Systematic characterization of immunoglobulin loci and deep sequencing of the expressed repertoire in the Atlantic cod (Gadus morhua)

Abstract

Background: The Atlantic cod is a prolific species in the Atlantic, despite its inconsistent specific antibody response. It presents a peculiar case within vertebrate immunology due to its distinct immune system, characterized by the absence of MHCII antigen presentation pathway, required for T cell-dependent antibody responses. Thorough characterisation of immunoglobulin loci and analysis of the antibody repertoire is necessary to further our understanding of the Atlantic cod's immune response on a molecular level.

Results: A comprehensive search of the cod genome (gadmor3.0) identified the complete set of IgH genes organized into three sequential translocons on chromosome 2, while IgL genes were located on chromosomes 2 and 5. The Atlantic cod displayed a moderate germline V gene diversity, comprising four V gene families for both IgH and IgL, each with distinct chromosomal locations and organizational structures. 5'RACE sequencing revealed a diverse range of heavy chain CDR3 sequences and relatively limited CDR3 diversity in light chains. The analysis highlighted a differential impact of V-gene germline CDR3 length on receptor CDR3 length between heavy and light chains, underlining different recombination processes.

Conclusions: This study reveals that the Atlantic cod, despite its inconsistent antibody response, maintains a level of immunoglobulin diversity comparable to other fish species. The findings suggest that the extensive recent duplications of kappa light chain genes do not result in increased repertoire diversity. This research provides a comprehensive view of the Atlantic cod's immunoglobulin gene organization and repertoire, necessary for future studies of antibody responses at the molecular level.

Keywords: Atlantic cod; Diversity; Gadmor3.0; Ig; Immunogenetics; Repertoire.

Fig. 1

Schematic representation of IGH and IGL loci in the genome of the Atlantic cod. A Schematic (top) and detailed (bottom) map of the IGH locus on chromosome 2 (11,595–12,377 kb). In the schematic the distances are not to scale and the number of blue bars is about one half of the number of V-genes. Numbers in the brackets indicate the total number of genes including pseudogenes. All genes are in the forward orientation indicated by the arrows. The V-genes are in blue of which the pseudogenes are in a lighter shade. D-genes are in red, and J-genes are green. IGHM genes are light brown and IGHD genes are dark brown. In the detailed map the numbers refer to V-gene names unless otherwise specified. Names of V-genes in families other than the largest family 3 are depicted in light blue for easier identification. B Schematic (top) and detailed (bottom) map of the IGL loci on chromosome 2 (5,085–5,170 kb) and chromosome 5 (4,120–4,140 kb and 17,350–19,690 kb). In the schematic the distances are not to scale. Numbers in the brackets indicate the total number of complete sets of V-J-C mini-clusters. In the detailed map the V-genes are in blue of which the pseudogenes are in a lighter shade of blue. J-genes are green, C genes are dark brown, and pseudogenes are light brown. The numbers refer to V-J-C mini-clusters unless otherwise specified. Names of V-genes in family V1-2 are depicted in light blue for easier identification. Thick arrows indicate the prevalent orientation of the V-genes (blue), J-genes (green) and C-genes (brown) in the locus, whereas thin arrows indicate genes that have diverging orientation from the main rule

Fig. 2

Number of unique receptors based on sequencing depth and abundance-based receptor rank and frequency for IgH and IgL iota-1 (kappa) chains. Number of unique receptors identified at the CDR3 amino-acid sequence level in relation to mapped sequencing reads for IgH (A) and IgL kappa (B) receptors. Subsamples of reads were created from mapped reads for each sample. Sampling depth was adjusted for each sample according to total number of mapped reads. Abundance-based receptor rank and proportion of repertoire of IgH (C) and iota-1 (kappa) light chains (D). Each point represents an individual receptor, stars denote D50 values for each sample. All axes are logarithmic. D50 values and corresponding vertical lines were jittered slightly for better visibility

Fig. 3

CDR3 length distribution of immunoglobulin heavy (A) and iota-1 (kappa) light chains (C) and relationship between V-gene germline and expressed CDR3 lengths (B and D, respectively). All samples were combined for calculating CDR3 length distributions. Germline V-gene CDR3 length was calculated by taking the number of CDR3 nucleotides from germline sequences and dividing them by 3. No significant correlation was found between IgH germline V-gene and expressed CDR3 lengths (Pearson’s R = 0.06, P = 0.63), while very strong correlation was observed in case of kappa chains (Pearson’s R = 0.96, P < 2.2*10^–16). Small jitter was added to plots B and D for better visibility

Fig. 4

Proportion of the repertoire unique to each fish and the proportion of the repertoire shared between two, widely shared between several fish and shared by all per immunoglobulin isotype. Colours denote the proportion of the levels of sharedness of the repertoire

Fig. 5

V gene usage in immunoglobulin heavy chains. Genes are ordered based on family and gene identifiers. All samples were used in calculation of gene usage, error bars represent standard deviation between individual fish