this actin isozyme, was used to prepare an isozyme-specific pep- tide antibody. SaN peptide was purified from chicken breast mus- cle actin by preparative ...
Proc. NatL Acad. Sci. USA Vol. 80, pp. 1506-1510, March 1983 Biochemistry
Peptide antibody specific for the amino terminus of skeletal muscle a-actin (muscle development/human myoblasts/actin isozymes/HPLC)
JEANNETTE CHLOE BULINSKI*t, SANTOSH KUMARt, KOITI TITANI*t, AND STEPHEN D. HAUSCHKA* *Department of Biochemistry, SJ-70, University of Washington, and tHoward Hughes Medical Institute Laboratory, Seattle, Washington 98195 Communicated by Earl P. Benditt, December 3, 1982
ABSTRACT The NH2-terminal peptide of skeletal muscle aactin (SaN peptide), which contains a primary sequence unique to this actin isozyme, was used to prepare an isozyme-specific peptide antibody. SaN peptide was purified from chicken breast muscle actin by preparative reverse-phase HPLC and was coupled to hemocyanin. This complex was used to immunize rabbits in order to elicit actin antibodies specific for the skeletal muscle a-actin isozyme. The antibody obtained, called SaN antibody, was reactive with SaN peptide and with skeletal muscle a-actin as well as with cardiac muscle a-actin. SaN antibody did not react with either of the actin isozymes present in smooth muscle (smooth muscle a and v) or in brain (nonmuscle 13 and y). SaN antibody was used to detect muscle-specific actin in differentiating mouse and human myoblasts by using immunoblots of myoblast extracts and immunofluorescent staining of fixed cells.
vertebrates show >90% overall sequence homology, the NH2terminal regions are much less homologous. Each isozyme has a unique sequence in the 18 residues of the NH2 terminus (Table 1); in this region, actin isozymes show only 50-80% sequence homology (5). The presence of many carboxylic acids in the NH2 terminus of actin accounts for its low isoelectric point, and variations in the NH2-terminal portion confer characteristic isoelectric points on the various isozymes (5, 10). Recently, several groups have prepared antibodies to selected small peptides which represent parts of larger proteins (15-23). In these cases, peptides 6-20 amino acids long were chemically synthesized and coupled to carrier proteins for use as immunogens. In many cases the antipeptide antibodies reacted specifically with the protein from which the peptide was derived. The success of any particular attempt seemed to depend on whether the peptide contained hydrophilic amino acid residues (20, 21). Presumably, a region rich in hydrophilic residues would be located on the exterior of the protein, causing it to be antigenic and also allowing reaction of antibody with that peptide in the native protein. Theoretical calculations based upon hydrophilicity numbers assigned to each amino acid residue have allowed accurate prediction of many primary sequence antigenic sites (21), and these can be used to predict the antigenicity of a particular peptide. The technology of antipeptide antibody production seems to be readily applicable to the study of actin isozymes. Because the NH2 terminus of each isozyme has a unique sequence and because hydrophilic amino acids are grouped in that region, one or more probable antigenic sites specific to muscle a-actin exist on the NH2-terminal peptide. Rather than synthesize the desired peptide, we sought to utilize the NH2-terminal tryptic peptide to prepare an isozyme-specific anti-actin antibody. In this paper we describe the production, characterization, and use of such an antibody.
Actin is a major cytoskeletal component of all eukaryotic cells and serves as an important part of the specialized contractile apparatus of skeletal muscle cells in higher vertebrates. All actins are remarkably similar in their physical properties and in vitro functions; but isoelectric variants have been identified in different tissues (1-4), and these isozymes may account for some of actin's tissue-specific functions. For example, the most prevalent isozymes in higher animals, the a-actins, function in muscle contraction. Three slightly different a-actin isozymes function in skeletal, cardiac, and smooth muscle (5). In addition, another smooth muscle isozyme, classified as a y-type muscle form, is a minor component of mammalian vascular smooth muscle and a major component of chicken gizzard muscle (5). In contrast, two isoelectric variants, called the nonmuscle (3 and y isozymes, are present in all nonmuscle tissues examined. Immunolocalization and electron microscopy have been utilized to study the distribution of microfilaments in various cell types (for recent reviews, see refs. 6 and 7); however, in no case has the specific localization of any of the six actin isozymes been reported. Similarly, although functional specialization of actin isozymes seems likely, there are no known examples of differences in in vivo function or in distribution of the isozymes. In myoblasts, for example, the nonmuscle isozymes ( and y are present before myogenesis whereas a-actin begins to be synthesized during the differentiation process. The a isozyme makes up most or all of the actin in fully differentiated muscle cells. It is not known whether the three coexistent isozymes copolymerize into filaments, nor is it known whether the a, 3, and y isozymes exhibit different functions or cellular distributions during myogenesis. Extensive amino acid sequence information has been used to identify the actin isozymes and to determine their relationship to one another (5, 8-14). Although the actins present in higher
MATERIALS AND METHODS Purification of Actin Isozymes and of the NH2-Terminal Peptide of Sarcomeric a-Actin (SaN Peptide). Skeletal muscle a-actin (Sa-actin) was purified from chicken breast muscle acetone-powder by the procedure of Spudich and Watt (24). Cardiac a-actin and smooth muscle a- and y-actins from bovine heart and aorta, respectively, were also purified according to Spudich and Watt (24). Bovine brain actin was purified according to Pardee and Bamburg (25) except that a Sephacryl S-200 column was substituted for the Sephadex G-150 column specified. All anAbbreviations: KLH, keyhole limpet hemocyanin; Sa-actin, skeletal muscle a-actin; SaN peptide, NH2-terminal tryptic peptide of Sa-actin; SaN antibody, rabbit antibody against SaN peptide; ELISA, enzyme-linked immunosorbent assay. t Current address: Dept. of Biology, Univ. of California, Los Angeles,
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Table 1. NH2-Terminal sequence difference among vertebrate actin isozymes Isozyme type Amino acid sequence Species analyzed Skeletal muscle a Rabbit (5, 8,9), cattle (5), chicken (12) Ac-Asp-Glu-Asp-Glu-Thr-Thr-Ala-Leu-Val-Cys-Asp-Asn-Gly-Ser-Gly-Leu-Val-Lys Cardiac muscle a Cattle (5, 13) Ac-Asp-Asp-Glu-Glu-Thr-Thr-Ala-Leu-Val-Cys-Asp-Asn-Gly-Ser-Gly-Leu-Val-Lys Smooth muscle a Cattle (5, 13), chicken (12) Ac-Glu-Glu-Glu-Asp-Ser-Thr-Ala-Leu-Val-Cys-Asp-Asn-Gly-Ser-Gly-Leu-Cys-Lys Smooth muscle y* Cattle, chicken (12) Ac-Glu-Glu-Glu-Thr-Thr-Ala-Leu-Val-Cys-Asp-Asn-Gly-Ser-Gly-Leu-Cys-Lys Nonmuscle fl Cattle (5,9-11), mouse (11), human (14) Ac-Asp-Asp-Asp- Ile -Ala-Ala-Leu-Val-Val-Asp-Asn-Gly-Ser-Gly-Met-Cys-Lys Ac-Glu-Glu-Glu- Ile -Ala-Ala-Leu-Val- Ile -Asp-Asn-Gly-Ser-Gly-Met-Cys-Lys Nonmuscle y Cattle (5, 9-11), mouse (11), human (14) * Smooth muscle yactin has been grouped with the a-type isozymes because, although its NH2-terminal peptide is more similar to a y-type isozyme, the bulk of its sequence more closely resembles a isozymes.
imal tissues were obtained from local abattoirs. For purification of SaN peptide, Sa-actin was oxidized with performic acid by the procedure of Hirs (26). The oxidized actin was dissolved in 0.5% ammonium bicarbonate and treated, at a 1:50 molar ratio, with TPCK-treated trypsin (Worthington) for 150 min at room temperature. Tryptic digests were added to 5.0% pyridine/0.5% acetic acid and the insoluble peptides were removed (5). Reversed-phase HPLC was used to purify the SaN peptide; a Synchropak RP-P column (Waters Associates) with 0.1% trifluoroacetic acid as solvent and a flow rate of 2 ml/min was used. A 0-30% acetonitrile gradient (in which the acetonitrile contained 0.08% trifluoroacetic acid) was used to elute peptides. Amino acid analysis, performed on a Dionex model D-500 amino acid analyzer, was used to analyze peaks obtained from HPLC for the presence of SaN peptide and also to measure its purity. Preparation of Antibody Directed Against SaN Peptide (SaN Antibody). SaN peptide was coupled to keyhole limpet hemocyanin (KLH; purchased from Calbiochem) by crosslinking with glutaraldehyde. SaN peptide (100 nmol) and KLH (1 nmol) were incubated with 5 ,umol of glutaraldehyde for 24 hr at room temperature. The reaction was terminated by the addition of 1 mg of sodium borohydride. SaN peptide-KLH complex was sedimented at 100,000 X g for 4 hr and the pellet was suspended in 2 ml of a 1:1 (vol/vol) mixture of phosphate-buffered saline and Freund's adjuvant (Miles). This mixture was emulsified by sonication and used for subcutaneous injection. Three injections, spaced at 2-week intervals, sufficed to produce antibody; blood was removed once per week and booster injections were administered every 6-9 weeks. The IgG fraction was purified from serum by the procedure of Garvey et aL (27) and was checked for purity by gel electrophoresis (28). In order to estimate the epitope density (29), in this case the molar ratio of SaN peptide to hemocyanin, the material not coupled to hemocyanin was measured. Supernatants from the 100,000 X g centrifugation were chromatographed on a Sephadex G-10 column (Pharmacia) in order to separate SaN peptide monomers and dimers from glutaraldehyde, and the peptide was then subjected to quantitative amino acid analysis with an internal standard of norleucine. SaN peptide was coupled in the same way to bovine serum albumin, with 5 mM glutaraldehyde, in this case by using 100 ,ug of SaN peptide and 1 mg of bovine serum albumin per ml. Immunological Assays. An enzyme-linked immunosorbent assay (ELISA) (30) was used to titer antisera and to quantify their reactivity with other antigens. In all cases the buffer used for dilution of each coating antigen was 20 mM Tris at pH 8.0. Protein concentrations were measured by the method of Lowry et at (31) with bovine serum albumin as. a standard. An immunoblot technique (32) was used to examine antibody reactivity. The procedure of Towbin et at (32) was followed except that 4chloro-1-naphthol (Sigma) at 1 mg/ml was used as the substrate for horseradish peroxidase. Cell Culture and Immunofluorescence Staining. Mouse MM14 myoblasts were grown and induced to differentiate by the procedures of Linkhart et aL (33), and extracts were pre-
pared for electrophoresis by scraping cells off dishes, washing twice by low-speed centrifugation in 50 mM Tris, pH 7.5/150 mM NaCi and boiling in NaDodSO4 gel sample buffer [prepared according to Laemmli (28)] at a concentration of 8 x 106 cells per ml.- Human myoblasts (strain H275) were isolated from 62-day human fetal limb muscle and were grown and induced to differentiate as described (34). Immunofluorescence staining and microscopy were performed as described (35) with a 1:50 dilution of SaN antibody, except that cells were fixed and stained in gelatin-coated plastic tissue culture dishes instead of on glass coverslips. RESULTS Preparation of Antibodies Directed Against SaN Peptide.
Tryptic digests of chicken Sa-actin were subjected to HPLC under conditions chosen to maximize separation of SaN peptide from others of similar charge and molecular weight. In order to determine the chromatographic behavior of SaN peptide, material from each of approximately 20 major peptide peaks was subjected to amino acid analysis. Table 2-shows amino acid composition data for the peak that was identified as that of SaN peptide. The amino acid composition of the isolated peptide is in good agreement with that predicted from the SaN peptide sequence (ref. 5; see also Table 1). In repeated preparative HPLC runs, we were able to isolate micromolar quantities of SaN peptide of sufficient purity for use as an antigen. Because antigens of Mr \
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FIG. 2. ELISA of SaN antibody. Antigen coatings were: Sa actin at 1 tg per well (curve a); oxidized Sa actin at 1 pg per well (curve b); tryptic "core" of Sa actin (37) at 10 pg per well (curve c); or preimmune rabbit IgG applied to Sa actin (1 jg per well) coating (curve d).
SaN antibody has been further characterized by immunoblotting (32)-i.e., immunostaining of proteins resolved on NaDodSO4/polyacrylamide gels and transferred electrophoretically to nitrocellulose sheets. Both one- and two-dimensional gels of actin isozymes and cell extracts were used to visualize the immunoreactive species present. No crossreactivity occurred between SaN antibody and either of the smooth muscle actins, a or y (Fig. 3). Also, there was no crossreactivity between nonmuscle ,B or 'yisozymes. ELISA showed significant reactivity of SaN antibody with smooth muscle and nonmuscle actins only at antibody dilutions of 1:100 or less, and at these antibody concentrations OD values for these isozymes were less than 1/6th of those for Sa or cardiac a isozymes (38). These results were predictable, given the similarity between skeletal and cardiac muscle NH2-terminal sequences and the dissimilarity of these to the smooth muscle and nonmuscle actins. Lanes e and
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FIG. 3. Immunoblot of SaN antibody. Triplicate lanes of samples to be analyzed were electrophoresed on 10% polyacrylamide gels. Lanes a-f represent proteins (stained with Buffalo blue-black). Lanes g-l were stained with SaN antibody IgG fraction (1:200 dilution), and lanes m-
stained with preimmune rabbit IgG (1:200 dilution). Lanes: a, and m, 1 ttg of chicken Sa-actin; b, h, and n, 4 ,g of bovine cardiac muscle a-actin; c, i, and o, 5 ,ug of bovine smooth muscle a- and y-actin; d,j, and p,4 ,ug of bovine brain and y-actin; e, k, and q, 25-,ug extract of undifferentiated mouse MM14 myoblasts; f, 1, and r, 15-,ug extract of differentiated MM14 cells. Protein concentrations in lanes e, k, and q were greater than in lanes f, 1, and r, that the actin bands would be of roughly equal intensity. Reactive material in lanes g-r was visualized by immunoperoxidase staining. A, gel mobility of Sa actin. r were
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FIG. 1. ELISA of SaN antibody. An IgG fraction of SaN antibody
was diluted as shown and assayed on polystyrene microtiter plates coated with: SaN peptide-bovine serum albumin complex at 1 ug per well (curve a); SaN peptide at 10 ,ug per well (curve b); or albumin at 1 ,ug
per well (curve c). An IgG fraction of preimmune serum from the same
rabbit was also assayed on wells coated with SaN peptide-albumin complex at 1 Mg per well (curve d).
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k clearly show the absence of crossreactive species in an extract of undifferentiated MM14 mouse myoblasts and its presence after differentiation had occurred (lanes f and 1). From other experiments in which the sensitivity of the immunoblot technique was examined, we estimate that undifferentiated cells contain Sa-actin representing
