Expression of smooth muscle and nonmuscle tropomyosins in Escherichia coli and characterization of bacterially produced tropomyosins

Biochimica etBiophysicaActa, 1162 (1993) 255-265 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4838/93/$06.00

255

BBAPRO 34441

Expression of smooth muscle and nonmuscle tropomyosins in Escherichia coli and characterization of bacterially produced tropomyosins Robert E. Novy

a,

Li-Fei Liu a, Ching-Shwun Lin b, David M. Helfman c and Jim Jung-Ching Lin a

a Department of Biological Sciences, University of Iowa, Iowa Oty, IA (USA), b Institute for Medical Research, San Jose, CA (USA) and c CoM Spring Harbor Laboratory, Cold Spring Harbor, N Y (USA)

(Received 3 August 1992)

Key words: Tropomyosin; Caldesmon; Cooperativity; N-terminal-deleted tropomyosin; Smooth muscle; Fibroblast

The cDNA encoding the /3-tropomyosin isoform of chicken smooth muscle (CSMfl) was constructed and expressed in Escherichia coli to produce recombinant, unacetylated fl-tropomyosin (rCSMfl) and a mutant (rCSMfl-7)with a 7-residue deletion at its amino-terminus. Furthermore, the cDNA coding for human fibroblast tropomyosin isoform 3 (hTM3) was also used to produce unacetylated hTM3 (called PEThTM3). All of bacterially-made tropomyosins were high t~-helical in structure as judged by CD analysis and resistant to heat denaturation. Both the rCSMfl and PEThTM3 exhibited saturable binding to F-actin with apparent binding constants of 1.14.106 and 2.78.106 M-1, respectively. The bacterially made, unacetylated smooth muscle tropomyosin (rCSMfl) appeared to have a comparable actin-binding affinity to that of gel-purified CSMfl homodimer (1.25 • 106 M -1) but significantly lower than that for native gizzard tropomyosin (CSM-TM) heterodimer (1.28.107 M - l ) . The aminoterminal deletion mutant rCSMfl-7 failed to bind to F-actin. Effects of gizzard caldesmon on the actin binding of these bacterially made tropomyosins were also examined. Under the binding condition containing 0.5 mM MgCI 2 and 30 mM KCI, caldesmon greatly enhanced the binding of rCSM/3 to F-actin. However, under the same condition, there was a slight enhancement in the aetin-binding for gel-purified CSM/3 or PEThTM3 (1.2-1.6-fold stimulation) and no enhancement for native gizzard tropomyosin. Neither the presence of caldesmon nor native gizzard tropomyosin induced detectable binding of the amino-terminal deletion mutant rCSMfl-7 to F-actin. These results clearly imply the importance of the amino-terminal 7 amino-acid residues of CSMfl in the actin binding and the caldesmon enhancement.

Introduction Tropomyosin (TM), a coiled-coil dimeric protein, binds along the length of actin filaments in a head to tail manner. Numerous tissue-specific isoforms of tropomyosin have been identified in vertebrates includ-

Correspondence to: J.J.-C. Lin, Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA. Abbreviations: TM, tropomyosin; CSM-TM, chicken smooth muscle TM (CSMa and CSMfl) purified from gizzard; CSMa, chicken smooth muscle a-TM; CSMfl, chicken smooth muscle fl-TM; rCSMfl, unacetylated CSMfl produced in E. coli; rCSM/3-7, 7 amino-acid residue deletion at the N-terminus of CSMfl produced in E. coli; hTM3, human fibroblast TM isoform 3 (one of human ot-TM gene products); PEThTM3, unacetylated hTM3 produced in E. coli; IPTG, isopropyl-fl-D-thiogalactopyranoside;PBS, phosphate-buffered saline (137 mM NaCI, 2.7 mM KCI, 1.5 mM KH2PO4, 8.0 mM NaEHPO4 (pH 7.3)); TCP, total cell proteins; HRS, heat-resistant supernatants.

ing 2 - 3 isoforms in striated muscle, 2 isoforms in smooth muscle and 4 - 6 isoforms in nonmuscle cells [1-3]. Tropomyosin diversity is generated by multiple genes and by tissue specific alternative splicing of the gene transcripts [4-10]. Comparisons of vertebrate c D N A and genomic clones has revealed that amino-acid variability is primarily restricted to three alternatively spliced exon regions; an amino-proximal/terminal region, an internal exon and a carboxy-terminal exon. Thus, these variable exon regions may encode aminoacid sequences which delineate isoform specific function. The regulatory role of tropomyosin in striated muscle contraction is well defined in contrast to what is known about the functional role(s) of tropomyosin isoforms in smooth and nonmuscle ceils. Several investigators [11-13] have suggested that tropomyosin in these cells may play a role in a Ca2+-calmodulin, caldesmon, tropomyosin, actin system postulated to regulate actinmyosin interactions at the level of the actin filament. In

256 addition, a variety of indirect evidence suggests that tropomyosin isoforms participate in the stabilization and organization of actin filament supramolecular structures in nonmuscle cells [14-16]. Indeed, a link may exist between the disruption of the actin cytoskeleton observed in transformed cells and the correlative perturbation of tropomyosin isoform protein levels [17-19]. Clearly, in light of the numerous isoforms expressed in nonmuscle cells, elucidation of isoform specific functions presents a complex challenge. Several investigators [20,21] reported the production of full length, amino-terminal unacetylated skeletal muscle tropomyosin in Escherichia coli using the expression vector pKK233-2. The unacetylated mutant was unable to polymerize head to tail in vitro and its actin affinity was drastically reduced relative to native, acetylated tropomyosin [21,22]. At the present time, there was no report concerning the importance of the N-terminus on smooth muscle and fibroblast tropomyosin molecules. In this report, we have characterized the expression products obtained when cDNAs encoding in chicken smooth muscle /3-tropomyosin (CSM/3, one of chick/3 gene products, previously designated by Sanders and Smillie [23] as /3 and by Hellman et al. [24] as a) and a nonmuscle human tropomyosin isoform 3 (hTM3, one of human a-TM gene products) [6,25] were subcloned into the E. coli expression vectors pKK233-2 and pET-8c. Analysis of the expression products revealed that the full-length tropomyosins had unacetylated amino-termini (called rCSM/3 and PEThTM3, respectively). In contrast to the bacterially made skeletal muscle tropomyosin, both mutants bound F-actin reasonably well, as assayed by a co-sedimentation experiment. In addition, we also obtained a 7-residue deletion at the amino-terminus of CSM/3 tropomyosin (called rCSM/3-7) and showed the failure of this rCSM/3-7 tropomyosin to bind F-actin detectably. The effects of caldesmon on the actin-binding of these bacterially made tropomyosins were also examined. Materials and M e t h o d s

cDNA and expression vector subcloning. The expression vector pKK233-2 was purchased from Pharmacia (Piscataway, N J, USA). The expression vector pET-8c and host strain BL21(DE3)LysS were a generous gift from Dr. W. Studier (Brookhaven National Laboratory, Brookhaven, NY, USA). Both expression vectors carry the ampicillin resistance marker and LysS designates a plasmid conferring chloramphenicol resistance. The entire coding region of the CSMfl eDNA was obtained from the plasmid pSMT-10 [24] via a partial NcoI digest. This 990-bp NcoI fragment was subcloned into the NcoI site of pKK233-2 and pET-8c to yield pKKCSMfl and pETCSM/3, respectively (Fig. 1). The

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Fig. 1. TropomyosineDNA-expressionvector constructs. Vector derived promotor,Shine-Delgarno(S/D) and transcription termination elements are indicated by black boxes. TropomyosineDNA coding regions are indicatedby striped boxes and the 3' noncodingregions are indicatedby unmarkedboxes. Relevantrestriction enzymesites are also indicated. Part A illustrates the recombinant vectors pKKCSM/3 and pKKhTM3and part B illustrates the recombinant vectors pETCSM/3and pEThTM3.

entire coding region of the hTM3 cDNA was obtained from the plasmid pSPThTM3 [25] (hTM3 eDNA subcloned into the EcoRI site of pSPT-18 (Pharmacia)) via NcoI/HindlII or NcoI/BamHI digests. A 1335-bp NcoI/BamHI fragment was subcloned into the corresponding sites of pET-8c to yield pEThTM3 (Fig. 1B). A 1399-bp NcoI/HindlII fragment was subcloned into the corresponding sites of pKK233-2 to yield pKKhTM3 (Fig. 1A). These subclones were verified by restriction-enzyme-site analysis, insert size and nucleotide sequencing of the terminus. Purification of bacterially produced tropomyosin. Bacterial cultures were grown at 37°C in LB containing 100 / ~ g / m l of ampicillin (pKKCSM/3 and pKIChTM3/JM109 transformants) or in LB containing 100/~g/ml of ampieillin and 25/zg/ml of ehloramphenicol (pETCSM/3 and pEThTM3/BL21(DE3)LysS transformants). After reaching an OI)600 greater than 0.6, the cultures were induced for 1.75 h with 0.4 mM IPTG in the case of pKKCSM/3/JM109 and

257 pKKCSM/3/BL21(DE3)LysS or for 2-3 h with 0.4 mM IPTG in all other cases. The shorter time of induction for the pKKCSM/3/JM109 transformant was necessary due to substantial cell lysis at longer induction times. Bacteria were harvested by centrifugation (4000 x g), washed with PBS, repelleted and then resuspended in 120 ml extraction buffer (0.3 M KCI, 50 mM imidazole (pH 7.0), 0.1 mM EGTA and 0.5 mM MgCI 2) per liter of original culture. The bacteria were lyzed by a sonicator and subsequently placed in a boiling water bath so that the lysate temperature exceeded 85°C for 10 min. The lysate was incubated on ice and insoluble materials subsequently removed by centrifugation (12 000 x g, 10 min). The supernatant was subjected to two consecutive ammonium sulfate precipitations at 30 and 65% saturation, respectively. The 65% precipitate was resuspended in and dialyzed against DE-52 column buffer (30 mM NaCI, 10 mM imidazole (pH 7.0), 0.1 mM DT-F, 0.1 mM EGTA) and then fractionated on a DE-52 ion-exchange column (Whatman, Clifton, N J, USA) using a 30-380 mM NaCI gradient. Tropomyosin fractions were pooled, concentrated and then fractionated on a hydroxyapatite column (Bio-Rad, Richmond, CA, USA) using a 20-300 mM phosphate gradient. T h e yields of p u r i f i e d t r o p o m y o s i n from pKKCSMfl/JM109, pETCSM/3/BL21(DE3)LysS and pEThTM3/BL21(DE3)LysS were 3.2 mg/liter of culture of rCSM/3-7, 9.5 mg/liter of culture of rCSM/3 and 3.7 mg/liter of culture of hTM3, respectively. Amino-acid sequencing. Amino-acid sequencing was performed by the Protein Core facility, University of Iowa. Sequencing was accomplished on an Applied Biosystems 470A gas-phase sequencer [26] with an online 120A PHT analyzer and 900A data station. The sample was applied directly to a preconditioned filter containing 1.5 mg of Biobrene. Standard RUN470-1 cycles were used for both the reaction and conversion cycles. Determination of carboxy-terminal sequences of bacterially made tropomyosin was performed as follows. Purified tropomyosin (2.5 mg/ml) in 100 mM ammonium carbonate buffer (pH 8.4) was digested with 0.04 mg/ml of carboxypetidase A (Sigma, St. Louis, MO, USA) at 37°C. At a designated time (0 min, 5 min, 20 min, 40 min, 1.5 h, 2 h and 3 h), an aliquot (40/~1) of the reaction mixture was removed and immediately stopped the digestion by the addition of 3 ml 88% formic acid. The released amino acids in each sample were lyophilized and analyzed on a Beckman 6300 high-performance ion-exchange amino-acid analyzer. SDS-PAGE and Western blotting. 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as previously described [27,28]. Estimations of the percentage of total bacterial cell protein constituted by the tropomyosin bands were obtained by scan-

ning the Coomassie blue stained gels with a Hoefer densitometer (GS300). Two-dimensional gel electrophoresis was performed according to a modified procedure of O'Farrell [29] as previously described [28]. Ampholines pH 4-6 were used for tropomyosin analysis. Protein immunoblotting was performed as described [30]. Replicate transfers to nitrocellulose were performed and one replicate blot stained with amido black to assess transfer quality. The monoclonal antibody CG1 and CG/36 were used here to detect rCSM/3 and PEThTM3, respectively. Their characterizations have been reported previously [31,32]. Actin binding assay. The actin binding assay was based on the co-sedimentation method [33] and performed on a Beckman airfuge rotor A-100/18. Actin (9.3 /zM) and various amounts of tropomyosin were mixed in 10 mM imidazole buffer (pH 7.0), 10 mM MgCI 2 100 mM KC1, 0.1 mM EGTA and 0.1 mM DTT. After incubation for 30 min at room temperature, the samples were centrifuged at 26 psi for 20 min in the airfuge. Aliquots of both pellets and supernatants were analyzed by 12.5% SDS-PAGE with trypsin inhibitor as an internal control for gel loading and scanning variability. After electrophoresis, the proteins were stained with Coomassie blue and scanned for the quantitation. Native smooth muscle tropomyosin (CSM-TM) was purified from chicken gizzard as previously described [31]. The individual isoforms (CSM/3 and CSMa) were separated by SDS-PAGE and isolated by electroelution of the appropriate gel bands as described previously [31] and used in the study for comparison. Caldesmon was isolated from chicken gizzard as previously described [34]. Actin was purified from acetone powder of rabbit skeletal muscle [35]. Acetone powder was prepared by the method of Ebashi and Ebashi [36]. Protein concentrations were determined by the method of Lowry et al. [37] with bovine serum albumin as standard. Amino-acid composition analysis was occasionally used to correct the protein concentration. V'~cocity measurement and conformation analysis. Viscosity measurement was carried out at 25°C using a Cannon-Manning semimicro viscometer (model 100, El50) in which buffer has a flow time of 45 s. The specific viscosity was calculated as described [38]. The circular dichroism spectra (190-260 nm) of bacterially made tropomyosins were determined on an Aviv 60 circular dichroism spectrophotometer in 100 mM NaC1, 30 mM sodium phosphate buffer (pH 7.0) and 0.1 mM DTT at 6°C. The molar residue ellipticity and the a-helix content were calculated according to Greenfield and Fasman [39]. Under this condition, all of bacterially made tropomyosins had comparable conformations to that of native gizzard tropomyosin (CSMTM) or gel-purified CSM/3 (a-helix content of > 90%).

258 Results

Expression of CSM[3 in E. coli E. coli strain JM109 was transformed with pKKCSM/3 and strain BL21(DE3)LysS transformed with pETCSM/3. Ampicillin resistant colonies were examined for tropomyosin expression by colony immunoscreening and western blot analysis. Fig. 2 demonstrates the results of IPTG induction. Total bacterial cell protein (TCP) and the fraction resistant to heat denaturation (HRS) were loaded in adjacent wells. Comparison of the pKK233-2/JM109 background with pKKCSM/3/JM109 (Fig. 2A, lanes 1-2 and 3-4, respectively) reveals the expression of two IPTG-induced bands. Two bands were more clearly seen in the gel loaded with less amounts of sample. The bottom, apparent lower molecular mass band was designated rCSMfl-7 and the top band designated rCSMfl. In the case of pETCSMfl/BL21(DE3)LysS, only one major induced band is expressed relative to its vector/host background (Fig. 2A, lanes 9-10 and 7-8, respectively). Since the E. coli strain BL21 lacks two proteinase activities Ion and ompT present in JM109, the production of rCSMfl-7 in JM109 may have been due to this proteinase background. To test this possibility, the pKKCSMfl plasmid was expressed in the BL21(DE3) LysS background (Fig. 2A, lanes 5-6). As can be seen the pKKCSMfl/BL21(DE3)LysS combination pro-

duced two bands similar to pKKCSM/3/JM109. Since pETCSM/3 directed the expression of large quantities of rCSM/3 but only very small quantities of rCSM/3-7 in the same BL21 background, it seemed very unlikely that proteolysis mediated the production of rCSM/3-7. All of the IPTG-induced bands were resistant to heat denaturation, a characteristic property of tropomyosins. Furthermore, in each case the induced bands proved to be immunoreactive with the antitropomyosin antibody CG1 (Fig. 2B, lanes 3-4, 5-6 and 9-10). Comparisons of the TCP and HRS fractions reveal that the majority of tropomyosin remains soluble in E. coli, since equivalent amounts of samples were loaded in the TCP and HRS lanes. Thus, high levels of soluble rCSMfl tropomyosin were expressed with both vector/host. In order to further characterize the nature of the expression products, two-dimensional gel analysis was performed. Both pKKCSM/3 and pETCSM/3 migrated to a slightly more basic position than native CSM/3 (Fig. 3B-C and E-F, respectively). Native CSMfl is known to be acetylated at its amino-terminus [23]. Full length CSMfl lacking this terminal acetyl group would have one additional positive charge due to the unblocked NH~- group thereby accounting for the slight basic shift. The presence of a minor spot labelled CSM/3 in Fig. 3B, which comigrates with native CSM/3 (Fig. 3C) suggests that a small fraction of rCSM/3 may

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Fig. 2. CSM/3 tropomyosinexpression from the vectors pKKCSM/3 and pETCSM/3. Part A represents a SDS-PAGE analysis of total cell proteins (TCP) and the heat-resistant supernatant (FIRS) fractions of IPTG induced bacteria. Total cell proteins were prepared by adding an equal volume of 2 × sample buffer to concentrated bacterial cells. The HRS fractionwas prepared by sonicating concentrated bacteria, heat treating the sonicate and removing insoluble protein by centrifugation. Lanes 1,2, TCP, HRS of pKK233-2/JM109; lanes 3,4, TCP, HRS of pKKCSM/3/JM109; lanes 5,6, TCP, HRS of pKKCSM/~/BL21(DE3)LysS;lanes 7,8, TCP, HRS of pET-8c/BL21(DE3)LysS;lanes 9,10, TCP, HRS of pETCSMfl/BL21(DE3)LysS.Part B represents a Western blot analysisof lanes 1-10, Part A. The anti-tropomyosinantibodyCG1 was used to detect bacterially-expressedCSM/3.

259 A combination of heat extraction, DE-52 column chromatography and hydroxyapatite column chromatography was used to purify rCSM/3-7 and rCSMfl (as described in Materials and Methods). The yields were 3.2 mg and 9.5 mg per original liter of culture, respectively, with a purity of at least 90%. Amino-acid sequencing the 20 amino-terminal residues of rCSM/3 verified that it represented amino-terminally unacetylated CSM/3. Similar amino-acid sequencing of rCSM/3-7 purified from pKKCSM/3/JM109 revealed that rCSMfl-7 actually consisted of two CSM/3 deletion products. One deletion product which constituted approx. 80% of the amino-acid sequencing yield corresponded to CSMfl with an amino-terminus at methionine, amino-acid position 8. The other deletion product which constituted approx. 20% of the yield corresponded to CSM/3 with an amino-terminus at lysine, amino-acid position 12. The carboxy-terminal sequence analysis revealed that both rCSMfl and rCSM/3-7 had an intact carboxy-terminus (data not shown).

be amino-terminally acetylated by the JM109 host. This may not be unreasonable in that N"-acetyl transferase has been reported in E. coli [40]. rCSM/~-7 migrated to a slightly more acidic isoelectric point and with a decreased apparent molecular mass relative to native CSM/3 (Fig. 3A-D). A very small amount of rCSM/3-7 was also expressed from pETCSMfl/BL21(DE3)LysS (Fig. 3E). A cluster of 3 basic Lys residues at amino-acid positions 5-7 exists in CSM/3 [24]. An amino-terminally truncated CSM/3 lacking these residues (and Glu at position 2) would be expected to migrate similarly to rCSM/3-7. Based on an examination of the CSMfl cDNA nucleotide sequence, we believed that a seven amino-acid, amino-terminally truncated product could result from unexpected translation initiation at a downstream methionine codon, amino-acid position number 8.

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Fig. 3. Two-dimensional gel analysis of pKKCSMfl and pETCSMfl expression products. The HRS fractions from Fig. 2 and native CSM-TM from chicken gizzards were analyzed. (A), Native CSMa and CSMfl tropomyosin isolated from chicken gizzards; (B), pKKCSMfl/JM109 expression products; (C), gel-purified CSMfl mixed with panel B; (D), pKKCSMfl/BL2t(DE3)LysS expression products; (E), pETCSM/3/BL21(DE3)LysS expression products; (F), native CSM-TM mixed with panel E. The acidic and basic ends of the isoelectric focusing dimension correspond to the left and right of the panels, respectively, and the top of each panel corresponds to the top of the SDS-PAGE dimension.

Expression of h TM3 in E. coli Plasmids pKKhTM3 and pEThTM3 were transformed into E. coli strains JM109 and BL21(DE3)LysS, respectively, and screened for tropomyosin expression. Fig. 4A, lanes 3-4 and 7-8 demonstrate the IPTG-induced, heat-resistant bands expressed in pKKhTM3/ JM109 and pEThTM3/BL21(DE3)LysS, respectively. In both cases, these induced bands were immunoreactive with the anti-tropomyosin antibody CG/36 (Fig. 4B, lanes 3-4 and 7-8). CG/36 also crossreacts with a higher molecular mass bacterial protein (labelled a) present in the TCP lanes which is not heat resistant. As before, the induced proteins were resistant to heat denaturation and remained soluble in E. coll. Again more than one tropomyosin expression product was observed. In this case the lower molecular mass band migrated with the same mobility as native hTM3 protein on SDS-PAGE. The higher molecular mass band may represent bacterial specific modifications of amino-acid side chains, such as Met and Cys, in hTM3. Despite this difference in electrophoretic mobility, the purified PEThTM3 containing two bands had a expected single amino-terminal Met residue and a single carboxy-terminal Met residue, as revealed from amino-acid sequencing of the 20 amino-terminal residues and carboxy-terminal amino-acid sequencing the four carboxy-terminal residues. Furthermore, as will be described below, both PEThTM3 bands are capable of binding to actin filaments. Irtscosity of bacterially-made tropomyosins It is known that muscle tropomyosins undergo headto-tail aggregation to form a linear polymer at low ionic strength. The head-to-tail polymerization of bacterially made tropomyosins was then examined by a

260 TABLE I Viscosity measurements of tropomyosin solutions Tropomyosin was dialyzed against 30 mM NaCI, 10 mM imidazole buffer (pH 7.0), 0.I mM EGTA and 0.1 mM dithiothreitol. Viscosity measurements were made at 25°C and 40/~M of tropomyosin solution. CSMa and CSMfl used here were purified from preparative SDS-PAGE gels as described previously [31], Tropomyosin

Specific viscosity a

CSM-TM CSM/3 CSMa rCSM/3 rCSM/3-7 PEThTM3

35.83 2.25 3.27 0.60 0.08 0.65

Data calculated from five independent measurements.

viscosity measurement. As can be seen in Table I, both rCSMfl and P E T h T M 3 had very low but detectable specific viscosity (0.6-0.65) at 40 m M of protein solution in 30 m M NaC1, 10 m M imidazole buffer ( p H 7.0), 0.1 m M E G T A and 0.1 m M D T T , indicating that unacetylated tropomyosins were capable of having head-to-tail interaction. However, the extent of polymerization was greatly reduced as c o m p a r e d to that for

native CSM-TM or gel-purified CSM/3 homodimer. In contrary, the a m i n o - t e r m i n a l l y d e l e t e d m u t a n t (rCSM/3-7) appeared to lose completely its head-to-tail interaction under the same measuring condition (Table I). It should be also noted that viscosity of native CSM-TM (heterodimer) is greater than that of gelpurified CSM/3 homodimer. This is consistent with the previous report by Graceffa [41]. Actin binding anaylsis In order to assess what effect the characterized mutations would have on tropomyosin binding to actin, the unacetylated mutants rCSM/3 and P E T h T M 3 and the deletion mutant rCSM/3-7 were analyzed by a F-actin co-sedimentation assay. The effects of Mg 2+ and KC1 on the binding of rCSM/3 and P E T h T M 3 to actin filaments were first examined and the results were shown in Figs. 5 and 6, respectively. At 10 mM KC1, native CSM-TM (heterodimer), gel-purified CSM/3 homodimer and P E T h T M 3 required greater than 2 mM Mg 2+ to obtain maximal binding, whereas the rCSM/3 needed at least 4 m M Mg 2+ for maximal binding (Fig. 5). Similarly, at 0.5 m M Mg 2+ condition, rCSM/3 required more KC1 (90120 mM) for maximal binding than native tropomyosins and P E T h T M 3 (60-120 mM) did (Fig. 6).

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Fig. 4. hTM3 expression from the vectors pKKhTM3 and pEThTM3. Part A represents a SDS-PAGE analysis of the TCP and HRS fractions from IPTG induced cultures. Lanes 1,2, TCP, HRS of pKK233-2/JM109; lanes 3,4, TCP, HRS of pKKhTM3/JM109; lanes 5,6, TCP, HRS of pET-8c/BL21(DE3)LysS; lanes 7,8, TCP, HRS of pEThTM3/BL21(DE3)LysS. Part B represents a Western blot analysis of lanes 1-8, Part A using the anti-tropomyosin antibody CGfl6. The band labelled 'a' is a cross-reacting bacterial protein.

261

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Fig. 5. Effect of Mg2+ concentration on the binding of gizzard tropomyosin (CSM-TM, [] ), gel-purified CSM/3 (e), rCSM/3 (*) and PEThTM3 (o) to F-actin. 0.39 mg/ml (9.3 /zM) skeletal actin was mixed with 0.15 mg/ml (2.3 /xM) various types of tropomyosins in buffer containing 10 mM KCI, 10 mM immidazole buffer (pH 7.0), 0.1 mM EGTA, 0.1 mM DTT and various amounts of MgCIz at room temperature. Binding was determined by a co-sedimentation assay. W h e n the binding assay was p e r f o r m e d at an optimal condition with 10 m M Mg 2÷ and 100 m M KCI (Fig. 7) apparent binding constants for rCSM/3 and gel-purified CSMI3 were very similar (1.14.106 and 1.25.106 M -1, respectively). However, this affinity is about 1 0 times lower than that for native gizzard tropomyosin C S M - T M (1.28- 107 M - ] ) . The difference in binding affinity may result from the difference between the heterodimer for CSM-TM [42] and the homodimer for rCSM/3 or gel purified CSM/3. T o test

Fig. 6. Effect of KCI concentration on the binding of gizzard tropomyosin (CSM-TM, n), gel-purified CSM/3 (*), rCSM/3 (*) and PEThTM3 (o) to F-actin. Binding assay conditions included 0.39 mg/ml (9.3 ~M) skeletal actin, 0.15 mg/ml (2.3/~M) various types of tropomyosins, 0.5 mM MgC12, 10 mM imidazole buffer (pH 7.0), 0.1 mM EGTA, 0.1 mM DTI" and various amounts of KCI at room temperature. this possibility, rCSMfl was mixed with gel-purified C S M a and subject to denaturation (100°C, 10 min) and renaturation at 37°C overnight to facilitate the formation of heterodimer [43]. Actin binding analysis was then performed with this resulting tropomyosin. The binding affinity for these mixed tropomyosins (apparent binding constant 1.82.106 M -1) was slightly but not significantly stronger than that for rCSM/3 alone. Therefore, the heterodimer formation cannot totally account for the different binding observed for the rCSM/3 and CSM-TM. However, it is possible that gel-purified C S M a is not fully native in terms of actin

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Free Tropomyosln (~M)

Fig. 8. Binding of bacterially-made tropomyosin rCSM/3 and PEThTM3 to F-actin. The binding condition was exactly the same as in the legend of Fig. 7. The same data for rCSM/3 as in Fig. 7 were used here for the easy comparison.

binding activity. It is reasonable to conclude that unacetylated rCSMfl has 10-times less affinity to F-actin than its native counterpart. Under the same optimal condition, the amino-terminal deletion mutant rCSM/3-7 did not bind to actin filaments at all (Fig. 7), suggesting an essential role for the amino-terminal 7 amino-acid residues in actin binding. Furthermore, the affinity of PEThTM3 to F-actin was 2-fold stronger than that of rCSM/3 (Fig. 8). The apparent binding constant (2.78. 106 M -1) for PEThTM3 appears to be 2.4-times smaller than the reported value for native high molecular mass isoform mixture of rat fibroblast tropomyosins (6.7.106 M -1) [44]. It should be noted that two variants found in PEThTM3 do not have any detectable difference in actin binding ability. In summary, unacetylated smooth muscle /3-TM (rCSM/3) and human fibroblast TM (PEThTM3) had reasonable-well binding affinity to F-actin, unlike the reported unacetylated skeletal muscle tropomyosins [20,21], which appeared to drastically lose their affinity to F-actin.

Effects of caldesmon on the binding of bacterially made tropomyosins to F-actin We have examined the effects of gizzard caldesmon on the actin binding of rCSM/3, rCSM/3-7 and PEThTM3 tropomyosins under the buffer condition containing 0.5 mM Mg 2+ and 30 mM KCI (Fig. 9). In the absence of caldesmon, rCSM/3 appeared to bind poorly to F-actin. Only 1 mol of rCSMfl was bound to 435 mol of actin. Under these conditions, the addition of gizzard caldesmon promoted the binding of rCSM/3 to actin by as much as 50-fold (Fig. 9) and the rCSM/3to-actin molar ratio was increased to 1 : 7.6. However, under the same condition, there was no enhancement in the actin-binding for the amino-terminally deleted

mutant rCSMfl-7, and the stimulation in the actin-binding for native gizzard tropomyosin (CSM-TM), gelpurified CSMfl homodimer and PEThTM3 appeared to be very small, about 1.1-1.6-fold (Fig. 9). Therefore, the difference in caldesmon enhancement of actinbinding abilities of rCSMfl and CSMfl may implicate an important role for the acetylation at the aminoterminal residue of smooth muscle tropomyosin. Discussion

In this study, we have characterized the actin-binding ability of three bacterially-expressed TM, including

I

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0

0.2

0.4

0.6

0.8

1.0

1.2

1,4

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Fig. 9. Effect of gizzard caldesmon on the binding of gizzard tropomyosin (CSM-TM, ~), gel-purified CSM/3 (e), rCSM/3 (<>) and PEThTM3 ( o ) to F-actin. Actin and tropomyosin concentration were 0.39 m g / m l (9.3 ~M) and 0.12 m g / m i (1.8 p.M), respectively, while gizzard caldesmon concentration was varied. Binding conditions were 10 mM imidazole buffer (pH 7.0), 0.5 mM MgCI 2, 30 mM KCI, 0.1 mM EGTA and 0.1 mM DTT.

263 an N-terminal unacetylated chicken smooth muscle /3-TM (called rCSM/3), a 7 amino-acid residue deletion at the N-terminus of chicken smooth muscle /3-TM (called rCSM/3-7) and an N-terminal unacetylated human fibroblast TM isoform 3 (called PEThTM3). rCSM/3 has comparable affinity to actin filaments as that of the gel-purified CSM/3. However, this affinity is about 10-times weaker than that for native CSM-TM, which is composed of CSMa and CSM/3 heterodimer. Surprisingly, the PEThTM3 exhibits only 2.4-times less affinity to actin filaments than the published data for the high molecular mass isoform mixture of rat fibrolast TM [44]. These characteristics are very different from the bacterially expressed skeletal muscle TM, including chicken skeletal a-TM [20,45] and human skeletal fl-TM [21]. Both of N-terminal unacetylated skeletal muscle TMs show a drastic reduction (30100-fold) in their binding constants as compared to their respective authentic counterparts. These observations together suggest that the lack of acetylation at the N-terminus of TMs may alter the actin-binding property to a different extent for different TM isoforms, with a drastic reduction on skeletal muscle TM, a moderate effect on smooth muscle TM and a slight effect on nonmuscle high molecular mass TM isoform. Such a difference may reflect the need of different regulation for specific TM isoforms in different tissues. This possibility is further supported by previous reports that in human red blood cells, chicken brain and chicken enterocytes, each of these tissues possesses a unique set of TM isoforms with distinct actin-binding properties [46,47]. Moreover, our preliminary analysis on the actin-binding property for a nonmuscle low molecular mass TM isoform produced in E. coli has revealed that the unacetylation of this isoform may in fact increase its binding affinity to F-actin by about 10-fold (data not shown). Similar strong affinity to F-actin has been also found for one of low molecular mass isoform TM5b of rat fibroblast tropomyosins, expressed in bacteria [48]. Therefore, the posttranslational modification, acetylation at the N-terminal residue, may play a vital role in regulating the TM's actin-binding ability and then their functions. The present work also demonstrates, for the first time, that a 7 amino-acid residue deletion at the Nterminus of CSM/3 results in a total loss of its actinbinding ability. Moreover, neither smooth muscle caldesmon nor CSM-TM/CSMot detectably enhance rCSMfl-7's actin-binding. It should be of interest to examine whether the newly-identified smooth muscle regulatory protein, caponin, [49] can exert any effect on rCSM/3-7's actin binding. Cho et al. [50] have recently demonstrated that a deletion of the first 9 amino-acid residues from chicken skeletal a-TM appears to result in the loss of its actin-binding ability, the failure of troponin complex to enhance its binding to F-actin and

the impairment on its regulatory function for actomyosin S1 ATPase. Our results for the 7-residue deletion of smooth muscle CSM/3 are not only consistent with but also extend their observation. This effect of N-terminal deletion is in contrast to that observed for the 9- or ll-residue deletion at the C-terminus of skeletal muscle TM, in which troponin complex will restore its actin-binding ability [51,52]. Therefore, the N-terminus of skeletal and smooth muscle TM isoforms contributes to a major proportion to its overall affinity to F-actin, although seven actin-binding sites have been proposed along the muscle TM molecules [22,53,54]. Under the condition of 0.5 mM MgCI 2 and 30 mM KC1, caldesmon appears to greatly enhance the binding of rCSM/3 to F-actin. However, no enhancement by caldesmon could be detected if the binding experiment was performed at the optimal condition (10 mM MgC12 and 100 mM KC1). This characteristic is also different from the enhancement of binding for unacetylated skeletal TM by troponin complex [21,45]. Although the mechanism of caldesmon's enhancement of actin binding is not known, it has been recently shown that the primary caldesmon's binding domain in a solution is located on the amino-acid residues 143-227 of smooth muscle TM [55]. Under the same measurement condition, chicken gizzard TM, rCSM/3 and rCSM/3-7 are found to bind caldesmon with the same affinity (K a = 22-106 M -1) [55]. However, caldesmon cannot improve rCSM/3-7's binding to F-actin. This result may suggest that TM must bind F-actin before caldesmon can bind and lock it in place. Alternatively, the incorrect folding of rCSM/3-7 may also be an explanation of the data. It is worth noting that rCSM/3-7 is capable of forming dimer as judged by the elution position on a Sephacryl S-300 column and that there is no detectable difference in the circular dichroism profiles between rCSM/3 and rCSM/3-7 (data not shown). Amino-acid sequence comparisons between rCSM/3 [24] and unacetylated human skeletal /3-TM [56] or between PEThTM3 (one of a gene product [25]) and unacetylated chicken skeletal a-TM [57] reveal that these two pairs of TM isoforms differ significantly only on the two alternative exons near the C-terminus of TM, i.e., exon 6 (residues 189-213) and exon 9 (residues 258-284). Nearly identical amino-acid sequences are found in regions from residue 1 to 188 and from residue 214 to 257. However, the affinity of rCSM/3 or PEThTM3 to F-actin is approx. 10-times higher than its respective striated muscle counterparts. In order to assess the relative importance of exon 6 or 9 in determining the actin binding affinity, we have constructed and expressed a variant of PEThTM3, called PEThTM2, which contains identical exon 6 sequence to skeletal a-TM. The apparent binding constant for this PEThTM2 (2.5 • 106 M -1) is very close to

264 that for PEThTM3. Analogy to human fibroblast tropomyosins, Pittenger and Helfman [48] recently reported that bacterially expressed tropomyosin isoforms TM-2 and TM-3 (a-gene products) of rat fibroblast have comparable binding affinity to F-actin. Thus, the difference in overall affinity to F-actin between PEThTM3 and unacetylated skeletal a-TM is likely due to the difference in the exon 9 sequence. This very C-terminal sequence is involved in the head-to-tail interaction, which may be also important in determining the extent of the cooperativity inherited for individual TM isoforms. Consistently, bacterially made skeletal muscle/3-TM with the deletion of exon 9 lacks the affinity to F-actin even in the presence of troponin complex [21]. Binding isotherms and cooperativity analysis in terms of the binding affinity for the first TM molecule and extent of cooperativity for the subsequent TM molecules may provide the valuable information for understanding the difference in the overall actin affinity and the effect of unacetylation at the N-terminus of TM.

Acknowledgements We would like to thank Dr. F.W. Studier for making the pET expression system available to us. This work was supported in part by grants (HD18577, GM40580 to J.J.-C.L. and GM43049, CA46370 to D.M.H.) from the National Institutes of Health and by grants (to J.J.-C.L.) from the Muscular Dystrophy Association and the Pew Memorial Trust. Dr. J.J.-C. Lin is a recipient of a Pew Scholarship in Biomedical Sciences from the Pew Memorial Trust.

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