Myofibril-bound Serine Proteinase Research Articles

Unlocking the secrets of crude myofibril-bound serine protease from grass carp: The role in degrading myofibrillar proteins

Grass carp (Ctenopharyngodon idella) are used as raw material for conventional surimi products in Southern China. However, endogenous serine proteases deteriorated the texture of the surimi gel. To unlock the mechanism behind, the present study isolated the crude myofibril-bound serine protease (cMBSP) in grass carp and studied its effects on surimi gel. The cMBSP activity was the highest at 40 °C and pH 8.0, and it remained stable at 20–55 °C neutral pH. Additionally, it was susceptible to serine protease inhibitors and high concentrations of Na+. The maximum degradation of myosin heavy chain by cMBSP was observed at 50 °C. Protein unc-45 homolog B (a myosin chaperone) is one of the apparent degradation products according to mass spectrometry. The cMBSP caused lower water holding capacity and deteriorated texture in the surimi gel. This study expanded insights about the mechanism of surimi gel degradation by cMBSP, which provided theoretical basis for enhancing surimi quality.

Dihydromyricetin as a potential enhancer for surimi gels with anti-modori activity: A molecular docking and experimental validation

Modori is a major quality issue in surimi gels, and the development of safe and effective food ingredients to prevent this phenomenon is highly desirable. In this study, molecular docking was used to identify compounds that could bind to the predicted myofibril-bound serine proteinase (MBSP). Three compounds- Epigallocatechin gallate (EGCG), phytate, and dihydromyricetin (DMY)- were selected for investigation. The effects of these compounds on surimi gels containing 1.5% salt were studied, and a degradation analysis was conducted after incubation at 55 °C. Phytate did not inhibit the degradation of myosin heavy chain, whereas both EGCG and DMY promoted the formation of macromolecular aggregates associated with certain thermal stability. The addition of 0.4% of both polyphenols significantly improved the resistance of surimi gels to modori. However, when 3% salt was applied, EGCG displayed a primary promotion effect on gel deformation but reducecd breaking force. In contrast, DMY exhibited favorable impacts on breaking force, deformation, hardness, gumminess, and chewiness of resulting gels. Among the three covalent cross-linking bonds detected, Lys-DMY-Lys, Lys-DMY-Cys or Cys-DMY-Lys, and Cys-DMY-DMY-Cys, were formed between oxidized DMY and myofibrillar protein. DMY at 100 μM significantly inhibited the activity of MBSP, thus supporting the results obtained from the molecular docking study. Overall, these results suggest that dihydromyricetin is a better gel enhancer for surimi than other candidates tested.

Site-directed mutagenesis of myofibril-bound serine proteinase from Crucian carp: possible role of Pro95, A127 and I130 on thermal stability

The myofibril-bound serine proteinase (MBSP) from the skeletal muscle of fish belongs to a class of trypsin-like serine proteinases with higher thermal stability than trypsin. To investigate the mechanism responsible for the thermal stability of MBSP from Crucian carp, mutagenesis of key residues were identified by amino acid sequence alignments and Protein Mutant Stability Prediction online prediction software. Meanwhile, five mutants (MutP95T, MutR125N, MutA127S, MutI130D, and MutA127S/I130D) were obtained using site-directed mutagenesis. The mutants and wild-type enzymes were expressed in Pichia pastoris (SMD1168), the secondary structure of the mutants of MBSP was approximately similar to that of wild-type MBSP by circular dichroism spectroscopy analysis. The thermal Inactivation parameters showed that the half-life for thermal inactivation (t1/2) of MutP95T was reduced by approximately 10min, whereas MutA127S, MutI130D and MutA127S/I130D were more stable than the wild-type and increased by approximately 18min, 15min and 24min, respectively. The unfolding free energy difference (ΔΔG) between the variants and wild-type of MutA127S, MutI130D and MutA127S/I130D was enhanced when compared with the wild-type. As a result, the mutants were efficiently produced with altered thermal stability, and MBSP could potentially be used as novel tool enzyme for mass spectrometric identification.

CDNA cloning of glucose-6-phosphate isomerase from crucian carp (Carassius carassius) and expression of the active region as myofibril-bound serine proteinase inhibitor in Escherichia coli

Glucose-6-phosphate isomerase (GPI) (EC 5.3.1.9) can act as a myofibril-bound serine proteinase (MBSP) inhibitor (MBSPI) in fish. In order to better understand the biological information of the GPI and its functional domain for inhibiting MBSP, the cDNA of GPI was cloned from crucian carp (Carassius carassius) with RT-PCR, nested-PCR and 3'-RACE. The result of sequencing showed that the GPI cDNA had an open reading frame of 1662bp encoding 553 amino acid residues. After constructing and comparing the three-dimensional structures of GPI and MBSP, the middle fragment of crucian carp GPI (GPI-M) was predicted as a functional domain for inhibiting MBSP. Then the crucian carp GPI-M gene was cloned and expressed in Escherichia coli. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the recombinant GPI-M (rGPI-M) with molecular mass of approximately 21kDa in the form of inclusion bodies. The rGPI-M was obtained at an electrophoresis level purity of approximately 95% after denaturation and dialysis renaturation.

Secretory expression and characterization of the recombinant myofibril-bound serine proteinase of crucian carp (Carassius auratus) in Pichia pastoris

The myofibril-bound serine proteinase (MBSP) is effective in the degradation of myofibrillar proteins, including myosin heavy chain (MHC), α-actinin, actin, and tropomyosin and was thus regarded as an important proteinase responsible for the metabolism of fish muscle in vivo. In order to better understand the characteristic differences between native MBSP and recombinant MBSP (rMBSP) and to obtain large quantity of MBSP for its application in protein science study, the crucian carp MBSP gene was cloned (669 bp) and expressed in Pichia pastoris (P. pastoris). The recombinant P. pastoris strain was cultured in shake flasks, and 66.85 mg rMBSP/L in the fermentation supernatant was obtained. SDS-polyacrylamide gel electrophoresis (PAGE) showed a main protein band with molecular weight of approximately 36 kDa. Substrate specificity analysis revealed that the rMBSP specifically cleaved substrates at the carboxyl side of lysine residue which differed from native MBSP that cleaved substrates at the carboxyl side of arginine and lysine residues. The optimum temperature and optimum pH range of the rMBSP were 55 °C and pH7.5, respectively. Furthermore, similar to native MBSP, the rMBSP also revealed high thermostability and pH stability and is effective in degradation of myofibrillar proteins from the skeletal muscle of crucian carp.

Purification and characterisation of cathepsin L from the skeletal muscle of blue scad (Decapterus maruadsi) and comparison of its role with myofibril-bound serine proteinase in the degradation of myofibrillar proteins

The modori phenomenon during surimi production is caused by endogenous proteinases, especially cathepsin L and myofibril-bound serine proteinase (MBSP). Cathepsin L from the skeletal muscle of blue scad (Decapterus maruadsi) was purified to homogeneity by ammonium sulphate fractionation and a series of column chromatographies and revealed a single band with molecular mass of 30 kDa on SDS–PAGE. Peptide mass fingerprinting (PMF) obtained three fragments with 48 amino acid residues, which were highly identical to cathepsin L from other fish species. Its optimal pH and temperature were 5.5 and 55 °C, respectively. Meanwhile, MBSP was purified from the skeletal muscle of blue scad, and the roles of cathepsin L and MBSP in the degradation of myofibrillar proteins were compared. The results indicated that MBSP is more effective than cathepsin L in promoting the degradation of myofibrillar proteins, especially myosin heavy chain (MHC), suggesting that MBSP plays a more significant role.

Mung Bean Trypsin Inhibitor Is Effective in Suppressing the Degradation of Myofibrillar Proteins in the Skeletal Muscle of Blue Scad (Decapterus maruadsi)

Mung bean trypsin inhibitor (MBTI) of the Bowman-Birk family was purified to homogeneity with a molecular mass of approximately 9 kDa on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 8887.25 Da as determined by matrix-assisted laser desorption/ionization-quadrupole ion trap-time-of-flight mass spectrometry (MALDI-QIT-TOF MS). Using blue scad myofibrillar proteins as targets, it was found that, in the absence of MBTI, proteolysis of myofibrillar proteins, especially myosin heavy chain (MHC), could be identified after incubation at 55 °C for 2 h, while in the presence of MBTI, with a final concentration of 25 ng/mL, proteolysis of these proteins was greatly suppressed even after incubation for 3 h. Although cysteine proteinase inhibitor E-64 was also effective in preventing protein degradation, inhibitors for metallo- and asparatic proteinases did not reveal obvious inhibitory effects. Our present results strongly suggested that the naturally occurring legume bean seed protein MBTI can be used as an effective additive in preventing marine fish blue scad surimi gel softening, which is quite possibly caused by myofibril-bound serine proteinase (MBSP).

EFFECT OF A MYOFIBRIL-BOUND SERINE PROTEINASE ON THE DEG OF GIANT PROTEIN TITIN AND NEBULIN

A simplified method has been adapted for the extraction and purification of the massive myofibrillar protein titin from the skeletal muscle of sea bream to homogeneity, and a specific polyclonal antibody against titin was generated in rat. The effect of myofibril-bound serine proteinase (MBSP) on the degradation of titin and nebulin from sea bream (Sparus latus Houttuyn) was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by Western blot. Analysis revealed that both titin and nebulin could be degraded by MBSP, with nebulin degrading faster whether at the optimum temperature of MBSP (55C) and cold storage temperature (4C). These results suggested that MBSP is an important enzyme not only involved in the degradation of myofibrillar proteins such as myosin heavy chain, α-actinin, actin and tropomyosin, but also giant proteins titin and nebulin during the postmortem stage. PRACTICAL APPLICATIONS Titin and nebulin are giant proteins with molecular masses of 3,000 kDa and 600–800 kDa, respectively. Until now, few research concerning the degradation of these two proteins of fish has been reported. In the present study, a simplified method was adapted to purify native titin, and a specific polyclonal antibody against it was prepared. The degradation effect of a myofibril-bound serine proteinase on titin and nebulin at two different temperatures (55C, 4C) was investigated. It is expected that the specific antibody against titin may be used to detect the degradation of titin during the postmortem stage of fish effectively.

Purification and partial characterization of a myofibril-bound serine protease from ostrich skeletal muscle

A myofibril-bound serine protease (MBSP) was partially purified from ostrich (Struthio camelus) skeletal muscle. MBSP was dissociated from the myofibrillar fraction by ethylene glycol treatment at pH 8.5, followed by partial purification via Toyopearl Super Q 650 S and p-aminobenzamidine column chromatographies. Ostrich MBSP revealed a major protein band of approximately 21 kDa on SDS-PAGE, showing proteolytic activity after casein zymography. Optima pH and temperature of ostrich MBSP were 8 and 40 degrees C, respectively. Substrate specificity analysis revealed that the enzyme cleaved synthetic fluorogenic substrates at the carboxyl side of arginine residues. Kinetic parameters (K(m) and V(max) values) were calculated from Lineweaver-Burk plots. The kinetic characteristics of ostrich MBSP were compared to values obtained for commercial bovine trypsin in this study, as well as those obtained for MBSP from mouse and various fish species. The results suggest that ostrich MBSP is a tryptic-like serine protease. Ostrich MBSP exhibited low sequence identity to commercial bovine trypsin (44%), MBSP from lizard fish skeletal muscle (33%) and trypsinogen from ostrich pancreas (22%).

Glucose-6-phosphate Isomerase Is an Endogenous Inhibitor to Myofibril-Bound Serine Proteinase of Crucian Carp (Carassius auratus)

Glucose-6-phosphate isomerase (GPI) was purified to homogeneity from the skeletal muscle of crucian carp ( Carassius auratus ) by ammonium sulfate fractionation, column chromatographies of Q-Sepharose, SP-Sepharose, and Superdex 200 with a yield of 8.0%, and purification folds of 468. The molecular mass of GPI was 120 kDa as estimated by gel filtration, while on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two subunits (55 and 65 kDa) were identified, suggesting that it is a heterodimer. Interestingly, GPI revealed specific inhibitory activity toward a myofibril-bound serine proteinase (MBSP) from crucian carp, while no inhibitory activity was identified toward other serine proteinases, such as white croaker MBSP and crucian carp trypsin. Kinetic analysis showed that GPI is a competitive inhibitor toward MBSP, and the K(i) was 0.32 microM. Our present results indicated that the multifunctional protein GPI is an endogenous inhibitor to MBSP and may play a significant role in the regulation of muscular protein metabolism in vivo.

PARTIAL PURIFICATION AND CHARACTERIZATION OF TROPOMYOSIN-BOUND SERINE PROTEINASE FROM THE SKELETAL MUSCLE OF YELLOW CROAKER (PSEUDOSCIAENA CROCEA)

A myofibril-bound serine proteinase (MBSP) in the skeletal muscle of yellow croaker (Pseudosciaena crocea) was isolated from myofibril by heat treatment and chromatographies on Sephacryl S-200, fast performance liquid chromatography (FPLC) on Mono Q column and high performance liquid chromatography (HPLC) using a Bio-Sil SEC-125 column. A single protein band with a molecular weight of 34 kDa was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimum temperature and pH of the purified protein was 55C and 8.0. Inhibitor susceptibility analysis indicated that the enzymatic activity was effectively suppressed by serine proteinase inhibitors such as Pefabloc 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and aprotinin, while inhibitors for other proteinases (namely cysteine, asparatic and metallo) did not show any inhibitory effect. At the last purification stage, the electrophoretic study revealed that the proteinase associated with tropomyosin, as the N-terminal amino acid sequence of the 34 kDa main band protein, exhibited high sequence homology (90%) to α-tropomyosin from white croaker, human and rat. This result suggested that yellow croaker MBSP is an α-tropomyosin-binding proteinase.

Purification, Characterization, and cDNA Cloning of a Myofibril-Bound Serine Proteinase from the Skeletal Muscle of Crucian Carp (Carassius auratus)

A myofibril-bound serine proteinase (MBSP) was highly purified from the skeletal muscle of crucian carp (Carasius auratus) by acidic treatment of myofibril solution and chromatographies on Q-Sepharose and benzamidine-Sepharose 6B. MBSP revealed a main protein band of approximately 28 kDa on SDS-polyacrylamide gel electrophoresis (PAGE) and was particularly inhibited by serine proteinase inhibitors. Substrate-specificity analysis revealed that the enzyme specifically cleaved at the carboxyl side of arginine and lysine residues, suggesting the characteristics of a trypsin-type serine proteinase. MBSP gene was cloned on the basis of the N-terminal sequence and the conserved active site peptide of serine proteinases together with 5'-rapid amplification of cDNA ends (5'-RACE) and 3'-RACE. The coding region gave an amino acid sequence of 242 residues including the initiation methionine and a signal peptide of 20 residues. Amino acid residues of His60, Asp106, and Ser196 consisting of the catalytic triad of serine proteinases were conserved in the sequence. Crucian carp MBSP shared relatively high identities with other serine proteinases, especially in well-conserved regions.

Myofibrillar proteolysis by myofibril-bound serine protease from white croaker Argyrosomus argentatus

The modori phenomenon is defined as heat-induced myofibrillar degradation caused by endogenous serine protease(s) of fish muscle during Kamaboko fish meat gel production. This study was undertaken to analyze myofibrillar proteolysis of white croaker Argyrosomus argentatus muscle, which is an ingredient of high quality Kamaboko, by myofibril-bound serine protease (MBSP) under conditions corresponding to the modori phenomenon. White croaker MBSP was stable between pH 2–11 and below 65°C, and about 60% of its initial activity remained after incubation for 2 h under the conditions at 65°C and pH 7.5. About 60% of the enzyme activity was suppressed by 0.5 M NaCl. White croaker MBSP degraded various myofibrillar proteins between 40 and 70°C and pH 6.0–9.0, and preferentially degraded myosin heavy chain rather than other myofibrillar proteins. The enzyme degraded the myosin heavy chain most strongly at 55°C and pH 7.0, and a major part of the bands of myosin heavy chain and its degradation products disappeared for a period of 2 h. These degradation characteristics are very similar to those observed during the modori phenomenon, indicating that MBSP could be a modori-inducing protease involved in the modori phenomenon of white croaker Kamaboko production.

PURIFICATION AND CHARACTERIZATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE FROM THE SKELETAL MUSCLE OF SILVER CARP

Recently, a myofibril-bound serine proteinase (MBSP) in the skeletal muscle of silver carp was identified. MBSP could be dissociated from myofibrils by treatment at pH 4.0. Following ultrafiltration concentration and chromatography on Sephacryl S-200, High Q ion-exchange and affinity column of Arginine Sepharose-4B, MBSP was partially purified. The enzyme with an estimated molecular weight of 28 kDa cleaves synthetic fluorogenic substrates specifically at the carboxyl sites of arginine and lysine residues. MBSP activity is suppressed by serine proteinase inhibitors such as Pefabloc SC, lima bean trypsin inhibitor and benzamidine; it is insensitive to Pepstatin, l-3-carboxy-trans-2, 3-epoxypropionyl-l-leucine-4-guanidinobutylamide and ethylenediaminetetraacetic acid, suggesting MBSP is a trypsin-like serine proteinase. Optimal profiles of pH and temperature of the enzyme are 8.5 and 55C, respectively. Hydrolysis of myofibrillar proteins such as myosin heavy chain, actin and tropomyosin by purified MBSP occurred especially at around 55C, consistent with our proposal that MBSP plays a significant role in the Modori phenomenon.

Degradation of myofibrillar proteins by a myofibril-bound serine proteinase in the skeletal muscle of crucian carp ( Carasius auratus)

A myofibril-bound serine proteinase (MBSP) in the skeletal muscle of crucian carp ( Carasius auratus) was identified. Hydrolysis of myofibrillar proteins by the endogenous MBSP was studied. Myosin heavy chain (MHC) was degraded markedly when crucian carp myofibril was incubated at 55 °C, as shown by SDS-PAGE. Prolonged incubation of myofibril at 55 °C also caused the obvious degradation of tropomyosin, while the decomposition of other myofibrillar proteins, such as α-actinin and actin, was slight, as detected by Western blotting. The results suggest the existence of an endogenous myofibril-associated proteinase in crucian carp myofibril, which efficiently cleaves MHC and tropomyosin. Serine proteinase inhibitors (Lima bean trypsin inhibitor, PMSF and benzamidine) greatly suppressed the degradation of MHC, caused by the enzyme, while inhibitors for cysteine, metallo-, and aspartic proteinases showed only partial or incomplete inhibitory effects, indicating that the endogenous proteinase is a serine proteinase. Substrate specificity analysis, using partially purified crucian carp MBSP, suggested that the enzyme is a trypsin-like serine proteinase.

A novel type of myofibril-bound serine protease from white croaker ( Argyrosomus argentatus)

Myofibril-bound serine protease (MBSP) was purified from the myofibril fraction of white croaker ( Argyrosomus argentatus) muscle and its enzymatic properties were compared with other fish MBSPs. White croaker MBSP was extracted by the heat treatment of myofibrils and then purified by a series of column chromatographies on Q-Sepharose, Sephacryl S-300, hydroxyapatite and Benzamidine Sepharose. The purified MBSP migrated as a single protein band at 67 kDa in SDS–PAGE under both reducing and non-reducing conditions. It was inhibited by Pefabloc SC, soybean trypsin inhibitor (STI), aprotinin and benzamidine, and was not affected by E-64, pepstatin A and EDTA. The enzyme was most active against Boc–Phe–Ser–Arg–MCA at pH 7.0 and 50 °C, and preferentially hydrolyzed Boc–Val–Pro–Arg–MCA and Boc–Asp–Pro–Arg–MCA. Unlike other marine fish MBSPs, white croaker MBSP considerably hydrolyzed Boc–Val–Leu–Lys–MCA and Boc–Glu–Lys–Lys–MCA. Some enzymatic characteristics including the molecular structure and the substrate specificity for a lysine residue at the P 1 position are quite different not only from other fish MBSPs but also from soluble serine protease obtained from white croaker muscle (MSSP). White croaker MBSP could be therefore classified into a novel type of fish muscle MBSP.

The effect of soybean trypsin inhibitor on the degradation of myofibrillar proteins by an endogenous serine proteinase of crucian carp

The effect of soybean trypsin inhibitor (STI) on the degradation of crucian carp myofibrillar proteins caused by an endogenous myofibril-bound serine proteinase (MBSP) was studied. Soybean trypsin inhibitor was purified to high homogeneity and mixed with myofibrils and its inhibitory effect on myofibrillar protein degradation was investigated. In the absence of STI, proteolysis of myofibrillar proteins including myosin heavy chain, α-actinin, actin and tropomyosin could be identified after incubation at 55 °C for 5–20 min depending on the kind of the protein. In contrast, in the presence of STI, with final concentration of 0.75 μg/ml, proteolysis of these proteins was greatly suppressed even after incubation for 1 h, suggesting STI is an effective inhibitor in preventing myofibrillar protein degradation caused by a serine proteinase that is quite possibly MBSP. Though STI has disadvantages for food digestion, as a native food grade inhibitor, it is safe as a food additive, especially at low concentration. Because in surimi production, the decrease of elasticity is always accompanied with the degradation of myofibrillar proteins, thus, the present result suggested the possibility that STI could be applicable in surimi production in order to enhance the elasticity that is the quality of the final products.

Erratum to ‘‘Purification and characterization of myofibril-bound serine protease from lizard fish (Saurida undosquamis) muscleʼʼ[Comp. Biochem. Physiol. 137B, 139–150

Erratum to ‘‘Purification and characterization of myofibril-bound serine protease from lizard fish (Saurida undosquamis) muscleʼʼ[Comp. Biochem. Physiol. 137B, 139–150

IDENTIFICATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE IN THE SKELETAL MUSCLE OF SILVER CARP

Myofibril-bound serine proteinase (MBSP) in the skeletal muscle of silver carp was characterized. Myosin heavy chain (MHC) degraded markedly when silver carp myofibril was incubated at 55-60C as shown by SDS-PAGE. Prolonged incubation of myofibrils also caused the degradation of other myofibrillar proteins such as α-actinin, actin and tropomyosin to some degree. The results suggest the existence of an endogenous myofibril associated proteinase. Serine proteinase inhibitors (Pefabloc SC and Lima bean trypsin inhibitor) greatly suppressed the degradation of myosin heavy chain, while inhibitors for cysteine, metallo, and aspartic proteinases did not show any effect, indicating that the endogeneous proteinase is a myofibril-bound serine proteinase.

Purification and characterization of myofibril-bound serine protease from crucian carp Carassius giberio langsdorfi muscle

Purification and characterization of myofibril-bound serine protease from crucian carp Carassius giberio langsdorfi muscle