Characterisation of a novel cold-adapted calcium-activated transglutaminase: implications for medicine and food processing.

Transglutaminases are a family of enzymes that catalyse the cross-linking of proteins by forming covalent bonds between lysine and glutamine residues in various polypeptides. Cross-linking reactions are involved in blood clots, skin formation, embryogenesis and apoptosis. Clinically, these enzymes appear to be implicated in neurodegenerative diseases, tumours and coeliac diseases. Transglutaminases have great potential for use in the food industry because of their ability to cross-link proteins that are not normally linked. Here, a gene coding for transglutaminase from Atlantic cod was cloned into a bacterial expression vector and used to transform protein expression in a strain of Escherichia coli. The successful expression of recombinant transglutaminase protein from Atlantic cod (AcTG-1) as a soluble protein upon induction at low temperature was confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, immunoblotting and mass spectrometry analysis. Biochemical characterisation demonstrated that the transglutaminase was active between 0 and 65 °C, but was completely inactivated after 20-min incubation at 70 °C. Interestingly, the enzyme displayed cold-adapted features, such as temperature instability combined with high catalytic efficiency at low temperatures (8-16 °C). In addition, the enzyme had optimal activity at 50 °C, a new feature for a cold-adapted enzyme. AcTG-1 was active in the pH range from 6 to 9, with an optimum at pH 8, and required 5 mm calcium for maximum activity. Potential calcium-binding sites in the enzyme were predictable, making the enzyme an appropriate model for studying structure-function relationships in the calcium-dependent transglutaminase family. In vitro gel analysis revealed that transglutaminase cross-linked casein, collagen and gelatin. The binding of fish fillets in the presence of recombinant AcTG-1 provided further macroscopic proof for the potential application of AcTG-1 as a biological cross-linker in the food industry. Once binding occurred, fish fillets withstood further processing such as frying, boiling, freeze-thawing and chilling. The low-temperature activity and new enzymatic properties of AcTG-1 appear to offer advantages over commercially available enzymatic glues in the food industry.

Proteolytic activity assayed by subcellular localization switching of a substrate.

An approach to assay proteolytic activity in vivo by altering the subcellular localization of a labelled substrate was demonstrated. The assay included a protein shuttling between different cellular compartments and a site-specific recombinant protease. The shuttle protein used was the human immunodeficiency virus type 1 (HIV-1) Rev protein tandemly fused to the enhanced green fluorescent protein (EGFP) and the red fluorescent protein (RFP), while the protease was the site-specific protease VP24 from the herpes simplex virus type 1 (HSV-1). The fluorescent proteins in the Rev fusion protein were separated by a cleavage site specific for the VP24 protease. When co-expressed in COS-7 cells proteolysis was observed by fluorescence microscopy as a shift from a predominantly cytoplasmic localization of the fusion protein RevEGFP to a nuclear localization while the RFP part of the fusion protein remained in the cytoplasm. The cleavage of the fusion protein by VP24 was confirmed by Western blot analysis. The activity of VP24, when tagged N-terminally by the Myc-epitope, was found to be comparable to VP24. These results demonstrates that the activity and localization of a recombinantly expressed protease can be assessed by protease-mediated cleavage of fusion proteins containing a specific protease cleavage site.

Isolation and characterisation of two cDNAs encoding transglutaminase from Atlantic cod (Gadus morhua).

Two cDNAs encoding transglutaminase (TG) were identified in a subtractive cDNA library prepared from the head kidney of poly I:C stimulated Atlantic cod (Gadus morhua). Full-length TG-1 and TG-2 cDNA were cloned from the head kidney by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The deduced amino acid (aa) sequence for TG-1 was 695 aa with an estimated molecular mass of 78.3 kDa, while TG-2 was a 698 aa protein with an estimated molecular mass of 78.8 kDa. The two proteins were named TG-1 and TG-2 and both possess transglutaminase/protease-like homologous domains (TGc) and full conservation of amino acids cysteine, histidine, and aspartate residues that form the catalytic triad. Sequence analysis showed high similarity (93.1%) with Alaska pollock TG, and the TGs were grouped together with TGs from chum salmon, Japanese flounder, Nile tilapia, and red sea bream in addition to Alaska pollock in phylogenetic analysis. Interestingly, they showed different tissue distribution with highest constitutive expression in reproductive and immunological organs, indicating important roles in these organs. Furthermore, the up-regulation of TG-1 and TG-2 in head kidney after stimulating Atlantic cod with poly I:C suggested a role of TGs in immune response in Atlantic cod.

Immune response of Atlantic salmon to recombinant flagellin.

Many viral vaccines used in aquaculture are unable to stimulate an appropriate level of immunity to withstand infection. By targeting specific components of the immune system it may be possible to trigger stronger, more effective responses to antigens. Flagellin has the ability to stimulate both the soluble and membrane-bound forms of toll-like receptor 5 (TLR5) in salmon leading to a proinflammatory response and activation of both the innate and adaptive immune system. In this study flagellin (FlaD from Vibrio anguillarum) was recombinantly produced in two forms, full-length (FDL) and a truncated form (FDS) with portions of the N- and C-termini removed to prevent polymerization. FDS was used to produce an antibody that was able to bind both forms of flagellin in immunoblot analysis. In cell culture using COS-7 cells, FDL was shown to stimulate the NF-κB pathway more effectively than FDS. Both forms of flagellin were used as an adjuvant with the antigen LPH (Hemocyanin from Limulus polyphemus hemolymph) in an immunization dose-response study. FDS and FDL stimulated the innate immune system of salmon inducing proinflammatory effects on days 2, 4 and 7 and the gene expression of important cytokines such as TNFα, IL-6, IL-8, and IL-1ß were significantly up-regulated (p<0.05) in the spleen. TLR5S was more highly up-regulated than TLR5M indicating that the soluble form of TLR5 may play an important role in the innate immune response in salmon. ELISA analysis showed that the use of flagellin as an adjuvant with LPH was not able to significantly induce flagellin or LPH antibodies. This study shows that flagellin has the potential to be a highly effective adjuvant for salmon immunization, but further research is needed.

Molecular cloning and characterization of bloodthirsty from Atlantic cod (Gadus morhua).

The tripartite motif (TRIM) proteins are involved in a variety of cellular functions including cell proliferation, differentiation, development, oncogenesis, apoptosis and antiviral activity. In this study, we report the identification and characterization of an Atlantic cod tripartite motif-containing protein named bloodthirsty from a poly I:C subtractive cDNA library. The Atlantic cod bloodthirsty (Acbloodthirsty) has a predicted open reading frame of 541 amino acids, encoding a putative 64-kDa protein. The N-terminal region contains the three motifs typical of TRIM proteins, a RING finger, one B-box, and a coiled-coil domain, which together form the TRIM motif found in this large family of proteins, whereas the C-terminal region contains a PRYSPRY domain. The intracellular localization of Acbloodthirsty in CHSE-214 cells showed mostly diffuse staining with some discrete compartments in the cytoplasm. The induction of bloodthirsty transcripts by poly I:C was seen in spleen using quantitative reverse transcriptase PCR (RT-qPCR). The expression pattern indicates that Acbloodthirsty is involved in antiviral immune response in Atlantic cod.