Unraveling the evolution of the Atlantic cod's (Gadus morhua L.) alternative immune strategy

Abstract

Genes encoding the major histocompatibility complex (MHC) have been thought to play a vital role in the adaptive immune system in all vertebrates. The discovery that Atlantic cod (Gadus morhua) has lost important components of the MHC II pathway, accompanied by an unusually high number of MHC I genes, shed new light on the evolution and plasticity of the immune system of teleosts as well as in higher vertebrates. The overall aim of this study was to further investigate the highly expanded repertoire of MHC I genes using a cDNA approach to obtain sequence information of both the binding domains and the sorting signaling potential in the cytoplasmic tail. Here we report a novel combination of two endosomal sorting motifs, one tyrosine-based associated with exogenous peptide presentation by cross-presenting MHCI molecules, and one dileucine-based associated with normal MHC II functionality. The two signal motifs were identified in the cytoplasmic tail in a subset of the genes. This indicates that these genes have evolved MHC II-like functionality, allowing a more versatile use of MHC I through cross-presentation. Such an alternative immune strategy may have arisen through adaptive radiation and acquisition of new gene function as a response to changes in the habitat of its ancestral lineage.

Figure 1. Classical and alternative pathways for antigen presentation.

A) Classical antigen presentation pathways. MHC class I molecules assemble in the ER together with dedicated chaperones (like tapasin) that retain the MHC class I molecules until peptide binding. Ubiquitinated antigens are degraded by the proteasome, and the resulting peptides are transported via the transporters associated with antigen presentation (TAPs) into the ER lumen. Here the peptides are loaded onto MHC class I, tapasin is released and the peptide-MHC class I complex is transported through the Golgi to the cell surface where they are recognized by specific CD8+ T cells. MHC class II molecules also assemble in the ER with the dedicated chaperone Invariant chain (Ii). Ii mediates trafficking of MHC class II from the ER, through the Golgi, and via the cell surface to the endosomal pathway. Ii is exchanged for degraded exogenous antigenic peptides in specialized MHC class II loading compartments (MIIC). Peptide-loaded MHC class II molecules are released from the endosomal compartment to the cell surface where they are recognized by specific CD4+ T cells (reviewed in . B) Alternative (Cross-presentation) pathway for exogenous derived peptides by MHC I molecules. MHC class I molecules carrying signal motifs in the cytoplasmic tail are transported to the endosomal pathway where endocytosed antigens are degraded. Peptides can then be loaded directly in the endosomes in a TAP-independent manner, or the antigens can translocate to the cytosol for proteasomal degradation. The processed antigens can then either be loaded on MHC class I in the ER, or transported back via TAP transporters that have been recruited to the endosomal membrane (reviewed in [35]). Peptide-loaded MHC class I molecules are subsequently released to the cell surface for antigen presentation to CD8+ T cells.

Figure 2. Phylogeny of MHC I diversity in Atlantic cod.

A) Unrooted polar cladogram of all unique cDNA sequences of MHC Ia and Ib in Atlantic cod, based on amino acid sequence alignment. Elongated branches illustrate sequences originating from at least two independent PCR reactions. B) Subset of sequences highlighted in a), rooted with additional teleost Ia and Ib sequences from Ensembl. Maximum likelihood (ML) and Bayesian posterior probabilities are shown for the basal branches. Scale bar represents number of amino acid substitutions pr site.

Figure 3. Conserved amino acid sites in typical MHC I molecules.

WebLogo presentation of important selected structural and functional sites for subset of MHC I sequences from Atlantic cod. Letter size indicates the probability of the particular amino acids at the given site. Coloring scheme follows standard presentation in MEGA 5.05, reflecting amino acid properties. Numbering is based on consensus sequence, starting at the α1 domain (exon 2).

Figure 4. Anchoring sites in MHC I sequences.

Amino acids found at conserved anchoring sites are shown for the selected subset of MHC Ia and Ib sequences in Atlantic cod. Conserved teleost amino acids are shown on top. Dots indicate coherence with conserved amino acid, while letters indicate substitute amino acids at each position for each contig. Gray branches represent Ib contigs containing six or fewer conserved sites. Numbering is based on consensus sequence, starting at the α1 domain (exon 2).

Figure 5. Signal motifs in cytoplasmic tail of MHC I.

Amino acid sequences for a manually curated ClustalW nucleotide alignment for a selected subset of MHC Ia and Ib sequences in Atlantic cod. Targeting motifs are boxed (ExxxLA and YxxL), and sequences containing these are indicated with round terminal branches. Open and filled triangles indicate the position of a stop codon (*) in clade 1 and 2 respectively. The “−“ represent sequence gaps. The coloring scheme follows standard presentation in MEGA 5.05, reflecting amino acid properties. Numbering of amino acids is based on consensus sequence, starting at the α1 domain (exon 2).