cDNA cloning and characterization of two gelatinases from Japanese flounder

SUMMARY: Toughness is one of the most important elements that define the commercial value of the raw meat of fish. Degradation of the extracellular matrix is thought to be a cause of postmortem tenderization of fish meat. A previous study has suggested that this tenderization is caused mainly by metalloproteinases. The present study seeks to identify the proteinase(s) involved in tenderization; hence, cloned cDNA of two gelatinases from Japanese flounder, which showed high homology with mammalian matrix metalloproteinase (MMP)-2 and MMP-9, were designated as jfMMP-2 and jfMMP-9 , respectively. Northern blot analysis revealed that jfMMP-2 mRNA was expressed almost ubiquitously in adult tissues including the brain, muscle, gill, heart, gut, kidney, spleen, testis, and ovary. In contrast, the expression of jfMMP-9 mRNA was observed in those tissues which were abundant in blood cells, such as kidney, spleen, heart, and gill. Both recombinant proteins (jfMMP-2 and jfMMP-9) produced with the COS-7 cell system exhibited gelatin-degrading activity that was sensitive to 1,10-phenanthroline, a typical metalloproteinase inhibitor.     

Inhibitory effects of a monoclonal antibody (MAb-001) on in vitro oxygen consumption and multiplication of the pathogenic haemoflagellate, Cryptobia salmositica Katz

A monoclonal antibody (MAb-001), against a surface glycoprotein on Cryptobia salmositica inhibited the multiplication and oxygen consumption of both virulent and avirulent strains of the parasite. The classical cysteine proteinase inhibitor (E-64) and a cysteine proteinase activator (EDTA) affected the in vitro multiplication of C. salmositica . Concentrations of E-64 higher than 10 μ M reduced the multiplication of C. salmositica while 5 m M of EDTA enhanced its multiplication. We propose that the cysteine proteinase is an important metabolic enzyme in C. salmositica and that binding of MAb-001 to the enzyme inhibited parasite multiplication and reduced oxygen consumption.     

A serine proteinase from the sarcoplasmic fraction of red sea bream Pagrus major is possibly derived from blood

Collagen degradation is known to be involved in the post mortem tenderization of fish muscle. A serine proteinase that is assumed to be related to collagen degradation after fish death was purified from the sarcoplasmic fraction of red sea bream Pagrus major by ammonium sulfate fractionation and column chromatography on Sephacryl S-300, Q Sepharose and Phenyl Sepharose CL-4B. The enzyme hydrolyzed gelatin and was obtained as a protein band of approximately 38 kDa upon sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. The N-terminal amino acid sequence of the enzyme was determined for 32 residues. A protein that had the same N-terminal amino acid sequence as the enzyme for ten residues was purified from the serum of red sea bream and showed the same characteristics as the enzyme. Therefore, it is suggested that the serine proteinase migrates from the blood to muscle and degrades muscle proteins after the death of the fish.     

Characterization of gelatinolytic enzymes in the skeletal muscle of red sea bream Pagrus major

Gelatinolytic enzymes were partially purified from the skeletal muscle of red sea bream Pagrus major and characterized to obtain information on post mortem tenderization of fish muscle. Four gelatinolytic activities, G1 (90 kDa), G2 (65 kDa), G3 (60 kDa), and G4 (100 kDa), were detected in the Q Sepharose column. G1, the major gelatinolytic enzyme, and G4 were identified as serine proteinases from results of inhibitor spectrum and substrate specificity. By contrast, G2 and G3 were found to be metalloproteinases since these were inhibited by ethylenediamine tetraacetic acid and o-phenanthroline, and activated by 4-aminophenylmercuric acetate. The optimum pH and temperature of these enzymes were in the ranges of 7–9 and 20–40°C, respectively.     

鲤肌肉肌原纤维结合型丝氨酸蛋白酶的分子克隆

肌原纤维结合型丝氨酸蛋白酶(myofibril-bound serine proteinase,MBSP)是最近发现的一种蛋白酶。该酶参与肌原纤维蛋白的降解及鱼糜制品弹性的下降。但是,对该酶一级结构的研究,迄今为止,未有报道。本文根据已测定的鲤MBSP N-末端氨基酸序列以及丝氨酸蛋白酶活性中心保守序列设计兼并引物,结合RT-PCR技术实现了MBSP基因片段的扩增。再根据克隆到的MBSP片段序列设计基因特异引物,用于MBSP基因的5′和3′末端快速扩增。综合以上结果,鲤MBSP的全长被确定。序列分析表明,MBSP cDNA含有一732 bp的开放阅读框,编码243个氨基酸残基,其中信号肽长度为21个氨基酸残基。组成丝氨酸蛋白酶活性中心的氨基酸残基(His61,Asp107和Ser197)在MBSP中保守存在。成熟MBSP含有222个氨基酸残基,分子量为24.5 ku,比其天然蛋白的分子量30 ku略小。成熟MBSP的等电点为10.43。鲤MBSP与鲫MBSP,猪胰蛋白酶,牛胰蛋白酶,美洲鲽胰蛋白酶的同源性分别为80.6%,55.8%,55.3%和53.9%。而与仓鼠肌肉中具有胰凝乳蛋白酶性质的蛋白酶的同源性为39.2%。MBSP有高含量的赖氨酸残基(11.93%),此特性可能与该酶的肌原纤维结合特性有关。     

Inhibition of post-mortem muscle softening following in situ perfusion of protease inhibitors in tilapia

ABSTRACT:     A method of introducing protease inhibitors into fish muscle through the bulbus arteriosus was developed using an in situ perfusion technique. Perfusion efficiency was initially tested using eosin and [³⁵S]-methionine. Visible fluorescence was observed in the gill, liver, intestine and dorsal muscle of the eosin-treated tilapia, and the occurrence of eosin in the blood vessels of the dorsal muscle was confirmed under a fluorescence stereoscopic microscope with ultraviolet light. The radioactivity of [³⁵S]-methionine was taken into the dorsal muscle and liver at a concentration of 7.8 Bq/g and 70.2 Bq/g, respectively, after perfusion with 1000 Bq/mL solution. Using the perfusion technique with four kinds of protease inhibitors dissolved in physiological saline, the type of proteases implicated in the post-mortem muscle softening in tilapia (867 ± 195 g, n  = 10/protease inhibitor) was investigated. After the perfusion of leupeptin (serine and cysteine protease inhibitor), benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk; caspase inhibitor), chymostatin (serine protease inhibitor) and ο -phenanthroline (metalloprotease inhibitor), the breaking strength of the perfused muscle was measured as a parameter of the meat toughness and compared with that of the control fish, which were perfused with physiological saline only. The reduction of breaking strength during storage was inhibited by the perfusion of leupeptin and Z-VAD-fmk.     

Autolysis and Characterization of Sarcoplasmic and Myofibril Associated Proteinases of Oxeye Scad (Selar boops) Muscle

ABSTRACT

The autolytic profile of oxeye scad mince was characterized. Mince showed higher proteolytic activity than washed mince. The highest autolysis was observed at 65 and 60°C for mince and washed mince, respectively. Both mince and washed mince showed the optimum pH for autolysis at pH 9.0, and their activities decreased with increasing NaCl concentration (0–3.5%). Autolysis of washed mince was markedly inhibited by soybean trypsin inhibitor (SBTI), suggesting that myofibril-associated proteinase was serine proteinase. Sarcoplasmic proteinase was characterized to be heat-activated alkaline proteinase having the optimal pH and temperature of 9.0 and 60°C, respectively. The activities were stable at pH range of 8.0–11.0 at 20–40°C. The crude proteinase was inhibited by N-p-tosyl-L-lysine chloromethyl ketone, SBTI, and phenylmethanesulfonyl fluoride, suggesting the predominance of serine proteinases, especially trypsin. NaCl suppressed the activity while β-mercaptoethanol, dithiothreitol, and CaCl₂ activated the activity. Therefore, trypsin-like proteinase is a major endogenous proteinase responsible for autolysis in oxeye scad muscle. The present results can be used as scientific guidelines to predict the gel strength of surimi made from oxeye scad muscle.     

Necrotic myositis of rainbow trout, Oncorhynchus mykiss (Walbaum): proteolytic characteristics of a crude extracellular preparation from Flavobacterium psychrophilum

A crude extracellular preparation (CEP) from a strain of Flavobacterium psychrophilum recovered from a case of necrotic myositis affecting rainbow trout was capable of causing severe muscle necrosis in rainbow trout following intramuscular injection. Cell wall-associated preparations, however, were unable to produce similar lesions in experimentally injected fish. The CEP degraded gelatin and type II collagen but not type I or type IV collagen. Furthermore, the CEP did not degrade 2-furanacryloyl- l -leucylglycyl- l -prolyl-alanine (FALGPA), chondroitin sulphates A, B or C, heparan sulphate, keratan sulphate, hyaluronic acid, elastin or rainbow trout erythrocytes. The addition of the protease inhibitors 1,10-phenanthroline, ethylenediamine-tetraacetic acid (EDTA) and EGTA to the CEP halted its ability to degrade gelatin in vitro and to produce muscle necrosis in rainbow trout in vivo . In vitro and in vivo activity was restored following the addition of 1 m m zinc chloride to the protease inhibitor-treated CEP, suggesting that this strain of F . psychrophilum secretes a protein complex with zinc metalloprotease-like activity. This protein complex, therefore, appears to be involved in the pathogenesis of necrotic myositis in rainbow trout.     

中国明对虾serpin型丝氨酸蛋白酶抑制剂基因的重组表达及活性分析

Serpin家族成员作为主要的丝氨酸蛋白酶抑制剂,通过调节一系列丝氨酸蛋白酶和半胱氨酸蛋白酶的活性来调节蛋白酶级联反应,进而参与了包括血液凝结、补体激活、纤维蛋白溶解、炎症反应、肿瘤抑制和激素转运等大量的基本生物过程,在调节机体免疫及其它重要生理功能方面发挥着重要作用。我们在前期研究中,从中国明对虾血细胞中克隆得到一个serpin型丝氨酸蛋白酶抑制剂基因（Fc-serpin, GenBank注册号：DQ318857）,初步研究显示它在转录水平参与了病原菌和白斑杆状病毒（WSSV）引发的对虾先天免疫应答反应;本研究利用原核重组表达系统,成功获得了重组的对虾serpin型蛋白酶抑制剂（rFc-serpin）,纯化复性后得率为0.3g/L;活性分析显示,重组目的蛋白（rFc-serpin）对多种病原菌的生长均有一定抑制作用,研究结果进一步证实它参与病原微生物引发的对虾防御应答过程,为探讨甲壳动物先天免疫防御应答机制提供了有价值的参考。     

Characterization of Gelatinolytic Activity in Seminal Plasma of Some Teleost Fish

Gelatin-containing SDS-PAGE combined with the incubation of gels in buffers containing protease inhibitors was performed for the visualization and characterization of proteolytic activity in teleost fish seminal plasma. To demonstrate the class of detected enzymes we used serine protease inhibitor – benzamidine or EDTA which inhibits metalloproteases activity. Additionally the effects of calcium ions on protease activity were investigated. Multiple gelatinolytic activities in seminal plasma of 10 teleost fish species from three orders (Cypriniformes, Salmoniformes, Perciformes) were found. Most proteases were either stimulated by Ca²⁺ and inhibited by EDTA or inhibited by benzamidine. This suggests that metalloproteases and serine proteases are major gelatinolytic proteases of fish seminal plasma. In cyprinid species we found a common profile of two gelatinolytic activities; the first band (60–66 kDa) belonged to metalloproteases, and the second one (76–81 kDa) belonged to serine proteases. Other bands were also visible and they represented mostly serine protease activity. Species from the Salmoniformes order showed a similarity in metalloproteases with molecular weights of about 64 and 75 kDa. Salmonid species also had similar serine proteases with molecular weights of about 102 and 165 kDa. In European grayling seminal plasma we found metalloproteases with molecular weight of 51, 57, 64, 70 kDa and two serine proteases activities of 35 and 125 kDa. Percid species had metalloproteases activities of 53 and 63 kDa and serine protease activity of 100 kDa. Protease of other, presently unknown classes were also found in seminal plasma of asp, chub, European grayling and pikeperch. The physiological role of seminal plasma proteases is still unknown.