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

Fisheries Science 64(5), 812-819 (1998) Determination of Primary Structure of Heavy Meromyosin Region of Walleye Pollack Myosin 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 ƒÉgt11-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 myosins (both 79%). The sequences of the regions for the puta tive ATP-binding, actin-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 protein 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 skeletal muscle 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 proteins 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.

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