Production and Characterization of Anti-MBP-mTTP Serum—
For antigen production, MBP-mTTP was expressed in Escherichia coli
and purified to near-homogeneity as described (6
). The results of this purification are shown after staining with Coomassie Blue () and immunoblotting with an anti-MBP antibody (). The Mono Q fractions with the highest purity of MBP-mTTP (, lane 6
) were pooled, concentrated, and used as antigen.
Fig. 1 Purification of MBP-mTTP from E. coli and characterization of antibodies. MBP-mTTP was purified from E. coli transformed with plasmid pMBP-mTTP. MBP-mTTP was initially purified from a 10,000 × g supernatant by amylose resin chromatography, followed (more ...)
The resulting anti-MBP-mTTP serum at a 1:10,000 dilution was able to detect as little as 1 ng of the purified MBP-mTTP, using the SuperSignal detection reagent and a 5-s exposure of the blot (). We also tested the antiserum using extracts from 293 cells expressing mouse TTP (3
). The antiserum at 1:10,000 dilution was able to detect mouse TTP when as little as 1 μg of total protein from the transfected 293 cells was loaded into the gel (). This antiserum recognized mouse TTP but not human TTP or mouse Zfp36L1 under these immunoblotting conditions (data not shown). However, it could recognize human TTP when much more protein was used or when the blot was exposed to film for longer times (see below).
Identification of TTP in Mouse Cells and Tissues—
Using the present antiserum at 1:10,000 dilution, TTP could be detected in Western blots of normal spleen when 5 mg of protein per gel lane and the SuperSignal detection reagent were used (). TTP in cells and tissues occurs as multiple bands of Mr
about 40–50,000, probably a mixture of differentially phosphorylated species and degradation products (1
). The TTP bands were absent in spleen taken from TTP KO mice (). As a positive control, we used 2 μg of protein extract from transfected 293 cells ().
Fig.2 Identification of endogenous TTP in mouse spleen. A, 2 μg of protein from 293 cell extracts expressing HA-mTTP, and 5 mg of protein from 20,000 × g supernatants of spleen homogenate from WT and TTP KO mice, were separated by SDS-PAGE and (more ...)
The 20,000 × g
spleen supernatant was separated into supernatant and pellet fractions by centrifugation at 100,000 × g
. TTP was found primarily in the 100,000 × g
supernatant, with very little immunoreactivity in the membrane pellet (). No TTP signal was detected in the 20,000 × g
pellet (data not shown). Because purified recombinant MBP-mTTP (10 ng, or 0.134 pmol of the 74.9-kDa fusion protein) was probed on the same blot as the spleen cytosol (1.7 mg of total protein), we used this blot to estimate the approximate concentration of TTP in normal mouse spleen. In these studies, the average spleen weight was 95.2 mg (n
= 14), the average soluble protein content per spleen was 16.5 mg/spleen (n
= 14), and adult mouse spleen water content was ~86% (26
). As shown in , the TTP immunoreactivity in 1.7 mg of spleen extract protein appeared to be about 10% of that of 10 ng (0.134 pmol) of the recombinant protein, or 0.0134 pmol. Therefore, TTP was expressed at ~0.0134 pmol per 1.7 mg/16.5 mg/spleen, or 0.1265 pmol/spleen. Because average spleen water content was 86% of an average wet weight of 95.2 mg/spleen, the final estimated TTP concentration was 0.1265 pmol/81.87 μl, or about 1.5 nM.
Distribution of TTP in Mouse Tissues—The anti-MBP-mTTP serum (1:10,000 dilution) cross-reacted with proteins of the appropriate size in spleen, lung, liver, large intestine, and thymus, and with proteins of different sizes from several other tissues (). To distinguish between true positive and false positive signals, we analyzed tissues from mice deficient in either TNFR1 and WT for TTP () or deficient in both TNFR1 and TTP (). These genotypes were used, because the absence of the TNFR1 largely prevents the development of the inflammatory TTP-deficiency syndrome, which might have altered the expression of nonspecific bands in the tissues. When compared with tissues from the TTP KO mice (), true positive TTP signals were seen in spleen, lung, liver, and large intestine (), whereas nonspecific immunoreactive bands of various sizes were detected in brain, heart, pancreas, and skin from both the TTP WT and KO mice (, compare B and C). These data showed that spleen expressed the highest concentration of TTP of any tissue examined.
Fig. 3 Expression of TTP in mouse tissues. A supernatant (10,000 × g) was prepared from tissues from WT and TTP KO mice and was used for immunoblotting. Transfected 293 cell extracts expressing HA-mTTP (1 μg of total protein) were used as positive (more ...)
Induction of TTP in Cultured Cells—To address the specificity of this antiserum in cultured cells, BMM from WT and TTP KO mice were exposed to LPS (1 μg/ml) for 2 h, and proteins from 10,000 × g supernatants were separated by SDS-PAGE and probed with the antiserum at a 1:10,000 dilution. The multiple bands of TTP were clearly detected when 200 μg of cellular protein was used per gel lane from the LPS-induced WT BMM, but were not seen in the macrophages from the TTP KO mice (). Similar results were obtained using 500 μg of protein per lane in the 10,000 × g supernatants from WT but not KO MEF, which had been stimulated with 10% FCS for 2 and 3 h (). As also shown in , TTP was readily detected in RAW 264.7 cell extracts when only 50 μg of protein was used per lane from the 10,000 × g supernatant of cells stimulated with 0.1 μg/ml LPS.
Fig.4 TTP expression in primary mouse macrophages, RAW 264.7 cells, and MEF. A, primary mouse macrophages from TTP WT and KO mice were treated with LPS (1 μg/ml) for 2 h, and soluble cellular extracts were prepared. Equal amounts of protein from these (more ...)
Time Course of Induction and Stability of TTP in Cultured Cells—To investigate the patterns of TTP protein accumulation in cultured cells, BMM were collected following LPS (0.1 μg/ml) stimulation. TTP was undetectable at time 0 but readily detectable after 2 h, reaching a peak of expression at about 4 h (). Immunoreactive TTP in 500 μg of BMM protein was compared with only 50 μg of protein from RAW 264.7 cells stimulated with LPS for 1.5 and 2 h ().
Fig. 5 Time course of induction and stability of TTP in mouse cells. RAW 264.7 cells and primary mouse macrophages were stimulated with 0.1 μg/ml LPS, and MEF were stimulated with 10% FCS, for various times as indicated. To measure protein stability, (more ...)
Similar induction kinetics were seen in MEF stimulated with 10% FCS (). Again, TTP was undetectable in the serum-deprived cells at time 0, but accumulated dramatically to reach peak levels at ~2–3 h. These peak levels, detected with 200 μg of cellular protein per lane, were comparable to the levels seen in LPS-stimulated RAW 264.7 cells when only 10 μg of protein was loaded into the gel lane (). Interestingly, there was a continued shift upward in the apparent molecular weight of the protein with still longer times of exposure to FCS (), compatible with increasing phosphorylation. Protein was still readily detectable at 5–6 h.
To estimate the stability of the newly synthesized protein in these cells, the cells were stimulated for 2 h with 10% FCS and then treated with CHX (50 μm) for a further 4 h (). Remarkably, there was very little apparent disappearance of the protein over 4 h following treatment with CHX (), at concentrations that completely inhibited protein synthesis in this cell type (data not shown).
Because RAW 264.7 cells produced much more TTP than the other cell types we tested, we analyzed the induction kinetics in more detail in these cells. As shown in (C and D), TTP was undetectable in unstimulated cells and in cells stimulated with LPS for 15 and 30 min, using 50 μg of cytosolic protein and a relatively short autoradiographic exposure (, C and D). TTP could be detected in the 0.5-h samples if much more protein and/or much longer autoradiographic exposure were used (data not shown). TTP was detectable following induction for 45 min (). At 1 h, TTP was detected as two major bands of about Mr 38,000 and 42,000 (). As TTP continued to accumulate, there were continued increases in the apparent molecular weight to 40–50,000, which stabilized by 2 h (, C and D). Significant amounts of immunoreactive TTP were still detectable in samples collected after 24 h of LPS induction (). We also calculated TTP concentration in the stimulated RAW 264.7 cells with 0.1 μg/ml LPS for 2 h using known concentrations of the purified MBP-mTTP (1–100 ng). The immunoreactivity in 50 μg of RAW 264.7 cell 10,000 × g supernatant was approximately the same as that of 10 ng (0.134 pmol) of the recombinant protein. This represents about 340 times the concentration in normal spleen, or ~0.5 μm.
The stability of TTP in RAW 264.7 cells was further evaluated following LPS stimulation for 2 h (0.1 μg/ml), followed by treatment with CHX (50 μM). No TTP was detected in RAW 264.7 cell extracts 1, 1.5, 2, 3, 4, or 5 h after both LPS and CHX were added to the culture medium at the same time, indicating that this concentration of CHX was effective at inhibiting protein synthesis under these experimental conditions (data not shown). Despite the inhibition of protein synthesis, there was only a modest decrease of TTP immunoreactivity over the next 4 h, although there was a continued shift to apparently greater molecular weight between 2 and 3 h after LPS, even in the presence of CHX (). Similar slow rates of protein disappearance were observed in cells stimulated with LPS for 2.5 h followed by LPS “washout” and CHX treatment (data not shown).
Monomeric Nature of TTP in RAW 264.7 Cells—The 10,000 × g supernatant from RAW 264.7 cells stimulated with LPS (0.1 μg/ml) for 2 h was subjected to size exclusion chromatography following ammonium sulfate concentration. TTP could be detected in a range of column fractions, but the peak of TTP was detected in fraction #31 (), which corresponded to a monomer size of about 40 kDa when compared with a standard curve (). Small amounts of TTP were detected in earlier fractions (), suggesting that a small proportion of the protein might exist in RAW 264.7 cells as oligomers or in complexes with other proteins under these conditions.
When LPS-stimulated RAW 264.7 cells were fractionated into cytosolic and nuclear fractions, most of the TTP was in the cytosolic fraction, with little if any associated with the nuclear fraction (); there was no evidence of higher molecular weight oligomers or aggregates under these conditions. There was also some degree of cross-reactivity with the human TTP protein expressed in and purified from E. coli
(, lane 5
). This figure also illustrates the extent of the inhibited SDS-PAGE migration caused by, presumably, phosphorylation in the RAW 264.7 cells. Human TTP has a predicted molecular weight of 34,086, and the protein purified from E. coli
migrated as a single band at Mr
~36,000, as estimated with protein standards (lane 5
), as we have shown previously (6
). However, the mouse protein in LPS-stimulated RAW 264.7 cells, with a calculated molecular weight of 33,613, migrated as multiple bands of apparent molecular mass 40–50 kDa. The migration of TTP was increased following dephosphorylation of mTTP in the RAW 264.7 cell 10,000 × g
supernatant with CIAP treatment (). Incubation with longer time or with more CIAP resulted in a faster migration of the protein, although the sizes of those dephosphorylated proteins were still larger than that of hTTP purified from E. coli
cells (data not shown). This change in SDS-PAGE migration appears to be largely due to phosphorylation, because the multiband complex collapses into a single protein band of lower apparent molecular weight upon dephosphorylation with alkaline phosphatase (27
Immunostaining of TTP in Intact Cells and Tissues—To further localize TTP in RAW 264.7 cells, they were treated with 0.1 μg/ml LPS for 3 h, followed by immunostaining with anti-MBP-mTTP (1:8,000 dilution). As shown in , confocal microscopy revealed bright cytosolic immunofluorescence in LPS-treated RAW 264.7 cells, whereas minimal fluorescence was detected in controls treated in parallel with LPS induction and pre-immune serum; without LPS induction but with anti-MBP-mTTP serum; or without LPS treatment but with pre-immune serum. The cytosolic staining was in a vesicular pattern (). There were occasional foci of nuclear staining, but these were seen also in the cells not treated with LPS; they were more prominent with immune serum than in the other two negative controls (), suggesting that they may represent true foci of nuclear TTP. Serial sections of a single RAW 264.7 cell following LPS induction showed that the vesicular pattern of fluorescence was almost exclusively cytosolic, with little if any signal in the nucleus ().
Fig. 7 Confocal microscopy detection of TTP in RAW 264.7 cells. A, confocal microscopy of RAW 264.7 cells. Cells were treated with either LPS (0.1 μg/ml) or PBS for 3 h, then fixed and stained with either the anti-MBP-mTTP serum (I) or preimmune serum (more ...)
Consistent with the immunoblotting results, TTP was barely detectable under unstimulated conditions but peaked in the cytosol by 2–3 h following LPS stimulation (). Significant immunostaining was still visible in the RAW 264.7 cells after 5 h, although the signal was somewhat decreased relative to the earlier time points (). Although there was minimal detectable fluorescence in the unstimulated cells in (A and C), we occasionally noticed increased fluorescence in the cytosol of cells undergoing division, even in the absence of LPS ().
We also evaluated the use of the antibody in rat spleen, after the intraperitoneal injection of LPS (5 mg/kg). 2 h after the injection, there was prominent TTP staining in macrophages and stromal cells in the spleen periphery (), whereas no specific staining was seen in spleen from a PBS-injected rat (C) or with preimmune serum staining of spleen from either an LPS-injected (B) or PBS-injected (D) rat. The light microscopic images in A–D were obtained and processed under identical conditions. Of interest was the negative staining of the white pulp in the center of (arrow) (see “Discussion”). This immunostaining was seen with the use of Bouin's fixed, paraffin-embedded sections, but not with spleens fixed in 4% paraformaldehyde and processed for frozen sections (data not shown).
Fig.8 TTP immunostaining in rat spleen. Rats were injected intraperitoneally with LPS (5 mg/kg; A and B) or PBS (5 ml/kg, C and D), and spleens were removed and used for immunostaining of TTP with either the anti-MBP-mTTP serum (I) or pre-immune serum (PI) (more ...)