(Received for publication, August 1, 1995; and in revised form, October 27, 1995)
From the
The trace interferon--induced protein, p36, was induced in
Raji cells in association with lupus inclusions. It was solubilized in
a nonionic detergent buffer, enriched by differential centrifugation
and by preparative isoelectric focusing, and purified to homogeneity on
two-dimensional protein gels. Failure to obtain N-terminal amino acid
sequence, however, suggested a blocked
-amino group. Sequences of
six tryptic peptides, 13-19 amino acids in length, were obtained
after digestion, microbore-high performance liquid chromotography
purification, and chemical sequence analysis. None of the six
sequences, which represented approximately 25% of the entire protein,
shared any meaningful homologies with entries in protein sequence
repositories. Raji-cell p36 was shown in Western blots with antipeptide
antibodies to be induced at least 400-fold and by immunofluorescence
microscopy to co-localize with the endoplasmic reticulum resident
protein, protein disulfide isomerase. These results show that p36 is a
new interferon-
-induced protein that localizes in the endoplasmic
reticulum, the cell region in which the lupus inclusions form, and that
p36 is probably physically associated with the lupus inclusions.
Interferons (IFNs) ()are a family of cytokines (1, 2) with a variety of biological
activities(3, 4) . They are named for their originally
discovered activity of interfering with the infection of cells by
virus(5) . Their use in the treatment of tumors is derived
primarily from their ability to inhibit the growth of cells and to
modulate cellular differentiation(6) . They are also known to
affect every component of the immune system(7) , and long-term
IFN-
therapy has induced systemic
autoimmunity(8, 9, 10, 11, 12, 13) .
The mechanism for this is in part mediated through the induction of
other cytokines that also act to modulate the immune
system(14) .
The biological activities of IFNs and other
cytokines are mediated through binding and activation of specific
receptors of the cytokine receptor superfamily(15) . The cell
response occurs by the phosphorylation of selected STAT (signal
transducers and activators of transcription) proteins by specific Jak
kinases(16) . The phosphorylated proteins form complexes, which
migrate to the nucleus, bind to a specific promoter
(interferon-stimulated response element for IFN-s), and activate
the corresponding genes. Specificity of cell activation is attributed
to the particular STAT proteins that are phosphorylated. These are
determined by the STAT's particular phosphotyrosine-binding
domain for the receptor rather than by the Jak kinases that associate
with the receptor(16) .
The most studied IFN-induced
proteins, such as MX, P1/eIF-2 protein kinase, and 2`5`-oligo (A)
synthetases, are those believed to be necessary for establishing an
antiviral state(17, 18) . The cytokine tumor necrosis
factor is also known to be induced by IFN-
(14) . Most
other induced proteins have been identified on two-dimensional gels
only as protein spots(19, 20, 21) . Their
functions and biological significances are unknown. Many of these
proteins are synthesized constitutively at low levels, and they are
enhanced in response to both
- and
-IFNs(17, 18, 21) .
p36 is an
IFN--induced protein that migrates on two-dimensional gels to an
estimated molecular mass of 36 kDa and an isoelectric point of
5.6(22) . It is unlike many other identified IFN-induced
proteins because it is not detected without IFN-
treatment, and it
is not induced by
-IFN(17, 18, 21) . It
is a trace protein that forms only in cells, such as Raji and
Daudi(22) , that readily make human lupus inclusions (LI, also
called tubuloreticular structures or tubuloreticular inclusions). These
cell lines synthesize p36 de novo and secrete it. Current
interest in p36 derives from its IFN-
-regulated expression, its
association with LI, and LI's association with SLE (23, 24) and AIDS(25, 26) .
LI
formation in endothelial and mononuclear cells is a prognostic marker
for disease progression in individuals with
AIDS(26, 27) , and their incidence reflects the
disease activities of individuals with SLE (24) . These
structures are not detected in the cells of healthy
individuals(24) . An unusual acid-labile IFN- is present
continuously in the circulation of individuals with SLE and
AIDS(28, 29) . This is in contrast to a typical viral
infection, in which IFN-
is produced as a burst for a 24-h period
only. The unusual acid-labile IFN-
in sera from individuals with
SLE and AIDS has an extraordinary ability to induce
LI(24, 30) . LI are known to be products of normal
cells abnormally stimulated with IFN-
because peripheral blood
mononuclear cells from healthy adult Red Cross blood donors (31) and umbilical-cord bloods from routine births (32) form LI when cultured with IFN-
.
LI, which are
also synthesized de novo in response to
IFN-(33) , are composed of ribonucleoprotein and membrane
complexes with carbohydrate and no DNA. In electron micrographs they
appear like myxoviruses(34) . They form in a restricted region
of the endoplasmic reticulum (ER) that makes contact with adjacent
regions of the outer nuclear envelope and the Golgi
apparatus(35) . The function of LI is not known. The ER
location of LI, however, suggests that they may affect the established
ER functions of membrane biogenesis, the trafficking of proteins to the
plasma membrane or to cytoplasmic vesicles, or the processing of
proteins for secretion.
The cell lines WISH, MDBK, and GM2504, which
are commonly used in antiviral assays, neither form LI (30) nor
produce p36, which suggests that neither p36 nor LI are involved in the
antiviral activities of IFN. Also, neither p36 nor LI seem to be
involved in the growth inhibition properties of IFN since both p36 and
LI form in Raji cells, which are not growth inhibited by
IFN-(22, 30, 31) . LI and p36 distribute
evenly between the nuclear and cytoplasmic fractions of
IFN-
-induced Raji cells extracted in reticulocyte standard buffer
(RSB)(22) . The appearance and disappearance of p36 and LI
coincide with the addition and removal of IFN-
from these
cultures. Both p36 and LI persist as long as IFN-
is present, in
contrast to oligoadenylate synthetase, which is synthesized only
transiently in these cultures in the continued presence of
IFN-
(22) . Altogether these results suggest that p36 and
LI induction share a common intracellular activation pathway and a
physical association.
In the present study, p36 was purified to apparent homogeneity, partially sequenced, and shown to be a novel protein, as a sequence data bases search failed to indicate homology or identity with any of the entries. It was shown to localize in the ER, where LI form, by in situ immunofluorescence with anti-p36-peptide antibodies. The partial sequence and specific antisera to p36 provide important new tools to study p36 further and to determine its importance in SLE and AIDS.
Biochemicals, enzymes, and antibody reagents were obtained from Sigma unless indicated otherwise.
All animal procedures have been approved by the Institutional Animal Use Committee of the Wadsworth Center.
The
antiviral titer of the purified recombinant human IFN-, IFLrA
(Hoffman-La Roche, Nutley, NJ), was assayed on WISH cells challenged
with vesicular stomatitis virus (Indiana strain) (36) relative
to the National Institutes of Health human IFN-
standard
(GO23-901-527, kindly provided by the Antiviral Substances Program of
the NIAID, National Institutes of Health). 1 unit/ml of IFN provides
50% protection for the WISH cell monolayers.
LI and p36 were induced
by culturing Raji cells at an initial density of 0.063 10
cells/ml for 72 h with 100 units of IFLrA/ml. For the one
experiment in which Raji cells were induced for only 2 h, the initial
density was 0.25
10
cells/ml. As detailed below,
cells were pelleted and overlaid with glutaraldehyde for electron
microscopy, washed, and precipitated with trichloroacetic acid for one-
or two-dimensional gel analyses or were used to prepare slides for
immunofluorescence.
Samples for
two-dimensional analyses were run on 3-mm inner diameter and 12-cm-long
focusing cylinders. Cylinders were focused at 400 V for 16 h and then
at 800 V for 1 h in a Polyanalyst apparatus (Haake, Inc., Paramus, NJ)
using 0.1 M HPO
(anode) and 0.1 M NaOH (cathode). Equilibrated cylinders (5 ml of 3% SDS (Bio-Rad),
6.2%
-mercaptoethanol, 62.5 mM Tris, pH 6.8, for 15 min
at 4 °C) were attached to 0.75-mm-thick SDS slabs with a minimal
volume of agarose. Low molecular weight markers (Pharmacia
Biotechnology Inc.) were used for size calibration, and pH measurement
was obtained with 0.5-cm-long slices of duplicate focused cylinders
equilibrated in 1 ml of water. The vertical slab apparatus for SDS-PAGE
(SE500, Hoefer Scientific Instruments, San Francisco, CA) was run at 10
°C and 110 V for 30 min, followed by 220 V, until the bromphenol
blue dye marker reached the bottom of the slab (
3 h). Slab gels
were stained with either Coomassie Brilliant Blue or
silver(40) .
For fractionation on the
Rotofor cell (Bio-Rad), the soluble supernatant was prepared in 2.06%
ampholines (Pharmacia Biotechnology Inc.) (1:10 mixture of pH
3.5-10 and pH 5-7 ampholines), 3.18% Triton X-100, 0.07%
SDS, 8.2 M urea, 0.79% -mercaptoethanol, 1.35 mM Tris-HCl, pH 7.4, 0.68 mM MgCl
, 6.75
µg/ml pancreatic RNase, 3.65 µg/ml pancreatic
DNase(38) . Samples reached equilibrium (defined by a constant
current for 30 min) by 5 h of electrofocusing at 4 °C and 12 watts
of constant power, at which time the 20 fractions were harvested. The
protein components in the Rotofor fractions were determined on
two-dimensional gels that were stained by the silver
method(40) . Samples for two-dimensional gel analysis were
prepared by combining 20 µl of a Rotofor fraction with 4 µl of
the following mixture: 13.3% of a 1:10 mixture of pH 3.5-10 and
pH 5-7 ampholines, 1.67% SDS, 35% Triton X-100, 18.8%
-mercaptoethanol(38) .
Enriched p36 in Rotofor
fractions was purified to homogeneity from 400-µl aliquots run on
two-dimensional gels. The slab gels were lightly stained with 0.1%
Coomassie Brilliant Blue, 10% methanol (without acetic acid) and
destained in 10% methanol. Spots of pure p36 were cut out and
equilibrated in sample buffer (5% -mercaptoethanol, 50 mM Tris, pH 6.8, 1% SDS, 10% glycerol) for SDS-PAGE. Collected spots
of pure p36 were concentrated into a single band of p36 on a
1.5-mm-thick one-dimensional SDS-PAGE slab (double the thickness of the
two-dimensional gels), and electrotransferred onto either PVDF
(Millipore, Bedford, MA), or nitrocellulose (Bio-Rad), membrane with a
Transphor apparatus (Hoefer Scientific Instruments) (192 mM glycine, 20% methanol, 25 mM Tris, pH 8.3).
For internal sequence determination, the band of p36 on
nitrocellulose was stained with Ponceau S (Fluka, Ronkonkoma, New York)
(0.1% in 1% acetic acid and destained with 1% acetic acid), excised and
further processed as described previously(43) , with
modifications. Briefly, in situ proteolytic cleavage was done
using 0.5 µg of trypsin (Promega, Madison, WI) in 25 µl of 100
mM NHHCO
(supplemented with 0.3% Tween
80) at 37 °C for 3 h. The resulting peptide mixture was reduced and S-alkylated with, respectively, 0.1%
-mercaptoethanol
(Bio-Rad) and 0.3% 4-vinylpyridine (Aldrich), and fractionated by
reversed phase HPLC. An enzyme blank was done on an equally sized strip
of nitrocellulose. HPLC solvents and system configuration were as
described previously (44) except that a 2.1-mm Vydac C4
(214TP54) column (The Separations Group, Hesperia, CA) was used with
gradient elution at a flow rate of 100 µl/min. Fractions were
collected by hand, kept on ice for the duration of the run, and then
stored at -70 °C before analysis. Chemical sequencing of
selected peptides was done using a model 477A instrument (Applied
Biosystems, Foster City, CA) with ``on-line'' analysis (120A
HPLC system with 2.1
220 mm phenylthiohydantoin C18 column;
Applied Biosystems). Instruments and procedures were optimized for fmol
level phenylthiohydantoin-derivative analysis as described
previously(45) . Peptide sequences were compared with entries
in various sequence data bases using the National Center for
Biotechnology Information BLAST program(46) .
Glutaraldehyde at a final concentration of 1% was used to couple each of the synthetic peptides to the carrier proteins KLH and ovalbumin (OVA)(48) . Equimolar mixtures of these conjugates were used to immunize rabbits for the production of polyclonal antibodies reactive with p36. Preimmune serum samples were taken from each of six rabbits. Two rabbits were inoculated with the mixture of five synthetic peptides, two with the mixtures of synthetic peptides conjugated to KLH, and two with the mixtures of synthetic peptides conjugated to OVA. Sera were decomplemented by heating at 56 °C for 30 min, sterile filtered (0.45 µm HAWP filters, Millipore, Bedford, MA), and stored at -70 °C.
Antibody titers were determined by enzyme-linked immunosorbent assay (49) . For test antigen, the synthetic peptides were conjugated to bovine serum albumin by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, which prevented cross-reactivity to KLH, OVA, and the linkages created by glutaraldehyde. Titers were determined relative to readings for a 1:100 dilution of the corresponding preimmune serum. Second antibody was goat anti-rabbit conjugated to alkaline phosphatase, and color development was with p-nitrophenyl phosphate. Plates were read on a BioTek (Winooski, VT) EL 340 BioKinetics Reader at a 405-nm wavelength.
Affinity purification (50) of rabbit antibodies (antiserum
raised against KLH-conjugated peptides) reactive with p36 peptides was
achieved on a column of CNBr-activated Sepharose 4B (Pharmacia
Biotechnology Inc.), which was coupled to the mixture of the five
synthetic peptides conjugated by
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to bovine serum albumin.
Bound p36-specific antibodies were eluted with 100 mM glycine,
pH 3.0, into 1 M Tris, pH 8.0. Aliquots were stored in 0.02%
NaN at -70 °C.
For quantitative immunoprecipitation, p36 in
the 100,000 g supernatant from 3
10
IFLrA-induced Raji cells was first precipitated with
trichloroacetic acid (7.5%), extracted with ethyl ether, solubilized
(21 µl of 0.1 N NaOH), neutralized (20 µl of 0.1 N HCl plus 75 µl of 0.1 M Tris, pH 7.0) and brought to
150 µl with H
O. It was then reacted overnight with 20
µl of antipeptide serum and with 50 µl of Pro-A-Sepharose
(Pharmacia Biotechnology Inc.) beads for an additional hour. The beads
were pelleted and washed. The p36 adsorbed and not adsorbed to the
beads was determined by Western analysis.
For immunofluorescence,
glass microscope slides of Raji cell suspensions were prepared on a
Cytospin (Shandon Scientific Ltd., Cheshire, UK) (400 g for 5 min), air-dried, fixed for 10 min in a 50:50 mixture of
methanol and acetone, and washed in PBS just prior to use. The slides
were incubated for 1 h with antibodies of either affinity-purified
rabbit anti-p36 or mouse monoclonal-anti-protein-disulfide isomerase
that were diluted 50-fold in 2% bovine serum albumin, 0.1% saponin
(Baker Inc., Phillipsburg, NJ), PBS. The slides were washed in this
antibody buffer and reincubated with the appropriate goat second
antibody (Cappel, Organon Teknika Corp., West Chester, PA)
(fluoresceinlabeled to detect p36 and rhodamine-labeled to detect
protein-disulfide isomerase). The slides were again washed, mounted in
Vectashield H-1000 medium (Vector Laboratories, Burlingame, CA),
examined in a Nikon (Melville, NY) Optiphot microscope equipped with
epifluorescence optics and a Nikon 100
oil objective lens, and
photographed on Ilford-XP2 (Ilford Limited, Cheshire, UK) 400 ASA film.
LI frequencies are based on a binomial model(51) . The ratio of the mean diameter of LI to the whole cells is the probability (d) of observing an LI in a random thin section of a given cell that contains an LI. Factoring in the probability that an LI exists in the cell (p) gives a probability of observing LI in a cell population (p*), or p* = dp. For each sample, 400 independent cell sections were examined. Thin sections separated by greater than the 20-µm diameter of the cells were used to attain random sampling. A 10% frequency (p*) (40 out of 400 independent cell sections) is consistent with one 2.0-µm diameter LI (p = 1) per cell of a 20-µm diameter (d = 2.0 µm/20 µm = 10%). The corresponding 95% confidence interval of LI is 7-13%(51) .
Figure 1:
A
LI in an IFN- induced Raji cell. Raji cells were grown for 72 h
with 100 units/ml of IFLrA. LI in these cells appeared as a complex of
microtubular elements 20-28 nm in diameter that localized in the
lumen of the endoplasmic reticulum. Labeled structures are the LI and
the nucleus, N. The section was stained with uranyl acetate
and then with lead citrate and examined in a Philips 301 electron
microscope. Bar = 1 µm.
Two-dimensional analyses of 400-µg amounts of protein from untreated and IFLrA-induced Raji cells failed to reveal any differences resulting from the IFLrA treatment when these gels were stained with Coomassie Brilliant Blue. Silver staining of gels with IFLrA-induced Raji cells made p36 apparent (Fig. 2a). This trace protein with an estimated molecular weight of 36 kDa, and an isoelectric point of 5.6 was greatly intensified by silver staining when compared with neighboring proteins.
Figure 2:
Purification of p36. Two-dimensional
protein gels showed p36 (marked by a triangle) in
IFLrA-induced Raji cells (a), the 100,000 g supernatant of the cytoplasmic fraction (b), and the 12th
Rotofor fraction, pH 5.7, of this 100,000
g supernatant sample (c). p36 in enriched Rotofor fractions
was purified to homogeneity on two-dimensional gels for use in
microsequencing. Molecular weight markers note the vertical scale, and
isoelectric pH values the horizontal scale. The gels were stained by
the silver method.
Acid hydrolysis
and amino acid analysis showed that 89 pmol of pure p36 was obtained
from 2,000 10
IFLrA-induced Raji cells. This amount
of sample required 90 IEF cylinders to be run on 30 SDS slabs (i.e. three central portions of IEF cylinders applied to a single SDS
slab). The 90 spots of p36 were cut out, collected in SDS sample
buffer, and concentrated into a single band on an SDS slab of double
thickness, and electrotransferred to PVDF or nitrocellulose membranes.
Direct microsequencing of 89 pmol of p36 did not yield any result, indicating the likelihood of a blocked N terminus. Tryptic digestion of approximately 100 pmol of nitrocellulose bound p36 was then carried out, giving peptides that were fractionated by microbore-HPLC and successfully sequenced (Table 1).
Computer searches of these
six p36 peptides, which represent an estimated 29.5% of the entire
amino acid sequence of p36 (93 amino acids/315 amino acids estimated
for the 36-kDa mass of p36), provided either no matches or ones without
any statistical significance (Table 1) with all five of the
protein data bases contained in the network service BLAST (46) (Brookhaven Protein Data Bank, Kabat Sequences of Proteins
of Immunological Interest, PIR, Swiss-Prot, and Swiss-Prot Weekly
Update). On the basis of these findings, it was concluded that p36 is a
new IFN--induced protein.
Reaction of these antibodies with p36 in Raji cells was assayed by Western blots. The six rabbit antisera reacted specifically with p36 in induced Raji cells when tested at a 1:100 dilution and developed with alkaline phosphatase, with no reaction occurring at this molecular weight location in the untreated cells. Additional nonspecific staining was common to both uninduced and induced Raji cells. The nonspecific bands were unique for each rabbit, and they occurred both with preimmune serum as well as the serum that contained high antibody titers. Results from one of the antisera prepared against the p36 peptides that were conjugated to KLH were used in the figures for this work.
p36 and
nonspecific bands were stained in whole cell samples of IFLrA-induced
Raji cells (Fig. 3, lane 2) while only the nonspecific
bands were stained (Fig. 3, lane 1) in the untreated
cells (1:1000 dilution). It was detected in the nuclear supernatant (Fig. 3, lane 6), and the supernatant of the 100,000
g pellet (Fig. 3, lane 8), but not in
the nuclear (Fig. 3, lane 4), or 100,000
g (Fig. 3, lane 10), pellets. p36 was not detected
in any of the corresponding control fractions (Fig. 3, lanes
3, 5, 7, and 9). Equal loading of
protein in corresponding sample wells was shown by the equal staining
of the nonspecific bands. As shown by these results IFN-
-induced
p36 was located solely in the cytoplasm.
Figure 3:
Localization of p36 in cell fractions.
Alkaline phosphatase-developed Western blots of Raji cells and their
fractions (serum dilution of 1:1000) showed that p36 (marked with an arrow) was present in the cytoplasm-plus-membrane (lane
6) and the 100,000 g supernatant (lane
8) fractions. Samples of whole cells (lanes 1 and 2), their nuclei (lanes 3 and 4),
cytoplasm-plus-membrane (lanes 5 and 6), 100,000
g supernatant (lanes 7 and 8), and
100,000
g pellet (lanes 9 and 10)
were prepared from uninduced (U) and IFLrA-induced (I) Raji cells. Cell fractions were prepared by differential
centrifugation of Raji cell extracts prepared with RSB made 0.1% in
Tween 40. The soluble supernatant and the insoluble membrane fraction
were prepared by centrifuging the cytoplasm-plus-membrane fraction at
100,000
g for 1 h. The samples were run on a 12.5%
SDS-PAGE, electrotransferred to a PVDF membrane, and reacted with an
antiserum prepared against the synthetic peptides that were conjugated
to KLH.
In addition to p36 in the
cytoplasm of 72-h IFLrA-induced Raji cells, these cells also secreted
sufficient p36 by an additional 24 h of culturing in serum-free medium
(containing 100 units/ml of IFLrA) for its detection by Western
blotting (Fig. 4, lane 2). The amount of p36 applied was
obtained from 1 10
Raji cells. The size of
secreted p36 was the same as intracellular p36, and the portion of p36
that was secreted was estimated to be 10% of that present in 1
10
cells. No p36 was seen in the corresponding serum-free
medium from untreated Raji cells (Fig. 4, lane 1).
These results showed that untreated Raji cells neither produced nor
secreted p36, and that any post-translational changes that may
accompany p36 secretion from IFLrA-induced cells did not significantly
alter its size according to its migration in SDS gels.
Figure 4:
Detection of secreted p36. Serum-free
medium containing 100 units/ml of IFLrA and conditioned for 24 h by
72-h induced Raji cells contained p36 protein (lane 2, marked
with an arrow) in contrast to the corresponding sample from
untreated Raji cells (lane 1). The samples were concentrated
25-fold by precipitation with acetone and washing with ethanol before
solubilization in SDS sample buffer for Western blot analysis (1:100
dilution of serum, and alkaline phosphatase development). The p36
assayed was secreted by 1 10
cells. The nonspecific
bands were derived from the 25-fold concentration of residual fetal
calf serum and the 1:100 dilution of antipeptide
serum.
Figure 5:
Immunoprecipitation of p36. Soluble p36
(marked with an arrow; 100,000 g supernatant
from IFLrA-induced Raji cells) that reacted with antipeptide antibodies
and protein A beads was determined by Western blot. p36 (lane
1, 3
10
cell equivalents) was well
immunoprecipitated after denaturation (lane 2, trichloroacetic
acid precipitation and resolubilization; lane 3, 8 M urea), but poorly immunoprecipitated without prior denaturation (lane 4). These findings were confirmed by the p36 that
remained in the supernatants following protein A pelleting (lane
5, following trichloroacetic acid denaturation; lane 6,
left untreated). For these assays, 20 µl of antiserum was mixed
overnight with 150 µl of 100,000
g supernatant of
IFLrA-induced Raji cells (3
10
cell equivalents),
followed by 50 µl of protein A-Sepharose beads for another hour
before processing for electrophoresis and Western blot analysis (1:100
dilution of serum, and alkaline phosphatase
development).
Amplified alkaline phosphatase
development of Western assays of the 100,000 g supernatant fractions provided a clear specificity for p36. No
background staining occurred with the corresponding control fraction
between antiserum dilutions of 1:50,000 and 1:1.5
10
. p36 was readily detected at the antibody dilution of
1:1.5
10
in 0.2 and 0.5
10
IFLrA-induced Raji cells (Fig. 6, lanes 1 and 2, respectively). In IFLrA-induced cells p36 was increased at
least 400 times as measured by the detection of p36 out to a 400-fold
dilution. Specificity of the antiserum reaction with p36 (Fig. 6, lane 2) was shown by its blockage upon
preincubation with the mixture of unconjugated synthetic peptides (Fig. 6, lane 3), and no effect from preincubation with
myoglobin (Fig. 6, lane 4). Cell samples reacted only
with the streptavidin-biotinylated alkaline phosphatase complex stained
the high molecular weight band of biotin that was endogenous to Raji
cells.
Figure 6:
Specificity of p36 antipeptide antibodies.
Amplified alkaline phosphatase developed Western blots of induced (lanes 1 and 2) Raji cells (at 0.2 and 0.5
10
cells, respectively) showed specific labeling of p36
(marked with an arrow) with little background staining at the
serum dilution of 1:1.5
10
. Preabsorption of
antibody with the mixed synthetic peptides blocked this reaction with
0.5
10
cells (lane 3), while absorption
with the same concentration of myoglobin had no effect (lane
4). The samples were run on a 12.5% SDS-PAGE, electrotransferred
to PVDF membrane, and reacted with an antiserum prepared against the
synthetic peptides conjugated to KLH. The intense high molecular weight
band was not related to the antipeptide serum as shown by its
appearance upon reaction with the biotin-alkaline
phosphatase-streptavidin complex only.
Figure 7:
Immunofluorescent localization of p36.
Affinity purified antipeptide antibodies reacted specifically with the
cytoplasm of IFLrA-induced Raji cells. Shown (a) is an equal
mixture of induced and uninduced Raji cells with the uninduced cells
having no fluorescence. FITC-labeled second antibody contributed no
fluorescence (b, induced Raji cells). The affinity-purified
anti-p36-antibody was used at a dilution of 1:50, second antibody was
FITC-conjugated goat anti-rabbit IgG (1:200 dilution). Pictures were
taken on a Nikon diaphot microscope with epifluorescence optics and a
Nikon 100 oil objective lens.
The location of p36 in the cytoplasm, and the particular region of the cytoplasm in which p36 localized, was determined by double immunofluorescence (Fig. 8) with anti-p36 and a monoclonal antibody to protein-disulfide isomerase; protein-disulfide isomerase is an ER-resident protein(52, 53) . Antibodies to protein-disulfide isomerase stained (rhodamine-labeled second antibody) the same cytoplasmic region of untreated (Fig. 8a) and IFLrA-induced (Fig. 8b) Raji cells. This showed that the distribution of protein-disulfide isomerase was not altered by IFLrA treatment. Micrographs of the same IFLrA-treated cells showed the colocalization of p36 in the same cytoplasmic region as protein-disulfide isomerase (Fig. 8c, FITC-labeled second antibody), the ER, and the location of the cytoplasm and nuclei in phase contrast (Fig. 8d). These results showed that p36 in IFLrA-induced Raji cells was restricted to the ER, the same region in which LI formed.
Figure 8:
Localization of p36 in the endoplasmic
reticulum. Protein disulfide isomerase (protein-disulfide isomerase) is
an endoplasmic reticulum resident protein(52, 53) .
Its distribution in a region of the cytoplasm of untreated (a)
and IFLrA-induced (b) Raji cells was determined with rhodamine
second antibody staining. No apparent alteration in protein-disulfide
isomerase distribution with IFLrA treatment occurred. In the same
IFLrA-induced cells (c, stained with a FITC-labeled second
antibody to detect p36) p36 was shown to localize to the same cell
region as protein-disulfide isomerase, the endoplasmic reticulum. The
nuclear and cytoplasmic regions of these cells are also shown (d, in phase contrast). Affinity-purified anti-p36 antibody
was used at a dilution of 1:50, the anti-protein-disulfide isomerase
monoclonal antibody was used at 1:25, second antibody for p36 was
FITC-conjugated goat anti-rabbit IgG (1:200), and the second antibody
for protein-disulfide isomerase was rhodamine-conjugated goat
anti-mouse IgG (1:400 dilution). Pictures were taken on a Nikon diaphot
microscope with epifluorescence optics and a Nikon 100 oil
objective lens.
Immunofluorescence microscopy also showed that p36 was just detectable by 45 min of IFLrA treatment of a culture of Raji cells in exponential growth with a doubling time of 18 h. If p36 expression was cell cycle-dependent, then only a proportion of the cells at 2 h of induction would stain for p36, like a mixture of induced and uninduced Raji cells. If p36 expression was independent of cell cycle stages, then all of the cells would stain for p36, similar to the pattern seen for 72-h IFLrA-induced Raji cells, but with a lesser intensity of fluorescence. Immunofluorescent staining showed that p36 was being expressed by all of these cells (Fig. 9), and with a lesser intensity than Raji cells that were induced for 72 h with IFLrA. Thus p36 expression was not restricted to a single cell cycle phase.
Figure 9:
Expression of p36 in Raji cells treated
for two hours with IFLrA. Raji cells in an exponentially-growing
culture induced with IFLrA (100 units/ml) for 2 h revealed that p36 was
being expressed in the cytoplasm of all of the cells. The
affinity-purified anti-p36 antibody was used at a dilution of 1:50, and
second antibody was FITC-conjugated goat anti-rabbit IgG (1:200
dilution). Pictures were taken on a Nikon diaphot microscope with
epifluorescence optics and a Nikon 100 oil objective
lens.
LI are IFN--induced (51) abnormal cytoplasmic
structures of unknown function that resemble myxovirus by
ultramorphology (Fig. 1, (34) ). In the human B
lymphoblastoid cell line, Daudi, LI were shown to be a
ribonucleoprotein and membrane complex with carbohydrate and no
DNA(33) . Formation of LI in the cytoplasm of endothelial and
mononuclear cells is associated with active disease and elevated
IFN-
in SLE (23, 24, 29) and
AIDS(25, 26, 27, 28) . In a previous
study of human lymphoblastoid cell lines, p36 was shown on
two-dimensional protein gels to be a trace protein induced by IFN-
and to form in association with LI(22) . It was of interest to
determine whether the p36 protein is of known function and how it is
related to LI.
For the present study, p36 was purified by
two-dimensional gel electrophoresis in approximate 100-pmol amounts for
further characterization. In O'Farrell's original work on
two-dimensional gels(37) , sample buffers with or without SDS
were used. Our preliminary studies showed that p36 was not detected
unless SDS was included in the IEF sample buffer (Fig. 2a). This applied even to the soluble (e.g. 100,000 g supernatant) fraction used to purify
p36 in this study (Fig. 2b). The SDS-buffer system had
to be modified for use with Rotofor fractionation to enrich p36 (32) (Fig. 2c) prior to purification to
homogeneity.
Attempts to determine the N-terminal amino acid
sequence of p36 failed, which suggested that the N terminus was
blocked. Blockage of the N terminus is a typical occurrence for more
than 80% of eukaryotic proteins (43, 54) due to N-acetylation and other post-translational modifications. We
succeeded in obtaining internal sequence by cleavage of p36 with
trypsin into peptides that were purified by HPLC. A search in the BLAST (46) protein data bases of the microsequences of six p36
trypsin peptide fragments (Table 1) established that p36 has no
significant homology with any of the proteins previously reported, and
it is therefore a new IFN--induced protein.
High titered p36-specific polyclonal rabbit antisera were prepared against synthetic peptides that were formulated from the p36 peptide microsequences (Table 1). In Western blots, these antisera detected no p36 in untreated Raji cells (Fig. 3). With IFLrA treatment, an estimated 400-fold increase in p36 occurred coincident with the formation of the 11.75% LI frequency, both of which were in the cytoplasmic fraction ( Fig. 1and Fig. 3). By immunofluorescence, affinity-purified anti-p36 peptide antibodies showed that p36 co-localized with protein-disulfide isomerase, a protein that is specifically found in the ER (52, 53) (Fig. 8). This is also the cell region where LI form(35) , and it suggests that p36 is physically associated with LI. The location of LI in a restricted region of the ER that makes contact with the outer nuclear envelope and the Golgi apparatus suggests their functioning in membrane biogenesis, the trafficking of protein to the plasma membrane or to cytoplasmic vesicles, or the processing of protein for secretion(35) . A role for LI in protein secretion is supported by the secretion of p36 by IFLrA-induced Raji cells (Fig. 4).
It was also shown in
our earlier study that IFLrA induces p36 in an exponentially growing
culture of Raji cells by 2 h(22) . From this radiolabeling
study and analysis of two-dimensional gels, it was not possible to
determine whether all, or only a subpopulation, of the cells were
synthesizing p36. A subpopulation of cells would be expected especially
if p36 synthesis was cell cycle-dependent. By immunofluorescence it is
now shown that all of these cells stained positive for p36 by 2 h (Fig. 9). Estimates of the duration of Raji cell cycle stages
are 9.5, 3.8, and 3.7 h for G, S, and G
,
respectively(55) , and 1 h for M from an estimated mitotic
index of 3%. This suggests that p36 is synthesized in all stages with
the possible exception of M, which is shorter than the 2-h period of
induction, so that all cells transitioning M phase would also have been
in G
or G
phases. This concurs with other
IFN-
-induced proteins that were recently shown to be induced in
Daudi cells by IFN-
during the G
, S, and G
phases(56) .
In contrast to the results obtained by
immunofluorescence, no signal was obtained with the same antibodies by
immunoelectron microscopy. The 80 amino acids in the peptides used to
generate the polyclonal antibodies represent approximately 25% of the
entire p36 protein (estimated to be 315 amino acids in length), and
therefore only a limited number of the epitopes of p36 are involved in
these antibody reactions. The epitopes recognized by the antipeptide
antibodies could be folded into the interior of the protein. This was
supported by the immunoprecipitation of p36 from the 100,000 g supernatant fraction by the antipeptide antibodies only
after p36 was unfolded by trichloroacetic acid precipitation and
resolubilization (Fig. 5). p36 unfolded by SDS in SDS-PAGE (Fig. 3Fig. 4Fig. 5Fig. 6) or by methanol
and acetone (Fig. 7Fig. 8Fig. 9), used with
immunofluorescence light microscopy, gave positive results in Western
and immunofluorescence assays, respectively. Methanol and acetone
destroyed the ultrastructural appearance of the cell organelles, which
made this approach not useful for immunoelectron microscopy.
Formaldehyde-fixed IFLrA-induced Raji cells failed to provide any
immunofluorescent signal, which suggested that the reactive epitopes
were not accessible subsequent to this treatment. Antibodies prepared
against intact p36 protein should facilitate in situ immunoelectron microscopy studies as well as the
immunopurification of native p36.
In preliminary studies, cDNA
prepared from total RNA isolated from IFLrA-induced Raji cells, and
amplified with degenerate oligonucleotide primers for the p36 peptides
T48 and T50(1/2), has provided a single polymerase chain reaction band
of approximately 560 base pairs. Its sequence of 186 amino acids codes
for three of the six peptides of p36(T28, T37.8, and T29) in addition
to T48 and T50(1/2), which were used in the design of the degenerate
primers. This confirms the correctness of the amino acid sequences of
these five p36 tryptic peptides and the conclusions drawn from their
sequences. Moreover, both IFLrA and the IFN- endogenous to SLE and
AIDS induce p36 in peripheral blood mononuclear cells purified from
healthy Red Cross blood donors, in peripheral blood mononuclear cells
from individuals with SLE and AIDS, and in Raji and Daudi cells (work
in progress). Our work with p36 and LI in lymphoblastoid cells is
thereby shown to be relevant to primary lymphocytes and more
importantly to individuals with SLE and AIDS.
A data base search of
the 186 amino acids coded by this polymerase chain reaction product
provided no significant homology with any of the proteins in the BLAST (46) protein data bases. However, a highly homologous (P(N) = 1.4 e)
gene segment from Caenorhabditis elegans (gp/U23172/CELF25B5
4, submitted March 21, 1995) was identified in the CDS translations
from GenBank® Release 88.0, April 15, 1995. Three of the six p36
peptides (Table 1) T29, T48, and T50(1/2) gave P(N) values of 0.73, 0.46, and 0.59, respectively,
for this same gene segment. Thus p36 appears to be highly conserved
from at least the time of C. elegans, and such conservation
suggests a function of biological importance.
While the discovery of
a new IFN--induced protein and its association with LI is
interesting, it leaves unanswered many important questions concerning
the function of both p36 and LI. The primary cell types in which LI
have been identified are T, B, monocytes, and endothelial
cells(57) . p36 is secreted, and all of these cells secrete
cytokines. The identification of LI and p36 with these immune cells in
SLE and AIDS suggests that p36 may be a soluble factor or a cytokine (58) that participates in the modulation of immune cell
interactions in response to a long term stimulation with
IFN-
(6, 7, 8, 9, 10, 11, 12, 13, 59) .
The definition of the complete sequence of p36 and its expression in
substantial amounts are critical for further studies of p36.
Elucidation of the structure of p36, its possible cytokine function,
and its relationship with LI will provide new understanding about
functions of IFN-
in SLE and AIDS at the cell and systemic levels.