Fisheries Science 64(5), 812-819 (1998)

Determination of Primary Structure of Heavy Meromyosin Region of Walleye Pollack Heavy Chain by cDNA Cloning

Takao Ojima, *1 Nagako Kawashima, *1 Akira Inoue, *1 Akiko Amauchi, *1 Marie Togashi,*2 Shugo Watabe,*3 and Kiyoyoshi Nishita*1,•õ

*1Department of Marine Bioresources Chemistry , Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido, 041-8611, Japan *2Laboratory of Physiological Chemistry and Metabolism , Graduate School of Medicine, The University of Tokyo, Bunkyo, Tokyo, 113-8657, Japan *3Laboratory of Aquatic Molecular Biology and Biotechnology , Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo, 113-8657, Japan (Received January 26, 1998)

Primary structure of heavy meromyosin region of walleye pollack Theragra chalcogramma myosin heavy chain was determined by cDNA cloning. By using one PCR product and five cDNA clones isolat ed from a ăgt11-cDNA library for the pollack dorsal muscle, a nucleotide sequence of 3,923 by compris ing 60 by of 5'-untranslational region and 3,863 by of coding region was determined. The deduced se

quence of 1,287 amino acids showed considerably high homology to the corresponding regions of carp myosin (83%) and chicken and rabbit (both 79%). The sequences of the regions for the puta tive ATP-binding, -binding, and regulatory light chain-binding were well conserved among the pol lack, carp, chicken, and rabbit myosins (83-100% homology). On the other hand, relatively low se

quence homologies were seen in the essential light chain-binding site (52-78%), junctions between 20 kDa and 50-kDa domains (27-33%) and 25-kDa and 50-kDa domains (53-57%) of subfragment-1. -

Key words: walleye pollack, myosin, HMM, cDNA cloning, primary structure

Myosin is a major muscular which possesses eins .9-14)Recently, it has been reported that cross-linking of three important functions, i.e., ATPase activity, actin myosin molecules by transglutaminase reaction and/or binding ability, and filament forming ability.'-') Vertebrate- hydrophobic interaction is intimately related to the setting myosin is hexameric protein consisting of of the surimi paste. 15-18)Since it is well known that proper two heavy chains with Mr 210,000 and four light chains ties and functions of are closely related to their with Mr 15,000-25,000. The N-terminal portion of the primary and higher order structures, investigations of the heavy chain forms a globular head which contains actin molecular mechanisms for aggregation of myosin and set and nucleotide-binding sites. While the C-terminal - por ting and gel-formation of surimi products basically need tions of the two heavy chains associate to form a coiled the amino acid sequence data of myosin. coil rod which is involved in filament formation under Up to now, primary structures of various myosin heavy physiological ionic conditions. chains and light chains have been determined by protein se Fish myosins possess substantially the same properties quencing and cDNA or genomic DNA cloning.19-25)Recent as above. However, they are generally less thermo-stable ly, the primary structure of carp myosin heavy chains", 27) than warm-blooded vertebrate myosins.4-6) So far as we and light chains") have been reported. However, no myo know, walleye pollack myosin is one of the most suscepti sin of fish for food processing materials has been deter ble myosin to heat-denaturation.6,7) In the previous mined up until now. paper,') we showed that the pollack myosin lost Ca-ATPase In the present paper, we describe the cDNA cloning for activity about 20% per day during ice-storage even in the heavy meromyosin region of the walleye pollack myosin presence of I M sorbitol, and the myosin filaments once heavy chain, and determination of the nucleotide and formed under the physiological ionic conditions readily ag deduced amino acid sequences. gregated to each other. On the other hand, walleye pollack "surimi" is one of Materials and Methods the most important materials for food processing indus tries since the surimi paste exhibits high gel forming ability Poly(A)+RNA and cDNA Library on setting followed by heating.9-11) For the gel formation Walleye pollack Theragra chalcogramma was generous of the surimi paste, myosin has been considered to be the ly supplied by Usujiri Fisheries Laboratory of Hokkaido protein most responsible among the myofibrillar prot- University. Dorsal muscle (4 g) was dissected from a living

•õ To whom correspondence should be addressed. Abbreviations: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Mr, molecular weight; PCR, polymerase chain reaction; DIG, digoxigenin. cDNA cloning for Walleye Pollack HMM 813 pollack (body length 20 cm) and total RNA was extracted matography with a linear gradient of 10-200 mm KCl in by the guanidium isothiocyanate method described in a the presence of 6 M urea. The heavy chain fractions were standard protocol.29) Poly(A)+RNA was separated from pooled, dialyzed against 50 mm NH4HCO3, and lyophi the total RNA with Oligotex-dT30 (Takara). Approximate lized. The heavy meromyosin heavy chain was subjected to ly 11.4pg of poly(A)+RNA was obtained from 3.5 mg of restricted digestion with lysyl endopeptidase or cyanogen the total RNA. Two ƒÊg of the poly(A)+RNA was used for bromide, and fragments produced were isolated using a synthesizing cDNA with a cDNA synthesis kit (Amers Hitachi L-6000 high performance liquid chromatograph ham) and 1.0 Mg of the cDNA was used for construction of equipped with Tosoh ODS-120T column (4.4 x 250 mm). the ƒÉgt11 cDNA library with a cDNA cloning kit (Amer The amino acid sequences of the fragments were deter sham). Escherichia coli, Y1090 was used as a host strain mined using a model 473A protein sequencer (Perkin El for the ƒÉgt11 phage. mer-ABI).

Polymerase Chain Reaction Comparison of Amino Acid Sequences Polymerase chain reaction was carried out in a 100 ƒÊl of Alignment of nucleotide and amino acid sequences and reaction medium containing 50 mm KCl, 10 mm Tris-HCl comparison of the amino acid sequences among various (pH 8.3), 2 mm each of dATP, dTTP, dGTP, and dCTP, myosins were performed by using a DNASIS program 1.2 mm MgCl2, 100 pmol primers, 1 ng/ml template DNA, (Hitachi). The sequence data of myosins were obtained and 0.05 units/ ml AmpliTaq DNA polymerase (Takara). from database of DNA Data Bank of Japan. A successive reaction at 92°C for 30 sec, 55°C for 60 sec, and 74°C for 90 sec was repeated 30 cycles. The amplified Results cDNAs were ligated to pCR-Script SK(+) plasmid (Stratagene) and subjected to determination of nucleotide Amplification of cDNAs by Polymerase Chain Reaction sequence and preparation of DNA probes. Escherichia At first, cDNAs for the pollack myosin heavy chain were coli, XL1-Blue (Stratagene) was used as a host strain for obtained by PCR. As shown in Fig. 1, two sets of PCR the pCR-Script. primers were designed on the basis of the nucleotide and amino acid sequences of conserved regions in various myo Screening the ƒÉgt11 Library sins.21-24.311Then, two cDNAs of approximately 550 by and The ƒÉgt11 library was screened by the plaque hybridiza 750 by estimated by agarose gel electrophoresis were am tion with cDNA probes which were prepared by labeling plified from the cDNA library by the PCR. The size of the with DIG-labeling system (Boehringer Mannheim) and cDNAs was coincided with the size expected from the ami hybridization was detected with DIG-detection system no acid sequence and nucleotide sequence of the other my (Boehringer Mannheim). The cDNA in the recombinant osins. Then, the cDNAs of 550 by and 750 by termed Pm1 gt11 was cut out from the phage DNA withƒÉ EcoRI or and Pm2, respectively, were ligated to pCR-Script SK(+) BamHI, and subcloned to pBluescript II KS(+) plasmid and sequenced. As a result, the Pml and Pm2 were found (Stratagene) for nucleotide sequencing. The XL1-Blue strain of Escherichia coli (Stratagene) was also used as a host strain for the pBluescript II.

Nucleotide Sequencing The nucleotide sequences of the cDNAs in both pCR Script and pBluescript II were determined with a - dye primer cycle sequencing kit (Perkin Elmer-ABI) using a model373A DNA sequencer (Perkin Elmer-ABI). If neces sary, unidirectional nested deletion was introduced to the cDNA with Kilo-Sequence deletion kit (Takara).

Preparation of Peptide Fragments and Protein Sequencing The pollack heavy meromyosin was prepared by the method for the pollack subfragment-130) with modifica tions as follows: pollack myosin B (5 mg/ml in 0.5 M KCl) was digested at 10°C for 15 min with 1/200 weight of a chymotrypsin in 0.5 M KCl, 20 mm Tris-HCl (pH 7.6), and Fig. 1. Oligonucleotide primers used for the amplification of DNAs by I MMEDTA, and the digests were precipitated with 35% PCR. saturation of ammonium sulfate. The precipitates were dis The primers were designed based on the amino acid sequences solved in 50 mm KCl, 20 mm Tris-HCl (pH 7.5), and 100 which are highly conserved among the myosins from carp,27) MMMgCl2. Immediately after the addition of 2 mm ATP to rabbit,21) chicken,22,24) and scallop.23) Upper and lower rows show the the mixture , the heavy meromyosin dissociated from F-ac nucleotide and the amino acid sequences, respectively. IF, forward tin was purified by ammonium sulfate fractionation (40 primer synthesized based on the amino acid sequence of residues 80 55% saturation). After the heavy meromyosin was dis 84 in the chicken myosin sequence. IR, reverse primer corre sponding to the residue 249-255 in the chicken myosin sequence. 2F, sociated to heavy chains and light chains in 6 M urea, 10 forward primer corresponding to the residues 245-252 in the chicken RIMKCl, 20 mm Tris-HCl (pH 7.5), the heavy chains were myosin sequence. 2R, reverse primer corresponding to the residues Purified by a DEAE-Toyopearl 650M column chro- 485-490 in the chicken myosin sequence. 814 Ojima et al.

Fig. 2. Summary of overlapping the cDNA for the pollack myosin heavy chain. Relative positions of Pm2, Cml, Cm4, and Cm6-8 are indicated as solid lines under the restriction map, while those of Pm1, Cm2, 3, 5 are as dotted lines. The numbers in parentheses indicate the relative nucleotide positions for cDNAs to the total sequence. Closed and an open boxes in dicate 5'-untranslated and coding regions, respectively. The positions for subfragment-1 and -2 were shown with arrows. Restriction sites: E, EcoRI; B, BamHI; P, PstI; H, HindIII. to consist of 528 and 735 nucleotides, respectively. The light chain-binding sites. The others were the cDNAs en deduced amino acid sequences showed approximately coding internal region of the Cm6. Therefore, another 80% homology to the sequence of chicken skeletal myo 3 •~ 103 recombinant phages were screened with Cm6 sin 21)at the positions 88-255 and 246-490, respectively (see cDNA. Accordingly, a positive clone named Cm7 was iso Figs. 2-4). From these results, we concluded that both the lated and its cDNA (811 bp) was found to encode the bind Pm1 and Pm2 are cDNAs encoding pollack myosin heavy ing sites of essential and regulatory light chains and N-ter chain. minal portion of the subfragment-2. Subsequently, Cm8 was isolated from 5 •~ 10° recombinant phages by using the Isolation of cDNAs from ƒÉgt11 library Cm7 cDNA as a probe, and its cDNA (883 bp) was rev Next, the ƒÉgt11 cDNA library was screened using the ealed to encode subfragment-2 region (see Fig. 2). DIG-labeled Pm1 and Pm2 to obtain longer cDNAs. Pm1 Consequently, a nucleotide sequence of 3,923 by consist and Pm2 were cut out from the pCR-Script by simultane ing of the 60 by of 5'-untranslated region and the 3,863 by ous digestion with NotI and XhoI, purified by 2% agarose of coding region was determined by overlapping the se gel electrophoresis, and labeled with DIG-labeling system. quences of Pm2, Cml, Cm4, and Cm6-8 (Figs. 2 and 3). By screening with the DIG-Pm1, three positive clones From the nucleotide sequence of coding region, an amino named Cml, Cm2, and Cm3 were isolated from the 8 •~ 103 acid sequence of 1,287 residues was deduced (Fig. 3). recombinant phages. While, two positive clones named Cm4 and Cm5 were isolated from the 5 •~ 103 recombinant Amino Acid Sequences of Peptide Fragments phages with DIG-Pm2. The cDNAs of Cm1-5 were then To confirm that the above deduced sequence is indeed a subcloned to pBluescript II, and their nucleotide se sequence of pollack myosin protein, partial amino acid se quences were analyzed. The sizes of cDNAs of Cml, Cm2, quences of the peptides prepared by digestions with both Cm3, Cm4, and Cm5 were 997, 948, 798, 564, and 405 bp, lysyl endopeptidase and cyanogen bromide were deter respectively. By overlapping the sequences of the cDNAs mined. As shown in Fig. 3, the deduced sequence from the obtained from Cml, Cm4, and Pm2, a nucleotide se cDNA was found to be completely consistent with the ami quence of 1,785 by was determined (Figs. 2 and 3). The cD no acid sequences of total 130 residues from the nine pep NAs of the Cm2, Cm3, and Cm5 were identified as inter tides. nal regions of the cDNA of Cml (see Fig. 2). Comparing the deduced amino acid sequences with the sequences of Discussion the other myosin heavy chains, the nucleotide sequence of 1,785 by was found to consist of 60 by of 5'-untranslation Coding Region of the Pollack Myosin cDNA al region and 1,725 by of coding region which encodes the Although no stop codon was found upstream of the first N-terminal 575 amino acids of the pollack myosin heavy ATG (nucleotide positions 61-63) of the pollack myosin chain. cDNA (Fig. 3), the ATG was regarded as the translational Next, newly 5 •~ 103 recombinant phages were screened start codon by following two reasons: Firstly, the sequence with DIG-Cm4 to obtain cDNAs encoding C-terminal por CCATCATGA at the nucleotide positions 56-64 is similar tions of the heavy chain, and four positive clones were ob to the consensus sequence for eukaryote translational start tained. Among those clones, one clone named Cm6 (1,218 site CCACCATGG32) (Fig. 3). Secondly, the deduced N bp) encoded a region including an actin-binding site and terminal sequence showed high homology to the N-termi- cDNA cloning for Walleye Pollack HMM 815

Fig. 3. The nucleotide and deduced amino acid sequences of the pollack myosin heavy chain for heavy meromyosin region. Residue numbers for both nucleotide and amino acid are indicated in the right of each row. Annealing sites of PCR-primers, IF, IR, 2F, and 2R (see in Fig. 1), are boxed. A putative transcription start site is double-underlined. Solid and dotted underlines indicate the amino acid se quences of peptides obtained by lysyl endopeptidase and cyanogen bromide digestions, respectively. nal sequences of heavy chains of chicken and rabbit myo proximately 2/3 portion of the pollack myosin heavy sins20-22,31)(Fig. 4). Therefore, the deduced sequence of chain from the N-terminus, i.e., heavy meromyosin 1,287amino acids was concluded to the sequence of an ap- region. 816 Ojima et al.

Fig. 4. Alignment of the deduced amino acid sequence of the pollack myosin with the sequences of carp, chicken, and rabbit myosins. The sequence of the pollack myosin was aligned with the sequences of carp (10•Ž-type),27) chicken,22) and rabbit.31) Pol, pollack myosin; Car, carp myosin; Chi, chicken myosin; Rab, rabbit myosin. Putative sites for ATP-binding, actin-binding, essential light chain (ELC)-binding, and regulatory light chain (RLC)-binding are boxed. Other sites closely related to the ATPase activity, such as the reactive lysine residue (RLR), SH-1, and SH-2 are also boxed. The junctions between 25-kDa and 50-kDa domains and 50-kDa and 20-kDa domains are represented as "25-50 K junc tion" and "50-20 K junction". "last Pro" indicates the position dividing the subfragment-1 and -2 region. •œ , initial amino acid of the 28 residue repeats for coiled-coil structure; +, skip residue. cDNA cloning for Walleve Pollack HMM 817

Heterogeneity in cDNA Clones major isoform in pollack muscle since the substitutions of On the sequence determination, some sequence heter amino acids are known to cause changes in thermostability ogeneities among the cDNAs were found (Table 1). The in several proteins. For example, in genetic variants A and nucleotide sequences of both the Cm3 and the Cm5 were B of ƒÀ-lactoglobulin,33) replacements of only two amino completely identical to that of the Cml. However, the se acid residues out of total 162 residues, that is, Asp and Gly quenceof the Cm2 showed some differences from that of at residue position 64 and Val and Ala at residue position the Cml. For example, "G" at nucleotide position 181 in 118 of A and B, respectively, result in great differences be the Cml was replaced by "A" in the Cm2, thus represent tween two variants in gelation temperature, gelling rate, ed as (GA). Similarly, following replacements were and viscoelastic properties. found between the Cml and Cm2; at the positions 182 of In the present study, we determined the amino acid se the Cml (CG), 250 (GA), 689 (AIC), 690 (TG), quences of some peptide fragments of walleye pollack myo 692(TA), 693 (AT), 695 (GC), and 696 (CA). Ac sin heavy chain by automated Edman method and cordingly, five amino acids in expressed myosin from the confirmed that the sequences coincide with a deduced se Cml were expected to be replaced at positions 41 (A -S), quence from the cDNA. Among the heterogeneous amino 64 (V I), and 210-212 (DVGDADA). In addition, from acids mentioned above, e.g., Val, Thr, and Phe at residues an overlapped region between Cm4 and Cm6, four nucleo 550, 551, and 557, respectively, were identical between the tide replacements were found at the positions 1,708 of the sequences of peptide and the Cm4 cDNA. However, the Cm4 (GT), 1,709 (TQ, 1,711 (AT), and 1,730 other heterogeneous residues have not yet been detected in (TA). These replacements cause three amino acid replace the peptides. To clarify which isoform is major in ex ments at residues 550 (VS), 551 (TS), and 557 (FY). pressed myosins, it is necessary to determine the further At present, physiological significance of the sequence heter amino acid sequences using peptide fragments. ogeneity is obscure in myosin heavy chain of the walleye pollack muscle, although it has been reported that multi Comparison with Carp, Chicken and Rabbit Sequences ple forms of myosin heavy chain mRNA are alternatively The amino acid sequence of the pollack myosin heavy expressed in carp by acclimation to different tempera chain was aligned with those of carp,27) chicken,22) and rab tures.26,271However, it is important to know which is the bit21,31)myosins (Fig. 4). Through the total region, the se quence of pollack myosin showed 83% homology to that of carp myosin and 79% homology to those of chicken Table 1. Heterogeniety among the cDNA clones and rabbit (Table 2). The subfragment-2 region has appar ently a more conserved sequence (90% for carp and 83% for chicken or rabbit) than the subfragment-1 region (79% for carp and 76% for chicken and rabbit). Highly con served regions are around putative ATP-binding sites and actin-binding sites (boxed sequences in Fig. 4). These sites showed 83-100% homology between pollack and the other myosins. Regions around a reactive lysine residue (RLR) and reactive cystein residues (SH-1 and SH-2) are also con servative. These conservations reflect structural impor tances for myosin functions. The binding site of regulato-

Table 2. Comparison of amino acid sequences of the pollack heavy meromyosin heavy chain and its internal portions with carp, chick en, and rabbit counterparts

*1 Residue numbers are shown in parentheses.* 2 defined as 11 residues including RLR in the middle position.*3 defined as 31 residues including SH1-SH2 region in the middle. 818 Ojima et al.

ry light chain of pollack myosin was also highly conserved 2) H. M. Warwick and J. M. Spudich: Myosin structure and function in cell motility. Annu. Rev. Cell Biol., 3, 379-421 (1987). (94-100% homology). However, the binding site of essen 3) H. E. Huxley: Sliding filaments and molecular motile systems. J. tial light chain showed lower homology (52-78%). In addi Biol. Chem., 265, 8347-8350 (1990). tion, considerable sequence diversities were seen in junc 4) K. Arai, K. Kawamura, and C. Hayashi: The relative thermostabili tions between 25-kDa and 50-kDa domains (27-33%) and ties of actomyosin ATPase from the dorsal muscles of various fish 50-kDa and 20-kDa domains (53-60%) of subfragment-1 species. Nippon Suisan Gakkaishi, 39, 1077-1085 (1973). region (Fig. 4). These junctions are known to consist of 5) I. A. Johnston, N. Freason, and G. Goldspink: The effect of en Gly and Lys rich flexible loops correlating with protease vironmental temperature on the properties of myofibrillar adeno sensitive regions of subfragment-1. It is noteworthy that sine triphosphatase from various species of fish. Biochem. J., 133, 735-738 (1973). three Pro residues are present in the junction between 50 6) A. Hashimoto, A. Kobayashi, and K. Arai: Thermostability of fish kDa and 20-kDa domains of the pollack myosin. myofibrillar Ca-ATPase and adaptation to environmental tempera However, only one Pro residue was found in the carp myo ture. Nippon Suisan Gakkaishi, 48, 671-684 (1982). sin and no Pro residue in the chicken and rabbit myosins. 7) 1. Kimura, M. Takahashi, E. Nagahisa, and T. Fujita: Preparation In the case of several vertebrate myosins,31) no Pro residue of monomeric myosin from Alaska pollack frozen surimi. Nippon was also found in the junction between 50-kDa and 20 Suisan Gakkaishi, 48, 251 (1982). kDa domains. Thus, the presence of three Pro residues in 8) T. Ojima, S. Yoshikawa, and K. Nishita: Isolation and characteriza tion of myosin from walleye pollack surimi. Fisheries Sci., 63, 811 the junction between 50-kDa and 20-kDa domains seems 815 (1997). one of the characteristic features in the pollack myosin 9) N. Kato, A. Hashimoto, H. Nozaki, and K. Arai: Effect of tempera and may cause some structural differences of this region ture on the rate for the setting of meat pastes from Alaska pollack, from the corresponding regions of other myosins. white croaker and tilapia. Nippon Suisan Gakkaishi, 50, 2103-2108 On the other hand, the sequence of subfragment-2 (1984). region showed a typical feature which is common with 10) T. Numakura, N. Seki, I. Kimura, K. Toyoda, T. Fujita, K. Takama, and K. Arai: Cross-linking reaction of myosin in the fish other myosins. It shows a seven-residue repeat pattern and paste during setting (SUWARI). Nippon Suisan Gakkaishi, 51, a superimposed 28-residue repeat pattern characteristic for 1559-1565 (1985). a-helical coiled-coil proteins") (Fig. 4). When the sequence 11) S. F. Noguchi: Dynamic viscoelastic changes of surimi (Mincedfish is arranged in 28-residue segments, the first segment starts meat) during thermal gelation. Nippon Suisan Gakkaishi, 52, 1261 from Met at residue 854, and a "skip residue", Gln was 1270 (1986). found at residue 1,162. This Gln was reported to modify 12) T. Yasui, M. Ishioroshi, and K. Samejima: Heat-induced gelation the pitch of the coiled-coil.") These data indicate that the of myosin in the presence of actin. J. Food Biochem., 4, 61-78 sequence of the pollack myosin subfragment-2 possesses a (1980). 13) K. Samejima, M. Ishioroshi, and T. Yasui: Relative roles of the common feature observed in many myosin sequences. head and tail portions of the molecule in heat-induced gelation of While, it was found that the amino acid at position 1,078 myosin. J. Food Sci., 46, 1412 (1981). of the pollack myosin and the corrrsponding amino acid 14) T. Yasui, M. Ishinomori, and K. Samejima: Effect of actomyosin of carp myosin are Ser, whose side chain is hydrophilic. on heat-induced gelation of myosin. Agric. Biol. Chem., 46, 1049 This position is ordinarily occupied by hydrophobic ami 1059 (1982). no acid. Indeed, the corresponding amino acid in rabbit 15) N. Seki, H. Uno, N. H. Lee, I. Kimura, K. Toyoda, T. Fujita, and K. Arai: Transglutaminase activity in Alaska pollack muscle and and chicken sequences is Leu. This substitution in fish myo surimi, and its raction with myosin B. Nippon Suisan Gakkaishi, sins may make the coiled-coil structure unstable to some 56, 125-132 (1990). extent. 16) Y. Tsukamasa and Y. Shimizu: Setting property of sardine and As described above, the heavy meromyosin region of pacific mackerel meat. 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