©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification, Microsequencing, and Immunolocalization of p36, a New Interferon--induced Protein That Is Associated with Human Lupus Inclusions (*)

(Received for publication, August 1, 1995; and in revised form, October 27, 1995)

Steven A. Rich (1) (2)(§) Mahuya Bose (1)(¶) Paul Tempst (3) (4) Ulrich H. Rudofsky (1) (2)

From the  (1)Laboratory of Cell Regulation, Wadsworth Center for Laboratories and Research, New York State Department of Health and the (2)School of Public Health, State University of New York, The University at Albany, Albany, New York 12201-0509 and the (3)Molecular Biology Program, Memorial Sloan-Kettering Cancer Center and (4)Cornell University Graduate School of Medical Sciences, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The trace interferon-alpha-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 alpha-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-alpha-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.


INTRODUCTION

Interferons (IFNs) (^1)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-alpha 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-alphas), 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-2alpha 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-alpha(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 alpha- and -IFNs(17, 18, 21) .

p36 is an IFN-alpha-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-alpha 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-alpha-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-alpha 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-alpha is produced as a burst for a 24-h period only. The unusual acid-labile IFN-alpha 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-alpha 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-alpha.

LI, which are also synthesized de novo in response to IFN-alpha(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-alpha(22, 30, 31) . LI and p36 distribute evenly between the nuclear and cytoplasmic fractions of IFN-alpha-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-alpha from these cultures. Both p36 and LI persist as long as IFN-alpha is present, in contrast to oligoadenylate synthetase, which is synthesized only transiently in these cultures in the continued presence of IFN-alpha(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.


EXPERIMENTAL PROCEDURES

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.

IFN-alpha Induction of Raji cell p36 and LI

The human B lymphoblastoid cell line, Raji, is of Burkitt lymphoma origin (American Type Culture Collection, CCL 86). It was maintained in exponential growth as a suspension culture at densities between 0.025 and 1.0 times 10^6 cells/ml with <5% nonviable cells by resuspension in fresh RPMI 1640 medium, 10% fetal calf serum (Life Technologies, Inc.), 100 units of penicillin, and 100 µg of streptomycin/ml. The doubling time of the cells was 18 h. Tissue culture flasks (Bellco Glass, Vineland, NJ) were kept in a humidity-controlled incubator with a 5% CO(2) atmosphere at 37 °C. Cell densities were determined in duplicate on a model F cytometer (Coulter Cytometry, Hialeah, FL), and the percentage of nonviable cells was determined by trypan blue exclusion.

The antiviral titer of the purified recombinant human IFN-alpha, 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-alpha 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 times 10^6 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 times 10^6 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.

One- and Two-dimensional Protein Maps

Two-dimensional gels (isoelectric focusing (IEF) cylinders in the first dimension and SDS-PAGE slabs for the second dimension) were run according to the procedure of O'Farrell(37, 38) . The second dimension SDS-PAGE (12.5% acrylamide; Bio-Rad) by itself was used for one-dimensional protein maps(39) .

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 H(3)PO(4) (anode) and 0.1 M NaOH (cathode). Equilibrated cylinders (5 ml of 3% SDS (Bio-Rad), 6.2% beta-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) .

Purification of p36

500 times 10^6 IFLrA-induced Raji cells were resuspended in 40 ml of RSB, 0.1 mM phenylmethylsulfonyl fluoride, 0.1% Tween 40 at 4 °C(22, 38, 41) . After 10 min on ice, the cells were gently vortexed until lysed. Nuclei were pelleted at 1500 times g for 10 min. The cytoplasm-plus-membrane fraction (the nuclear supernatant) was further fractionated into a 100,000 times g pellet and a soluble supernatant by centrifugation at 100,000 times g for 1 h.

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% beta-mercaptoethanol, 1.35 mM Tris-HCl, pH 7.4, 0.68 mM MgCl(2), 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% beta-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% beta-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).

Microsequencing of p36

p36 on PVDF membrane was stained with Coomassie Brilliant Blue (0.1% in 10% methanol with destaining in 10% methanol) and subjected to direct microchemical sequencing (42) and composition analysis. For amino acid analysis, the p36 on the PVDF membrane was hydrolyzed in constant boiling HCl at 110 °C for 22-24 h and analyzed for amino acids with a Beckman System Gold amino acid analyzer (Fullerton, CA).

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 NH(4)HCO(3) (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% beta-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 2.1times220 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) .

Rabbit Antipeptide Antibodies

Peptides of p36-derived sequence (T28, T37.8, T29, T48, and T50(1/2)) were prepared on an Applied Biosystems (Foster City, CA) 431A automated peptide synthesizer. Fmoc (N-(9-fluorenyl)methoxycarbonyl)/tert-butyl chemistry (47) was used with trifluoroacetic acid cleavage in the presence of appropriate scavengers. The final product was lyophilized from aqueous buffer following extraction with organic solvent.

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(3) at -70 °C.

Immunodetection of p36

Western analyses of p36 with the antipeptide antisera were performed on proteins electrotransferred onto PVDF membranes from one-dimensional SDS-PAGE gels. Membranes were blocked for 90 min with 5% nonfat dry milk, 1% normal goat serum in Tris-buffered saline (10 mM Tris, pH 7.4, 150 mM NaCl), 0.05% Tween 20, washed in Tris-buffered saline, 0.05% Tween 20, and incubated for 2-24 h with polyclonal anti-p36 antiserum. Bound antibodies were detected with alkaline phosphatase-conjugated goat anti-rabbit IgG (regular alkaline phosphatase) or with biotinylated goat anti-rabbit IgG followed by a conjugate of streptavidin-biotinylated alkaline phosphatase (amplified alkaline phosphatase). In both cases, 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium were used as substrate and color development reagents.

For quantitative immunoprecipitation, p36 in the 100,000 times g supernatant from 3 times 10^6 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(2)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 times 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 100times oil objective lens, and photographed on Ilford-XP2 (Ilford Limited, Cheshire, UK) 400 ASA film.

Electron Microscopy and Enumeration of LI

Cell samples were fixed in 3% glutaraldehyde (Sorensen's phosphate buffer, pH 7.4), postfixed in osmium tetroxide, dehydrated in graded ethanol, and embedded in Epon(51) . Thin (0.1 µm) sections were stained with uranyl acetate and then with lead citrate, and examined at 10,000 times on a Philips 301 electron microscope (Philips Electronic Instruments Company, Mahwah, NJ).

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) .


RESULTS

IFN-alpha Induction of LI and p36 in Raji Cells

Raji cells were maintained in an exponentially growing culture with a doubling time of 18 h. No LI were detected in 400 random thin sections of these cells. Growth of these cells in the presence of 100 units/ml of IFLrA for 72 h had no effect on their doubling time. These cells developed LI (Fig. 1) at a frequency of 11.75% (47 LI were detected in 400 random thin sections of Raji cells) by 72 h. This frequency translated (51) into a single LI of an average 2.35-µm diameter/cell of 20 µm average diameter (0.1175 times 20 µm). Earlier work (51) with untreated Raji cells failed to detect any LI in 15,000 random thin sections or less than one LI of an average 2.5-µm diameter/500 cells of an average diameter of 20 µm.


Figure 1: A LI in an IFN-alpha 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 times g supernatant of the cytoplasmic fraction (b), and the 12th Rotofor fraction, pH 5.7, of this 100,000 times 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.



Purification and Microsequencing of p36

A suspension of nuclei and cytoplasm plus membrane was prepared from IFLrA-induced Raji cells by gentle vortexing in the low ionic strength buffer RSB/Tween 40. Nuclei, cytoplasm plus membrane, 100,000 times g pellet, and 100,000 times g supernatant fractions were prepared by differential centrifugation of this suspension. p36 was detected only in the cytoplasm plus membrane and the 100,000 times g supernatant fractions (Fig. 2b). The 100,000 times g supernatant contained 38% of the total cell protein. Rotofor fractionation of this soluble supernatant prepared from 500 times 10^6 IFLrA-induced cells typically focused p36 among fractions 11, 12, and 13 (Fig. 2c) with isoelectric points of 5.6, 5.7, and 5.8, respectively. p36 in these fractions focused near the middle of the IEF cylinders used in two-dimensional gels to purify p36 to homogeneity.

Acid hydrolysis and amino acid analysis showed that 89 pmol of pure p36 was obtained from 2,000 times 10^6 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-alpha-induced protein.

Immunolocation of p36 in Raji Cell Fractions

Six rabbits (Flemish giant Chinchilla Cross, Nys-{SG}) that were immunized with the mixture of synthetic peptides formulated to match the p36 trypsin fragments T28, T37.8, T29, T48, and T50(1/2) (T42 was not included because its sequence had not been determined at the time this work was begun) made high titered antisera that were specific for p36. The 80 amino acids in these five peptides represent approximately 25% of the amino acids in the entire p36 (80 amino acids/315 amino acids estimated for the 36-kDa mass of p36). Enzyme-linked immunosorbent assay-determined antibody titers of 9600 and 25,600 were obtained for the two rabbits that were inoculated with unconjugated peptides. The two rabbits that were injected with peptides conjugated to OVA produced antibody titers of 51,200 and 102,400. Both rabbits that were injected with peptides conjugated to KLH produced antibody titers of 102,400. The animals immunized with unconjugated peptides not only produced antibodies that were of lower titer, but their response to make antibodies was much delayed when compared with the animals that were injected with the conjugated peptides.

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 times g pellet (Fig. 3, lane 8), but not in the nuclear (Fig. 3, lane 4), or 100,000 times 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-alpha-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 times 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 times g supernatant (lanes 7 and 8), and 100,000 times 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 times 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 times 10^6 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 times 10^6 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 times 10^6 cells. The nonspecific bands were derived from the 25-fold concentration of residual fetal calf serum and the 1:100 dilution of antipeptide serum.



Specificity of p36 Antipeptide Antibodies

Western analysis showed that p36 in the 100,000 times g supernatant fraction was poorly precipitated by overnight reaction with p36 antipeptide antiserum and protein A beads (Fig. 5, lane 4) in comparison with the same amount of p36 applied directly to an SDS slab (Fig. 5, lane 1). Near complete immunoprecipitation of p36 in solution was accomplished by denaturation of the 100,000 times g supernatant sample by trichloroacetic acid precipitation and resolubilization (Fig. 5, lane 2). A diminished reaction with antiserum was achieved in 8 M urea (Fig. 5, lane 3). In agreement with these results, only a trace amount of p36 denatured by trichloroacetic acid remained unbound to the antibodies (Fig. 5, lane 5), while without denaturation an estimated amount greater than 80% remained unbound (Fig. 5, lane 6). These results suggest that the epitopes recognized by the antipeptide antibodies are folded into the interior of the native protein.


Figure 5: Immunoprecipitation of p36. Soluble p36 (marked with an arrow; 100,000 times 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 times 10^6 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 times g supernatant of IFLrA-induced Raji cells (3 times 10^6 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 times 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 times 10^6. p36 was readily detected at the antibody dilution of 1:1.5 times 10^6 in 0.2 and 0.5 times 10^6 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 times 10^6 cells, respectively) showed specific labeling of p36 (marked with an arrow) with little background staining at the serum dilution of 1:1.5 times 10^6. Preabsorption of antibody with the mixed synthetic peptides blocked this reaction with 0.5 times 10^6 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.



Localization of p36 in the Endoplasmic Reticulum

Immunofluorescence microscopy confirmed that p36 was present only in the 72-h IFLrA-induced Raji cells (Fig. 7a). p36 antibodies, purified on an affinity column of bound p36 synthetic peptides, demonstrated p36 specific immunofluorescence of IFLrA-induced Raji cells when they were fixed and permeabilized with methanol and acetone, and the antibodies were applied in saponin buffer.


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 100times 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 100times 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 100times oil objective lens.




DISCUSSION

LI are IFN-alpha-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-alpha 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-alpha 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 times 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-alpha-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(1), S, and G(2), 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(2) or G(1) phases. This concurs with other IFN-alpha-induced proteins that were recently shown to be induced in Daudi cells by IFN-alpha during the G(1), S, and G(2) 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 times 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-alpha 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-alpha-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-alpha(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-alpha in SLE and AIDS at the cell and systemic levels.


FOOTNOTES

*
This work was supported by Grant AR 41619 from the National Institutes of Health (to S. A. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Wadsworth Center for Laboratories and Research, New York State Dept. of Health, Empire State Pl., P. O. Box 509, Albany, NY 12201-0509. Tel.: 518-474-6389; Fax: 518-474-7992.

A postdoctoral fellow during the course of this work.

(^1)
The abbreviations used are: IFN, interferon; LI, human lupus inclusions; SLE, systemic lupus erythematosus; ER, endoplasmic reticulum; IFLrA, pure recombinant human interferon-alpha A; IEF, isoelectric focusing; p36, 36-kDa IFN-alpha-induced protein; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; HPLC, high performance liquid chromatography; KLH, keyhole limpet hemocyanin; OVA, ovalbumin; FITC, fluorescein isothiocyanate; RSB, reticulocyte standard buffer.


ACKNOWLEDGEMENTS

We thank Dr. Sidney Pestka (Robert Wood Johnson Medical School) for providing the IFLrA; Dr. Claudia Kent (The University of Michigan Medical School) for providing the monoclonal antibody VB(3)B(5) against the protein disulfide isomerase (the hybridoma for the VB(3)B(5) monoclonal was developed by the late P. T. Varandani from Wright State University); Dr. Paul Masters (Wadsworth Laboratories) for the amplification, cloning, and sequencing of the partial p36 cDNA; and Dr. Dilip Vakharia (Wadsworth Laboratories) for reading the manuscript and providing many helpful suggestions. p36 digestion, microbore-HPLC purification, and chemical sequence analysis were performed by Lynne Lacomis at the Sloan-Kettering Microchemistry Facility under the directorship of Dr. Paul Tempst (supported by the Irma T. Hirschl Foundation and National Science Foundation Grant BIR-9420123 and National Cancer Institute Cancer Center Core Grant 5 P30 CA08748-29 to the Sloan-Kettering Microchemistry Facility), and with the expert advice of Scott Geromanos on analytical and chromatographic instrumentation. Mr. Ulrich Rudofsky (Wadsworth Laboratories) prepared the rabbit p36 anti-peptide antibodies. The authors gratefully acknowledge the use of Wadsworth Center's Peptide Synthesis, Amino Acid Analysis, Peptide Sequencing, and Molecular Genetics core facilities, the expert technical assistance of William E. Gibbons in purifying the p36, and the ready assistance of Ivan Auger with the data base searches.


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