Japanese basket clams (C. japonica), blood clams (Anadara broughtonii), Asiatic hard clam (Meretrix lusoria), Japanese littleneck (Ruditapes philippinarum), Japanese oysters (Crassostrea nippona), hard clam (Mercenaria mercenaria), Japanese scallop (Mizuhopecten yessoensis), Mediterranean mussel (Mytilus galloprovincialis), banana prawn (Fenneropenaeus merguiensis), and snow crab (Chionoecetes opilio) were obtained from the supermarket.
Formation of cloudy liquid
One hundred grams of sample was placed in 60 mL of boiling distilled water for 3 min. The sample was filtered using a filter paper (Qualitative filter papers No. 2, Advantec, Tokyo, Japan) and centrifuged at 8,000 × g for 10 min at 4 °C.
The sample was ultrafiltered using an Amicon Pro Purification System with a 100-kDa cut-off (Merck Millipore, Tokyo, Japan) by centrifuging at 8,000 × g for 90 min at 4 °C.
Equal volumes of 1% ninhydrin solution (Fujifilm Wako Pure Chemical, Osaka, Japan) and the sample were mixed and heated in a boiling water bath for 10 min and then allowed to cool to 25 °C. The formation of colour was observed.
Trichloroacetic acid (TCA) precipitation
Sample and 20% TCA were mixed in equal amounts, stirred, placed on ice for 10 min, and centrifuged at 10,000 × g for 10 min at 4 °C.
A laser pointer (635 nm, 200-LPP029, Sanwa Supply Inc., Okayama, Japan) was used to observe the Tyndall effect.
The boiled soup was stained using a negative staining method. First, one drop of the soup was dropped onto a microgrid covered with a carbon support film hydrophilized via ion sputtering. The microgrid was allowed to stand for 10 min. Next, a 2% solution of uranyl acetate was added dropwise to the microgrid, and the excess solution was absorbed using a filter paper. The microgrid was dried naturally and observed using a transmission electron microscope (TOPCON Co., Ltd., Tokyo, Japan; EM-002B) at an acceleration voltage of 80 kV.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzymatic proteolysis
Protein concentrations were measured using the Bradford method, with bovine serum albumin (Fujifilm Wako Pure Chemical) as the standard. Proteins (5 µg) were separated on a 10% (w/w) polyacrylamide gel at a constant current of 25 mA/gel and stained for 60 min with Coomassie Brilliant Blue R-250 (Fujifilm Wako Pure Chemical) and acetic acid solution. Trypsin (50 ng/µL) (Promega Madison, WI, USA) was used for digesting all samples. Depending on the volume of the gel pieces, 2–5 µL of enzyme solution was added and incubated at 37 °C for 16 h.
Trypsin-digested samples were mixed with α-cyano-4-hydroxycinnamic acid and applied to a spot on a mass spectrometry (MS) target plate. MS data were obtained using a 5800 MALDI-TOF/TOF analyzer (ABSciex, Concord, Canada) according to the manufacturer’s instructions. A monoisotopic precursor for MS/MS was selected using automatic precursor selection with an interpretation method of the DynamicExit algorithm (AB Sciex). The MS/MS data were analyzed using the ProteinPilot™ software (version 3.0) and the Paragon protein database search algorithm (AB Sciex). Each MS/MS spectrum was searched against the database constructed by AB Sciex.
The absorption spectrum from 200 nm to 300 nm was measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Cleveland, OH, USA).
Inductively coupled plasma (ICP)-MS analysis
The boiled soup was filtered using a 100-kDa ultrafiltration spin column and precipitated using TCA; the pellet was treated with nitric acid and then analyzed using ICP-MS. The S, Na, Mn, Ti, As, Fe, Ni, Cu, Li, Mg, Al, Si, P, K, Ca, Co, Zn, Rb, and Cs contents were determined by ICP-MS (8800 Triple Quadrupole ICP-MS; Agilent, CA, USA), according to the manufacturer’s instructions.
Isolation of the tropomyosin gene from C. japonica
Total RNA was extracted from C. japonica using the ISOSPIN Plant RNA kit (Nippon Gene, Tokyo, Japan) and digested with DNase I (NipponGene Co., Ltd., Tokyo, Japan) according to the manufacturer’s instructions. A 3-µg aliquot of total RNA was used to synthesize single-stranded cDNA using the ReverTra Ace qPCR RT master mix (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer’s instructions. For PCR amplification, the cDNA was denatured at 98 °C for 3 min in the first cycle and then for 10 s in subsequent cycles. Primer annealing and extension were performed at 55 °C and 72 °C for 5 s and 10 s, respectively. Thirty cycles of PCR were performed using the following oligonucleotide primers: 5¢-GCCATCAAGAAGAAGATGCAGGCAATGG-3¢ and 5¢-CCAGCCAATTCAGCAAA-3′. The PCR products were fractionated on a 1% (v/w) agarose gel, and DNA fragments from agarose gel slices were purified using a DNA fragment purification kit (MagExtractor, Toyobo Co., Ltd.). The purified reverse transcription-polymerase chain reaction (RT-PCR) products were directly sequenced using the BigDye termination kit ver 3.1 (Applied Biosystems, Foster City, CA, USA).
Construction of recombinant Escherichia. coli expressing tropomyosin
The C. japonica tropomyosin gene was amplified using PCR with the appropriate primers (5¢-CATCATCATCATCATATGGATGCCATCAAGAAGAA-3¢ and 5¢-TATCTAGACTGCAGGTTAATACCCAGCCAATTCAG-3′). The amplicon was cloned into the pColdII expression vector to create pColdII-tropomyosin with a His-tag using the In-fusion ® Snap Assembly master mix (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. The construct was sequenced for confirmation and then transformed into E. coli BL21 (DE3) cells for the production of the His6-tagged (N-terminus) C. japonica tropomyosin.
Purification of recombinant proteins
The transformants were cultured at 37 °C in Luria Bertani medium containing ampicillin (50 µg/L) (Fujifilm Wako Pure Chemical) until the OD600 was approximately 0.5. Next, the temperature was changed to 15 °C to induce cold-shock promoters, and incubation was continued for 24 h. The cells were harvested via centrifugation (6,000 × g, 10 min, 4 °C) and suspended in 1 mL of protein extraction reagent (Integral Co., Tokushima, Japan). One microliter of 1,000 U/mL recombinant DNase I (Takara Bio Inc.) and 0.2 mg/mL lysozyme (Fujifilm Wako Pure Chemical) were added to each sample and incubated for 15 min at room temperature. Then, the samples were centrifuged (13,000 × g, 20 min, 4 °C). Recombinant proteins were isolated using the His-Spin protein miniprep kit (Zymo Research Co., Irvine, CA, USA), according to the manufacturer’s instructions.
Elemental analysis of tropomyosin
Boiled soup was purified using an Amicon Pro Purification System with a 100-kDa cut-off (Merck Millipore). Samples were collected following TCA precipitation. Subsequently, nitric acid was added to the pellet, and the mixture was incubated at 100 °C for 12 h. The degraded product was suspended in 0.1 N nitric acid solution and analyzed using ICP-MS (8800 Triple Quadrupole ICP-MS; Agilent).
Amino acid composition of all human proteins
Amino acid sequence data of all human proteins were downloaded from Uniprot (https://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/reference_proteomes/Eukaryota/UP000005640/UP000005640_9606.fasta.gz). Only the canonical entries were used for the following analysis. The amino acid composition was calculated using the Perl script (https://www.nntp.perl.org/group/perl.beginners/2010/12/msg114941.html) with a slight modification (Supplementary Data 1).