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• W2000732040 abstract "Paranemin was initially found to copurify with the intermediate filament (IF) proteins vimentin and desmin from embryonic chick skeletal muscle and was described as an IF-associated protein (IFAP). We have purified paranemin from embryonic chick skeletal muscle, prepared antibodies, and demonstrated that they label at the Z-lines of both adult avian and porcine cardiac and skeletal muscle myofibrils. We determined the cDNA sequence of paranemin by immunoscreening a λgt22A cDNA library from embryonic chick skeletal muscle. Northern blot analysis revealed a single transcript of 5.3 kilobases, which is much smaller than predicted from the size of paranemin (280 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The derived amino acid sequence of paranemin (1,606 residues; 178,161 kDa) contains the conserved IF rod domain (308 amino acids), which has highest homology to the rod domains of nestin and tanabin. Thus, paranemin is an IF protein rather than an IFAP. Sequence analysis also revealed that the partial cDNA sequences of two proteins, namely EAP-300 and IFAPa-400, are almost identical to regions of the cDNA sequence of paranemin. The complete paranemin cDNA was expressed in a cell line (SW13) with, and without, detectable cytoplasmic IFs. Antibody labeling of these cells suggests that paranemin does not form IFs by itself, but rather is incorporated into heteropolymeric IFs with vimentin. Paranemin was initially found to copurify with the intermediate filament (IF) proteins vimentin and desmin from embryonic chick skeletal muscle and was described as an IF-associated protein (IFAP). We have purified paranemin from embryonic chick skeletal muscle, prepared antibodies, and demonstrated that they label at the Z-lines of both adult avian and porcine cardiac and skeletal muscle myofibrils. We determined the cDNA sequence of paranemin by immunoscreening a λgt22A cDNA library from embryonic chick skeletal muscle. Northern blot analysis revealed a single transcript of 5.3 kilobases, which is much smaller than predicted from the size of paranemin (280 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The derived amino acid sequence of paranemin (1,606 residues; 178,161 kDa) contains the conserved IF rod domain (308 amino acids), which has highest homology to the rod domains of nestin and tanabin. Thus, paranemin is an IF protein rather than an IFAP. Sequence analysis also revealed that the partial cDNA sequences of two proteins, namely EAP-300 and IFAPa-400, are almost identical to regions of the cDNA sequence of paranemin. The complete paranemin cDNA was expressed in a cell line (SW13) with, and without, detectable cytoplasmic IFs. Antibody labeling of these cells suggests that paranemin does not form IFs by itself, but rather is incorporated into heteropolymeric IFs with vimentin. Intermediate filaments (IFs), 1The abbreviations used are: IF, intermediate filament; IFAP, intermediate filament-associated protein; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s); RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; bp, base pair(s). 1The abbreviations used are: IF, intermediate filament; IFAP, intermediate filament-associated protein; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s); RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; bp, base pair(s). together with microfilaments and microtubules, comprise the three major classes of cytoskeletal filaments in cells of nearly all differentiated, multicellular eukaryotes (1Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1123) Google Scholar, 2Robson R.M. Curr. Opin. Cell Biol. 1989; 1: 36-43Crossref PubMed Scopus (58) Google Scholar, 3Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1273) Google Scholar, 4Klymkowsky M.W. Curr. Opin. Cell Biol. 1995; 7: 46-54Crossref PubMed Scopus (102) Google Scholar, 5Fuchs E. Annu. Rev. Genet. 1996; 30: 197-231Crossref PubMed Scopus (147) Google Scholar). Much is now known about the cellular distribution, structure, and assembly of IFs (for recent reviews, see Refs. 3Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1273) Google Scholar, 4Klymkowsky M.W. Curr. Opin. Cell Biol. 1995; 7: 46-54Crossref PubMed Scopus (102) Google Scholar, 5Fuchs E. Annu. Rev. Genet. 1996; 30: 197-231Crossref PubMed Scopus (147) Google Scholar, 6Foisner R. BioEssays. 1997; 19: 297-305Crossref PubMed Scopus (61) Google Scholar). Most IF proteins can be grouped into five major types or classes based upon their sequence and structure (1Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1123) Google Scholar, 2Robson R.M. Curr. Opin. Cell Biol. 1989; 1: 36-43Crossref PubMed Scopus (58) Google Scholar, 3Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1273) Google Scholar, 5Fuchs E. Annu. Rev. Genet. 1996; 30: 197-231Crossref PubMed Scopus (147) Google Scholar), with a small number of IF proteins possibly comprising additional classes (3Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1273) Google Scholar). The IFs, in general, are often considered to play an important role in the mechanical integration of cellular space (7Lazarides E. Nature. 1980; 283: 249-256Crossref PubMed Scopus (1443) Google Scholar, 8Goldman R.D. Khuon S. Chou Y.H. Opal P. Steinert P.M. J. Cell Biol. 1996; 134: 971-983Crossref PubMed Scopus (309) Google Scholar, 9Chou Y.H. Skalli O. Goldman R.D. Curr. Opin. Cell Biol. 1997; 9: 49-53Crossref PubMed Scopus (69) Google Scholar), and their more specific cellular functions recently are becoming evident as well (3Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1273) Google Scholar, 4Klymkowsky M.W. Curr. Opin. Cell Biol. 1995; 7: 46-54Crossref PubMed Scopus (102) Google Scholar, 5Fuchs E. Annu. Rev. Genet. 1996; 30: 197-231Crossref PubMed Scopus (147) Google Scholar, 10Maniotis A.J. Chen C.S. Ingber D.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 849-854Crossref PubMed Scopus (1322) Google Scholar). Intermediate filaments in mature striated muscle cells are composed primarily of desmin (7Lazarides E. Nature. 1980; 283: 249-256Crossref PubMed Scopus (1443) Google Scholar, 11Huiatt T.W. Robson R.M. Arakawa N. Stromer M.H. J. Biol. Chem. 1980; 255: 6981-6989Abstract Full Text PDF PubMed Google Scholar, 12O'Shea J.M. Robson R.M. Hartzer M.K. Huiatt T.W. Rathbun W.E. Stromer M.H. Biochem. J. 1981; 195: 345-356Crossref PubMed Scopus (28) Google Scholar). They are present in a collar-like arrangement around the myofibrillar Z-lines where they appear to connect adjacent myofibrils together, and possibly help link the peripheral layer of myofibrils to costameric sites along the muscle cell membrane (4Klymkowsky M.W. Curr. Opin. Cell Biol. 1995; 7: 46-54Crossref PubMed Scopus (102) Google Scholar, 13Richardson F.L. Stromer M.H. Huiatt T.W. Robson R.M. Eur. J. Cell Biol. 1981; 26: 91-101PubMed Google Scholar, 14Tokuyasu K.T. Maher P.A. Dutton A.H. Singer S.J. Ann. N. Y. Acad. Sci. 1985; 455: 200-212Crossref PubMed Scopus (34) Google Scholar, 15Yagyu M. Robson R.M. Stromer M.H. Proc. 12th Int. Congr. Electr. Micros. 1990; 3: 454-455Google Scholar, 16Price M.G. Adv. Struct. Biol. 1991; 1: 175-207Google Scholar). Paranemin, ∼280 kDa by SDS-PAGE, was first identified in embryonic chick skeletal muscle (17Breckler J. Lazarides E. J. Cell Biol. 1982; 92: 795-806Crossref PubMed Scopus (44) Google Scholar). Paranemin has been considered an IF-associated protein (IFAP) (1Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1123) Google Scholar, 18Foisner R. Wiche G. Curr. Opin. Cell Biol. 1991; 3: 75-81Crossref PubMed Scopus (94) Google Scholar) because it copurified with the type III IF proteins vimentin and desmin from embryonic muscle (17Breckler J. Lazarides E. J. Cell Biol. 1982; 92: 795-806Crossref PubMed Scopus (44) Google Scholar), and colocalized with the major IF proteins vimentin and desmin at the periphery of avian myofibrillar Z-lines (17Breckler J. Lazarides E. J. Cell Biol. 1982; 92: 795-806Crossref PubMed Scopus (44) Google Scholar, 19Price M.G. Lazarides E. J. Cell Biol. 1983; 97: 1860-1874Crossref PubMed Scopus (77) Google Scholar). Its immunolocalization indicated a developmentally regulated expression in chick myogenic cells, and a more restricted expression in adult chicken muscle cells (17Breckler J. Lazarides E. J. Cell Biol. 1982; 92: 795-806Crossref PubMed Scopus (44) Google Scholar, 19Price M.G. Lazarides E. J. Cell Biol. 1983; 97: 1860-1874Crossref PubMed Scopus (77) Google Scholar). As shown herein, paranemin contains the ∼310-amino acid rod domain characteristic of IF proteins. Therefore, paranemin is a member of the IF protein superfamily rather than an IFAP. By sequence comparisons with other IF proteins, we have found that paranemin shares some homologies to other IF proteins, but significant differences as well. We also show in this report that regions of paranemin's sequence are almost identical to the partial cDNA sequences reported for two other proteins, EAP-300 (20Kelly M.M. Phanhthoutath C. Brees D.K. McCabe C.F. Cole G.J. Dev. Brain Res. 1995; 85: 31-47Crossref PubMed Scopus (18) Google Scholar) and IFAPa-400 (21Simard J.-L. Cossette L.J. Rong P.-M. Martinoli M.G. Pelletier G. Vincent M. Dev. Brain Res. 1992; 70: 173-180Crossref PubMed Scopus (10) Google Scholar). The functional ability of paranemin to assemble into IFs was tested by cell transfection of the complete paranemin cDNA. Expression of paranemin cDNA in SW13 cell clones that either do, or do not, express vimentin, suggests that paranemin by itself is unable to form homopolymeric IFs, but that it can coassemble with vimentin into heteropolymeric IFs. Approximately 100 g of thigh and breast muscles were dissected from 144 14-day-old chick embryos and homogenized in 235 ml of 130 mm KCl, 5 mm EDTA, 20 mmTris-HCl, pH 7.5, as described (17Breckler J. Lazarides E. J. Cell Biol. 1982; 92: 795-806Crossref PubMed Scopus (44) Google Scholar). The resulting homogenate was centrifuged for 15 min at 20,700 × g. The supernatant was filtered through glass wool and centrifuged for 90 min at 125,000 × g. The resulting supernatant, referred to as crude paranemin, was separated into four equal volumes (∼50 ml, ∼10 mg/ml), and each was applied to a 2.6 cm × 110-cm Bio-Gel A-5m (Bio-Rad) column previously equilibrated with 100 mm NaCl, 0.1 mm EDTA, 20 mm Tris-HCl, pH 7.5 (17Breckler J. Lazarides E. J. Cell Biol. 1982; 92: 795-806Crossref PubMed Scopus (44) Google Scholar). Elution was carried out in equilibration buffer at a flow rate of 24 ml/h. All column fractions for the entire purification were collected in the presence of N α-p-tosyl-l-argininamide methyl ester to reduce proteolysis. Paranemin-enriched fractions (as determined by Western blotting) eluted near the column void volume, and were collected from the four column elutions, pooled, dialyzed against 6 m urea, 1 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, 10 mmTris-HCl, pH 7.5, and subjected to hydroxyapatite chromatography on a 2.6 cm × 33-cm column of HA-Ultrogel (Sepracor) previously equilibrated in the dialysis buffer. The column was eluted in the dialysis buffer with a linear 0–500 mm potassium phosphate gradient, pH 7.5, programmed on a Gradifrac System (Pharmacia Biotech) at a flow rate of 24 ml/h. Paranemin-containing fractions eluted from the column at ∼200 mm potassium phosphate, and were pooled, dialyzed against 6 m urea, 1 mmdithiothreitol, 5 mm EDTA, 0.2 mmphenylmethylsulfonyl fluoride, 20 mm Tris-HCl, pH 7.5, and loaded onto a 2.6 cm × 20-cm DEAE-cellulose (Whatman, DE-52) column previously equilibrated with the dialysis buffer. The column was eluted in the dialysis buffer with a linear 0–2 m NaCl gradient at a flow rate of 24 ml/h. Paranemin-containing fractions eluted from the column at ∼1 m NaCl, and were pooled and dialyzed against column equilibration buffer. The sample of purified paranemin could be stored at −70 °C for at least 4 weeks without detectable degradation. Monoclonal antibodies were prepared following procedures described by Hemken et al. (22Hemken P. Guo X.-L. Wang Z.-Q. Zhang K. Gluck S. J. Biol. Chem. 1992; 267: 9948-9957Abstract Full Text PDF PubMed Google Scholar). Fusions with SP2/0-Ag14 (ATCC CRL 1581) myeloma cells were performed essentially according to published protocols (23McKearn T.J. Fitch F.W. Smilek D.E. Sarmiento M. Stuart F.P. Immunol. Rev. 1979; 47: 91-115Crossref PubMed Scopus (50) Google Scholar). Enzyme-linked immunosorbent assays were performed to screen the monoclonals as described (22Hemken P. Guo X.-L. Wang Z.-Q. Zhang K. Gluck S. J. Biol. Chem. 1992; 267: 9948-9957Abstract Full Text PDF PubMed Google Scholar). Paranemin bands from SDS-PAGE gels, each containing ∼1 mg of protein, were minced and homogenized in 1-ml aliquots of phosphate-buffered saline and emulsified with equal volumes of TiterMax (Vaxcell, Inc.) adjuvant. New Zealand White specific pathogen-free rabbits were injected with purified paranemin at several sites subcutaneously over the back and at one site intramuscularly in the thigh. The rabbits were boosted twice after the initial injection at 4–6-week intervals. Titers were determined by Western blotting. Purified paranemin was subjected to SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes (Micron Separations) (24Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar). For amino acid analysis, the 280-kDa paranemin band was excised from the washed blots and hydrolyzed in vacuo in 6 n HCl at 150 °C for 1, 2, and 3 h (25Moore S. Stein W.H. Methods Enzymol. 1963; 6: 819-831Crossref Scopus (1565) Google Scholar). Amino acid composition was determined with an Applied Biosystems Amino Acid Analyzer at the Iowa State University Protein Facility. For amino acid sequencing, deblocking using trifluoroacetic acid was necessary (26Wellner D. Panneerselvam C. Horecker B.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1947-1949Crossref PubMed Scopus (95) Google Scholar). The sample was analyzed with a 477A Protein Sequencer/120A Analyzer (Applied Biosystems Inc.). SDS-PAGE and Western blotting were performed essentially as described previously (22Hemken P. Guo X.-L. Wang Z.-Q. Zhang K. Gluck S. J. Biol. Chem. 1992; 267: 9948-9957Abstract Full Text PDF PubMed Google Scholar). Purified paranemin and synemin (27Becker B. Bellin R.M. Sernett S.W. Huiatt T.W. Robson R.M. Biochem. Biophys. Res. Commun. 1995; 213: 796-802Crossref PubMed Scopus (40) Google Scholar, 28Bellin R.M. Sernett S.W. Robson R.M. Kreis T. Vale R. Guidebook to the Cytoskeleton and Motor Proteins, Second Edition. Oxford University Press, Oxford1998Google Scholar), and paranemin that had been partially digested with purified m-calpain (0.9 units/mg for 10 min) in 2 m urea, 5 mm CaCl2, 10 mm β-mercaptoethanol, and 10 mm Tris-HCl, pH 7.4, were run on SDS-polyacrylamide gels and transferred to nitrocellulose (29Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44841) Google Scholar, 30Burnette W.N. Anal. Biochem. 1981; 112: 195-203Crossref PubMed Scopus (5889) Google Scholar). The blots were probed with monoclonal (4D3, 4C7, or 3B12 culture supernatant) or polyclonal antibodies to paranemin. Secondary antibodies included alkaline phosphatase-labeled sheep anti-mouse IgG (Sigma) and goat anti-rabbit IgG (Sigma), and horseradish peroxidase-labeled goat anti-mouse IgG (Sigma). Antibody labeling for the antibody specificity experiments was visualized by using the substrate nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. Labeling for the paranemin purification, β-galactosidase fusion protein experiments, and the expression of full-length paranemin in Escherichia coli was visualized by chemiluminescence (ECL, Amersham). Total RNA was isolated from skeletal muscle of 14-day embryonic chicks by extraction with guanidinium thiocyanate at 4 °C (31Han J.H. Stratowa C. Rutter W.J. Biochemistry. 1987; 26: 1617-1625Crossref PubMed Scopus (349) Google Scholar). Poly(A)+ RNA was purified by chromatography on an oligo(dT)-cellulose column (Collaborative Biomedical Products) (32Aviv H. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 1408-1412Crossref PubMed Scopus (5179) Google Scholar). Ten μg of poly(A)+ RNA were used to construct an oligo(dT)-primed, directional cDNA expression library in λgt22A using the Lambda SuperScript system (Life Technologies, Inc.). By immunoscreening of the λgt22A cDNA library using monoclonal antibody 4D3 to paranemin and the ProtoBlot detection system (Promega Corp.), 132 positive plaques were identified. Of these, 20 were purified to homogeneity. For sequencing, the two clones having the largest inserts, 9 (3.2 kb) and 40 (2.7 kb), were subcloned into pBluescript II SK(+) vector (Stratagene) (33Sambrook J. Fritsch E.G. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The cDNA library was rescreened by hybridization using a 205-bp BstXI restriction enzyme-generated cDNA probe from the 5′ end of clone 9 and a 2.2-kb cDNA probe generated by 5′-rapid amplification of cDNA ends (5′-RACE). For the 2.2-kb 5′-RACE probe, reverse-transcription of a poly(A)+ RNA sample was performed with a gene-specific primer spanning paranemin cDNA positions 2148–2165. PCR amplification of the target cDNA was performed using a paranemin-specific primer spanning cDNA positions 2130–2147 and the Anchor Primer (Life Technologies, Inc.). The λgt22A library was screened with both probes using a Digoxigenin DNA Labeling and Detection Kit (Boehringer Mannheim). A total of 200 positive plaques were chosen, 100 from each probe. PCR was used to screen λ phage mixes of all 200 positive plaques for sequence overlapping clone 9 by using a gene-specific primer spanning paranemin cDNA positions 2130–2147 and the λgt11 Upstream Amplimer Primer (CLONTECH). Twenty clones that contained a 2.2-kb product by PCR screening were purified to homogeneity. Clones 3, 24, 89, and 169 were subcloned into pBluescript II SK(+) vector for sequencing. Northern blots were performed essentially as described (34Wiche G. Becker B. Luber K. Weitzer G. Castañon M.J. Hauptmann R. Stratowa C. Stewart M. J. Cell Biol. 1991; 114: 83-99Crossref PubMed Scopus (162) Google Scholar, 35Davis L.G. Dibner M.D. Battey J.F. Methods in Molecular Biology. Elsevier Science Publishing, New York1986: 143-146Google Scholar) on poly(A)+ RNA samples from skeletal muscle cells of 14-day embryonic chick with synthetic RNA markers (Life Technologies, Inc.) for size determination. Hybridization was performed overnight at 42 °C using 32P random prime-labeled cDNA probes diluted to a specific activity between 1.7 × 104 and 1.7 × 105 Bq/ml. Unbound probe was removed by washing the filter at a final stringency of 0.2 × SSC and 0.1% (w/v) SDS at 50 °C for 30 min. Filters were exposed to Hyperfilm-MP (Amersham) for 3 days at −70 °C. Synthetic RNA markers were visualized by staining in 0.04% (w/v) methylene blue in 0.5 m sodium acetate, pH 5.2. Purified phages of clone 9 were tested with monoclonal antibodies 4C7 and 3B12, and with rabbit polyclonal antiserum to paranemin. Bacterial lawns were infected with approximately 500 plaque forming units for each 150-mm plate. Incubations and visualization of antibody binding were done identically to immunoscreening of the library with monoclonal antibody 4D3. Crude lysates from recombinant lysogens in E. coli Y1089r− were prepared as described (36Huynh T.V. Young R.A. Davis R.W. Glover D.M. DNA Cloning: A Practical Approach. 1. IRL Press Limited, Oxford, UK1985: 76-78Google Scholar) for subsequent SDS-PAGE analysis and Western blotting. For analysis of the protein expressed from the full-length paranemin sequence, clone 24 was inserted into the pProExHTb vector (Life Technologies, Inc.) and expressed in E. coli XL-I Blue. Bacterial lysates, along with paranemin purified from muscle tissue, were subjected to Western blotting using the paranemin polyclonal antibodies. Nucleotide sequencing was conducted at the Iowa State University DNA Sequencing and Synthesis Facility using automated sequencers (373A and 377 DNA Sequencers, Applied Biosystems Inc.). Both strands of overlapping clones 9 and 89 were sequenced at least one time. Sequences at both 5′- and 3′-ends and at least three internal regions were also obtained for clones 3, 24, 40, and 169, which spanned the following paranemin cDNA positions: 3 (−24 to 2853), 24 (−14 to 5261), 89 (−14 to 2164), 169 (−9 to 2574), 9 (2035 to 5255), and 40 (2575 to 5255). Analysis of the nucleotide and predicted amino acid sequences was done using the GCG software package (Program Manual for the Wisconsin Package, Version 8) (37Devereux J. Haekerli P. Smithies O. Nucleic Acids Res. 1984; 12: 387-395Crossref PubMed Scopus (11534) Google Scholar). Dot matrix comparisons were performed with COMPARE, and the output was displayed by DOTPLOT (38Maizel Jr., J.V. Lenk R.P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7665-7669Crossref PubMed Scopus (345) Google Scholar). Multiple sequence alignment was done using PILEUP. Sequences of the rod domains from the following IF proteins were aligned: frog tanabin (39Hemmati-Brivanlou A. Mann R.W. Harland R.M. Neuron. 1992; 9: 417-428Abstract Full Text PDF PubMed Scopus (51) Google Scholar), human nestin (40Lendahl U. Zimmerman L.B. McKay R.D.G. Cell. 1990; 60: 585-595Abstract Full Text PDF PubMed Scopus (2765) Google Scholar), human keratin 14 (41Marchuk D. McCrohon S. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1609-1613Crossref PubMed Scopus (104) Google Scholar), chicken synemin (27Becker B. Bellin R.M. Sernett S.W. Huiatt T.W. Robson R.M. Biochem. Biophys. Res. Commun. 1995; 213: 796-802Crossref PubMed Scopus (40) Google Scholar), chicken vimentin (42Zehner Z.E. Li Y. Roe B.A. Paterson B.M. Sax C.M. J. Biol. Chem. 1987; 262: 8112-8120Abstract Full Text PDF PubMed Google Scholar), mouse NF-M (43Levy E. Liem R.K.H. D'Eustachio P. Cowen N.J. Eur. J. Biochem. 1987; 166: 71-77Crossref PubMed Scopus (106) Google Scholar), and chicken lamin A (44Peter M. Kitten G.T. Lenher C.F. Vorburger K. Bailer S.M. Maridor G. Nigg E.A. J. Mol. Biol. 1989; 208: 393-404Crossref PubMed Scopus (81) Google Scholar). The percent identity was calculated by individual alignments of each rod domain using GAP. The IF signature was identified with MOTIFS. Secondary structure predictions were analyzed according to the methods of Chou and Fassman (45Chou P.Y. Fasman G.D. Adv. Enzymol. 1978; 47: 45-148PubMed Google Scholar) and Garnier et al. (46Garnier J. Osguthorpe D.J. Robson B. J. Mol. Biol. 1978; 120: 97-120Crossref PubMed Scopus (3410) Google Scholar) via PEPTIDESTRUCTURE and PLOTSTRUCTURE. A restriction enzyme map and isoelectric point of paranemin sequence were predicted by using MAPSORT and ISOELECTRIC, respectively. Basic Local Alignment Search Tool (BLAST) searches were performed using the NCBI BLAST E-mail server (47Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69652) Google Scholar) to search both nucleotide (PDB, GBUpdate, GenBank, EmblUpdate, and EMBL) and peptide (PDB, SwissProt, PIR, SPUpdate, GenPept, and GPUpdate) sequence data bases. The 5′-RACE system Life Technologies, Inc.) was used to synthesize cDNA corresponding to the 5′-end of the paranemin transcript (48Frohman M.A. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8998-9002Crossref PubMed Scopus (4329) Google Scholar). Reverse-transcription of a poly(A)+ RNA sample was performed with gene-specific primers spanning paranemin cDNA positions 1011 to 1028 and 2130 to 2147, and the SuperScript II reverse transcriptase (Life Technologies, Inc.). After removal of excess dNTP and primers, the cDNA was tailed with dCTP and terminal deoxynucleotidyl transferase. Amplification of target cDNA was performed with Taq DNA polymerase (Promega), the anchor primer, and nested gene-specific primers spanning paranemin cDNA positions 455 to 472 or 1011 to 1028. PCR reactions of clones 3 and 24 were performed with the same nested gene-specific primers used for the amplification of the target cDNA and the M13 forward primer (Life Technologies, Inc.). The 5′-RACE and PCR products were analyzed on a 1% (w/v) agarose gel. Myofibrils were prepared according to Goll et al. (49Goll D.E. Stromer M.H. Robson R.M. Luke B.M. Hammond K.S. Tregear R.T. Insect Flight Muscle. Elsevier/North-Holland Inc., New York1977: 15-40Google Scholar) from adult chicken heart papillary and thigh muscle, and adult pig heart papillary and mixed neck muscle samples removed immediately after exsanguination. Myofibrils were placed on coverslips, and the coverslips were washed thoroughly to remove unbound myofibrils, incubated with undiluted culture supernatant of anti-paranemin monoclonal antibody 4D3 at 4 °C overnight, washed extensively, and then incubated for 30 min with a 1:50 dilution of fluorescein isothiocyanate-labeled goat anti-mouse (Sigma). The coverslips were then washed thoroughly, mounted on glass slides with FITC-Guard (Testog), and examined with a Zeiss Photomicroscope III equipped for epifluorescence using × 67 and × 100 planapochromat objectives. Paranemin clone 24 was subcloned into the mammalian expression vector pRC/RSV (Invitrogen) at NotI and SalI sites. The expression construct was transfected into SW13cl.1Vim+ and SW13cl.2Vim− cells (obtained from Dr. W. Ip, University of Cincinnati, Cincinnati, OH; originally described by Sarria et al. (50Sarria A.J. Lieber J.G. Nordeen S.K. Evans R.M. J. Cell Sci. 1994; 107: 1593-1607PubMed Google Scholar)) (5 μg/∼75% confluent plate) via LipofectAMINE reagent (Life Technologies, Inc.). For vector control studies, the expression construct was replaced with the pRC/RSV vector with no insert. Proteins were visualized by double label immunofluorescence with anti-paranemin polyclonal antibodies and anti-vimentin monoclonal antibody AMF-17b (developed by Dr. A. B. Fulton, obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, and the Department of Biological Sciences, University of Iowa, under contract NO1-HD-2-3144 from the National Institute of Child Health and Human Development). Transfected cells were grown on coverslips for 48 h and then fixed with cold methanol for 6 min at −20 °C. Coverslips were blocked for 30 min with 5% goat serum before a 1-h (37 °C) incubation with a mixture of the primary antibodies, with dilutions in phosphate-buffered saline of anti-paranemin, 1:100, and anti-vimentin, 1:5. The coverslips were thoroughly washed in phosphate-buffered saline and incubated for 2 h (37 °C) with a mixture of fluorescein isothiocyanate-labeled goat anti-rabbit (1:200) and tetramethylrhodamine isothiocyanate-labeled goat anti-mouse (1:200). After incubation, the coverslips were again thoroughly washed in phosphate-buffered saline, mounted on glass slides with FITC-Guard (Testog), and observed on a Zeiss Photomicroscope III. The chromatographic purification of a representative (five total) paranemin preparation is shown in Fig. 1. A high molecular mass protein (∼280 kDa) was detected in the whole embryonic muscle homogenate (lane 1) and in the crude paranemin (lane 2), together with the IF proteins vimentin (54 kDa) and desmin (53 kDa), and many other proteins (17Breckler J. Lazarides E. J. Cell Biol. 1982; 92: 795-806Crossref PubMed Scopus (44) Google Scholar). Fractions eluting just after the void volume from the gel filtration column contained a complex of primarily paranemin, vimentin, and desmin (lane 3; vimentin and desmin are in same major band). Examination of negatively stained samples of these early fractions by electron microscopy revealed the presence of many long, ∼8–12 nm diameter filaments with irregular surface contour (data not shown), in agreement with results of Breckler and Lazarides (17Breckler J. Lazarides E. J. Cell Biol. 1982; 92: 795-806Crossref PubMed Scopus (44) Google Scholar). Only the first few fractions of the first peak collected immediately following the void volume were used for further purification. Hydroxyapatite chromatography was very effective in removing from the gel filtration, partially purified paranemin, small amounts of several proteins with molecular masses near that of paranemin, and near those of vimentin and desmin (lane 4). DEAE-cellulose column chromatography removed both the remaining vimentin and desmin and a trace of actin before elution of the purified paranemin (lane 5). The pooled fractions of purified paranemin are shown in Fig. 1 (lane 5). Paranemin made by this procedure also was examined by SDS-PAGE with higher percent acrylamide gels, and showed no evidence of lower molecular mass contaminants. Although Breckler and Lazarides (17Breckler J. Lazarides E. J. Cell Biol. 1982; 92: 795-806Crossref PubMed Scopus (44) Google Scholar) indicated the paranemin in their paranemin-enriched fractions migrated as two closely spaced polypeptides of very similar molecular mass by SDS-PAGE, we usually observed only one paranemin band, and sometimes one major band with a small trace of a band migrating just below it, which also labeled with monoclonal antibody 4D3 by Western blotting. The yield of paranemin from 100 g of embryonic skeletal muscle ranged from 1.2 to 2.5 mg with an average of 1.7 mg. Two monoclonal antibodies, 4D3 and 4C7, and the rabbit polyclonal a" @default.
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• W2000732040 title "Molecular Characteristics of the Novel Intermediate Filament Protein Paranemin" @default.
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