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.

Lil3 dimerization and chlorophyll binding in Arabidopsis thaliana.

The two-helix light harvesting like (Lil) protein Lil3 belongs to the family of chlorophyll binding light harvesting proteins of photosynthetic membranes. A function in tetrapyrrol synthesis and stabilization of geranylgeraniol reductase has been shown. Lil proteins contain the chlorophyll a/b-binding motif; however, binding of chlorophyll has not been demonstrated. We find that Lil3.2 from Arabidopsis thaliana forms heterodimers with Lil3.1 and binds chlorophyll. Lil3.2 heterodimerization (25±7.8 nM) is favored relative to homodimerization (431±59 nM). Interaction of Lil3.2 with chlorophyll a (231±49 nM) suggests that heterodimerization precedes binding of chlorophyll in Arabidopsis thaliana.

Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley.

The light-harvesting-like (LIL) proteins are a family of membrane proteins that share a chlorophyll a/b-binding motif with the major light-harvesting antenna proteins of oxygenic photoautotrophs. LIL proteins have been associated with the regulation of tetrapyrrol biosynthesis, and plant responses to light-stress. Here, it was found in a native PAGE approach that chlorophyllide, and chlorophyllide plus geranylgeraniolpyrophosphate trigger assembly of Lil3 in three chlorine binding fluorescent protein bands, termed F1, F2, and F3. It is shown that light and chlorophyllide trigger accumulation of protochlorophyllide-oxidoreductase, and chlorophyll synthase in band F3. Chlorophyllide and chlorophyll esterified to geranylgeraniol were identified as basis of fluorescence recorded from band F3. A direct interaction between Lil3, CHS and POR was confirmed in a split ubiquitin assay. In the presence of light or chlorophyllide, geranylgeraniolpyrophosphate was shown to trigger a loss of the F3 band and accumulation of Lil3 and geranylgeranyl reductase in F1 and F2. No direct interaction between Lil3 and geranylgeraniolreductase was identified in a split ubiquitin assay; however, accumulation of chlorophyll esterified to phytol in F1 and F2 corroborated the enzymes assembly. Chlorophyll esterified to phytol and the reaction center protein psbD of photosystem II were identified to accumulate together with psb29, and APX in the fluorescent band F2. Data show that Lil3 assembles with proteins regulating chlorophyll synthesis in etioplasts from barley (Hordeum vulgare L.).

An organellar nα-acetyltransferase, naa60, acetylates cytosolic N termini of transmembrane proteins and maintains Golgi integrity.

N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltransferases (NATs). NatF, or Nα-acetyltransferase 60 (Naa60), was recently identified as a NAT in multicellular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60-knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins and has a preference for N termini facing the cytosol. Moreover, NAA60 knockdown causes Golgi fragmentation, indicating an important role in the maintenance of the Golgi's structural integrity. This work identifies a NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus-far poorly characterized part of the N-terminal acetylome.

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.

Atlantic cod (Gadus morhua L.) possesses three homologues of ISG15 with different expression kinetics and conjugation properties.

Two new interferon stimulated gene 15 (ISG15) family members were identified in a subtractive cDNA library constructed from a mixture of head kidney and spleen of Atlantic cod (Gadus morhua) stimulated with polyinosinic:polycytidylic acid (poly I:C). Two full-length Atlantic cod (Ac) ISG15-2 and AcISG15-3 cDNAs were cloned with rapid amplification of cDNA ends (RACE). The cDNA sequence of AcISG15-2 encodes a 16.9kDa protein and AcISG15-3 encodes a 18.4kDa protein, both of which possess the characteristic structural features of two tandem ubiquitin-like domains and the LRGG motif necessary for conjugation. Furthermore, the AcISG15-3 protein is expressed with a C-terminal extension in common with the human ISG15 protein. Gene expression analysis using quantitative reverse transcriptase PCR (RT-qPCR) showed that AcISG15-1, AcISG15-2, and AcISG15-3 transcripts were up-regulated in head kidney after poly I:C stimulation, suggesting that these proteins may be involved in the cod immune response. However, transient expression of myc-tagged AcISG15 proteins revealed differences in their abilities to form conjugates in vitro. We show that AcISG15-2 forms covalent conjugates to a range of cellular protein as a response to poly I:C, recombinant Atlantic salmon IFNa1 (rSasaIFNa1) and infectious pancreatic necrosis virus (IPNV), whereas conjugation was absent for AcISG15-1 and AcISG15-3. Thus, these results suggest there are three ISG15 homologues in Atlantic cod and that the three proteins may play different roles in innate immunity.

Molecular characterisation and expression analysis of interferon gamma in Atlantic cod (Gadus morhua).

Interferon gamma (IFN-gamma) has important roles in both innate and adaptive immune responses. In this study, the cDNA and genomic sequences of Atlantic cod IFN-gamma were cloned and found to encode a putative protein containing 194 amino acids with a 24 amino acid signal peptide sequence. The gene is composed of four exons and three introns similar to IFN-gamma genes of other vertebrates. The cod IFN-gamma showed only 14-29% amino acid identity with other fish IFN-gamma and 9-17% identity with IFN-gamma from higher vertebrates. However, cod IFN-gamma possesses the typical IFN-gamma motifs in the C-terminal end of the protein and displays an alpha-helix structure similar to mammalian IFN-gamma. The promoter region contains a putative ISRE element indicating up-regulation by type I IFNs and dsRNA. Real time RT-PCR analysis confirmed that IFN-gamma gene expression was up-regulated in organs of cod injected with the dsRNA polyinosinic:polycytidylic acid (poly I:C), which is a strong inducer of type I IFNs. Injection of cod with formalin-killed Vibrio anguillarum also increased IFN-gamma expression in head kidney, but to a much lesser extent than poly I:C. The gene expression results thus indicate a role for IFN-gamma in innate immune response against both virus and bacteria in Atlantic cod.

HIV-1 Rev oligomerization is not obligatory in the presence of an extra basic domain.

BACKGROUND: The HIV-1 Rev regulatory protein binds as an oligomeric complex to viral RNA mediating nuclear export of incompletely spliced and non-spliced viral mRNAs encoding the viral structural proteins. However, the biological significance of the obligatory complex formation of Rev upon the viral RNA is unclear. RESULTS: The activity of various fusion proteins based on the negative oligomerization-defect Rev mutant M4 was tested using Rev dependent reporter constructs. An artificial M4 mutant dimer and an M4 mutant containing an extra basic domain from the HTLV-I Rex protein exhibited nearly full activity when compared to wild type Rev. CONCLUSION: Rev dimerization appears to be required to expose free basic domains whilst the Rev oligomeric complex remains bound to viral RNA via other basic domains.