From the Department of Immunology, Osaka Medical
Center for Cancer and Cardiovascular Diseases, Higashinari-ku,
Osaka 537 and the § Department of Molecular Immunology,
Nara Institute of Science and Technology, Ikoma,
Nara 631-0101, Japan
Received for publication, December 26, 2000, and in revised form, March 5, 2001
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ABSTRACT |
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LITAF and PIG7
encode an identical protein, and they have recently been reported
as lipopolysaccharide and p53-inducible genes, respectively. By using
the differential display approach, we identified a Mycobacterium
bovis BCG cell wall skeleton
(BCG-CWS)-inducible gene fragment from human monocytes, showing no
homology to any reported gene. Full-length cloning of this fragment
reveals the following. 1) The differential display product represents
the incomplete 3'-untranslated region of LITAF/PIG7. 2) The coding region of the transcript differs from LITAF/PIG7 due to an absence of a
single guanine residue, resulting in a potential translational frameshift. 3) The newly coded protein turns out to be 86% identical and 90% similar to an estrogen-inducible rat gene, EET-1.
Repeated analysis, expressed sequence tag search, comparison with
homologues, and genome sequence analysis confirmed the absence of the
single guanine residue. One interesting feature of this protein is that it possesses the RING domain signature and is predicted to be localized
in the nucleus. However, detailed analysis together with
experimental evidence suggests it is neither a RING family member nor a
nuclear protein. Comparison of a total collection of 18 proteins from
various species indicates that proteins of this family are small in
size and mainly conserved at the C-terminal domain with a unique motif.
We characterize this novel protein as an unglycosylated
small integral membrane
protein of the lysosome/late endosome (SIMPLE) whose expression is elicited in monocytes
by live and heat-killed BCG, BCG cell wall complex, lipopolysaccharide, and tumor necrosis factor- Fighting off microbial infection has been one of the oldest wars
waged by man. In order to develop more potent vaccines and therapeutic
agents, it has become essential to understand the mechanism of
pathogenesis at the point of host-microbial interaction. Despite the
identification of a number of host genes involved in protective
responses and those exploited by the microbe for pathogenesis, the
evolving nature of pathogens and ranges of their variant and invariant
components offer a virtually unlimited challenge. We focused on a
microbial component derived from the cell wall complex of
Mycobacterium bovis Bacillus Calmette-Guerin
(BCG),1 a non-pathogenic
vaccine strain of tuberculosis. The cell wall complex, which we call
BCG-CWS, is a highly purified fraction with immunotherapeutic potential
(1, 2) and is mainly composed of peptidoglycan, arabinogalactan, and
mycolic acid. The conserved microbial structures that are relatively
invariant within a class of microorganisms such as LPS, peptidoglycan,
lipoteichoic acid, and lipopeptides are recognized by Toll-like
receptors (TLR) leading to the induction of immune and inflammatory
genes (3, 4). It has been reported recently from our laboratory that
BCG-CWS is recognized by both TLR2 and TLR4, and it effectively induces dendritic cell (DC) maturation (5) through TNF- Preparation and Treatment of Monocytes with BCG-CWS and
BCG--
Peripheral blood mononuclear cells were isolated by standard
density centrifugation with Ficoll-Paque (Amersham Pharmacia Biotech)
from 400 ml of citrate phosphate dextrose-supplemented human
blood. CD14+ monocytes were separated from peripheral blood mononuclear
cells by anti-CD14-coated microbeads and a magnetic cell sorting column
(Miltenyi Biotec GmBH). Cells were cultured overnight in 10-cm dishes
in the presence of RPMI supplemented with 10% FBS and 2% human AB
serum. The next day cells were treated for 8 h with 15 µg/ml
BCG-CWS prepared in emulsion buffer (PBS containing 1% Drakeol
6VR and 1% Tween 80). Cells in the control plates were treated
with 15 µl/ml emulsion buffer for the same time. For heat-killed and
live BCG treatment M. bovis BCG Tokyo strain has been used
at a concentration of 1 bacillus/monocyte for 8 h. In some
experiments immature dendritic cells (iDC) have been used to stimulate
with BCG-CWS, and the preparation of iDC was essentially the same as
described previously (5).
Differential Display RT-PCR--
Total RNAs from
BCG-CWS-stimulated and unstimulated human monocytes were isolated using
TRIZOL (Life Technologies, Inc.) reagent according to the
manufacturer's instructions. Two micrograms of total RNA from control
and from stimulated cells were reverse-transcribed using Superscript RT
(Life Technologies, Inc.) by 12 types of T12MN (M = A/C/G; N = A/T/C/G) primers. Arbitrary primer (AP) sets were selected from
RNAmap Kit I and II (Gene Hunter Corp., Nashville, TN), and PCR
amplification was performed using Takara PCR kit components (Tokyo,
Japan) and [32P]dCTP (Daiichi Pure Chemicals, Tokyo,
Japan). PCR conditions, gel run parameters, fragment elution, and
further amplification of PCR products were described previously (15).
Fragments extracted from the gel were sequenced after TA cloning
(Invitrogen, Carlsbad, CA) in an ABI-373 Sequencer (PE Applied
Biosystems, Foster City, CA) using Dye terminator ABI sequencing Kit
(PE Applied Biosystems).
Cell Cultures--
HeLa, COS-7, RK13, DLD-1, THP-1, and RAW264.7
cells were obtained from the Japanese Cell Resource Bank (Osaka,
Japan). DLD-1 and THP-1 cells were maintained in RPMI1640 medium
supplemented with 10% heat-inactivated FBS. HeLa, COS-7, RK13 and
RAW264.7 cells were maintained in Dulbecco's modified Eagle's medium
(Life Technologies, Inc.) supplemented with 10% heat-inactivated
FBS.
Northern Blot Analysis--
Total RNAs from unstimulated and
stimulated monocytes and other cells were isolated, and at least 10 µg of total RNA/lane was loaded on 1% formamide gel. RNA was
transferred onto Hybond N+ membranes (Amersham Pharmacia Biotech);
membranes were prehybridized for 30 min and hybridized for 1 h at
65 °C in rapid hybridization buffer (Qiagen, Valencia, CA) in the
presence of appropriate 32P-labeled cDNA probe (SIMPLE
ORF, GCAP2, PCR-amplified human and murine TNF- Purification of Escherichia coli-expressed SIMPLE and
Immunization--
The open reading frame of SIMPLE cDNA was
amplified by 5'-gga tcc atg tcg gtt cca gga cct ta-3' with
BamHI site (bold) and 5'-aag ctt cta caa acg ctt
gta ggt gc-3' with HindIII site (bold) and ligated into the
E. coli expression vector pQE-30 (Qiagen) in frame with
N-terminal His tag and used to transform competent E. coli
M15[pREP4] cells (Qiagen). N-terminal (His)6-tagged SIMPLE was purified under denaturing condition by
nickel-nitrilotriacetic acid-agarose (Qiagen) chromatography according
to the manufacturer's instructions. Polyclonal antibody against
purified SIMPLE was generated by immunizing a rabbit following standard methods.
Constructs and Transfection--
The coding region of SIMPLE
(234-719 bp) was cloned into two mammalian expression vectors pEGFP-C1
(CLONTECH) and pcDNA3 (Invitrogen), respectively. The primer combinations used for cloning into
XhoI and HindIII sites of pEGFP-C1 were
5'-ctc gag cca cca tgt cgg ttc cag gac ctt a-3' and
5'-aag ctt cta caa acg ctt gta ggt gc-3'. Primers containing
BamHI and EcoRI sites, 5'-gga tcc cca
cca tgt cgg ttc cag gac ctt a-3' and 5'-gaa ttc cta caa acg
ctt gta ggt gc-3', respectively, were used for cloning into pcDNA3.
All DNA constructs were checked by sequencing and transfected into
various mammalian cell lines (HeLa, COS-7, and RK13) with Lipofectin
reagent (Life Technologies, Inc.).
Immunoblotting--
For detection of SIMPLE protein,
SIMPLE-transfected RK13 cells, human monocytes, HeLa, THP-1 and DLD-1
cells were lysed in cell lysis buffer containing 1% Nonidet P-40, 10 mM EDTA, 140 mM NaCl, 20 mM
Tris-HCl, pH 7.4, 1.0 mM phenylmethylsulfonyl fluoride, and
5 mM iodoacetamide. The cell lysates were solubilized
either in reducing or non-reducing sample buffer and resolved in a
12.5% SDS-PAGE and transferred onto polyvinylidene difluoride
membranes (Millipore, Bedford, MA). The blots were blocked with 10%
non-fat milk and treated with rabbit anti-SIMPLE polyclonal antibody at a dilution of 1:5000. After washing, the blots were incubated with
horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) and
developed with ECL (Amersham Pharmacia Biotech).
Subcellular Fractionation--
THP-1 cells were harvested by
centrifugation at 1000 × g and then washed 3 times
with PBS. Cell pellets were washed once with hypotonic buffer (10 mM KCl, 20 mM Hepes, pH 7.4, 1.5 mM
MgCl2, 0.1 mM EDTA) and pelleted at 1000 × g for 10 min. The resulting cell pellet were then
resuspended in hypotonic buffer and placed on ice for 20 min. Lysis of
the cell suspension was accomplished with 50 strokes in a Dounce
homogenizer and sequentially centrifuged at 1000 × g
to pellet nuclei, 8000 × g to pellet mitochondria and
lysosome, and 100,000 × g to pellet endoplasmic
reticulum, Golgi, and membranes. The supernatant after the last
centrifugation was kept as the cytosolic fraction. The pellets,
resuspended in homogenizing buffer, and the cytosolic fraction were
analyzed directly by immunoblotting using rabbit anti-SIMPLE antibody
or mouse anti-LAMP-1 antibody (PharMingen, San Diego, CA).
Triton X-114 Extraction and Deglycosylation Assay--
THP-1
cells (~5 × 106 cells) were extracted in 0.5 ml of
0.5% Triton X-114 in PBS for 1 h on ice, and the nuclei and cell
debris were removed by centrifugation at 1800 × g for
10 min at 4 °C. The proteins were then partitioned into the
detergent and aqueous phases according to the method of Bordier (35).
The separated detergent and aqueous phases were analyzed directly by
immunoblotting. THP-1 cells or RK13 cells expressing SIMPLE were
solubilized in 20 mM Tris maleate buffer, pH 6.0, containing 1% Nonidet P-40, 0.1% SDS, 1.0 mM
phenylmethylsulfonyl fluoride, and 5 mM iodoacetamide. To
remove the debris, solubilized sample was centrifuged at 10,000 × g for 30 min at 4 °C. After centrifugation, the
supernatant was incubated with 100 microunits of neuraminidase (Sigma)
for 1 h at 37 °C followed by 4.5 milliunits of
O-glycanase (endo- Confocal Microscopy--
HeLa cells incubated in 4-well glass
slides (Nunc Inc., Naperville, IL) were fixed for 30 min with 1%
paraformaldehyde in PBS and were permeabilized with 0.5% saponin, 1%
BSA/PBS for 30 min, washed four times with PBS. After soaking in 1%
BSA/PBS, the cells were treated for 1 h at room temperature with
rabbit anti-SIMPLE polyclonal antibody or pre-immune rabbit antiserum in 1% BSA/PBS at a dilution of 1:500. The cells were washed with 1%
BSA/PBS and treated for 30 min with fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG (1:100) (Organon Teknika
Corp., West Chester, PA) diluted in 1% BSA/PBS. Cells were washed 4 times by 1% BSA/PBS and incubated for 1 h with mouse anti-LAMP-1
antibody (5 µg/ml) in 1% BSA/PBS, washed again, and incubated for 30 min with rhodamine B-conjugated goat anti-mouse IgG (Organon Teknika
Corp.) (1:100). Visualization of acid pH compartments was performed by
staining cells with 75 nM Lysotracker Red DND-99 (Molecular
Probes, Leiden, The Netherlands) for 1 h at 37 °C. Single- or
double-stained cells were examined with a confocal laser scanning
microscope (Olympus FLUOVIEW). Colocalized green (fluorescein
isothiocyanate) fluorescence and red (Lysotracker or Rhodamin-B)
fluorescence appeared yellow in the merged images.
BCG-CWS-induced Differential Display RT-PCR Product, GCAP2, Defines
the 3' End of LITAF/PIG7--
Differential display RT-PCR has been
performed between unstimulated and BCG-CWS-stimulated monocyte RNA. A
combination of pairs of primers, T12GC (3' anchor) and AP2 (5'
arbitrary), produced a distinct band designated as GCAP2 in BCG-CWS
induced RNA (Fig. 1A).
Amplification and sequencing of this band identified a 373-bp fragment
having identity, with a few acceptable mismatches, to the paired
primers at the ends (Fig. 2, boxed
sequence). This fragment did not show any homology with any known
genes in the data base; however, we found several human ESTs.
Furthermore, the fragment when used as a probe showed a distinct signal
on Northern blots, suggesting the differential display product belongs to a 2.4-kb transcript (Fig. 4). Next, we employed the in
silico cloning strategy (16, 17) to generate an extended virtual contig with a group of EST clones that were directly, or through another overlapping EST, linked to the GCAP2 sequence. This procedure identified a human EST (AW022014) that bridged between the GCAP2
fragment and ESTs denoted by the LITAF/PIG7-end sequence. Sets of EST
contigs for this zone are also currently available under a TIGR
transcript (THC116999, October, 2000) at NCBI. In order to verify this
link, RT-PCRs with 5' primers from LITAF and 3' primers from the GCAP2
fragment and 5'-rapid amplification of cDNA ends from GCAP2 were
conducted. Various combinations of RT-PCR primer pairs yielded the
expected size bands that were further verified by sequencing and
confirmed the link. A representative result of RT-PCR is in Fig.
1B. The newly identified 3' region exhibited a
polyadenylation signal (Fig. 2) located 18 bp upstream to the beginning
of the poly(A) tail shown. No such polyadenylation signal has been
detected in the published LITAF sequence or in the deposited PIG7
sequence. The poly(A) tail consisting of 19 A residues described for
LITAF does not seem to be the true poly(A) tail, rather the sequence
corresponds to a stretch of 19 As (1754-1777 bp) interrupted by a G in
the SIMPLE cDNA (Fig. 2). The presence of the same A stretch in
both cDNA and in genomic sequences (exon 4, Fig.
3C) further emphasizes that
the long A-tailing starting at 1755 bp in the LITAF sequence is an
intra cDNA region, whereas the poly(A) tail shown for SIMPLE was
not derived from the genome. Thus, the GCAP2 fragment defines the 3'
end of the LITAF/PIG7 transcript, which in turn leads us to conclude
that LITAF/PIG7 is a differentially expressed gene in
BCG-CWS-treated human monocytes.
Identification and Verification of a Translational Frameshift in
LITAF/PIG7--
During sequencing of several RT-PCRs and 5'-rapid
amplification of cDNA ends products from human monocyte RNA, we
consistently noticed a single G was missing from a stretch of 5 consecutive guanine residues present in the coding region of LITAF
(608-612 bp; GenBank/EMBL/DDBJ accession number NM_004862 or
U77396)/PIG7 (454-458 bp; GenBank/EMBL/DDBJ accession number
AF010312). Absence of this G residue created a translational frameshift
(Fig. 3B) yielding a protein of 161 amino acids, whereas
LITAF/PIG7 encoded a protein of 228 amino acids. This raised a
potential question whether there were two types of transcripts
producing two different proteins. In order to verify this further,
first we used Pfu polymerase to amplify the coding region
from various RNA sources that included monocytes from different
donors' blood, THP-1 monocytic cells (from which LITAF has been
isolated), and DLD-1 colon carcinoma cells (from which PIG7 has been
cloned). After cloning the PCR products in each case, we sequenced the critical region for 10-12 independent clones and detected the stretch
of 4 Gs (Fig. 3A, top) instead of 5 Gs in all of them. Second, we looked for ESTs specific for that region because many tissues express the gene, and if the zone is polymorphic that could be
represented by some of the ESTs. We found many ESTs (Fig. 3A) with 4 Gs but could not detect any human or murine EST
showing 5 Gs for that particular region, neither in the EST data base nor in the pooled EST set for LITAF/PIG7 under UniGene collection (Hs.76507, October, 2000). Third, we wanted to verify the region from
the genomic sequence. For this purpose, we utilized the information from the unfinished genome sequencing project through the HTG data base
at NCBI. We partially succeeded, but a few contigs remain yet to be
aligned, in identifying the genomic boundaries (Fig. 3C) for
the entire SIMPLE transcript from a chromosome 16 clone, RP11-547D14
(accession number AC007616.3). The astonishing observation was that the
region of dispute, 4 Gs or 5 Gs, falls into a potential splice junction
(Fig. 3, B and C). According to the splice
junction donor acceptor rule (AG/GT), it is clearly in favor of the
presence of 4 Gs in the cDNA. Finally, we focused on the deduced
amino acid sequence of the transcript. If the protein with the changed
C-terminal amino acid truly represented a conserved protein, then we
might find other homologues and orthologues in the data base. Due to
the advantage of the small size of SIMPLE, we easily could find
full-length or near full-length proteins from various species described
below (Fig. 9). The C-terminal domain was found to be the most
conserved region in a number of proteins from different species,
indicating the evolutionarily conserved feature of this domain. On the
other hand, we failed to generate any such data for the C-terminal
domain specific for LITAF/PIG7. Based on the above four lines of
evidence, we conclude that the 161-amino acid sequence coding the
SIMPLE gene is the natural and the most abundant transcript. For
clarity, from this point, the 161-amino acid-coded 2368-bp transcript
will be referred as SIMPLE. For comparison the first base number of
SIMPLE cDNA (Fig. 2) has been kept the same as LITAF.
SIMPLE Is a Widely but Variably Expressed Transcript--
Multiple
tissue Northern blots from CLONTECH were
independently hybridized with full-length cDNA, the coding region,
and with the GCAP2 fragment. Three types of hybridizations successively detected a single transcript of the same size on multiple tissues. The
result obtained by hybridizing with the coding region of SIMPLE is
presented in Fig. 4. Most of the human
tissues except testis expressed SIMPLE abundantly, and this was also
consistent with the fact that there was a vast collection of human ESTs
(UniGene Hs.76507, October, 2000) from various organs and a single EST (AA62566) of testis origin. In respect to the RNA size marker provided
on the multiple tissue Northern blot, the message size fits reasonably
with the full-length SIMPLE (~2.4 kb). The message size appeared to
be the same in a number of human cell lines, and in 12 paired
tumor-normal colon carcinoma samples (data not shown), suggesting there
is no aberrant or variant transcript that could be detectable by size
difference on Northern blot. The tissue distribution pattern also
suggests SIMPLE probably has a more generalized function rather than
playing a unique role for the sole benefit of the monocyte/macrophage
lineage. However, there is a great variability in the relative
expression level among the tissues tested; peripheral blood
lymphocyte showed the highest level of expression: little in brain and,
compared with ovary, little in testis. Inter-library comparative
profile of the SAGE-tag, TGAATACTAC (Fig. 2), indicates that the
expression could be regulated by a hormone in the human reproductive
tissues: as evident in LNCaP with DHT versus LNCaP without
5 Expression of SIMPLE Is Induced by BCG-CWS and BCG--
Often
differential display fragments gives rise to false positives, so it was
essential to reverify the differential expression in response to
stimulation. In different batches of monocytes and iDC, BCG-CWS
enhanced the expression of SIMPLE about 2-fold at 8 h of induction
(Fig. 5A), the same incubation
time for the differential display study. The expression has also been
checked in response to heat-killed and live M. bovis BCG;
both are found to be potent inducers of this gene (Fig. 5B)
at 8 h of infection. M. bovis BCG doubling time in
monocytes was ~20 h, suggesting the induction was
replication-independent. However, a detailed study is required to
confirm whether the expression was phagocytosis-dependent or the expression undergoes alteration with bacilli growth.
SIMPLE Versus TNF- Sequence Analysis of SIMPLE--
Next we focused on structural
analysis of the deduced amino acid sequence of SIMPLE to characterize
its possible function. One easily noticeable feature of the deduced
amino acid sequence of SIMPLE is its proline-rich N terminus and the
cysteine-rich C terminus (Fig. 2). The total proline content is 15%
which is about 3 times higher than the proline content typically found in eukaryotic proteins. The majority (87%) of the prolines are concentrated in the N-terminal half; the proline content in this region
exceeds 22% and is completely devoid of any cysteine. Similarly the
C-terminal half of 68 amino acids lacks proline abundance and possesses
11 cysteines. Unlike many proline-rich proteins, it does not have many
glutamines; instead, it is often punctuated by serine-threonine and to
some extent by tyrosine, and the majority of these residues are present
in the N-terminal half. Proline-rich proteins with repetitive or
non-repetitive motifs are found to be involved in varieties of
functions (18, 19). However, the overall pattern of proline in
combination with serine, threonine, and especially with tyrosine,
SIMPLE shows similarity with tyrosine-hydroxyproline-rich extensin
family of plant cell wall proteins elicited during infection and
wounding (20).
The most striking feature of SIMPLE is the C-terminal domain; motif
search and profile scan indicate that the amino acid residues 96-152
of SIMPLE are similar to C3H4 type zinc RING finger (21, 22). As shown,
cysteines (Fig. 9A, light green vertical bars) and
possible loop1 and loop2 regions of SIMPLE can be aligned with a group
of known RING finger proteins (Fig. 9, A and B). The alignment fits well with the consensus of RING and allied zinc
finger motifs (23) such as FYVE and FYVE related fingers (24, 25). The
protein does not have any known nuclear localization signal sequence,
but the k = 9/23 in k-NN prediction (PSORT) suggests a 52%
probability for nuclear localization. The presence of
proline-cysteine-rich domains are the characteristics of several RING
proteins including many transcription factors (26-28), and they are
found to be involved in diverse cellular functions through RNA or DNA
binding, protein-protein interaction, or both (23, 29-31).
Apparently SIMPLE has all the features to qualify as a RING protein and
to be involved in transcriptional regulation. But the hydropathy
profile (Fig. 8A) suggests SIMPLE has a potential TM domain,
and the region lies within the predicted RING structure (Fig.
9A). The sequence has been analyzed through several
transmembrane prediction programs available at the Expasy site and is
detected as an integral membrane protein with the same TM domain. The
SOSUI-predicted TM-spanning region of 23 amino acid (Fig. 2) residues
is long enough to conform stable integration in the membrane. Another intriguing feature of this C-terminal domain is the presence of a
di-leucine motif and a YXX Subcellular Localization of SIMPLE--
We first took the routine
approach of constructing GFP fusion proteins and prepared N-terminally
GFP-tagged versions of SIMPLE as it lacks the signal sequence.
Expression in 3 different cell lines, COS-7, NIH3T3, and HeLa, produced
comparable results. A representative example of expression in COS-7
cells is shown in Fig. 7A;
N-terminally GFP-fused SIMPLE was concentrated in a paranuclear position with a number of vesicles surrounding the nucleus. Expression of the GFP-only vector showed diffuse cytoplasmic and nuclear fluorescence (data not shown). A paranuclear localization
position is mainly suggestive of Golgi, however, and the trans-Golgi
network, newly synthesized transport vesicles, and lysosomes are also
present around this location, and overexpression makes this
particularly difficult to ascertain. Therefore, a polyclonal antibody
was generated against bacterially expressed His6-SIMPLE and
used, after checking the specificity, for immunostaining in HeLa cells.
Results of confocal analysis are shown in Fig. 7B; SIMPLE is
distributed with LAMP-1 and Lysotraker-stained vesicular compartments,
indicating SIMPLE is associated with perinuclear lysosomes and late
endosomes. Cells with or without permeabilization were also analyzed by
fluorescence-activated cell sorter; no definitive staining was observed
in the plasma membrane or cells stained with preimmune serum, whereas
permeabilized cells showed a strong fluorescence shift (data not
shown).
In parallel, subcellular fractionation was carried out to check whether
the immunostaining pattern corresponds. As evident from the Western
blot of various subcellular fractions, the lysosome-enriched 8,000 × g pellet (Fig. 7C, lane 2, top) contained
SIMPLE; the same fraction showed the strongest signal for the presence
of LAMP-1-positive vesicles (Fig. 7C, lane 2, below).
Absence of any cell surface expression further suggests that SIMPLE is
a lysosomal/late endosomal residence protein rather than a recycling receptor. This work clearly demonstrated that SIMPLE is not a nuclear
or cytosolic protein even though most of the RING proteins are found to
be nuclear or multicomplex cytosolic proteins.
SIMPLE Is an Integral Membrane Protein of the Lysosome--
RING
proteins like EEA1 are localized in early endosomes and utilize their
RING domain for peripheral membrane anchoring (34). To see whether
SIMPLE is an integral membrane protein of the lysosome, we have
analyzed the partition of the protein during phase separation in a
solution of Triton X-114 according to Bordier (35). In this method
integral membrane proteins with an amphiphilic nature are recovered in
the detergent phase, whereas peripheral and cytosolic proteins remain
exclusively in aqueous phase. After two rounds of Triton X-114
extraction, the protein was completely recovered in the detergent phase
(Fig. 8A), demonstrating that
SIMPLE represented an integral membrane protein of the lysosome.
Simultaneously, a duplicate blot also has been tested with a control
7-TM integral membrane protein to monitor the efficiency of extraction
(data not shown).
Lysosomal membrane proteins are in general heavily
N-glycosylated to be protected from unwanted degradation
inside the lumen. SIMPLE does not have any N-glycosylation
sites, although it has several O-glycosylation sites as
predicted by the NetOglyc 2.0 program (Fig. 8B). In order to
verify its glycosylation status, we used THP-1 cells as a natural
source of the protein and SIMPLE cDNA-transfected rabbit RK13 cells
for expressed protein. Expressed SIMPLE or THP-1-derived protein
remained unaffected by O-glycanase (Fig. 8B;
THP-1 data not provided). Under similar conditions the deglycosylation
status was observed for a known O-glycosylated protein
(CD46), suggesting that SIMPLE is an unglycosylated protein, which is
consistent with the fact that the O-glycosylation sites are
poorly defined and not necessarily used (36). The protein is
approximately 24 kDa (in both reduced and non-reduced condition) as
detected in THP-1, human monocytes, HeLa, DLD-1, and in
RK13-transfected cells. However, the unglycosylated molecular size (24 kDa) of SIMPLE is slightly higher than its unmodified calculated mass of 17 kDa; the slower migration could be attributed to the
phosphorylation status or due to the proline richness of the protein as
noted for other proteins such as Zyxin, Krupple, and TESK-1 (37,
38).
Assignment of SIMPLE into a New Family--
As mentioned above,
the C-terminal domain of SIMPLE resembles the RING structure but
interrupted by a TM region. That is an unusual structural feature
unable to satisfy RING family characteristics, yet unique by itself. We
wanted to know whether proteins containing this structural feature
compose a new family, and we searched data bases to collect SIMPLE
homologues and related proteins (see Fig.
9). The homology among human (SIMPLE),
rat (EET-1; accession number U53184), and murine (TBX1; accession
number AF171100) is high (90-91%). A considerable degree of homology
is present with Zebrafish (53%; EST AW184464) and chicken (72%; EST
AI979890), and the most conserved region appeared to be the C-terminal
domain 70-75 residues long (N-terminal alignment for these proteins
has not been shown). Based on this region of SIMPLE, we performed TBLASTN and BLASTP searches against several data bases and 2 rounds of
PSI-BLAST iteration. Several hits appeared in these query modes, and
simple visual inspection could identify that they have a pattern. An
alignment of 18 sequences from all the species (2 human, 2 rodent, 1 fish, 1 avian, 5 insects, 8 nematode, and 1 from plant) is provided in
Fig. 9C. Transmembrane prediction analysis was done for each
protein, and strong TM regions were underlined whenever detected. It is
now more convincing that this domain has a consensus (shown below the
alignment profile) to be clearly distinguishable from the RING-like
domain, yet borrowing the first and last pair of cysteines conserved
among the zinc finger family. Between the cysteine dyad there is a long
variable region that often harbors the membrane-spanning region. The TM
region is preceded and followed by two unique consensus sequence
signatures, CPXCX5T and
#X3#X2HXCX2C, respectively (Fig. 9C). The majority of the proteins in this
family seem to be small in size (around 160 aa); however, we can see the domain and the motifs in proteins of larger sizes such as C16orf5
(261 aa) and DmCG13515 (283 aa). The domain is not necessarily restricted to the C terminus of all proteins as in the case of C16orf5
(39) and CeT26805 (see TL/CL), suggestive of a module domain that could
be utilized by a variety of proteins at different locations but serving
a common function. In this connection Caenorhabditis elegans
protein CeT26805 of 386 amino acids can be mentioned. The N-terminal
region residues 31-98 of this protein show the SIMPLE-like domain
signature, and the rest of the sequence (105-386 residues) is 70%
similar to the WD repeat region of human The identification of SIMPLE, which is similar to LITAF or PIG7
transcripts but with a different coding potential, was an accidental
finding. We confirmed in various ways the presence of SIMPLE as a
single transcript and protein. Our main findings are as follows: 1)
identity of SIMPLE with LITAF/PIG7 at the nucleotide level but not at
the level of coded protein; 2) perfect agreement of all exon-intron
junctions in the SIMPLE transcript but not in LITAF/PIG7; 3) lack of
evidence for the presence of 5 Gs in the coding region sequences from
monocytes, THP-1, and DLD-1 cells; and 4) an abrupt change (Fig.
9A) in the amino acid sequence in LITAF/PIG7, compared with
SIMPLE, EET-1, and TBX1, supports a frameshift in the LITAF due to the
misincorporation of an additional G residue. However, the region of
dispute corresponds to a splice junction; aberrant splicing or allelic
polymorphism of 4 Gs versus 5 Gs may still create a
LITAF/PIG7-coded protein, and it could act as a dominant negative form
against the natural version, SIMPLE.
Similarity with RING Family--
The next question is whether
SIMPLE can be considered as a variant of the zinc RING proteins because
it has a similar sequence motif. We have not examined the zinc binding
potential of this protein due to the difficulties of purifying its
native form, and in addition, the predicted RING region has been found
to be disrupted by a single potential TM domain. Our experimental
evidence, including the phase separation, supports that the protein is
tightly fastened in the intracellular membrane compartment, suggesting the predicted TM domain within the RING is the anchoring region. The TM
domain signature also corresponds to the BLOCK pattern of the 5th TM
domain of the Srg family 7-transmembrane receptor (40) of C. elegans, which further supports the integral nature of the domain.
The presence of this TM domain dampens the possibility of considering
this protein as a RING protein or even to be a divergent type. However,
it is apparent from the alignment that the C-terminal domains of SIMPLE
family genes are nicely bracketed by a pair of CXXC motifs
(Fig. 9C, green bars), resembling the first and last pair of
cysteines in zinc finger proteins. This feature, together with the
fairly well spaced additional cysteines within the bracket, can easily
be mistaken as a RING-like contour. We conclude from the alignment
profile that the proteins under this family share a domain that has
similarity in organization with the RING domain; however, the presence
of the TM domain limits further comparison.
Comparison with Major Lysosomal Membrane Proteins--
SIMPLE is a
new lysosomal membrane protein, a motif search showed that the
N-terminal 14 amino acids (residues 10-23) of SIMPLE have homology
with the LAMP block. We compared SIMPLE with major integral membrane
proteins of lysosomes (41), LAMPs, LIMPs, and also with Endolyn (42,
43). ClustalW alignment showed patches of sequence similarity with
those groups mainly due to the proline, serine, and threonine (Pro > Ser=Thr) richness of the N-terminal domain of SIMPLE. The partially
matched regions correspond to the mucin-like domains (Ser-Thr-rich) of
Endolyn, CD168, and DC-LAMP and the hinge regions
(Pro-Ser/Ser-Thr-rich) of LAMPs. There is little architectural
similarity between SIMPLE and classic bipartite or semi-bipartite
patterns of the extracellular domain of LAMP or the Endolyn family (41,
43). Mucin-like domains and hinge regions of the above families of
proteins are heavily N- and O-glycosylated (41,
44), whereas SIMPLE is completely devoid of glycosylation. The
C-terminal domain also does not show any similarity except for the
presence of dileucine (LL) and YXX Induction of SIMPLE by Microbial Components--
BCG cell wall
components and LPS are potent effectors exerting maturation and
survival signals for monocytes and dendritic cells, and these pathways
are intimately linked to TLR2- and TLR4-mediated signaling (49). TLR2
has been characterized recently as a novel death receptor (50) that
implies an analogous mechanism to the TNF receptor family; the
signaling events of these receptors are bifurcated downstream, and they
can modulate life and death upon ligand activation during infection.
The genes regulated through these pathways leading to the final
cellular response are mainly unknown. The putative promoter region of
SIMPLE contains AP1- and p53-like binding motifs; if those are
functional this could explain how SIMPLE could be induced through
TLR/TNF receptor and p53-mediated pathways.
Relevance of SIMPLE Induction by M. bovis--
During microbial
infection one of the most predominant innate responses exerted by an
immunocompetent host is the induction of apoptosis of the infected
cells thereby minimizing the spread and restricting the infection. The
similarity of SIMPLE with PIG7, which is more than 10-fold increased in
a p53-mediated apoptotic environment, and its localization in
lysosomes, which play role in the process of cell self destruction,
suggest SIMPLE could be involved in host cell apoptosis. In
vitro studies have shown that apoptosis is responsible for
intracellular killing of mycobacteria (51), and down-regulation of
anti-apoptotic genes has been observed due to BCG or heat-killed
Mycobacterium tuberculosis (52). Since promoting apoptosis
is not beneficial for the growth of the bacilli, the components of
programmed cell death are also impaired. It is evident from recent work
that both live and heat-killed M. bovis BCG were capable of
increasing the viability of monocytes through up-regulation of an
anti-apoptotic gene A1 (53). Hence, it is not unlikely that many genes
in apoptotic and anti-apoptotic pathways could be altered during
mycobacterial infection. Several lines of evidence suggest that
avirulent strains of Mycobacterium are most active in
eliciting the apoptotic response, whereas the virulent strain bypasses
this, and cells remain less apoptotic (54, 55). If the expression of
SIMPLE is potentially connected to elicit the host cell apoptosis, the
expression could be affected differentially by virulent and avirulent strains.
At this point, there is no direct evidence that PIG7 or SIMPLE is
involved in apoptosis because their mechanism of action is unknown.
However, SIMPLE as a lysosomal membrane protein and being proline-rich
may raise an interesting possibility in the view of lysosomal and
ubiquitin-mediated intracellular protein degradation pathways (56-58).
Lysosomal membrane protein LAPTM5 (59) that is specifically expressed
in hematopoietic cells possesses a proline-rich carboxyl-terminal
domain, and the domain has been found to interact with precursors of
ubiquitin, leading to the concept that LAPTM5 mediates degradation of
ubiquitinated protein in the lysosome. Another lysosomal membrane
protein LAMP2/LGP96 (60) has been found to be a receptor for selective
uptake of proteins into the lysosome and subsequent degradation.
Recently, a unique sequence motif has been detected in the cytosolic
tail of the LAMP2a isoform, which is required for the binding of
substrate protein and is proposed to be important for
chaperone-mediated autophagy by the receptor (61). Structural analysis
of the proline-rich domain and the identification of interacting
proteins for SIMPLE may provide important clues about the function of
this gene. It is also intriguing that the rat homologue, EET-1, was
rapidly induced by estrogen treatment in the rat uterus. Programmed
cell death is an essential feature of normal ovarian and uterine cycles (62, 63), and increased lysosomal activity is known during endometrial/luteal degeneration. Studying rodent reproductive tissue
may reveal the functional aspect of SIMPLE as it lacks estrogen-responsive elements in the 3'-untranslated region, and its
regulation by estrogen in human has yet to be defined.
In summary, identification of SIMPLE revealed that LITAF/PIG7 could
encode the same protein as EET-1 provided a G residue was absent from a
specific region of the coding sequence. We confirmed the absence of the
G residue in an identical transcript, SIMPLE. SIMPLE belongs to a new
family of proteins having a unique domain with two conserved sequence
motifs. The gene was induced in antigen-presenting cells upon
activation by microbial components, and a dramatic induction occurred
during p53-mediated apoptosis. There is little information regarding
the role of lysosomal membrane proteins in apoptosis and their
alteration during infection. Characterization of SIMPLE as a novel
member of lysosomal membrane proteins certainly puts forward this gene
as a promising candidate defining its novel role in programmed cell
death and in host-microbial interaction.
. To our knowledge this is the first report of pathogen-associated
molecular pattern (PAMP)-induced differential
expression of a lysosomal membrane protein presumably involved in apoptosis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
production, which is
comparable to the heat-killed or live BCG-induced maturation profile.
These findings suggest that the component can be used to explore other
potential genes regulated downstream of the signaling cascade,
including those genes that could also possibly change during live
mycobacterial infection. In a combined effort of mRNA differential
display, suppression subtractive hybridization, and cDNA array
analysis, several differentially expressed genes have been reported (6,
7). We mainly focused on identifying those gene fragments that are
segments of new genes or those that are not yet defined as a part of
known genes, with an aim of generating a comparative expression profile
for a set of PAMPs (8, 9). During our study, one of these genes
appeared to be significantly similar to the previously identified human
genes, LITAF (10) and PIG7 (11). LITAF is a novel
protein binding to a critical region of human TNF-
promoter and is
reported to be involved in TNF-
expression during LPS induction (10,
12). The same gene was found to be induced severalfold during
p53-mediated apoptosis (11, 13) and was described as PIG7. Our findings
raise the possibility that the gene we found to be induced by BCG-CWS
and named SIMPLE, which is also a human homologue of rat EET-1 (14), could actually be the LITAF/PIG7. Thus, the induction of this gene in
various contexts is intriguing and may not be simply a coincidence;
here we describe the detailed analysis of the SIMPLE transcript,
including its expression, localization, genomic organization, and its
assignment under a new family.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ORF regions, and EST
as murine SIMPLE/TBX1). Blots were washed at high stringency (65 °C,
0.2× SSC, 0.1% SDS) and exposed to the films for different times. The
mRNA expression level was estimated from radioactivity of the
hybridized probe by phosphorimaging or autoradiography.
-N-acetylgalactosaminidase; Genzyme, Cambridge, MA) treatment for 16 h at 37 °C.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, identification of a differentially
expressed cDNA, GCAP2, in BCG-CWS-treated monocytes. Total RNA
obtained from human monocytes treated with BCG-CWS or vehicle control
(emulsion buffer) for 8 h was subjected to differential display
RT-PCR using 3' anchor primer (5'-T12GC-3') and 5' arbitrary primer AP2
(5'-GACCGCTTGT-3'). The PCR products were separated on standard
sequencing gels and visualized by autoradiography. The arrow
indicates a differentially expressed band of 343 bp (GCAP2) in
BCG-CWS-RNA lane. B, human EST, AW022014,
identifies the link between LITAF/PIG7 and GCAP2. Schematic
presentation at the top shows the RT-PCR strategy to confirm the link
between GCAP2 and the 3' end of LITAF/PIG7. The result of the RT-PCR
using total RNA from BCG-CWS-treated monocytes has been shown
below the diagram. The sequences of primer F and primer R
are (5'-gggccttcctcagcaccatc-3') and (5'-gaatcctggcttgctgcttg-3'),
respectively. The expected amplified product of 1790 bp is indicated by
arrow for lane 2; control lane
represents an RT-PCR performed in the absence of reverse
transcriptase.
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Fig. 2.
The 2.4-kb SIMPLE cDNA
(GenBankTM AB034747) and deduced amino
acid sequence. The cDNA region (GCAP2) identified by
differential display is shown within a box. The large
shaded region represents the connecting sequence between LITAF and
GCAP2, which also overlaps with the EST AW022014 sequence. At and near
the end of 3'-untranslated region, the poly(A) tail and polyadenylation
signal are indicated in uppercase, boldface letters. The
poly(A) stretch, which corresponds to the 3' end of the LITAF sequence,
is shown in lowercase, boldface letters.
Underlined sequences in the 3'-untranslated region represent
SAGE tags, and the vertical bars correspond to the potential
exonic junctions. In the amino acid sequence, a putative transmembrane
region is double underlined; proline and cysteine residues
are inside squares and circles, respectively; the
dileucine and YXX motifs are hatched, and
potential phosphorylation sites are marked by asterisks
(CKC2) or diamonds (PKC).
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Fig. 3.
A, the presence of four guanine
residues in the coding region of SIMPLE, which differs from the
reported sequence of LITAF/PIG7. The coding region was amplified from
human monocyte RNA by RT-PCR using primers, forward
(5'-atgtcggttccaggacctta-3') and reverse
(5'-ctacaaacgcttgtaggtgc-3'); translational start and stop
sites are underlined in the primer sequences. Amplified PCR
products from THP-1 and DLD-1 cell RNA also showed the same sequence,
and the EST clones representing the sequence are indicated below.
B, presence of 4 Gs in the coding region leads to a 161 amino acid (aa) protein, SIMPLE, whereas the presence of 5 Gs leads to the 228-amino acid protein, LITAF/PIG7. The region is a
junction between exon 3 and exon 4; G residues are
underlined, and splice junction acceptor donor sequences are
shown in small, boldface letters. C, genomic
organization of SIMPLE on a chromosome 16 HTG clone has been presented
schematically. Possible splice junctions are shown inside the
box; exon (Ex) sequences are in capital
letters; intron sequences are shown in small letters,
and AG/GT sequences at the splice junctions are
underlined.
-dihydrotestosterone or MCF7 3 h versus
MCF7-estradiol 3 h. The SAGE data further demonstrate that the
transcript might be differentially expressed in certain malignancies,
such as prostate, breast, and ovary.
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Fig. 4.
SIMPLE is expressed as a single transcript of
2.4-kb in human multiple tissue Northern blots.
PBL, peripheral blood lymphocyte.
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Fig. 5.
A, confirmation of the BCG-CWS-mediated
induction of SIMPLE in human monocytes and iDC cells. Monocytes or iDCs
of various batches were stimulated by BCG-CWS or emulsion buffer
(control), and after 8 h total RNA was harvested for
Northern analysis. B, monocytes were treated with either
heat-killed or live M. bovis BCG at a concentration of 1 bacillus/monocyte, and after 8 h post-infection cells were washed,
and RNA was prepared for Northern hybridization. Hybridization signals
were quantitated by NIH image program, and normalized values were
plotted.
Induction in Human and Murine
Monocytes--
BCG-CWS and BCG are both potent inducers of TNF-
,
and LITAF has been described as a novel regulator of TNF-
expression
during LPS stimulation. We wanted to see if there was any correlation of SIMPLE and TNF-
expression in our experimental condition. Human
monocytes and murine RAW cells were stimulated by LPS and BCG, and at
different time points RNA was prepared. The blots were first probed
with species-specific SIMPLE cDNA and then stripped and reprobed
with species-specific TNF-
cDNA (Fig.
6). In human monocytes BCG-CWS as well as
LPS both enhanced the SIMPLE expression, but the induction was faster
in the case of LPS as evident from the peak expression levels, 2 versus 4 h by LPS and BCG-CWS, respectively (Fig.
6A). The induction of TNF-
expression was prior to the SIMPLE induction, suggesting TNF-
itself could be an inducer. This
point was further verified by stimulating monocytes with recombinant
TNF-
(Fig. 6B); however, the induction was not as robust
as found in LPS treatment. In the case of a murine macrophage cell
line, RAW, expression of SIMPLE gradually increased with a peak level
at 24 h by BCG-CWS, whereas induction by LPS was observed at early
time points (Fig. 6C). Despite the degradation of RNA in the
control lane of LPS, it was not difficult to conclude that the
expression of TNF-
was prior or concomitant to the SIMPLE induction
as observed in human monocytes. Again, rapid induction by LPS and
gradual induction by BCG-CWS for SIMPLE message has been reflected in
RAW cells, suggesting PAMPs derived from mycobacteria and Gram-negative
bacteria differentially modulate the expression of SIMPLE.
Interestingly, RAW cells showed a second transcript of ~1.5 kb and
that was also altered due to the induction. We also tested THP-1 cells
under similar conditions, but we could not detect significant
differences in SIMPLE expression between stimulated and unstimulated
cells. THP-1 cells itself had a good basal expression level that we
found to be reduced upon phorbol 12-myristate 13-acetate treatment and
cannot be elicited further by LPS/BCG-CWS treatment (data not
shown).
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Fig. 6.
A, time course of SIMPLE and TNF-
induction by BCG-CWS (15 µg/ml) and LPS (0.1 µg/ml) in human
monocytes. For Northern analysis RNA was prepared from control and
stimulated cells at the indicated time points and hybridized with
different probes (hSIMPLE, hTNF-
, and
-actin) successively. Results obtained for
BCG-CWS and LPS treatments are summarized in the left and
right panels, respectively. The signals from the Northern
blots were quantified by PhosphorImager analysis, and values were
normalized and plotted (top panels). B, induction
of SIMPLE expression by treating human monocytes with TNF-
(100 ng/ml) for 4 h. C, time course of SIMPLE and TNF-
induction by BCG-CWS (15 µg/ml) and LPS (0.1 µg/ml) in the murine
macrophage cell line, RAW264.7. Conditions for the Northern analysis is
the same as those mentioned above except murine cDNA sequences
(mSIMPLE and mTNF-
) were used for the probe preparation and
hybridization. Ethidium bromide (Eth.Br.)-stained gel
pictures indicate the amount of RNA per lane in the RAW cell
blots.
motif (Fig. 2), where
X is any amino acid and
is a bulky hydrophobic amino
acid. These motifs are known to interact with a family of adapter
protein complexes during intracellular sorting of the transmembrane
proteins and membrane receptors and also to function in
lysosomal/endosomal and trans-Golgi network targeting (32, 33). The
above analysis prompted us to examine whether SIMPLE is a member of the
nuclear or cytoplasmic RING family proteins or a completely new type of
membrane protein localized in the cell surface or intracellular
vesicular compartment.
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Fig. 7.
A, localization of N-terminal GFP-fused
SIMPLE in transiently transfected COS-7 cells. B, analysis
by confocal microscopy of the subcellular localization of SIMPLE in
HeLa cells. Fixed and permeabilized cells were stained with anti-SIMPLE
antibody (green, panels 1 and 4) followed by
staining with either Lysotracker (red, panel 2) or LAMP-1
antibody (red, panel 5). The merged color (yellow,
panels 3 and 6) is indicative of colocalization.
Juxtanuclear and perinuclear vesicular distribution of SIMPLE is
positive for lysotracker and LAMP-1. C, immunolocalization
of SIMPLE and LAMP-1 in THP-1 cell fractions. Subcellular fractions
were performed as described under "Materials and Methods."
Approximately 60 µg of proteins from each fraction was separated by
SDS-PAGE in 12.5 (top panel) and 7.5% (bottom
panel) gels followed by immunoblotting with anti-SIMPLE antibody
and LAMP-1 antibody, respectively. Specified second antibodies
conjugated with alkaline phosphatase were used for chemiluminescent
detection (ECL, Amersham Pharmacia Biotech). Lanes 1-4
indicate 1000 × g, 8000 × g,
100,000 × g and cytosolic fractions,
respectively.
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Fig. 8.
A, Triton X-114 extraction of SIMPLE
protein from THP-1 cells. Equal amounts of proteins from detergent and
aqueous phases were separated in an SDS-PAGE, and duplicate blots were
prepared. One blot was immunostained with anti-SIMPLE antibody and the
other with pre-immune serum. SIMPLE protein was recovered as an
integral membrane protein exclusively in the detergent phase. Predicted
membrane spanning region of SIMPLE has been underlined in
the Kyte-Doolittle hydropathy plot shown next to the immunoblots.
B, O-glycanase treatment of SIMPLE expressed in
RK13 cells. Solubilized SIMPLE expressing RK13 cells was treated
without ( ) or with (+) O-glycanase, and the samples were
analyzed in duplicate immunoblots as mentioned above. The NetOgly 2.0 program-predicted O-glycosylation profile of SIMPLE is
presented below the immunoblots. Vertical lines
represent the O-glycosylation potentials of serine and
threonine residues, and the horizontal line indicates the
threshold values. C, the apparent molecular mass of SIMPLE
is 24 kDa. Solubilized cell extracts were prepared from RK13 cells;
RK13 cells were transfected with SIMPLE, human monocytes, THP-1 cells,
HeLa cells, and DLD-1 cells. Samples were separated in a 12.5%
SDS-PAGE under reducing conditions, transferred onto the membranes, and
detected with anti-SIMPLE antibody.
COP. CeT26805 is a
hypothetical protein, yet it is a good example where the SIMPLE domain
has been fused to the WD domain generating a new protein. Based on our
analysis above we propose to designate this family, the domain, and the
motif by the name of SIMPLE. A plant protein has been shown as
an example, just below the consensus, to show that the hypothetical
protein follows the consensus pattern and can be considered as a member
of this new family (Fig. 9C).
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Fig. 9.
A, ClustalW alignment of SIMPLE with
LITAF/PIG7, EET-1 (rat homologue), and TBX1 (murine homologue).
Identical residues are in red with an asterisk;
strongly similar residues are in green with a colon,
and weakly similar residues are in blue with a single
dot. From residue 127 of LITAF/PIG7 that corresponds to the region
of 5 Gs in the coding sequence, there is an abrupt change (sequence in
black italics) in amino acid sequence. B,
cysteine residues in the C-terminal domain of SIMPLE, EET-1, and TBX1
correspond to the conserved positions (numbered in magenta
text, 1-8) of cysteines and histidine in the RING domain. The aligned
RING proteins were collected from Refs. 24 and 64. Below
these sequences the consensus of RING family and related zinc finger
domains (23, 25) are also shown in magenta. The number of
amino acid residues between the conserved cysteines and histidine is
omitted for the alignment purpose and is shown as dashed
lines. C, alignment of the C-terminal domain of SIMPLE with sequences from other species. Sequences
were mainly obtained by searching protein data bases through BLASTP and
PSI-BLAST programs available at NCBI home page. Some sequences are also
obtained by running the TBLASN program against EST data bases using the
C-terminal domain of SIMPLE (residues 91-161) as a query. Multiple
alignment of protein sequences has been performed using MultAlin
sequence alignment program and by manual adjustment. The consensus
shown just below the alignment includes residues conserved
in the majority (>90%, uppercase in red;
>50%, lowercase in blue) of the aligned
sequences. The 1st right-hand column indicates the total
length (TL) known for each protein versus the
sequence length selected for the comparison (CL), and the
last right-hand column shows the % identity
(I)/% positive (P) values obtained by BLOSUM62
matrix setting for each sequence with respect to the C-terminal domain
of SIMPLE. Accession numbers for SIMPLE (Hum), TBX1
(Mus; Mus musculus) and EET-1 (Rat),
and for chicken and Zebrafish (Zeb) were cited in the text.
Gene identification or accession number has been included for
Drosophila melanogaster (Dm), C. elegans (Ce), and Arabidopsis thaliana
(At), and the EST for Bombyx mori
(Bombyx) is AU004815.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
motifs, one of which
is invariably present in the cytoplasmic domain of the above-mentioned
families. In the case of the LAMP family, YXX
is preceded
by a G and is known to be critical for direct delivery from trans-Golgi
network to lysosome (45). SIMPLE, EET-1, and TBX1 all possess the
YXX
motif with a GT prefix, although the most conflicting
and contradictory aspect is the predicted type II orientation of
SIMPLE. SIMPLE lacks the typical signal sequence and contains only one
potential hydrophobic stretch that presumably works as a stop transfer
signal. According to the charge difference rule (46) of TM topology the
(C-N) value is
2.5, indicating that the dileucine and YXX
motifs will be in the luminal side. The charge difference is not the
sole factor involved in membrane orientation; type II proteins may have
a type III configuration (47, 48), and in that case
LL/YXX
will be retained in the cytoplasmic tail, which
remains to be experimentally verified for SIMPLE.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Graham F. Barnard, University of Massachusetts Medical Center, for providing the colon carcinoma blots and for critical review of the manuscript. We thank Dr. A. Chakrabartty, University of Illinois, and Dr. I. Azuma, Hakodate National College of Technology for helpful discussions. We are grateful to Dr. A. Hayashi, Dr. H. Koyama, and Dr. M. Tatsuta (Osaka Medical Center) for valuable discussions. We also gratefully acknowledge the preparation of the photographic reprints by K. Okugawa. Dr. Nasim A. Begum conducted the main part of this work.
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Note Added in Proof |
---|
Type II topology of SIMPLE has been
confirmed (by proteinase K digestion of lysosomal fraction), which is
consistent with the recent finding that Nedd4 is interacting with the
N-terminal domain of mouse homologue (Jolliffe, C. N., Harvey, K. F.,
Haine
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FOOTNOTES |
---|
* This work was supported in part by Organization for Pharmaceutical Safety and Research, grant-in-aid from the Ministry of Education, Science, and Culture, and the Ministry of Public Welfare of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide (sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB034747.
¶ Supported by Japan Science and Technology Corp. fellowship.
To whom correspondence should be addressed: Dept. of
Immunology, Osaka Medical Center for Cancer and Cardiovascular
Diseases, Higashinari-ku, Osaka 537 Japan. Tel./Fax: 81 6 6973 1209;
E-mail: tseya@mail.mc.pref.osaka.jp.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M011660200
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ABBREVIATIONS |
---|
The abbreviations used are:
BCG, Bacillus
Calmette-Guerin;
LPS, lipopolysaccharide;
TNF-, tumor necrosis
factor-
;
kb, kilobase pair;
bp, base pair;
PCR, polymerase chain
reaction;
RT-PCR, reverse transcriptase-PCR;
TLR, Toll-like receptors;
DC, dendritic cell;
iDC, immature DC;
FBS, fetal bovine serum;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel electrophoresis;
BSA, bovine serum albumin;
GFP, green fluorescent protein;
AP, arbitrary primer;
ORF, open reading frames;
TM, transmembrane;
aa, amino acids.
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REFERENCES |
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