Characterization of Elongation Factor-1A (eEF1A-1) and eEF1A-2/S1 Protein Expression in Normal and wasted Mice
2001; Elsevier BV; Volume: 276; Issue: 25 Linguagem: Inglês
10.1074/jbc.m101011200
ISSN1083-351X
AutoresAbdelnaby Khalyfa, Denis Bourbeau, Edwin Chen, Emmanuel Petroulakis, Jie Pan, Suying Xu, Eugenia Wang,
Tópico(s)Cancer-related gene regulation
ResumoThe eEF1Α-2 gene (S1) encodes a tissue-specific isoform of peptide elongation factor-1A (eEF1A-1); its mRNA is expressed only in brain, heart, and skeletal muscle, tissues dominated by terminally differentiated, long-lived cells. Homozygous mutant mice exhibit muscle wasting and neurodegeneration, resulting in death around postnatal day 28. eEF1Α-2/S1 protein shares 92% identity with eEF1A-1; because specific antibodies for each were not available previously, it was difficult to study the developmental expression patterns of these two peptide elongation factors 1A inwasted and wild-type mice. We generated a peptide-derived antiserum that recognizes the eEF1Α-2/S1 isoform and does not cross-react with eEF1A-1. We characterized the expression profiles of eEF1A-1 and eEF1A-2/S1 during development in wild-type (+/+), heterozygous (+/wst), and homozygous (wst/wst) mice. In wild-type and heterozygous animals, eEF1A-2/S1 protein is present only in brain, heart, and muscle; the onset of its expression coincides with a concomitant decrease in the eEF1A-1 protein level. In wasted mutant tissues, even though eEF1A-2/S1 protein is absent, the scheduled decline of eEF1A-1 occurs nonetheless during postnatal development, as it does in wild-type counterparts. In the brain of adult wild-type mice, the eEF1A-2/S1 isoform is localized in neurons, whereas eEF1A-1 is found in non-neuronal cells. In neurons prior to postnatal day 7, eEF1A-1 is the major isoform, but it is later replaced by eEF1A-2/S1, which by postnatal day 14 is the only isoform present. The postdevelopmental appearance of eEF1A-2/S1 protein and the decline in eEF1A-1 expression in brain, heart, and muscle suggest that eEF1A-2/S1 is the adult form of peptide elongation factor, whereas its sister is the embryonic isoform, in these tissues. The absence of eEF1A-2/S1, as well as the on-schedule development-dependent disappearance of its sister gene, eEF1A, in wst/wstmice may result in loss of protein synthesis ability, which may account for the numerous defects and ultimate fatality seen in these mice. The eEF1Α-2 gene (S1) encodes a tissue-specific isoform of peptide elongation factor-1A (eEF1A-1); its mRNA is expressed only in brain, heart, and skeletal muscle, tissues dominated by terminally differentiated, long-lived cells. Homozygous mutant mice exhibit muscle wasting and neurodegeneration, resulting in death around postnatal day 28. eEF1Α-2/S1 protein shares 92% identity with eEF1A-1; because specific antibodies for each were not available previously, it was difficult to study the developmental expression patterns of these two peptide elongation factors 1A inwasted and wild-type mice. We generated a peptide-derived antiserum that recognizes the eEF1Α-2/S1 isoform and does not cross-react with eEF1A-1. We characterized the expression profiles of eEF1A-1 and eEF1A-2/S1 during development in wild-type (+/+), heterozygous (+/wst), and homozygous (wst/wst) mice. In wild-type and heterozygous animals, eEF1A-2/S1 protein is present only in brain, heart, and muscle; the onset of its expression coincides with a concomitant decrease in the eEF1A-1 protein level. In wasted mutant tissues, even though eEF1A-2/S1 protein is absent, the scheduled decline of eEF1A-1 occurs nonetheless during postnatal development, as it does in wild-type counterparts. In the brain of adult wild-type mice, the eEF1A-2/S1 isoform is localized in neurons, whereas eEF1A-1 is found in non-neuronal cells. In neurons prior to postnatal day 7, eEF1A-1 is the major isoform, but it is later replaced by eEF1A-2/S1, which by postnatal day 14 is the only isoform present. The postdevelopmental appearance of eEF1A-2/S1 protein and the decline in eEF1A-1 expression in brain, heart, and muscle suggest that eEF1A-2/S1 is the adult form of peptide elongation factor, whereas its sister is the embryonic isoform, in these tissues. The absence of eEF1A-2/S1, as well as the on-schedule development-dependent disappearance of its sister gene, eEF1A, in wst/wstmice may result in loss of protein synthesis ability, which may account for the numerous defects and ultimate fatality seen in these mice. peptide elongation factor-1A glutathioneS-transferase polymerase chain reaction Tris-buffered saline, 0.5% Tween 20 base pair(s) kilobase(s) Peptide elongation factor-1A (eEF1A-1)1 is an abundant protein, once thought to be ubiquitously expressed; its major role is to mediate the transfer of charged aminoacyl-tRNA to the A site of the ribosome during peptide elongation. We have previously shown that the mRNA expression of this gene declines in rat brain, heart, and skeletal muscle during development (1Lee S. Wolfraim L.A. Wang E. J. Biol. Chem. 1993; 268: 24453-24459Abstract Full Text PDF PubMed Google Scholar, 2Merrick W.C. Microbiol. Rev. 1992; 56: 291-315Crossref PubMed Google Scholar), to the extent that in adult skeletal muscle, there is an almost complete loss of eEF1A-1 mRNA expression; a sister gene, eEF1A-2/S1, is expressed instead (3Lee S. Francoeur A.M. Liu S. Wang E. J. Biol. Chem. 1992; 267: 24064-24068Abstract Full Text PDF PubMed Google Scholar). Furthermore, in contrast to the ubiquitous expression of eEF1A-1 in many cell types, eEF1A-2/S1 expression is limited to the terminally differentiated cells of the brain, heart, and skeletal muscle (3Lee S. Francoeur A.M. Liu S. Wang E. J. Biol. Chem. 1992; 267: 24064-24068Abstract Full Text PDF PubMed Google Scholar, 4Lee S. Duttaroy A. Wang E. Cellular Aging and Cell Death. Wiley-Liss, Inc., New York1996: 139-151Google Scholar). Recent evidence suggests that eEF1A-2/S1 is an alternative peptide elongation factor-1A for neurons and myocytes, because the amino acid homology between them is 92% (5Ann D.K. Moutsatsos I.K. Nakamura T. Lin H.H. Mao P.L. Lee M.J. Chin S. Liem R.K. Wang E. J. Biol. Chem. 1991; 266: 10429-10437Abstract Full Text PDF PubMed Google Scholar, 6Knudsen S.M. Frydenberg J. Clark B.F. Leffers H. Eur. J. Biochem. 1993; 215: 549-554Crossref PubMed Scopus (139) Google Scholar), and eEF1A-2/S1 can perform peptide elongation in vitro (7Kahns S. Lund A. Kristensen P. Knudsen C.R. Clark B.F. Cavallius J. Merrick W.C. Nucleic Acids Res. 1998; 26: 1884-1890Crossref PubMed Scopus (94) Google Scholar). The absence of eEF1A-2/S1 mRNA, as a result of a deletion in the promoter and first exon of the eEF1A-2/S1 gene, was recently identified in the mutant mousewasted (8Chambers D.M. Peters J. Abbott C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4463-4468Crossref PubMed Scopus (129) Google Scholar). wasted mice exhibit severe muscle wasting and degeneration of motor neurons, beginning at 3 weeks after birth and die by the age of 28 days (8Chambers D.M. Peters J. Abbott C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4463-4468Crossref PubMed Scopus (129) Google Scholar, 9Shultz L.D. Sweet H.O. Davisson M.T. Coman D.R. Nature. 1982; 297: 402-404Crossref PubMed Scopus (81) Google Scholar, 10Lutsep H.L. Rodriguez M. J. Neuropathol. Exp. Neurol. 1989; 48: 519-533Crossref PubMed Scopus (25) Google Scholar, 11Inoue T. Tezuka H. Kada T. Aikawa K. Shultz L.D. Basic Life Sci. 1986; 39: 323-335PubMed Google Scholar). This coincides with the decline of eEF1A-1 expression in brain, heart, and skeletal muscle, without the normal concordant appearance of its sister gene; because neither form of elongation factor-1A is found, the consequential loss of protein synthesis ability is the likely cause of death. Distinguishing between eEF1A-1 and eEF1A-2/S1 protein expression was long hampered by the absence of isoform-specific antibodies; for example, it was impossible to determine the proportion of eEF1A-1versus eEF1-A2/S1 in any given skeletal muscle biopsy. This is specifically true with regard to a recent report, which concludes that there is no change in eEF1A in young and old muscle (12Welle S. Thornton C. Bhatt K. Krym M. J. Gerontol. A Biol. Sci. Med. Sci. 1997; 52: B235-B239Crossref PubMed Scopus (10) Google Scholar); this conclusion was based on a study using a pan-monoclonal antibody that recognizes both isoforms and thus cannot discriminate either qualitatively or quantitatively between the two sister forms of this gene family. To investigate the presence of eEF1A-2/S1 protein and differentiate its level from that of its sister, during mouse development, we generated a polyclonal antiserum that specifically recognizes the eEF1A-2/S1 isoform and does not cross-react with eEF1A-1. Using it along with another antiserum that specifically recognizes eEF1A-1, we characterized the protein presence of both peptide elongation factor-1A isoforms during development in normal and wasted mutant mice. A highly purified, sequence-specific peptide of eEF1A-2/S1 protein, CB5 (see Fig.1 A), was obtained by high performance liquid chromatography on a C18 reverse-phase column, using an acetonitrile and trifluoroacetic acid gradient. CB5 protein was solubilized in 20 mm phosphate buffer and coupled to keyhole limpet hemocyanin carrier protein (Pierce) through amino-terminal cysteine residues. Antibodies were generated by multiple injections of 200 μg of the peptide in Freund's incomplete adjuvant (Pierce), followed by two booster injections given at 3-week intervals with the same amount of antigen. One week after the final injection, the rabbits were sacrificed, and blood was collected. The eEF1A-1-specific antibody was raised against eEF1A-1 protein-specific peptide HT7 (see Fig. 1 B), as previously described (13Liu C.H. Liu S. Wang E. Biochem. Biophys. Res. Commun. 1993; 195: 1371-1378Crossref PubMed Scopus (6) Google Scholar). Brain, heart, muscle, liver, and spleen samples were obtained from mouse, rat, and human autopsy tissues. Tissue extraction and preparation were performed as previously described (14Upreti G.C. Ratcliff R.A. Riches P.C. Anal. Biochem. 1988; 168: 421-427Crossref PubMed Scopus (13) Google Scholar), with minor modifications. All tissues were extracted by homogenization in a 2-ml tissue grinder (Wheaton, Millville, NJ) with 1 ml of homogenizing buffer (300 mm sucrose, 150 mm KCl, 30 mm Tris-HCl, 5 mmMgCl2, 1.5 mm dithiothreitol, 1 mmEDTA, pH 8.0, and 1.5% Triton X-100) containing protease inhibitors (10 μg/ml aprotinin, 2 μg/ml leupeptin, 10 μg/ml pepstatin, and 2 mm phenylmethylsulfonyl fluoride) (ICN Biochemicals Canada, Montréal, Quebec). Protein extracts were then incubated with 150 μg/ml DNase I (Roche Molecular Biochemicals) on ice for 1 h and sonicated for 1 min in an ice bath, followed by centrifugation at 3000 rpm for 15 min. The resulting pellet was discarded, and the supernatant was designated as total protein. Protein concentration was assayed by a Bio-Rad protein assay reagent (Bio-Rad), using bovine plasma globulin as a standard. The construction of recombinant plasmids and expression of eEF1A-2/S1 protein in the glutathione S-transferase (GST) system using the pGEX-2T plasmid vector (Amersham Pharmacia Biotech) were reported previously, and the recombinant plasmid was referred to as GST-eEF1A-2/S1 (13Liu C.H. Liu S. Wang E. Biochem. Biophys. Res. Commun. 1993; 195: 1371-1378Crossref PubMed Scopus (6) Google Scholar). We also amplified the mouse eEF1A-1 cDNA by polymerase chain reaction (PCR) and subcloned it into the pGEX-2T expression vector, resulting in the GST-eEF1A-1 construct. The recombinant vectors were used to transform Escherichia coli. Individual clones were isolated, and the constructs were analyzed by DNA sequencing to identify those carrying the desired fragment, without any mutations that might have been introduced by PCR. Extraction and purification of GST and GST fusion proteins (GST-eEF1A-2/S1 and GST-eEF1A-1) using glutathione-Sepharose 4B beads were performed as previously described (15Smith D.B. Johnson K.S. Gene. 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). SDS-polyacrylamide gel electrophoresis was carried out on 10 or 12% gels using a discontinuous Laemmli system (16Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). For each assay of protein profile determination, 50 μg of total protein extract was loaded onto the SDS-polyacrylamide gel. After electrophoresis, the proteins were either stained with Coomassie Brilliant Blue R-250 or transferred onto nitrocellulose paper. The nitrocellulose membranes were stained with Ponceau S (Sigma) to visualize the electrophoretic pattern of total protein and subsequently destained with Tris-buffered saline (50 mm Tris-HCl, 500 mm NaCl) containing 0.5% Tween 20 (TBST), followed by blocking with 5% milk in TBST for 1–2 h. The membranes were probed with the anti-eEF1A-1 or anti-eEF1A-2/S1 polyclonal antibodies for 2 h at room temperature in TBST at a 1:2000 dilution. The membranes were then washed four times with TBST for 10 min each, incubated with goat anti-rabbit immunoglobulin conjugated to horseradish peroxidase (Cappel, Durham, NC) for 1 h at room temperature in a 1:20,000 dilution, and washed four times as described above. Nitrocellulose blots incubated with horseradish peroxidase-conjugated secondary antibodies were developed using enhanced chemiluminescence detection (ECL) (Amersham Pharmacia Biotech) according to the manufacturer's protocols and exposed to x-ray film. eEF1A-1 or eEF1A-2/S1 was immunoprecipitated from total protein extracts of adult rat brain, liver, and skeletal muscle, using a monoclonal anti-EF1A antibody (17Kaur K.J. Ruben L. J. Biol. Chem. 1994; 269: 23045-23050Abstract Full Text PDF PubMed Google Scholar) (Upstate Biotechnology, Lake Placid, NY) that recognizes both proteins. One mg of total protein extracts was pre-cleared with 50 μl of protein G-agarose (Sigma) for 30 min at 4 °C. Of the pre-cleared tissue extract, 720 μg was incubated with 3 μg of anti-EF1A monoclonal antibody for 17 h at 4 °C. Immune complexes were collected with 100 μl of protein G-agarose (Sigma) for 3 h at 4 °C. The protein G-agarose/immune complex was washed four times with 1 ml of ice-cold lysis buffer containing 1 mmphenylmethylsulfonyl fluoride, resuspended in 100 μl of SDS-loading buffer, and heated at 95 °C for 10 min. Twenty-five μl of the immunoprecipitated mixture was used for Western blotting analysis using the monoclonal anti-eEF1A-1, polyclonal anti-eEF1A-1, or anti-EF1A-2/S1 antibodies. A pair of heterozygous mice obtained from the Jackson Laboratory was further cross-bred. When the F1 offspring reached the age of day 7, ∼0.5 cm of the tails of the pups was cut, and genomic DNA was isolated as described (18Laird P.W. Zijderveld A. Linders K. Rudnicki M.A. Jaenisch R. Berns A. Nucleic Acids Res. 1991; 19: 4293Crossref PubMed Scopus (1303) Google Scholar). Essentially, tails were incubated in 0.5 ml of lysis buffer (100 mm Tris-HCl, pH 8.5, 5 mm EDTA, 0.2% SDS, 200 mm NaCl, 100 μg of proteinase K/ml) overnight at 55 °C. DNA was extracted using phenol/chloroform, precipitated using isopropanol, and dissolved in TE buffer (10 mm Tris-HCl, pH 7.4, EDTA). One hundred μg of genomic DNA was amplified in 50 μl of reaction buffer containing 10 mm dNTPs, 100 ng of primers (designated P1), and 4 units of rTth DNA polymerase (PerkinElmer Life Sciences) using long distance polymerase chain reaction. Primers selected for identifying eEF1A-2/S1 gene presence were designed as follows: Primers 1 (P1), sense (5′-GACAGAGAAAGAGATAGTGAG-3′; 4–24 base pairs (bp)) and antisense (5′-CGCCATTCTTGTATTGTTTG-3′; 18814–18795 bp); primers 2 (P2), sense (5′-TAGTGGCTCCTTGGAACAG-3′; 15752–15770 bp) and antisense (5′-CTACTCTCCCTGAATGCCTT-3′; 16204–16187 bp). Long distance PCR reactions were carried out at 95 °C for 3 min, followed by 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 3 min, for a total of 28 cycles. Regular PCR reactions in buffer containing 200 μg of genomic DNA, 100 ng of primers (P2), 10 mm dNTPs, and 1 unit of Taq DNA polymerase were performed at 99 °C for 5 s, followed by 94 °C for 40 s, 55 °C for 30 s, and 72 °C for 40 s, for a total of 30 cycles. PCR products were separated by electrophoresis in a 1% agarose gel. Western blot analysis of protein patterns from wild-type, heterozygous, and mutant mice from embryonic day 18 (E18) and postnatal days 1 (P1), P7, P14, P20, and P28 were scanned and quantified on a Molecular Dynamics Densitometer SI scanner (Sunnyvale, CA). The band intensities were quantified by ImageQuaNTTM software 4.1. For each sample, means and standard deviations were derived from specimens isolated from three different animals. All mouse tissues processed for immunofluorescence analysis were immediately embedded in Optimum Cutting Temperature (OCT) compound (VWR, Montréal, Quebec) at −80 °C after dissection. Frozen sections (10 μm) were obtained using a Leica microtome type 2800 Frigocut. The tissue sections were incubated with either the anti-eEF1A-1 antibody (1:1000) or the anti-eEF1A-2/S1 antibody (1:100). The secondary antibody used was goat anti-rabbit fluorescein isothiocyanate-conjugated (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). For double labeling, serial sections of samples previously stained for eEF1A-1 or eEF1Α-2/S1 immunoreactivity were incubated with a mouse antibody against a 68-kDa neurofilament neuronal marker (NF-68) (Sigma), followed by incubation with rhodamine-conjugated goat anti-mouse antibody (Cappel). The histochemical preparations were examined using a Bio-Rad MRC-600 laser scanning confocal microscope, equipped with a × 60 oil immersion objective. Images were scanned on two channels (red and green) and merged to produce a signal profile. In this model, all regions exhibiting colocalization of red and green emitters produce yellow fluorescence. The images were collected using the COMOS software package (Bio-Rad) and stored on an optical disc. Because of the 92% amino acid homology between the eEF1A-1 and eEF1A-2/S1 proteins, antibodies to distinguish between them were not available. To resolve this matter, we generated an eEF1A-2/S1-specific antibody (anti-eEF1A-2/S1) by immunizing rabbits with a purified peptide composed of a sequence unique to eEF1A-2/S1, designated CB5 (Fig.1). In addition, an eEF1A-1-specific antibody (anti-eEF1A-1) was similarly produced elsewhere, using the HT7 peptide with sequence unique to eEF1A-1 (Fig. 1). To verify the specificity of these polyclonal antibodies against eEF1A-1 or eEF1A-2/S1 proteins, we subcloned the eEF1A-1 and eEF1A-2/S1 cDNAs in the pGEX-2t vector, resulting in a GST fusion protein, and produced recombinant proteins (referred to as GST-eEF1A-1 and GST-eEF1A-2/S1, respectively). After induction with isopropyl-1-thio-β-d-galactopyranoside, transformedE. Coli bacteria were harvested, and bacterial proteins were analyzed by SDS-polyacrylamide gel electrophoresis. In bacteria transformed with a GST vector alone, the 26-kDa GST protein is induced upon treatment with isopropyl-1-thio-β-d-galactopyranoside, whereas bacteria possessing the pGEX-eEF1A-1 or pGEX-eEF1A-2/S1 constructs expressed 76-kDa proteins representing the GST-eEF1A-1 and GST-eEF1A-2/S1 fusion proteins, respectively (Fig.2 A). These fusion proteins were further purified by affinity chromatography on Sepharose 4B beads and yielded purified proteins of 26 kDa for GST and 76 kDa for GST-eEF1A-1 and GST-eEF1A-2/S1 (Fig. 2 B). The size of both eEF1A-1 and eEF1A-2/S1 proteins after thrombin cleavage of the two recombinant fusion proteins, GST-eEF1A-1 and GST-eEF1A-2/S1, was 50 kDa, consistent with the predicted size of the open reading frame of the cDNA sequence for both proteins. To characterize the peptide-derived antibody for eEF1A-2/S1 protein, immunoblotting assays were performed to assess its reactivity with the purified GST-eEF1A-2/S1 and absence of reactivity with GST-eEF1A-1. In fact, anti-eEF1A-2/S1 recognizes only the GST-eEF1A-2/S1 fusion protein and does not react with GST-eEF1A-1 or GST proteins (Fig.2 C). The anti-eEF1A-1 antibody recognizes only GST-eEF1A-1 and does not cross-react with either GST or GST-eEF1A-2/S1 proteins (Fig. 2 C). Furthermore, we assessed the specificity of our antibodies by competition with soluble HT7 and CB5 peptides (Fig.2 D); the binding capability of anti-eEF1A-1 to the GST-eEF1A-1 fusion protein is competed by soluble HT7 peptide in a concentration-dependent manner, whereas it is unaffected by soluble CB5 peptide (Fig. 2 D). Conversely, the CB5 soluble peptide competes for the reaction of anti-eEF1A-2/S1 to GST-eEF1A-2/S1 fusion protein in a concentration-dependent manner, and the HT7 peptide does not compete for anti-eEF1A-2/S1 to GST-eEF1A-2/S1 (Fig. 2 E). Therefore, we conclude that the CB5 peptide-derived anti-eEF1A-2/S1 is specific for its intended substrate, eEF1A-2/S1, and the HT7 peptide-derived anti-eEF1A-1 is specific for eEF1A-1. We used a monoclonal antibody that recognizes both eEF1A-1 and eEF1A-2/S1 in an immunoprecipitation reaction against total protein extracts from adult rat tissues (liver, skeletal muscle, and brain) and determined the presence of eEF1A-1 and eEF1A-2/S1 in the immunoprecipitated products by Western blot analysis, using either anti-eEF1A-1 or anti-eEF1A-2/S1 antibodies. Fig.3 shows the total and immunoprecipitated protein extracts probed with the anti-eEF1A-1 monoclonal antibody (A), secondary antibody alone as a control (Panel B), and the anti-eEF1A-1 and anti-eEF1A-2/S1 antibodies (C and D, respectively). Evidently the monoclonal antibody for eEF1A-1 immunoprecipitates both eEF1A-1 and eEF1A-2/S1 proteins; probing the immunoprecipitates with this antibody detects the presence of both eEF1A isoforms, eEF1A-1 in liver and eEF1A-2/S1 in skeletal muscle and brain. The presence of eEF1A-1 (which has a slightly smaller molecular weight) was not detected in muscle and brain, because we used adult tissues, where the level of eEF1A-1 protein is drastically diminished in later postdevelopmental stages (described in this report; Figs. 4 and7). However, eEF1A-1 was detected in liver, an eEF1A-2/S1-negative tissue (Fig. 3 A). To verify this finding, we probed the immunoprecipitates with anti-eEF1A-1 and anti-eEF1A-2/S1 antibodies; we found eEF1A-1 only in the liver (and a small amount in the brain (Fig.3 D)) and eEF1A-2/S1 only in skeletal muscle, and the latter as a major band in brain (Fig. 3 C). These results verify the specificity of the anti-eEF1A-1 and anti-eEF1A-2/S1 polyclonal antibodies in tissue extracts and demonstrate that the commercially available monoclonal antibody to eEF1A-1 recognizes both isoforms of eEF1A indiscriminately.Figure 4eEF1A-1 and eEF1A-2/S1 proteins in adult mouse, rat, and human tissues. Immunoblotting analysis of eEF1A-1 and eEF1A-2 protein presence in mouse (3-months-old), rat (3-months-old), and human autopsy (68-year-old woman) tissues. Extraction was performed as described under "Materials and Methods." IA, various mouse tissues (brain (B), heart (H), skeletal muscle (M), liver (L), spleen (S)) were probed with eEF1A-1-specific antibody, revealing a 50-kDa protein band in abundance in liver and spleen. The 50-kDa protein is also present in moderation in brain and muscle but absent in heart (arrow).IB, on the other hand, probing the same mouse tissues with eEF1A-2/S1-specific antibody reveals a differentially expressed 50-kDa protein band in brain, heart, and muscle alone (arrow), in addition to two prominent nonspecific 66- and 45-kDa bands (*).IC, immunoblotting with rabbit preimmune serum verifies that the additional bands seen in IB (*) are the result of some pre-immune reaction in the rabbits used for immunization.IIA and IIB, in rat tissues, eEF1A-1 and eEF1A-2/S1 proteins exhibit the same pattern of expression;i.e. eEF1A-2/S1 protein is restricted to the same three tissues, brain, heart, and muscle, whereas eEF1A-1 protein is prominent in liver and spleen (arrow) but reduced in abundance in brain and skeletal muscle and absent in heart. The same nonspecific reactions associated with the eEF1A-2/S1-specific antibody are also observed (*). IIIA and IIIB, human autopsy tissues (68-year-old woman), also probed with the same antibodies, reveal a similar tissue-specific pattern of eEF1A-1 and eEF1A-2/S1 protein expression (arrow). Again, the same nonspecific antibody reaction is seen as with the preimmune serum (*).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Differential expression of eEF1A-1 and eEF1A-2/S1 proteins during development of wild-type, heterozygous, and mutant mice. Mouse tissues from brain (A), heart (B), skeletal muscle (C), and liver (D) obtained at ages of embryonic day 18 (E18) and postnatal (P) days 1-, 7-, 14-, 20-, and 28-days-old were used for immunoblotting assays from wild-type (+/+), heterozygous (+/−), and mutant (−/−) mice. Protein extracts of the four selected tissues were processed for immunoblot analysis with eEF1A-1- and eEF1A-2/S1-specific antibodies, as described under "Materials and Methods."View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our laboratory reported previously that eEF1A-2/S1 mRNA expression is restricted to three tissues: brain, heart, and skeletal muscle. The appearance of eEF1A-2/S1 mRNA is a late differentiation event, occurring after myotubes are formed and neuronal terminal differentiation is completed (1Lee S. Wolfraim L.A. Wang E. J. Biol. Chem. 1993; 268: 24453-24459Abstract Full Text PDF PubMed Google Scholar). Using anti-eEF1A-1 and anti-eEF1A-2/S1 antibodies, we investigated the presence of eEF1A-1 and eEF1A-2/S1 proteins in various tissues previously characterized for positive or negative expressions of eEF1A-2/S1 mRNA. Immunoblotting analyses were performed on various tissues isolated from adult mice, rats, and humans. A major band of 50 kDa in mouse liver and spleen was detected using the anti-eEF1A-1 antibody (Fig. 4 IA). The 50-kDa eEF1A-1 protein was also present at a very low level in heart and reduced in abundance in brain and skeletal muscle (Fig.4 IA). By probing with anti-eEF1A-2/S1, a 50-kDa protein was detected only in brain, heart, and muscle (Fig.4 IB). Anti-eEF1A-2/S1 also detected two other bands at 66 and 45 kDa; to determine whether these two additional bands were specific, a reaction with a rabbit antiserum obtained prior to immunization was compared with the anti-eEF1A-2/S1 reaction, in which the preimmune serum also yielded 66- and 45-kDa protein bands but lacked the specific reaction with the 50-kDa eEF1A-2/S1 protein (Fig.4 IC). We conclude that the 66- and 45-kDa bands detected using anti-eEF1A-2/S1 are the result of nonspecific reactions of the preimmune serum. Similar studies in adult rat (3 months) and human autopsy tissues (68-years-old) revealed the same pattern of expression found in mice. In rat and human tissue, eEF1A-2/S1 protein is restricted to brain, heart, and muscle (Fig. 4, IIA, IIB,IIIA, and IIIB). However, eEF1A-1 protein levels in these three eEF1A-2/S1-positive tissues vary among the three tested species, humans, mice, and rats, ranging from non-detectable to modest levels. This variation may be due to species differences. Nevertheless, results of these studies at the protein level are in overall agreement with the specific gene expression patterns examined at the message level, because eEF1A-2/S1 mRNA is only expressed in brain, heart, and skeletal muscle (3Lee S. Francoeur A.M. Liu S. Wang E. J. Biol. Chem. 1992; 267: 24064-24068Abstract Full Text PDF PubMed Google Scholar, 6Knudsen S.M. Frydenberg J. Clark B.F. Leffers H. Eur. J. Biochem. 1993; 215: 549-554Crossref PubMed Scopus (139) Google Scholar). We also investigated rat smooth muscle (bladder and stomach) for both eEF1A-1 and eEF1A-2/S1 proteins (Fig.5); lung and spleen were also studied, along with liver and skeletal muscle, which we know are negative and positive, respectively, for eEF1A-2/S1 protein. Fig. 5 Ashows that eEF1A-1 protein is present in all tested tissues, whereas eEF1A-2/S1 is only present in skeletal muscle but not in smooth muscle of aorta, bladder, or stomach or any other eEF1A-2/S1-negative tissues (including liver, lung, and spleen) (Fig. 5 B). The wastedmouse is a spontaneous mutant strain from the Jackson Laboratories (9Shultz L.D. Sweet H.O. Davisson M.T. Coman D.R. Nature. 1982; 297: 402-404Crossref PubMed Scopus (81) Google Scholar). The mutant phenotype was recently shown to correlate with a 15.8-kb deletion, including the promoter and first non-coding exon of the eEF1A-2/S1 gene (8Chambers D.M. Peters J. Abbott C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4463-4468Crossref PubMed Scopus (129) Google Scholar) (Fig.6 A). To detect the presence of the mutant allele, we designed oligonucleotide primers to amplify around the boundary of the deletion (the P1 fragment), with the forward primer upstream and the reverse primer downstream, of the 15.8-kb deleted region (Fig. 6 A). PCR using these two primers amplifies a 2.9-kb DNA fragment from both homozygous (−/−) and heterozygous (+/−) mice (Fig. 6 B, lanes 2 and4). This PCR product is not observed with the wild-type (+/+) mouse DNA, because the expected PCR reaction yields a product larger than 18 kb (Fig. 6 B, lane 3). To detect a wild-type allele, oligonucleotide primers were designed to amplify a 456-bp fragment within the 1.5-kb deletion (the P2 fragment). The absence of the fragment was used to identify the homozygous mutants (Fig. 6 B, lane 6), whereas the PCR product is readily obtained in the parallel PCR reactions of the wild-type and heterozygous animals (Fig. 6 B, lanes 7 and8). Using anti-eEF1A-1 and anti-eEF1A-2/S1 antibodies, we expanded the previous findings
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