Molecular Localization of Health-Promoting Peptides Derived from Fish Protein Hydrolyzates on Fish Muscle Proteins.

The four previously reported health-promoting dipeptides, valine-tyrosine, lysine-tryptophan, methionine-phenylalanine, and arginine-isoleucine, found in the fish muscle hydrolyzates, were mainly located in the myosin subfragment-1 heavy chain, whereas the health-promoting tripeptide, alanine-lysine-lysine, was found in the fibrous rod consisting of the myosin subfragment-2 and light meromyosin with a regular coiled-coil structure of α-helix, irrespective of the fish species. Furthermore, the localization of these peptides either in the random coil, ß-sheet, or α-helix was also examined in the three-dimensional image, showing no specific tendency. Surprisingly, the same trend was observed even for the mammalian rabbit fast muscle myosin heavy chain. Since a trade-off between myofibrillar ATPase and structural stability has been reported for fish living at low environmental temperatures, it is speculated that fish muscle proteins, when ingested, are easily digested by various proteases in the human digestive tract and provide various health-promoting peptides also in vivo. While fish actin contained only two dipeptides, methionine-phenylalanine and valine-tyrosine, glyceraldehyde 3-phosphate dehydrogenase, one of the major components of fish muscle water-soluble protein, contained all of the four dipeptides and one tripeptide mentioned above.

Peptidomic Analysis of a Disintegrated Surimi Gel from Deep-Sea Bonefish Pterothrissus gissu.

Surimi gel is a commonly found gelled product in Japan. Disintegration of the surimi gel is mainly caused by proteolytic degradation of the myosin heavy chain (MHC) under an inappropriate heating process. Many studies have reported the decrease in MHC in the disintegrated surimi gel but the mechanistic details of this degradation remain unclear. This study employed peptidomic analysis of disintegrated surimi gels from deep-sea bonefish Pterothrissus gissu to reveal the MHC cleavage causing gel disintegration. More peptides derived from an MHC rod were found in the disintegrated P. gissu surimi gels than in the integrated gel. Most MHC peptides were derived from the Src homology 3 domain or near the skip residues. The results of the terminome analysis suggest that the catalytic type of the proteases is responsible for light meromyosin cleavage activated at ∼35 °C. These results showed the temperature-dependent cleavage of the MHC rod, causing disintegration of the P. gissu surimi gel.

The effects of endogenous proteases within abdominal muscle parts on the rheological properties of thermally induced gels from white croaker (Pennahia argentata).

Three types of material meats were prepared from a so-called normal muscle part of white croaker (Pennahia argentata) containing 0, 4.2 and 8.4% of an abdominal muscle part. Thermally induced gels were then prepared from these materials by pre-heating at 65 °C for 30 or 60 min and subsequent heating at 85 °C for 20 min. The breaking strength and breaking strain rate of thermally induced gels decreased with increasing contamination levels of the abdominal muscle part, in which degradation of myosin heavy chains was observed. The proteolytic activity in the abdominal muscle part homogenate was highest at 62.5 °C. These results suggest that the abdominal muscle part contains proteases that induce the modori phenomenon. Technical experts assume that a contaminated abdominal muscle part leads to quality deterioration in surimi production industries. Our findings will aid the production of high-quality surimi-based products.

Deterioration of white croaker (Pennahia argentata) meat thermally-induced gel products caused by proteolytic enzymes in the contaminated intestine and kidney.

Thermally-induced gels were made from white croaker (Pennahia argentata) meat in the presence of its organ extracts by pre-heating at 40 and 65°C for 20 min and subsequent heating at 85°C for 20 min. The breaking strength of the gels decreased with increasing concentrations of the intestinal extracts accompanying decomposition of myosin heavy chains. However, no significant changes in the gel strength occurred when the kidney extract was added. The proteolytic activity in the intestinal extracts examined in the meat homogenate had a maximum at 60°C and pH 8.90. These results suggest that the intestinal rather than kidney proteolytic activities are responsible for gel softening known as a modori phenomenon. Thus, the removal of intestinal tracts is essential to maintain a high quality of surimi-based products.

The pepsin digestibility of thermal gel products made from white croaker (Pennahia argentata) muscle in associating with myosin polymerization levels.

Thermal gels were made from white croaker (Pennahia argentata) surimi at various polymerization levels of myosin heavy chains induced by suwari treatment at 38 °C for various time periods and subsequently heated at 85 °C for 20 min. Myosin heavy chain polymerization levels were also achieved in the presence of microbial transglutaminase (MTG) added at various concentrations in the surimi. The breaking strength and breaking strain rate were markedly increased during suwari treatment up to 60 min in accordance with the increased levels of myosin heavy chain polymerization. MTG enhanced myosin heavy chain polymerization during suwari treatment for 15 and 30 min, resulting in the increase of breaking strength. The solubilization in 8 M urea and pepsin digestibility of these gels as well as angiotensin I-converting enzyme (ACE) inhibitory activity of their pepsin digests were decreased with the increased levels of myosin heavy chain polymerization. These results suggest that myosin heavy chain polymerization affects not only rheological properties of thermal gels but also their functional properties for human health.

Effects of point mutations on the structural stability of tuna myoglobins.

Structural stabilities of myoglobin (Mb) from several tuna fish species significantly differ from each other, although the amino acid sequence identities are very high (>95%), suggesting that limited number of substitutions greatly affect the stability of Mb. To address this hypothesis, attempts were made to elaborate recombinant tuna Mbs with point mutations on the different residues among fish Mbs. The expression plasmid constructs were based on bigeye tuna Mb cDNA sequence, and the recombinant proteins were expressed as GST-fusion proteins in Escherichia coli. After removal of the GST segment and affinity purification, the stability of five Mb mutants, namely, A49G, T91K, K92Q, V108A, and H112Q, together with the wild type (WT) were measured, taking temperature dependency of alpha-helical content and denaturant (urea and guanidine-HCl) concentration dependency of Soret band absorbance as parameters. As a result, the mutant H112Q showed much higher stability than WT, while the structures of K92Q, T91K and A49G mutants were destabilized. No essential change in helical content was observed for V108A, but the mutant was found to be destabilized easier by the denaturants. These findings suggested that the highly conserved residues among tuna species are responsible for their stability of Mbs, but a few non-conserved residues dramatically give rise to the differences in stability of Mbs among species.

Cryptides: functional cryptic peptides hidden in protein structures.

Peptidergic hormones, neurotransmitters, and neuromodulators are extracellular signaling molecules that play central roles in physiological signal transmissions between various cells, tissues, and organs. These factors are primarily translated as inactive precursor proteins according to the genetic information. These precursor proteins are then cleaved by various proteases including signal peptidases and processing enzymes to produce matured bioactive factors. During these processes, various fragmented peptides are also produced from the same precursor proteins. Such fragmented peptides may have various unexpected biological activities that have not been identified yet because these peptides are considered to be produced and released along with mature factors at the same secretary pathways. Recently, we found that various fragmented peptides of mitochondrial proteins that are produced during the maturation processes, such as fragments of cytochrome c oxidase, activate neutrophils whose functions are distinct from their parent proteins. These findings suggest the existence of many different functional peptides whose functions have not been identified yet. These unidentified peptides may play a variety of roles in various regulatory mechanisms, and therefore, they are expected to provide novel regulatory and signaling mechanisms, "Peptide World".

Effect of amino acid replacements on the structural stability of fish myoglobin.

Structural stabilities of myoglobin (Mb) from several fish (scombridae) species differ significantly, although their amino acid sequence identity is very high (>95%), suggesting that only a few substitutions greatly affect the stability of Mb. Accordingly, recombinant Mbs with point mutation(s) derived from bigeye tuna Mb cDNA were expressed as GST-fusion proteins in the soluble fractions of Escherichia coli. After removal of the GST segment, the stability of five mutants, namely, P13A, I21M, V57I, A62G, and I21M/V57I, together with the wild type (WT) were investigated, taking temperature dependency of alpha-helical content and denaturant concentration dependency of Soret band absorbance as parameters. As a result, the stability of P13A against denaturants and its alpha-helical content at 10 degrees C was found to be the highest among the mutants, whereas those of A62G were the lowest. The stabilities of V57I and I21M/V57I were higher than that of WT, though that of I21M was nearly the same as WT. These findings suggest that the structural stability of fish Mb is tuned up only by the substitutions of a few amino acid residues located in the alpha-helical segments forming the hydrophobic heme pocket.

Structural stabilities of recombinant scombridae fish myoglobins.

An expression system of recombinant myoglobins (Mb) of 3 scombridae fish species was constructed. The stability of these Mbs was compared with native Mbs purified from slow skeletal muscle. The addition of hemin during the cultivation of an Escherichia coli strain harboring a pGEX-2T expression vector was found to be necessary to prevent recombinant Mb from degrading and to attain its proper folding. The stabilities of recombinant Mbs were generally lower than those of native Mbs, partly due to the absence of post-translational modification. The alpha-Helical content of bullet tuna recombinant Mb at 10 degrees C was the lowest (29.0%) among the recombinant Mbs examined (the values for bluefin tuna and bigeye tuna Mbs being 34.8 and 35.5%, respectively). On the other hand, the stabilities of recombinant Mbs of bluefin tuna and bigeye tuna against denaturants (urea and guanidine hydrochloride) were found to be similar, whereas bullet tuna recombinant Mb exhibited the lowest stability among these Mbs. The pattern of temperature-dependent decrease in the alpha-helical content supported these results.

Characterization of bullet tuna myoglobin with reference to the thermostability-structure relationship.

Myoglobin (Mb) was isolated from bullet tuna (Auxis rochei) skeletal muscle and characterized from the viewpoint of the thermostability-structure relationship. Differential scanning calorimetry (DSC) measurement showed that the thermostability of bullet tuna Mb was the lowest among all the scombridae fish Mbs so far examined. The highest value (72.8 degrees C) of melting temperature (Tm) was obtained at pH 6.52. alpha-Helical content at 10 degrees C was 34.5%, clearly lower than that of horse Mb (55.3%). The amino acid sequence was then deduced by cloning cDNA which encodes bullet tuna Mb. Bullet tuna Mb consisted of 147 amino acids, and the sequence identity was very close to that of skipjack (Katsuwonus pelamis) Mb (91.8%). A few amino acid substitutions, which could be involved in stability difference of Mb, were recognized. By mass spectrometry of lysyl endoproteinase digest of Mb, the N-terminus was found to be acetylated like that of other fish Mbs.