(Received for publication, June 6, 1994; and in revised form, October 20, 1994)
From the
LMP2 is one of the two proteasome subunits encoded by genes in
the major histocompatibility complex class II region. Here we report
the genomic organization of human LMP2 gene. Sequence analysis of
polymerase chain reaction-amplified cDNA from a number of
lymphoblastoid cell lines demonstrated two forms of LMP2 mRNA, one
(LMP2.l) complete and homologous to the published LMP2 genomic sequence
from cosmid clones, and the other (LMP2.s) a smaller transcript
resulting from splicing of a 30-base pair fragment from the first exon.
Antibodies to recombinant LMP2.s protein (22.3 kDa) were raised in
rabbits. This anti-LMP2.s serum recognized both recombinant proteins
(LMP2.l = 23.3 kDa and LMP2.s = 22.3 kDa) and a single
protein of 21.5 kDa molecular mass in lysates from human lymphoblastoid
cell lines. Pulse-chase experiments demonstrated that LMP2 polypeptide
also undergoes processing from 22.3- to 21.5-kDa protein when
incorporated into proteasomes. These data suggest that the processing
of human LMP2 subunit takes place both at the transcription and
post-translational levels. Northern blot analysis showed that the LMP2
mRNA is expressed in lymphoblastoid cell lines and in fibroblasts
following -interferon induction, but not in brain, smooth muscle,
fibroblasts (uninduced), and colon epithelial cells.
Cytotoxic T lymphocytes recognize peptides derived from
intracellular antigens presented at the cell surface in association
with MHC class I molecules. Before peptide presentation by
class I molecules, cytoplasmic antigenic proteins must be degraded in
cytosol and the peptides derived from them transported into endoplasmic
reticulum (Townsend and Bodmer, 1989; Monaco, 1992). Furthermore, it
has been demonstrated that bound peptide is important for the
stability, conformation, and cell-surface expression of class I
molecules (Townsend et al., 1989; Spies and DeMars, 1991;
Powis et al., 1991; Attaya et al., 1992). Four
tightly linked genes involved in generation of peptides and their
transport to the assembly site have been mapped within the MHC class II
region. Of these, two genes (TAP1 and TAP2) related
to the superfamily of the ATP-binding cassette transporters have been
identified in the class II region of human, rat, and mouse (Deverson et al., 1990; Monaco et al., 1990; Spies et
al., 1990; Trowsdale et al., 1990). Transfection of
mutant class I-deficient cell lines with TAP1 and TAP2 cDNA restored
the cell surface expression of class I molecules (Spies and DeMars,
1991; Powis et al., 1991; Attaya et al., 1992).
Two genes, LMP2 and LMP7, are tightly linked to
TAP genes and encode two of the subunits of a large cytoplasmic
structure called the LMP complex (Monaco and McDevitt, 1986; Martinez
and Monaco, 1991; Glynne et al., 1991; Kelly et al.,
1991). The predicted amino acid sequences of gene products of these
genes, mapping of these genes to the region of the MHC that encodes
genes for antigen processing, and the regulation of the LMP complex by
-IFN suggest that the MHC-encoded proteasome chains play some
distinctive role in protein degradation for antigen processing (Monaco
and McDevitt, 1986). Two reports, however, showed that mutant cell
lines with defective class I expression and lacking genes for both TAP
and LMP could be restored to normal functional expression of class I
molecules by transfection of TAP genes in the absence of LMP (Arnold et al., 1992; Momburg et al., 1992). Some recent
studies bring the LMP2 and LMP7 chains back into consideration by
demonstrating that the lack or presence of LMPs was associated with
changes in cleavage specificity of the proteasomes (Brown et
al., 1993; Driscoll et al., 1993; Gaczynska et
al., 1993). Indeed, it has been shown that the substrate
specificity of LMP2
7
proteasomes
differs from that of LMP2
7
proteasomes (Driscoll et al., 1993). In addition, it has
been demonstrated that the LMP2-deficient mice express normal or near
normal levels of class I molecules, but generate 5-6-fold fewer
cytotoxic T lymphocytes in response to influenza infection (quoted by
Brown et al., 1993).
The genomic structures of human LMP2 and LMP7 genes have recently been reported (Martinez and Monaco, 1991; Kelly et al., 1991; Früh et al., 1992). Western analysis and pulse-chase experiments have suggested that the polypeptide encoded by LMP2 gene undergoes proteolytic processing when incorporated into proteasome. In the present study, we report a second form of human LMP2 mRNA, which results from splicing of a 30-bp fragment from the first exon during transcription. In addition, the data suggest proteolytic processing of the LMP2 polypeptide at the post-translational level when incorporated into proteasome.
PCR-amplified DNA was ligated into Bluescript KSII vector. DH
competent cells (Life Technologies, Inc.) were
transformed. DNA extracted from at least two colonies in each case was
sequenced by the dideoxy chain termination method (Sanger and Coulson,
1975).
Figure 1: Electrophoretic analysis of PCR-amplified LMP2 cDNA from human lymphoblastoid cell lines. Lane 1 = JESTHOM, lane 2 = DEU, and lane 3 = unrelated subject JW.
Sequence analyses of these two LMP2 cDNAs from two different clones of a cell line JESTHOM are shown in Fig. 2. For comparison, it also shows nucleotide sequences (row 1) of LMP2 gene as described previously (Kelly et al., 1991; Früh et al., 1992). It is evident that two sets of LMP2 sequences were observed from two different clones from this cell line. Of these, one sequence (LMP2.l) showed identity with the published sequence (row 2), whereas the other one (LMP2.s) had a deletion of 30 bp (from 16 to 45 bp) in the first exon (row 3). Since nucleotide sequence CGGGCGGGA (position 7-15) is repeated again (position 37-45), it is likely that splicing takes place from bp 7 to 36 bp. Similarly, two sets of sequences for LMP2 cDNA (LMP2.l and LMP2.s) were observed in cell line DEU and each of the six lymphoblastoid cell lines from unrelated subjects.
Figure 2: Nucleotide sequences of LMP2. Row 1 = LMP2 gene sequence obtained from Früh et al.(1992); rows 2 and 3 = LMP2 sequences from two different clones obtained following PCR amplification of cDNA from a lymphoblastoid cell line JESTHOM. Dashes indicate identity to respective nucleotide in row 1, and the shaded area indicates deletion.
Figure 3:
Dot-blot analysis of PCR-amplified genomic
DNA and cDNA with sequence-specific oligonucleotide probes. Samples of
DNA were separately amplified with primers PR1 and PR3 and hybridized
with SSO probes; rows A and C = SSO-1, rows B and D = SSO-2. DNA and cDNA samples
were obtained from lymphoblastoid cell lines (1-4),
fibroblasts (5 and 6), smooth muscle (7),
colon epithelial cells (8), brain (9), and
-IFN-treated fibroblasts (10).
Dot-blot analysis of PCR-amplified genomic DNA from fibroblasts (5 and 6), smooth muscle (7), colon epithelial cells (8), and brain (9) also showed only one, i.e. LMP2.l, sequence (Fig. 3). No amplification, either for exon 1 or for the entire LMP2 sequence, was observed with cDNA from these tissues.
Figure 4:
Northern blot analysis of total RNA from
lymphoblastoid cell lines (1-4), -IFN-induced
fibroblasts (5), uninduced fibroblasts (6), smooth
muscle (7), colon epithelial cells (8), and brain (9) with human LMP2 cDNA probe (lower panel) or with
-actin cDNA probe (upper
panel).
Figure 5:
Western analysis of LMP2 subunit with
rabbit anti-LMP2.s serum raised in the present study (lanes
1-4, and 8), and with anti-LMP serum obtained from
Patel et al. (lanes 5, 6, and 7). Lanes 1 and 5 = recombinant LMP2.l protein generated from
LMP2.l mRNA, lanes 2 and 6 = recombinant
LMP2.s protein generated from spliced LMP2.s mRNA, lane 3 = lysate from a lymphoblastoid cell line, VAVY, lanes 4 and 7 = lysate from a lymphoblastoid cell line,
SLE005, and lane 8 = lysate from -IFN-treated
fibroblasts.
To identify the possible precursor of LMP2, we performed the pulse-chase experiments prior to immunoprecipitation with anti-LMP2.s antiserum. Cells from three homozygous typing cell lines (BM14, SLE005, and JESTHOM) were labeled for 50 min followed by 0-, 60-, and 120-min chase. Fig. 6shows the results from one such experiment. It can be seen that the anti-LMP2.s serum precipitated a 22.3-kDa protein (lane 1), whose intensity decreased during the chase period, and a protein of molecular mass of 21.5 kDa appeared (lanes 2 and 3) after 60- and 120-min chase. This shows that the low molecular mass form of LMP2 is derived from the precursor of 22.3 kDa, the product of the smaller transcript, i.e. LMP2.s mRNA.
Figure 6:
Pulse-chase analysis of LMP2.
Lymphoblastoid (SLE005) cells (lanes 1-3) and
-IFN-treated fibroblasts (lanes 4 and 5) were
metabolically labeled with [
S]methionine for 50
min followed by a 0-min (lanes 1 and 4), 60-min (lane 2), and 120-min (lanes 3 and 5) chase
with unlabeled methionine. After cell lysis, proteins were
immunoprecipitated with anti-LMP2.s serum and analyzed by
SDS-PAGE.
To elucidate the effect of -IFN on processing of LMP2 protein,
pulse-chase experiments were carried out with
-IFN-treated
fibroblasts. The results show that the anti-LMP2.s serum precipitated a
22.3-kDa protein (Fig. 6, lane 4). The intensity of
this band decreased during the chase period and a protein of 21.5 kDa
appeared after 120-min chase (lane 5). These data suggest that
the processing of protein in
-IFN-treated fibroblasts was similar
to that in untreated lymphoblastoid cell lines.
In the present article, we have analyzed the genomic organization of human proteasome LMP2 gene by sequencing the PCR-amplified cDNA from a number of human EBV-transformed lymphoblastoid cell lines. Nucleotide sequence analysis showed the presence of two transcripts; one homologous (LMP2.l) to the LMP2 sequence published earlier from the cosmid clone, U15 (Martinez and Monaco, 1991; Kelly et al., 1991; Früh et al., 1992), and the other representing a smaller transcript (LMP2.s). It is likely that this new form of LMP2 mRNA arises following deletion of a 30-bp fragment from the first exon.
Northern blot
analysis of lymphoblastoid cell lines and various human tissues
revealed differences in LMP2 mRNA levels in different tissues. The
results in the present study therefore confirm and extend the earlier
data in that LMP2 mRNA is abundant in lymphoblastoid cell lines, but no
detectable amounts are present in brain, smooth muscle, fibroblasts,
and colon epithelial cells (Früh et al.,
1992; Frentzel et al., 1993). Treatment of fibroblasts with
-IFN led to an increase in both LMP2.l and LMP2.s mRNA levels.
Pulse-chase experiment showed that the precursor protein in
-IFN-treated fibroblasts, as in lymphoblastoid cells, is the
product of LMP2.s transcript. In addition, processing of the precursor
protein in
-IFN-treated fibroblasts is similar to that in
lymphoblastoid cell lines, suggesting that
-IFN up-regulates
levels of LMP2 mRNA, but does not affect post-translational regulation
of LMP2 protein.
Cloning and expression of recombinant proteins to
the two forms of LMP2 mRNA resulted in proteins of expected sizes (23.3
and 22.3 kDa). Anti-LMP2.s sera raised against the 22.3-kDa recombinant
protein recognized a single protein of 21.5 kDa in cell lysates from a
number of human lymphoblastoid cell lines and from -IFN-treated
fibroblasts by the Western analysis. Reactivity of this antiserum with
T1 and mutant T2 cell lines, and comparison of results with anti-LMP2
serum from Dr. McDevitt (Patel et al., 1994), suggest a high
degree of specificity of the anti-LMP2.s serum raised in the present
study.
Investigations of the LMP2 subunits in cell lysates have been carried out utilizing anti-LMP2 and anti-proteasome antibodies (Früh et al., 1992; Frentzel et al., 1993, Patel et al., 1994). These studies demonstrated that rabbit anti-LMP2 antibodies recognized a single protein of approximately 21 kDa in mouse RMA cells (Früh et al., 1992; Frentzel et al., 1993). The rabbit anti-human LMP2.s serum in the present study also recognizes a single protein of approximately the same molecular mass in cell lysates from human lymphoblastoid cell lines.
Pulse-chase experiments have been performed to identify the possible precursors of LMP2 proteins (Früh et al., 1992; Frentzel et al., 1993). The results from these experiments suggest that the polypeptide encoded by LMP2 undergoes proteolytic processing from a protein of molecular mass of 23.3 kDa to a 21.5-kDa protein when incorporated into proteasomes. These experiments were, however, carried out with mouse RMA cells utilizing rabbit antiserum directed against mouse LMP2 (Frentzel et al., 1993) or human LMP2 protein (Früh et al., 1992). In the present study, we utilized rabbit anti-human LMP2.s serum to recognize LMP2 subunits in human lymphoblastoid cell lines. This antibody recognizes in cell lysates a single protein of a lower (21.5 kDa) molecular mass. Pulse-chase experiments demonstrated that this lower molecular mass protein (21.5 kDa) is derived from precursor protein of 22.3 kDa, the product of smaller transcript of LMP2, i.e. LMP2.s. The results in the present study, therefore, demonstrate that processing of LMP2 takes place both at the transcription and post-translational levels. These data suggest different mechanisms for processing of LMP2 subunits in mice and humans; LMP2 undergoes proteolytic processing when incorporated in proteasomes in mice, whereas in humans the processing of LMP2 takes place both at the transcription and post-translational levels.
Patel et al.(1994) utilized rabbit anti-LMP2 (LMP2) antibody
and a monoclonal anti-proteasome (mAb2-17) antibody and
demonstrated the existence of two forms of LMP2, LMP2a, and LMP2b.
Rabbit anti-LMP2 antibody recognized only LMP2a subunit, whereas
mAb2-17 immunoprecipitated both subunits. They suggested that
LMP2a is probably the precursor of mature LMP2 subunit, since anti-LMP2
antibody recognized protein of the expected (23.3 kDa) molecular mass.
However in the pulse-chase, the complex was still present after a 6-h
chase (Patel et al., 1994). In our laboratory, this antibody
recognized LMP2 polypeptide of 21.5 kDa, suggesting that this protein
is the processed LMP2 subunit. The reasons for differences in these two
investigations are not clear, however.
LMP gene products play an important role in processing antigens for class I-restricted presentation. It is likely that a distortion in the novel processing mechanisms of LMP2 described in the present report, i.e. splicing at the transcriptional level followed by processing at the post-translational level, for example by pathogens, will result in an aberrant immune response and may contribute to the development of autoimmune diseases. Furthermore, these data will facilitate to investigate if the MHC-associated diseases are caused by defective structural (sequence) and/or regulatory (processing) components of LMP2 gene (Früh et al., 1992).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) UO1025.
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