Molecular cloning of IgT from Atlantic salmon, and analysis of the relative expression of τ, μ and δ in different tissues

Abstract

In the present study, IgT genes of Atlantic salmon were cloned and characterised. Analysis of our sequence data as well as ESTs reported to the databases revealed three distinct IgT heavy chain sub-variants in salmon, as opposed to two of IgM and IgD. The IgT sub-variants in salmon are 76–80% identical to each other, and 75–82% identical to the reported rainbow trout sequences, whereas the similarity to the orthologous molecules in zebrafish, grass carp, mandarin fish, and grouper is 25–41%. The heavy chains of both secreted and membrane anchored forms of salmon IgT include four constant Ig domains, τ1–τ4. This parallels the IgM heavy chains in elasmobranch fish and higher vertebrates, but differs from IgM in teleost fish where the membrane anchored form include only three constant Ig domains, μ1–μ3. The similarity between τ1 and μ1 in salmon is relatively high (52%) when compared to the remaining part of the molecules (τ2–τ4 and μ2–μ4 are 13–24% similar). To compare τ, μ and δ expressions in different tissues (head kidney, thymus, spleen, gill, skin, hind gut, brain and muscle) of Atlantic salmon, RT-qPCR assays were designed and evaluated. The analyses revealed that IgM transcripts are most abundant (up to 200 times more than IgD) followed by IgT (up to 20 times more than IgD) in most tissues. Highest expression of IgM, IgT, and IgD was in head kidney and spleen.

Introduction

The teleost Ig heavy chain gene complex typically encodes three antibody classes: IgM, IgD and IgT (or IgZ). While transcripts of both membrane and secreted forms are generated from the IgT and IgM genes, most teleosts appear to express primarily (or only) membrane bound IgD (Hordvik et al., 1999, Stenvik and Jorgensen, 2000, Stenvik et al., 2001, Hordvik, 2002, Hirono et al., 2003, Saha et al., 2004), except in channel catfish which possesses both secreted and membrane forms of IgD, and lacks IgT (Bengten et al., 2002). Whereas IgD, and to some degree IgT, have been subjected to major structural divergence (Hordvik et al., 1999, Stenvik and Jorgensen, 2000, Zhao et al., 2002, Danilova et al., 2005, Hansen et al., 2005, Savan et al., 2005a, Rogers et al., 2006, Deza et al., 2009, Hu et al., 2010), the IgM monomer has been evolutionary stable; with four constant Ig heavy chain domains, μ1–μ4. However, IgM make up tetramers in teleost fish, in contrast to mammals and elasmobranch fish where IgM typically form pentamers (Rombout and Joosten, 1998, Bromage et al., 2006).

IgT of rainbow trout was reported simultaneously as the orthologous molecule in zebrafish, named IgZ (Danilova et al., 2005, Hansen et al., 2005). The secreted form of both of these molecules possesses four Ig domains, τ1–τ4 (ζ1–ζ4). As in mammalian IgM, τ4/ζ4 is included in the membrane anchored form of IgT/IgZ. This is in contrast to teleost IgM, where μ3 is fused directly to the membrane anchoring part. The special splicing pattern that leads to the exclusion of the entire fourth constant Ig domain from membrane anchored IgM has been introduced after bony fish branched off the phylogenetic tree: Membrane bound IgM in elasmobranch fish includes the fourth constant domain as in mammals (Kokubu et al., 1988), whereas both μ3-TM1 and μ4-TM1 splice variants are present in some primitive bony fish (Wilson et al., 1995).

In contrast to the IgM genes which consistently encode four constant Ig domains, the overall structure of the IgT/Z genes varies to some degree. While the rainbow trout IgT and zebrafish IgZ contain four constant Ig domains (Danilova et al., 2005, Hansen et al., 2005), the IgT genes in stickleback encode three constant Ig domains (Deza et al., 2009, Bao et al., 2010). In fugu, the IgZ gene encodes only two Ig domains, corresponding to zebrafish ζ1 and ζ4 (Savan et al., 2005a), and the ζ gene of common carp is a chimera of IgM–IgZ with two constant region domains made up of μ1 of carp and a domain similar to ζ4 of zebrafish (Savan et al., 2005b). Very recently, a membrane bound zebrafish IgZ homologue (named IgZ-2), was reported (Hu et al., 2010). This is an independent homologue (with 53.5% identity to IgZ), indicating that IgZ variants are encoded by different genes, in addition to alternative splicing (Hu et al., 2010).

In the present study, IgT genes of Atlantic salmon were cloned and characterised. Cognizant to this, we designed RT-qPCR assays and analysed the relative expression of IgT, IgM, and IgD in a series of tissues (head kidney, thymus, spleen, gill, skin, hind gut, brain, muscle, and liver). Designing gene expression assays for Atlantic salmon is challenging because of a whole genome duplication event that most likely occurred in the ancestor of salmonids; which subsequently resulted in two distinct heavy chain loci (Hordvik, 1998). Thus, to achieve optimum gene quantification, qPCR assays must be carefully designed and tested. This gets even trickier when it comes to designing assays that amplify the membrane and secreted forms of the Igs. Thus, the present work provides a basis for further studies involving Ig gene expression in Atlantic salmon.