Purification, characterization and molecular cloning of alpha-2-macroglobulin in cobia, Rachycentron canadum.

Alpha-2-macroglobulin (α-2-M) is a broad spectrum protease inhibitor which is abundant in the plasma of vertebrates and several invertebrates. The α-2-M was purified from cobia (Rachycentron canadum) plasma by a four-step procedure: poly ethylene glycol fractionation, affinity chromatography, hydrophobic interaction chromatography and ion exchange chromatography on Fast Protein liquid chromatography system in the present study. It migrated as one protein band with a molecular mass of about 360 kDa in the native state, whereas in SDS-PAGE it was about 180 kDa under non-reducing condition. This result revealed that the native protein was a dimer. In addition, it was cleaved into two different fragments of molecular mass about 93 and 87 kDa when reduced by dithiothreitol (DTT). The anti-protease activity of the purified α-2-M was apparently decreased as temperature elevated above 50 °C. The α-2-M exhibited highest protease inhibitory activity at pH 9. The results indicate that the α-2-M is a heat-labile and alkaline protease inhibitor. The purified α-2-M exhibited more than 50% protease inhibitory activity against extracellular products (ECP) of Vibrio alginolytius isolated from diseased cobia. It seems that the protease activities in ECP may be affected by the plasma α-2-M. The protease inhibitory activities of cobia plasma or purified α-2-M were decreased when incubated with 10 mM methylamine for 30 min. The α-2-M cDNA consisted of 4611 bp with an open reading frame of 4374 bp had been cloned from cobia liver. This sequence contained thioester domain (GCGEQ) and thirteen predicted N-linked glycosylation sites. In addition, the amino acid sequence of thioester domain and genes of adjacent regions of cobia α-2-M were further compared with sequences of known fish species in GenBank. The unweighted pair group method using arithmetic average (UPGMA) was employed to construct the phylogenetic trees of α-2-M among different fish species (freshwater fish, sea water fish and primitive fish), and all these fish species were then clustered into three groups. The cobia α-2-M was closer to that of sea water fish than that of freshwater fish compared basing on its similarity of amino acid sequence and phylogenetic analysis of the partial gene.

Characterization of alpha-2-macroglobulin from groupers.

Alpha-2-macroglobulin (α-2-M) is a protease inhibitor broadly present in the plasma of vertebrates and invertebrates, and is an important non-specific humoral factor in defence system of the animals. This study conducted the immuno-analysis and mass spectrometric analysis methods to investigate the characteristics of the protease inhibitor, α-2-M, among groupers and related species. Rabbit antiserum to the purified α-2-M of Epinephelus coioides was used in different immunological methods to determine the immune cross-reactions of the α-2-M in samples. Plasma of Epinephelus bruneus, Epinephelus fuscoguttatus, Epinephelus lanceolatus, and Epinephelus quoyanus exhibited high protease inhibitory activities by BAPNA-trypsin assay. To purify the α-2-M protein, plasma protein of grouper E. coioides was first precipitated by using PEG 6000, then Blue Sepharose 6 Fast Flow, DEAE Sephacel, Con A Separose 4B and Phenyl Sepharose High Performance columns were used on FPLC system for purification. The molecular mass of grouper plasma α-2-M was determined as a 180 kDa protein on non-reduced SDS-PAGE. In addition, it was determined as 97 and 80 kDa protein on reduced SDS-PAGE. Enzymatic and chemical deglycosylation of glycogen revealed that the contents of glycogen in 97 and 80 kDa subunits were 12.4% and 15%, respectively, and were all belonging to N-linked type. Only one precipitation arc was visualized in all plasma of Epinephelus spp. using the rabbit antiserum to the purified α-2-M of E. coioides, on crossed immunoelectrophoresis (CIE) gels. The plasma of Epinephelus spp. and seawater fish species showed stronger responses than freshwater fish species while that of other animal species showed no response by dot-blot assay. One single band was detected on Native PAGE-Western blotting assay, one single 180 kDa band was detected on non-reduced SDS-PAGE-Western blotting, and four bands (80, 97, 160, 250 kDa) were detected on reduced SDS-PAGE when various grouper plasma was performed respectivity. However, no band was detected using plasma from the freshwater fish species and other animal species. Thus, further indicates that the protein structure of α-2-M of Epinephelus spp. was closely related among seawater fish species. In addition the identity of the two subunits was identified using LC/MS/MS which was similar to α-2-M of grass carp (Ctenopharyngodon idella) and bluegill sunfish (Lepomis macrochirus) on the protein hit.

Purification of a toxic metalloprotease produced by the pathogenic Photobacterium damselae subsp. piscicida isolated from cobia (Rachycentron canadum).

The aim of the present study was to purify and characterize a toxic protease secreted by the pathogenic Photobacterium damselae subsp. piscicida strain CP1 originally isolated from diseased cobia (Rachycentron canadum). The toxin isolated by anion exchange chromatography, was a metalloprotease, inhibited by L-cysteine, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), 1,10-phenanthroline, N-tosyl-L-phenylalanine-chloromethyl ketone (TPCK), and N-alpha-p-tosyl-L-lysine-chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0-8.0 and an apparent molecular mass of about 34.3 kDa. The toxin was also completely inhibited by HgCl2, and partially by sodium dodecyl sulfate (SDS) and CuCl2. The extracellular products and the partially purified protease were lethal to cobia with LD50 values of 1.26 and 6.8 microg protein/g body weight, respectively. The addition of EDTA completely inhibited the lethal toxicity of the purified protease, indicating that this metalloprotease was a lethal toxin produced by the bacterium.

Erratum: Purification of a toxic cysteine protease produced by pathogenic Aeromonas hydrophila isolated from rainbow trout.

An extracellular lethal toxin produced by Aeromonas hydrophila strain RT860715K originally isolated from diseased rainbow trout (Oncorhynchus mykiss) was purified by using Fast Protein Liquid Chromatography system with hydrophobic interaction chromatography and anion exchange columns. The toxin was a cysteine protease, inhibited by L-cysteine, iodoacetic acid, N-ethylamleimide, P-chloromercuibenzene-sulfonic acid and N-α-p-tosyl-1-lysine-chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0. The molecular weight of the purified enzyme proved to be 94 kDa as estimated by SDS-PAGE. In addition, the toxin was also completely inhibited by HgCl2 but partially inhibited by ethylenediamine tetraacetic acid (EDTA) and CuCl2. Both the extracellular products of Aeromonas hydrophila RT860715K and the purified protease were lethal to rainbow trout (weighing 18 g) with LD50 values of 2.87 and 0.93 µg protein g-1 fish body weight, respectively. The addition of L-cysteine completely inhibited the lethal toxicity of the purified protease, indicating that this cysteine protease was a lethal toxin produced by the bacterium.

Purification of a toxic cysteine protease produced by pathogenic Aeromonas hydrophila isolated from rainbow trout.

An extracellular lethal toxin produced by Aeromonas hydrophila strain RT860715K originally isolated from diseased rainbow trout (Oncorhynchus mykiss) was purified by using Fast Protein Liquid Chromatography system with hydrophobic interaction chromatography and anion exchange columns. The toxin was a cysteine protease, inhibited by L -cysteine, iodoacetic acid, N -ethylamleimide, P-chloromercuibenzene-sulfonic acid and N-α-p-tosyl-1-lysine-chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0. The molecular weight of the purified enzyme proved to be 94 kDa as estimated by SDS-PAGE. In addition, the toxin was also completely inhibited by HgCl(2) but partially inhibited by ethylenediamine tetraacetic acid (EDTA) and CuCl2. Both the extracellular products of Aeromonas hydrophila RT860715K and the purified protease were lethal to rainbow trout (weighing 18 g) with LD50 values of 2.87 and 0.93 µg protein g⁻¹ fish body weight, respectively. The addition of L-cysteine completely inhibited the lethal toxicity of the purified protease, indicating that this cysteine protease was a lethal toxin produced by the bacterium.