
of the miniature swine class I transgene (PD1) mRNA, and a normal
level or less than a 2-fold reduction in H-2 mRNA, as compared with
that in the normal cell lines as exemplified by M1. The level of class
I mRNA in E1Ad5-transformed cells is either normal or enhanced.
Full restoration of cell surface expression level and transport of PD1
was achieved by re-expression of peptide transporter molecules in the
Ad12-transformed cell line VAD12.79 (12) but the expression level of
H-2Db was only partially restored, and the expression level
of H-2Kb remained very low in these TAP-reconstituted and
in TAP+LMP-reconstituted cells (12, 34). In order to examine whether
the selective suppression of H-2Kb expression was mediated
by H-2Kb-specific gene regulatory elements, the mRNA
levels of the individual H-2 genes were analyzed in the transformed
cells. Since efficient detection and quantitation of H-2 mRNA by
Northern blot analysis requires a probe that does not distinguish H-2K
from H-2D mRNA, we utilized a quantitative RT-PCR assay in order to
determine the relative amounts of mRNA encoded by each of these
genes. A total of 5-30 PCR cycles were performed for each of the
cDNAs that were reverse-transcribed from total RNA. The PCR
products were fractionated on agarose gels, blotted, hybridized to an
H-2 probe, and the radioactive signal was quantitated, following
various exposure periods. Fig. 2A shows the
results of the analyses of H-2 cDNA in one of the transformed cell
lines, using specific primers for H-2Kb and
H-2Db. The primers were complementary to the same region in
the two genes, thus minimizing possible differences that could result from DNA positioning or conformation. Since both sets of primers gave a
band of the same size, the specificity of the PCR reaction was proved
by the generation of the expected bands, using H-2Kb and
H-2Db plasmid DNA templates (Fig. 2B). Analysis
of the data showed a linear response curve when the number of PCR
cycles ranged between 10 and 25. Similar response curves were obtained
for E1Ad5-transformed (A5O1 and A5O5), Ad12-transformed (VAD12.79 and
VAD12.42), and the normal cell line M1. Using the linear part of the
curves, we compared the mRNA levels for each of the genes in the
different cell lines. A representative display of the results is shown
in Fig. 2B. The ratios in the levels between
H-2Kb and H-2Db were determined, and the values
are presented in Table I. These analyses showed that in
Ad12-transformed, as well as in the normal and E1Ad5-transformed cell
lines the ratio between H-2Kb and H-2Db
transcripts is about 1, implying that the level of H-2D and H-2K mRNA is about the same. Thus, in Ad12-transformed cells
H-2Kb is not selectively suppressed at the transcriptional
level.
Steady state level of class I heavy chains
and 2m in normal and Ad-transformed cell lines.
Electrophoresis on a formaldehyde/formamide agarose gel was carried out
with 20 µg of total RNA from the "normal" cell line (M1),
Ad12-transformed cell lines (VAD12.79, VAD12.42, VAD12.36, VAD12.25, VAD12.20, VAD12.54, and
VAD12.43), and Ad5E1-transformed cell lines (A5O1 and A5O5). RNA was
transferred to a nylon membrane and hybridized with probes specific for
PD1, H-2,
2m, and actin (A). The blot was
stripped between hybridizations, as indicated under "Experimental
Procedures." The results of the normalized densitometric analyses are
summarized in B and C. D. U.,
densitometric units; H. C., heavy chain.
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Since a limiting amount of 2m could be a factor of major
importance in class I assembly and transport, we next determined the
levels of
2m mRNA in the transformed cells. Fig. 1
(A and C) shows that the steady state levels of
2m mRNA varied among the Ad12-transformed cell
lines. In VAD12.79 (the cell line that was transfected with TAP and LMP
genes and yet demonstrated inefficient transport of H-2 molecules), the
2m mRNA level was 2.5-fold lower than that in the
normal cell line M1 and 4-fold lower than that in the E1Ad5-transformed
cell lines. The reduced levels of
2m mRNA in
Ad12-transformed cells might limit
2m synthesis to a level whereby competition for this molecule by different class I heavy
chains results in inefficient assembly and transport of particular
class I complexes.
As we were not able to
immunoprecipitate significant amounts of H-2 molecules, even with
antibodies directed against the 3 domain or the
cytoplasmic tail of class I antigens (33, 34), the possibility that the
class I heavy chains and
2m transcripts were poorly
translated was considered. Polysome-associated RNA from normal and
Ad12-transformed cells was fractionated and hybridized with several
probes. The control probes were chosen to represent mRNAs of
1.5-kilobase mRNAs (class I heavy chain mRNA was compared with
actin mRNA), and of 0.5-kilobase mRNAs (
2m
mRNA was compared with histone mRNA). Fig. 3
shows a representative experiment in which hybridization signals
obtained with polysomal RNA from VAD12.79 and M1 cell lines were
compared. The distribution of PD1, H-2, actin, and histone mRNAs
was identical in the two cell lines. The maximal level of class I and
actin mRNA was in fractions 2-4, and that of histone was in
fractions 5 and 6. These results are in agreement with the mRNA
sizes of these genes. The distribution of
2m mRNA
showed a slight shift toward the lighter polysome fraction in VAD12.79
(fractions 3-5 in VAD12.79, compared with fractions 2-4 in M1). While
these results were consistent in the VAD12.79 cell line, we did not
detect a similar shift in other Ad12-transformed cell lines (data not
shown). Therefore we conclude that both class I heavy chains and
2m mRNA are efficiently translated in
Ad12-transformed cell lines.
Overexpression of
Since the intracellular concentration
of 2m might affect the efficiency of assembly of class I
complexes, we attempted to induce assembly and transport by
overexpression of
2m molecules. TAP-reconstituted
VAD12.79 cells were transfected with a vector expressing mouse
2m from an actin promoter. Several individual clones
that expressed levels of
2m identical to the normal cell line were grown, and the transport of PD1 and H-2 molecules
was analyzed by immunoprecipitation with antibodies directed against PD1 or against the H-2 heavy chains, respectively. Fig.
4 (A and B) shows two
representative pulse-chase experiments performed with two of the
transfected clones (clones 2 and 9). The transport rate of PD1 was
enhanced in both clones, as evidenced by the rate of acquisition of
Endo H resistance especially following the 60-min chase period. This
augmentation was not seen with H-2 molecules analyzed in parallel (data
not shown). These results were supported by data obtained with
TAP-reconstituted Ad12-transformed-cells infected with recombinant
vaccinia viruses expressing
2m, and with cell extracts
to which excess purified human
2m was added (data not
shown). In all three cases, an excess of
2m resulted in
augmented assembly and transport of PD1/
2m, but did not
affect the assembly of H-2/
2m. Thus, even in VAD12.79,
in which the
2m levels are comparable or exceed levels
in normal cells, it somehow was not accessible for assembly of H-2
class I complexes.
Turnover of
Since increased amounts of newly synthesized
2m in the Ad12-transformed cell line did not affect the
assembly and transport of H-2 molecules, we next focused on the fate of
the
2m molecules in these cells. The level and half-life
of the assembled and the total
2m was assayed in
pulse-chase experiments with the relevant antibodies. The level of PD1
in M1 cells is 3-4-fold higher than that of H-2Kb or
H-2Db (12), and its maturation rate and half-life resembles
that of H-2Kb (33). Consequently, the turnover rate of
PD1-associated
2m is representation of the mean turnover
rate of class I-bound
2m in the cells. As can be seen in
Fig. 5 (A and B), the half-life of
PD1-assembled
2m in the normal cell line was greater
then 7 h, the half-life of total
2m molecules in
the normal and in E1Ad5-transformed cell lines was less then 100 min,
but in VAD12.79 there was minimal turnover of the
2m
during this time period. Identical results to that observed in VAD12.79
were obtained with two additional Ad12-transformed cell lines (VAD12.42
and VAD12.36; data not shown). Since the ratio of the heavy chain to
the light chain did not change during the chase periods, we feel that
the rapid turnover rate of free
2m in the normal and in
E1Ad5-transformed cell lines represents the true estimation of
2m half-life and does not result from an exchange
between labeled and non-labeled
2m molecules. Thus, our
data clearly demonstrate that both assembly of class
I/
2m (Refs. 12 and 33; see Fig. 7, B1) and
secretion or degradation of free
2m were both abrogated
in Ad12-transformed cells.
In order to determine whether poor assembly and the factors retarding
2m turnover are related, we determined the half-life of
total
2m in TAP-reconstituted VAD12.79 cells, which can
assemble and transport PD1 molecules (12, 34). Fig. 5 (A and
C) shows that in these cells, the half-life of total
2m was comparable with that of PD1-assembled
2m, and significantly longer than that of total
2m in the normal cell line. Pretreatment of
Ad12-transformed cells with interferons, which reconstitutes almost
completely the assembly and transport of both the PD1 and H-2 molecules
(34), enhanced the turnover of
2m molecules but did not
fully restore their normal turnover rate (Fig. 6,
A and B). These results imply that in
Ad12-transformed cells there is a mechanism that acts to retain
2m in the cells and that this retention is only
partially alleviated by reconstituting the assembly of class I
complexes.
In order to localize the
compartment where 2m is retained in Ad12-transformed
cells, we separated cell lysates into two fractions: one enriched for
soluble proteins, and the other for cell membranes. The latter fraction
was solubilized by detergent treatment, and both fractions were
immunoprecipitated with antibodies directed against
2m.
The results of two experiments are depicted in Fig. 7.
In the first (A), the cells were pulsed for 30 min in order to be able to detect molecules with short and long half-lives. In the
second (B), the cells were labeled for 15 h to enrich
for molecules that have a long half-life. In order to directly
determine which fraction was enriched with heavy chain-associated
2m molecules, we immunoprecipitated both total
(B2) and PD1-assembled
2m (B1). The immunoprecipitates were reduced or left untreated before
fractionation by polyacrylamide gel electrophoresis. The data were
quantitated and are summarized in Table II. The results
of both experiments show that the membrane fraction of the two cell
lines was enriched for
2m. Whereas in M1,
2m molecules are associated with class I heavy chains
and, therefore, were expected to reside in the membrane fraction, the
2m-enriched membrane fraction of VAD12.79 implies that
in these cells the
2m molecules are associated with another membrane component. Experiment 2 shows that PD1 resides within
the membrane fraction of M1 (B1, compare lanes 1 and 3), but, as expected, was absent from extracts of
VAD12.79 (B1, lanes 2 and 4). These
results confirmed previous data that there was no assembly of heavy
chain/
2m in VAD12.79 cells and assured us that there was
no contamination of the soluble fraction proteins with membrane-bound
proteins. The signal obtained for the PD1-associated
2m
in M1 was comparable with the signal obtained for total
2m in the membrane fraction of the same cells
(B1, lane 3 compared with B2,
lane 3). This result indicates that most of the
2m residing in the membrane fraction of M1 cells is
bound to class I heavy chains. Unlike M1 in which all membrane-bound
2m can be accounted for by class I heavy chain
associations, in VAD12.79 some other membrane component must be playing
this role. Moreover, the association of this membrane component with
2m is more stable than the
2m-association with class I heavy chains, as evidenced by the fact that after 15 h of labeling the amount of both soluble (B2, lanes
1 and 2) and membrane
2m (B2,
lanes 3 and 4), in VAD12.79 cells was 3-fold higher than in the M1 cells, whereas after a short pulse these signals
in the membrane fraction were almost identical in both cell lines
(A, lanes 3 and 4). The binding
between this yet to be identified membrane component and
2m is not via covalent disulfide bonds, since the same
results were obtained whether or not the immunoprecipitates were
reduced before fractionation (Fig. 7).
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The fact that VAD12.79 cells appear to posses a unique membrane
2m-binding component was further substantiated by the
finding that in the soluble fraction of the M1 cells, partial
degradation of
2m molecules was evident (B2,
lanes 1), while no such degradation was detected in the
soluble fraction of VAD12.79 cells (B2, lanes 2),
despite the fact that the
2m signal in these cells was
3.5-fold stronger. Degradation products of
2m molecules
were also detected in immunoprecipitates of total cell extracts from M1
cells, but not from VAD12.79 cells (B2, lanes 5 and 6, respectively).
The selective suppression in transport and cell surface expression
of H-2 molecules in Ad12-transformed cells that were reconstituted for
the expression of peptide transporter and LMP genes implied that there
was an additional factor(s) involved in the assembly and transport of
the class I complex. The following possibilities were considered:
(a) differences in the steady state level of H-2Kb and H-2Db mRNA, leading to
differences in the magnitude of their expression; (b)
differences in the translation efficiency of class I transcripts, resulting in differences in the amounts of proteins produced; (c) mutations affecting the conformation of the endogenous
class I molecules and, thus preventing their transport through cell organelles; and (d) competition among class I heavy chains
for limited amounts of 2m or inaccessible
2m molecules, leading to selective enrichment in
particular class I complexes.
Our data show that equal amounts of H-2Kb and
H-2Db genes were transcribed (Fig. 2). We also found that
PD1 and H-2 mRNAs were translated efficiently (Fig. 3). In one
Ad12-transformed cell line (VAD12.79), we observed some inhibition in
the translation of 2m mRNA as compared with that in
the normal cell line M1. However, even in this cell line,
2m mRNA was associated with the polysomal fraction
and the slight shift of its mRNA toward association with lighter
polysomes was not detected in other Ad12-transformed cell lines. Thus,
aberrant transcription or translation of the class I heavy chains or of
2m molecules cannot account for the low level of
assembled and transported H-2 molecules in the TAP and
LMP-reconstituted Ad12-transformed cells.
In order to determine whether inefficient assembly of specific class I
heavy chains resulted from mutations in the endogenous genes which
affect their conformation, we previously overexpressed either H-2 or
2m molecules via infection with recombinant vaccinia viruses and analyzed their assembly and transport (34). The results
were comparable with those obtained with the endogenous molecules,
i.e. the transport of H-2 molecules was inefficient, while
the transport of PD1 was completely restored. In the present study we
also found that TAP-reconstituted VAD12.79 cells stably transfected
with a
2m gene show enhanced assembly and transport of
PD1, but not of H-2 molecules (Fig. 4).
Collectively, the data suggested that one of the components
(2m or peptides) of the class I complex was not
available for assembly and led us to compare in greater detail the fate
of
2m in the normal versus the transformed
cells. Based on our previous immunoprecipitation data and on other
studies (45, 46, 47, 48, 49), it seemed likely that in normal cells most of the
2m molecules would be associated with class I heavy
chains and free molecules would either be degraded or secreted. Several
studies have shown that free
2m is secreted from cells
and that its secretion can be enhanced by various cytokines, growth
factors, and growth promoters (47, 48, 49). Dragmont et al. (45)
demonstrated that free
2m was secreted into the culture
media of cell lines independent of cell surface expression of class I
heavy chains.
2m has also been shown to be secreted from
endometrial cells (46), leukemic B cells (47), fibroblasts (48), and
hepatocytes (49). Much less is known about the degradation pathway of
free
2m within the cells. Since in Ad12-transformed
cells there is no assembly with class I heavy chains, it seemed likely
that most of the
2m would be secreted from the cells or
rapidly degraded. However, surprisingly, unlike normal and
E1Ad5-transformed cells in which free
2m was either
secreted or degraded, and class I-assembled
2m was
transported to the cell membrane, in Ad12-transformed cells, free
2m was retained in the cells (Fig. 5). Furthermore, we
found that most of the
2m molecules in the transformed
cells resided in the membrane-enriched fraction (Fig. 7) and were more efficiently retained, following an overnight labeling period, than in
the normal cell line. These data suggest that in the absence of
detectable assembly with class I heavy chains,
2m
molecules could bind other membrane-associated molecules, resulting in
the retention of these molecules within the cell. We cannot completely rule out the possibility of the existence of denatured class I heavy
chains in Ad12-transformed cells that have lost all epitopes recognized
by the antibodies used for immunoprecipitations and still have the
potential to bind
2m efficiently and to be retained in a
stable form in the ER. Nevertheless, this possibility seems highly
unlikely in view of the fact that none of the panel of antibodies
directed against epitopes on the
3 domain of
H-2Db (33), H-2Dd (34), PD1 (33, 34), and
antisera against peptide 8 of the H-2Kb molecules (data not
shown) detected significant assembly of the relevant heavy chain with
2m.
Since it has been documented that -interferon modulates the
secretion of
2m (47, 49) and the same cytokine also
reconstitutes the transport of H-2 molecules in Ad12-transformed cells
(Ref. 34, and data not shown), we pretreated the transformed cells with
interferons and determined the half-life of
2m. Indeed, interferon treatment significantly shortened the half-life of
2m, but it was still longer than in normal or
E1Ad5-transformed cells. Thus, we can conclude that in Ad12-transformed
cells, most
2m is retained in the cells via a
membrane-bound component and that it can be partially released by
assembly with a class I heavy chain/peptide or by interferon
treatment.
2m is known to bind at least two other non-MHC molecules
that have low significant homology with class I heavy chains: the neonatal Fc receptor (50, 51) and the CD1 class I-like molecule (51,
52). Neonatal Fc receptors mediate transfer of maternal IgG to the
newborn, providing the neonate with humoral immunity before the
development of a fully functional immune system. The neonatal Fc
receptor shows similarity to the class I heavy chains in the
organization of domains and their sequence, especially in the
3 domain (53), a fact that explains its binding to
2m molecules. On the other hand, the non-MHC-encoded CD1
family of molecules present antigens entirely distinct from MHC class
I, class II, or related molecules (51). The molecules are abundantly expressed on professional antigen-presenting cells and are associated with
2m, and their expression and function is
independent of the expression of peptide-transporter molecules (52).
Thus, it is clear that
2m can bind class I-related
proteins with apparently diverse functions. It is tempting to speculate
that
2m can function as a scaffold for multiple proteins
and promote their correct folding, as suggested by Solheim et
al. (54) for class I heavy chains. In the latter case
2m would participate in class I transport not only by
stabilization of the class I heavy chains, but also by induction of a
heavy chain conformation that enables it to transit to the cell
surface. The same group also demonstrated (55) that
2m
was associated with TAP in a class I negative cell line, supporting the
hypothesis that
2m can bind to multiple proteins within
the cell, either directly or via another molecule(s).
Since 2m could function to stabilize and maintain a
variety of proteins, the constitutive secretion of
2m by
a variety of cells may have an important physiological role. Indeed,
free
2m has been shown to have some unexpected
biological activities.
2m produced by synovial
fibroblasts stimulated by phorbol esters induced collagenase synthesis
in these cells (56).
2m also increased the number of
insulin-like growth factor I transcripts and polypeptides, as well as
their receptors, on cultured bone marrow cells (57). These data
suggested that
2m could be involved in the modulation of
connective tissue breakdown and bone remodeling.
2m
might also play a role in the migration of hematopoietic cell precursors from their site of emergence, the bone marrow, to their site
of differentiation, the thymus, since it is produced by a thymic
epithelial cell line and is able to induce oriented migration of
immature lymphoid bone marrow cells in vitro (58). Cytokines and growth factors that were reported to enhance
2m
secretion possibly could act via two mechanisms: enhancement of
2m transcription and induction of
2m
release from specific cellular proteins that might be similar to the
2m-binding proteins described in this paper.
Another system that is similar in several aspects to that described
above is the association of surrogate immunoglobulin light chains with
glycoproteins in cells that do not express the immunoglobulin heavy
chain (59). The complex of µ heavy chain/surrogate light chain is
expressed on pre-B cell lines and has been found to transmit biochemical signals to the cells (60, 61). However, the surrogate light
chain can be expressed on the cell surface in the absence of rearranged
heavy chain. Several proteins were suggested as potential candidates
for binding non-covalently the surrogate light chain and carrying it to
the surface of pre-B and pro-B cell lines (59). Unlike the surrogate
light chains, some classical immunoglobulin light chains, in the
absence of heavy chains, are retained and degraded within the ER (62).
The half-life of these molecules depends on their binding and
dissociation from the chaperone BiP. Our data suggest that, like the
surrogate light chains, 2m molecules can bind other
molecules in the absence of efficient assembly with class I heavy
chains. However, such molecules either have long half-life (>5 h)
and/or they are not transported to the cell surface and remain
associated with
2m molecules in the cells. Whether these
2m-binding molecules are expressed in normal cells, or
are known chaperons such as BiP or calnexin that bind misfolded
proteins, or are novel proteins, which are overexpressed in
Ad12-transformed cells, remains to be determined. The fact that in
Ad12-transformed cells the membrane fraction is enriched with
2m suggests that at least one binding protein is a
membrane-bound protein such as calnexin. However, calnexin is commonly
thought to be a chaperone that binds mostly glycoproteins (63),
although a different mechanism for its association with class I heavy
chains and class II invariant chains has been suggested (64, 65). Both
of these molecules associated with calnexin even when their glycosylation was inhibited, and in the latter case, calnexin association retained the invariant chain in the ER and prevented its
degradation. Thus, it is conceivable that
2m binds
calnexin or another molecule via a mechanism similar to that involved
in class II invariant chain association.
If Ad12-transformed cells overexpress a 2m-binding
protein, it might interfere with the assembly of class I heavy
chain/
2m/peptide. The ability of this protein to
successfully compete with class I heavy chains for
2m
binding could explain our inability to fully restore the assembly of
the H-2 complex in TAP+LMP-reconstituted cells. Thus, Ad12-transformed
cells appear to acquire a novel membrane-associated mechanism for
sequestering
2m. This mechanism may be unique to these
cells or may be induced in cells that do not express fully conformed
class I complexes.
We are grateful to Dr. John E. Coligan (NIAID, National Institutes of Health) for the critical reviewing of the manuscript.