Immunoglobulin T from sea bass (Dicentrarchus labrax L.): molecular characterization, tissue localization and expression after nodavirus infection

Abstract

Background: Immunoglobulins (Igs) are fundamental components of the adaptive immune system of vertebrates, with the IgT/IgZ isotype specific of Teleosts. In this paper we describe the identification of an IgT heavy chain from the European sea bass (Dicentrarchus labrax L.), its molecular characterization and tissue mRNA localization by in situ hybridization.

Results: Sea bass IgT consists of 552 aa (Accession Number KM410929) and it contains a putative 19 amino acids long signal peptide and one potential N-glycosylation site. The C-region consists of four C_H domains; each contains the cysteine and tryptophan residues required for their correct folding. Based on the recent sequencing of sea bass genome, we have identified five different genomic contigs bearing exons unequivocally pertaining to IgT (C_H2, C_H3 and C_H4), but none corresponded to a complete IgH locus as IgT sequences were found in the highly fragmented assembled genomic regions which could not be assigned to any major scaffold. The 3D structure of sea bass IgT has been modelled using the crystal structure of a mouse Ig gamma as a template, thus showing that the amino acid sequence is suitable for the expected topology referred to an immunoglobulin-like architecture. The basal expression of sea bass IgT and IgM in different organs has been analysed: gut and gills, important mucosal organs, showed high IgT transcripts levels and this was the first indication of the possible involvement of sea bass IgT in mucosal immune responses. Moreover, sea bass IgT expression increased in gills and spleen after infection with nodavirus, highlighting the importance of IgT in sea bass immune responses. In situ hybridization confirmed the presence of IgT transcripts in the gut and it revealed a differential expression along the intestinal tract, with a major expression in the posterior intestine, suggesting the hindgut as a site for the recruitment of IgT⁺ cells in this species. IgT transcripts were also found in gill filaments and parallel lamellae and, for the first time, we identified scattered IgT positive cells in the liver, with a strong signal in the hepatic parenchyma.

Conclusions: In conclusion, we performed a full molecular characterization of IgT in sea bass that points out its possible involvement in mucosal immune responses of this species.

Keywords: IgT; In situ hybridisation; Mucosal immunity; Sea bass; Tissue expression.

Fig. 1

Alignment of the predicted sea bass IgT heavy chain amino acid sequence with other known IgT molecules. The amino acids of the signal peptide in the sea bass sequence are in italics. The position of the framework (FR) and CDR regions for the sea bass IgT V-DOMAIN (following the IMGT numbering) is indicated above the sequences, together with the J region, the four C_H domains and the secretory tail; in this region the typical conserved amino acids in the sea bass sequence are in bold and italics. The N-linked glycosylation site in the sea bass IgT sequence is evidenced (see the C_H3 domain). The conserved amino acids are indicated with an asterisk below the sequences, while dot and semicolon showed amino acids with conserved physical and chemical properties. The conserved cysteine (in bold and underlined) and tryptophan (in bold) residues that are required for the correct folding of the immunoglobulin superfamily (IgSF) C_H domains are highlighted along the sequences. The conserved cysteine residues possibly involved in the disulphide bond with the IgT light chain and the ones related to the possible polymerization with other IgT heavy chains are evidenced in bold during the sequences. Accession numbers: Oncorhynchus mykiss (rainbow trout) AAW66978; Larimichthys crocea (large yellow croaker) XP_010754058; Thunnus orientalis (Pacific blue tuna) AHC31432; Dicentrarchus labrax (sea bass) KM410929; Siniperca chuatsi (mandarin fish) AAY42141; Epinephelus coioides (orange-spotted grouper) GU182366

Fig. 2

Phylogenetic tree showing the relationship between sea bass IgT, IgM and IgD sequence with other known Ig molecules from teleost fish. The tree was bootstrapped 10,000 times with 50,000 random seeds. The Accession Numbers are the same as indicated in a, except for: Salmo salar (Atlantic salmon) IgT ACX50292, IgM AAB24064, IgD AF141607; Danio rerio (zebrafish) IgT AY643752, IgM AAT67445; Ctenopharyngodon idella (grass carp) IgT DQ478943, IgM ABD76396, IgD ADK66818; Anguilla anguilla (European eel) IgM ACD76833; Gadus morhua (Atlantic cod) IgM CAA41680; Dicentrarchus labrax (sea bass) IgM KY173353, IgD KU132360; Takifugu rubripes (pufferfish) IgM BAD26619, IgD BAD34542; Oncorhynchus mykiss (rainbow trout) IgM AAB27359; Ictalurus punctatus IgD (channel catfish) ADF56020; Epinephelus coioides (orange-spotted grouper) IgD AFI33218; Paralichthys olivaceus (Japanese halibut) IgD BAB41204; Scophthalmus maximus (turbot) IgD AFQ38975; Lutjanus sanguineus (humphead snapper) IgD AIC33830; Siniperca chuatsi (mandarin fish) IgD ACO88906; 0.2 indicates the genetic distance

Fig. 3

Schematization of the three-dimensional model of sea bass IgT. N- and C-terminus of the model are indicated by arrows. The four structural domains are indicated, from left to right, as VH, C_H1-gamma, C_H2-gamma, and C_H3-gamma, respectively. Short helices are coloured in red, while beta strand are in cyan, and turns in green

Fig. 4

IgT and IgM sea bass basal expression in different tissues. IgT and IgM mRNA levels were expressed as a ratio relative to rRNA 18S levels in the same tissue after real-time PCR analysis using the brain as calibrator. Data were expressed as the mean ± SD

Fig. 5

IgT expression after infection with nodavirus. Sea bass IgT and IgM mRNA levels were expressed as a ratio relative to rRNA 18S in the same samples after real-time PCR analysis of spleen and gills leukocytes from four fish infected i.m. with nodavirus and normalized against one muscle from a time 0 control (not showed)

Fig. 6

IgT-expressing cells in sea bass gills. A IgT expressing cells in sea bass gill filaments and parallel lamellae (arrows). B Negative control with sense probe. Scale bars A, B 10 µm

Fig. 7

IgT-expressing cells in sea bass posterior, middle and anterior intestine. A IgT⁺ cells in the epithelium and lamina propria of posterior segment. B Higher magnification showing intraepithelial lymphocytes expressing IgT transcripts. C Negative control of posterior intestine with sense probe. D IgT⁺ cells in epithelium and lamina propria of the middle segment. E Anterior segment showing IgT-expressing cells localized in the lamina propria. Scale bars A, D, E 20 µm; B 10 µm; C 50 µm

Fig. 8

IgT transcripts in the liver. A IgT anti sense probe stains positive cells in the hepatic parenchyma. B Higher magnification of IgT⁺ cells. C No signal was revealed using m-RNA sense probe. Scale bars A, B 20 µm; C 50 µm