Characterization of an antiserum against Atlantic cod (Gadus morhua L.) g-type lysozyme.

In this study we describe the production and characterization of an antiserum against recombinant g-type lysozyme derived from Atlantic cod. This is also the first initial analyses of g-type lysozyme protein expression in tissues of Atlantic cod. Recombinant expression and purification of cod g-type lysozyme was used for immunization to rabbit and the rabbit sera were analysed for anti g-type lysozyme antibodies using enzyme-linked immunosorbent assay (ELISA), Western blot and immunohistochemistry. ELISA results showed that antibody titres were mounted between 12,800 and 25,600 as measured at an optical density corresponding to 50% of the maximal level. By Western blot analysis the rabbit immune serum detected a single ∼23 kDa band representing the size of the injected antigen, in both spleen and head kidney homogenates from the Atlantic cod. Immunohistochemisrty detected the native folded g-type lysozyme in tissues and revealed that g-type lysozyme positive cells were observed in haematopoietic tissue of the head kidney and in red pulp of spleen. In conclusion, the rabbit anti g-type lysozyme immune sera was developed and is effectively utilized for ELISA, Western analysis as well as for immunohistochemistry. This has allowed us to obtain new knowledge about this protein regarding localization and distribution in cod tissue.

Structural and functional analysis of the hemagglutinin-esterase of infectious salmon anaemia virus.

Infectious salmon anaemia virus (ISAV) is a piscine orthomyxovirus causing a serious disease in farmed Atlantic salmon (Salmo salar L.). The virus surface glycoprotein hemagglutinin-esterase (HE) is responsible for both viral attachment and release. Similarity to bovine and porcine torovirus hemagglutinin-esterase (BToV HE, PToV HE), bovine coronavirus HE (BCoV HE) and influenza C hemagglutinin-esterase-fusion (InfC HEF) proteins were exploited in a computational homology-based structure analysis of ISAV HE. The analysis resolved structural aspects of the protein and identified important features of relevance to ISAV HE activity. By recombinant expression and purification of secretory HE (recHE) proteins, receptor-binding and quantitative analyses of enzymatic activities displayed by ISAV HE molecules are presented for the first time. Three different recHE molecules were constructed: one representing a high virulent isolate, one a low virulent, while in the third a Ser(32) to Ala(32) amino acid substitution was introduced in the enzymatic catalytic site as inferred from the model. The three amino acid differences between the high and low virulent variants, of which two localized to the putative receptor-binding domain and one in the esterase domain, had no impact on receptor-binding or -release activities. In contrast, the Ser(32) amino acid substitution totally abolished enzymatic activity while receptor binding increased, as observed by agglutination of Atlantic salmon red blood cells. This demonstrates the essential role of a serine in the enzyme's catalytic site. In conclusion, structural analysis of ISAV HE in combination with selected recHE proteins gave insights into structure-function relationships and opens up for further studies aiming at dissecting molecular determinants of ISAV virulence.

Characterization of crustins from the hemocytes of the spider crab, Hyas araneus, and the red king crab, Paralithodes camtschaticus.

Crustins are distributed across the decapods and are believed to play a significant part in the humoral defense system of their host. In this study, two crustin isoforms from Hyas araneus hemocytes were purified and tested for antimicrobial activity against selected microorganisms. They show both antibacterial and antifungal activity, with highest activity against the Gram-positive bacteria Corynebacterium glutamicum. Sequencing of the transcripts showed them to have a mature peptide of 90 amino acids and differing in three positions in the mature peptide. They were named CruHa1 and CruHa2. Real-time RT-PCR revealed that they mainly are expressed in hemocytes. Screening a cDNA library detected a crustin sequence in Paralithodes camtschaticus hemocytes, coding for a mature peptide of 98 amino acids. It was named CruPc. Based on phylogenetic inference and primary structure, CruHa1 and CruHa2 were placed within the Type I group of crustins, while CruPc belongs to the Type II.

Heterologous expression and purification of the infectious salmon anemia virus hemagglutinin esterase.

This study presents the heterologous production and purification of a soluble and functional form of the hemagglutinin esterase (HE) of the infectious salmon anemia virus (ISAV) isolate 4 (Glesvaer/2/90). The HE possesses receptor binding and receptor destroying enzyme (RDE) activity and is probably involved in the infection process. The recombinant HE protein (recHE 4) was expressed in insect cells (Sf9) using the baculovirus expression vector system. Both the transmembrane region and the cytoplasmic tail were deleted, and a C-terminal His(6)-tag was attached to facilitate identification and purification of the recHE 4 protein. As determined by Western analysis the recHE 4 was secreted at 20 degrees C and not at 28 degrees C. By testing three HE constructs differing in their promoter and secretion signal sequences it was clear that the HE's own secretion signal sequence is more important than the promoter with respect to the amount of secreted recHE 4 obtained under the conditions used. A one-step purification by nickel-affinity chromatography resulted in a highly purified recHE 4, identified by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. Also, the recHE 4 is glycosylated and contains disulfide bridges within the molecule. Functional studies including the verification of the receptor destroying enzyme (RDE) activity as well as the binding to Atlantic salmon erythrocytes (hemagglutination) indicate that the recHE 4 has similar functions as its native counterpart. In conclusion, insect cells secrete a functional form of the ISAV 4 HE. This is suitable for further analyses on its function and immunogenicity.

Antibody repertoire development in teleosts--a review with emphasis on salmonids and Gadus morhua L.

The group of teleosts is highly diverse, comprising more than 23000 extant species. Studies of the teleost antibody repertoire have been conducted in many different species within different orders, though some species and families have been better characterised than others. The Atlantic cod (Gadus morhua L.) and several species within the Salmoninae (e.g. Salmo salar and Oncorynchus mykiss) are among the best-studied teleosts in terms of the antibody repertoire. The estimated size of the repertoire, the organisation of immunoglobulin (IG) gene segments, the expressed IG repertoire, the IgM serum concentration, and the serum antibody responses reveal some fundamental differences between these species. The serum IgM concentration of G. morhua is some ten times higher than that of S. salar, though G. morhua is characterised as a 'low' (or 'non') responder in terms of specific antibody production. In contrast, an antibody response is readily induced in S. salar, although the response is strongly regulated by antigen induced suppression. The IGHD gene of G. morhua has a unique structure, while the IGHM and IGHD genes of S. salar have a characteristic genomic organisation in two parallel loci. In addition, salmonids, express a broad repertoire of IGH and IGI V-region gene segments, while a single V gene family dominates the expressed heavy and light chain repertoire of G. morhua. Little is known about the developing antibody repertoire during ontogeny, in different stages of B-cell maturation, or in separate B-cell subsets. Information on the establishment of the preimmune repertoire, and the possible role of environmental antigens is also sparse.

The primary structure and specificity determining residues displayed by recombinant salmon antibody domains.

Previously, single chain fragments of salmon (Salmo salar L.) immunoglobulin variable regions (scFv) were isolated by reactivity towards trinitrophenyl (TNP) or fluorescein (FITC) using phage display technology. The fine specificity of six scFv clones were analysed by ELISA, while the primary structure was determined by DNA sequencing. In addition, preliminary models of one anti-TNP and one anti-FITC clone were built. Here, a follow-up analysis of the primary and tertiary structure of all six clones is focused on the structural basis for hapten specificity. Tertiary structure was analysed by molecular modelling of the antigen combining site. The analysis shows that reactivity to each hapten is maintained by a number of different combinations of VH, D, JH and VL sequences. Accordingly, various sizes of CDR3 on both the heavy and light chain and CDR2 of IgH may support TNP binding. Due to variability of the antigen combining site each clone probably has a distinct binding affinity. However, a feature common among the four scFv antibodies that recognise TNP is a positively charged Arg in CDR2 of either the heavy or light chain. In the majority of the anti-TNP clones localisation of this side-chain is stabilised by a negatively charged Asp in LCDR1. In addition, a Trp in LCDR3 is conserved in all the anti-TNP clones. Also, the anti-FITC clones display a Trp in the LCDR3, suggesting its participation in binding of FITC as well. In combination with a large aromatic amino acid near the N-terminus of HCDR2 and a positively charged Arg in CDR1, these residues probably determine both specificity and affinity towards the FITC moiety.

Analysis of two IgM isotypes in Atlantic salmon and brown trout.

Atlantic salmon (Salmo salar) possesses two distinct subpopulations of polymeric IgM which are separable by anion exchange chromatography. Consistent with this finding there are two isotypic IgM heavy chain genes, CmuA and CmuB, in the genome of this species, presumably as a result of ancestral tetraploidy. In the present study it was shown that IgM of brown trout (Salmo trutta) is also separated into two subpopulations by anion exchange chromatography, while IgM of rainbow trout (Oncorhynchus mykiss) and Arctic char (Salvelinus alpinus) are eluted in one peak. Molecular cloning of IgM heavy chain cDNAs from brown trout revealed messages of two distinct constant region genes, named CmuA and CmuB. As deduced from the translated cDNA sequences (and in agreement with isoelectric focusing of the corresponding proteins) the mean pI values of the heavy chains in brown trout differ with only 0.14 units, in comparison to a 0.67 unit difference in salmon. Based on the present sequence analysis we suggest that an additional cysteine near the C-terminus of CmuB is critical in relation to the fractionation of IgM by anion exchange chromatography, for example by altering the overall structure of the IgM polymer and the exposure of charged residues. Most likely, the Cmu subvariant with the characteristic extra cysteine residue arose in the ancestor of Atlantic salmon and brown trout, i.e. after the three genera Salmo, Oncorhynchus and Salvelinus radiated.

Characterisation of immunoglobulin light chain cDNAs of the Atlantic salmon, Salmo salar L.; evidence for three IgL isotypes.

By screening a cDNA library and analysis of DNA produced by a combined 3'RACE/5'-anchored PCR, we have isolated three isotypes of IgL in the Atlantic salmon. Two of the isotypes were homologous to rainbow trout IgL1 and L2 sequences, while the third represents a previously uncharacterised salmonid IgL. The novel type 3 CL region is homologous to spotted wolffish c1 and yellowtail sequences, while the VL region is more similar to channel catfish F class than to any other fish VL sequences. Southern analysis indicates that the gene segments of all three isotypes are organised in multiple clusters. In addition, the VL gene segments of type 3 are arranged in opposite orientation relative to the JL and CL segments, while gene segments in type 2 clusters are all in the same orientation. Although transcripts of type 1 and 3 were readily found in the spleen and head kidney, only minute amounts of type 2 transcripts were seen. The majority of type 3 messages were truncated, suggesting that spliced and full-length transcripts of this isotype probably are present at a low level compared to type 1 transcripts. The uniqueness of the type 3 VLJL sequences suggests that this isotype offers additional diversity to the antigen-binding site of Atlantic salmon immunoglobulins.

The antibody site in Atlantic salmon; phage display and modeling of scFv with anti-hapten binding ability.

Cloning and sequencing the cDNA of around 50 VH (VDJ) and 15 VL genes in Atlantic salmon demonstrated nine VH families (above 80% identity within each family) and one dominating but relatively diverse VL family in this species. The highest variability of the VH was seen in the CDR3, but CDR2 also expressed a modest variability. The 'whole' antibody repertoire was expressed as single chain Fv (scFv) in a phage display library by combining 12 VH and two VL specific primers (FR1/microl and FR1/CL, respectively). The PCR products (VH and VL) were ligated (with a G-rich spacer) into the lambda Surf-Zap (Stratagene) vector and expressed as a surface fusion protein on the M13 phage. Anti-TNP and anti-FITC specific scFv clones were isolated by panning using hapten-coated magnetic beads and the coding DNA sequenced. The specificities of the anti-TNP and anti-FITC clones were similar to mouse monoclonal antibodies. 3D-models of the active sites (CDRs) of the anti-TNP and anti-FITC clones suggest hapten-interacting structures of the salmon antibody site similar to mammalian antibodies.