Holostean genomes reveal evolutionary novelty in the vertebrate immunoproteasome that have implications for MHCI function

Abstract

Holosteans (gars and bowfins) have emerged as valuable models for understanding early vertebrate evolution, offering insights into diverse topics ranging from genomic architecture to molecular processes. These lineages also exhibit unusual features in their immune response, combining molecular elements seen in both tetrapods and ray-finned fishes. However, the immune repertoire of holosteans remains relatively unexplored. Here, we investigate the evolution of PSMB8, a core component of the immunoproteasome responsible for cleaving intracellular proteins into peptides for presentation by MHC class I molecules. We identify two holostean PSMB8 types-S type and K type-that are unique among vertebrates. These types likely cause significant biochemical changes to the S1 binding pocket involved in antigen cleavage which could result in the presentation of novel peptides by MHC class I. Integrating comparative analyses across major ray-finned fish lineages demonstrates that bowfins and gars independently evolved the PSMB8 S type within separate PSMB8 paralog lineages, while the PSMB8-K type is an evolutionary novelty found only in gars. Our results provide new perspectives into PSMB8 haplotypes and their role in peptide antigen processing, offering unique insights into the molecular evolution of the vertebrate immunity and antigen presentation.

Keywords: Actinopterygii; MHC; PSMB8; holostei; immunoproteasome; major histocompatibility complex.

Figure 1

Alignment of the amino terminus of PSMB8 sequences. Representative A lineage and F lineage PSMB8 sequences from major Actinopterygian lineages. Sequences were aligned using Clustal Omega (Sievers and Higgins 2021). Positions that are ≥70% identical are shaded black and those that are structurally related are shaded gray. The predicted cleavage site which produces the mature protein is indicated with an arrow. The 13th residue, in which M is described as predictive for the A lineage, and I as predictive for F lineage (Noro and Nonaka 2014) are shaded violet or blue, respectively. Residue 31, which defines the PSMB8 type is shaded as defined in the key with taxon names shaded correspondingly. Sequence identifiers for ruddy bowfin (Amical) and longnose gar (Leposs) are indicated with gray shading. Sequences previously reported by Noro and Nonaka (2014) are indicated by asterisks (*). All sequences and display identifiers are provided in Tables S1 to S4. Alignments of full-length sequences are provided in Figure S3.

Figure 2

Predicted 3D structures of multiple PSMB8 types from zebrafish, ruddy bowfin, and longnose gar. a) Structures of the PSMB8 S1 binding pocket were modeled (a) contrasting gar and zebrafish A types in the A lineage; b) contrasting bowfin and zebrafish F types within the F lineage c) evaluating the novel K type in longnose gar, and d) contrasting the S types that occur in gar A lineage and bowfin F lineage. The same view of the binding pocket is shown with three different reference color sets. “S1 pocket”—the six residues forming the S1 pocket are color-coded as shown previously (Tsukamoto et al. 2012) with orange (20th position), red (31st), yellow (35th), green (45th), cyan (49th), and magenta (53rd), respectively and the catalytic threonine (Thr1) in blue. “Ser/Thr”—serines and threonines are shaded blue. “Charge”—positively charged residues are shaded blue and negatively charged residues are shaded red. Background shading differentiates between figure panels and fish images indicate representative taxa within each column.

Figure 3

Phylogenetic relationships of PSMB8 sequences. Maximum likelihood tree topology of PSMB8 protein sequences across the evolutionary history of ray-finned fishes. Taxon names are color shaded based on the 31st position, with shadings corresponding to outer rectangles. Outerbands identify PSMB8F (gray) and PSMB8A (black) lineages. Holostean branches in the phylogeny are shaded to identify the most recent common ancestor of gar and bowfin PSMB8 sequences respectively. * in accession names indicate sequences from ENSEMBL. The functional equivalent of the 31st residue for denticle herring (Denticeps) PSMB8 is undefined (see main text and Figure S3). PSMB5 sequences from human (Homo), zebrafish (Danio), and lamprey (Petromyzon) are included as an outgroup. Dashed lines correspond to three PSMB8A clades discussed in the text: Teleostei, Acanthomorpha, and the PSMB8A clade containing numerous independent acquisitions of the PSMB8F allele (A→F reversion). Full length protein sequences are provided in Tables S1 and S4. Fish images correspond to representative taxa within the phylogeny.

Figure 4

Alignment of the amino terminus of gar and bowfin PSMB8 sequences reveals variation at local spatial scales. (a) F lineage PSMB8 sequences from ruddy bowfin collected in North Carolina (Amacal) and from eyetail bowfin collected in Louisiana (Amaoce). (b) Longnose gar (Leposs) sequences collected in either North Carolina or Tennessee, with additional gar sequences from spotted gar (Lepocu) and alligator gar (Atrspa) for comparison. Sequences were aligned using Clustal Omega (Sievers and Higgins 2021). Positions that are ≥70% identical are shaded black and those that are structurally related are shaded gray. The predicted cleavage site which produces the mature protein is indicated with an arrow. The 13th residue, in which M is described as predictive for the A lineage (Noro and Nonaka 2014), is shaded in violet. I is predictive for the F lineage (Noro and Nonaka 2014); however, no lineages possess an I. However, many sequences possess a V, which is structurally similar to I, and are shaded light blue. Residue 31, which defines the PSMB8 type is shaded as defined in the key with taxon names shaded correspondingly. Alignments of full-length sequences and a collection site details are provided in Figures S1, S2, and S9 and Tables S9 and S10.