An Ancient, MHC-Linked, Nonclassical Class I Lineage in Cartilaginous Fish

Abstract

Cartilaginous fishes, or chondrichthyans, are the oldest jawed vertebrates that have an adaptive immune system based on the MHC and Ig superfamily-based AgR. In this basal group of jawed vertebrates, we identified a third nonclassical MHC class I lineage (UDA), which is present in all species analyzed within the two major cartilaginous subclasses, Holocephali (chimaeras) and Elasmobranchii (sharks, skates, and rays). The deduced amino acid sequences of UDA have eight out of nine typically invariant residues that bind to the N and C termini of bound peptide found in most vertebrae classical class I (UAA); additionally, the other predicted 28 peptide-binding residues are perfectly conserved in all elasmobranch UDA sequences. UDA is distinct from UAA in its differential tissue distribution and its lower expression levels and is mono- or oligomorphic unlike the highly polymorphic UAA UDA has a low copy number in elasmobranchs but is multicopy in the holocephalan spotted ratfish (Hydrolagus colliei). Using a nurse shark (Ginglymostoma cirratum) family, we found that UDA is MHC linked but separable by recombination from the tightly linked cluster of UAA, TAP, and LMP genes, the so-called class I region found in most nonmammalian vertebrates. UDA has predicted structural features that are similar to certain nonclassical class I genes in other vertebrates, and, unlike polymorpic classical class I, we anticipate that it may bind to a conserved set of specialized peptides.

Figure 1.

Multiple amino acid alignment of the four distinctive MHC class I lineages in cartilaginous fish, UAA, UBA, UCA and the new lineage UDA. GenBank accession numbers are listed in Supplemental Table I. Dots indicate amino acids identical to Gici UAA, and dashes indicate gaps, respectively. Highly conserved residues in all class I proteins are shaded black (16, 28), s and h indicate the β-strands and α-helices, and the line connecting the two Cys in the α2 domain indicates the class I canonical disulfide bridge. P marks the invariant residues that bind to the N- and C-termini of the bound peptide in the classical class I molecules and p indicates the other 28 peptide binding residues. The Asn marks the Asparagine-linked glycosylation site, Asp/Glu indicates the typical Aspartic acid and Glutamic acid residues found in the connecting piece (Conn, light shade), and Tyr and Ser mark the conserved positions of Tyr and Ser in the cytoplasmic tail (also in light shade) of classical class I molecules.

Figure 2.

Phylogenetic tree of cartilaginous fish and other selected vertebrate class I molecules. The tree was constructed using the neighbor-joining (NJ) method with the α1 and α2 domains (PBR), and rooted with CD1. Bootstrap support values are shown as percentages on the branches. The boxes indicate the four distinctive MHC class I lineages in cartilaginous fish. GeneBank accession numbers for all the sequences are listed in Supplemental Table I, and the corresponding alignment is found in Supplemental Figure 1. Common names are shown followed by abbreviation of the scientific name (two letters of the genus and two letters of species). The scale bar indicates the number of amino acid differences per sequence.

Figure 3.

Unique tissue distribution of UDA lineage in nurse shark compared to UAA via northern blotting. Nucleoside-diphosphate kinase (NDPK) was used as a loading control (ubiquitous expression) although we found it to have lower expression in muscle and pancreas in the presence of similar amounts of 28S and 18S rRNA across samples. RNA marker size (kb) is shown on the left of the blot.

Figure 4:

Differential expression in nurse shark gill section between the classical UAA and UDA lineages, using in situ hybridization of full-length UAA and UDA region nurse shark riboprobe. A) H&E stained tissue section with the gill filament structures highlighted in the remainder images; B), C) and D) UAA antisense riboprobe; E) and F) UAA sense riboprobe; G), H) and I) UDA antisense riboprobe; J) and K) UDA sense riboprobe; B) E) G) and J) DAPI contain staining; C) H) F) and J) riboprobe with SA-AF647 signal detection and D) and I) riboprobe with signal detection into SA-AP and NBT/BCIP substrate. Note the highest expression of UAA in epithelial cells (white arrows), and of UDA in some pillar (blue arrows) and epithelial cells (white arrows). All images were taken at 20X magnification except the H&E that was taken at 10X magnification.

Figure 5.

Presence of the UDA lineage in Chondrichthyans via Southern blotting. BamHI-digested DNA from different species was hybridized with the nurse shark MHC class I UDA α3 domain probe under low stringency conditions. The Chondrichthyan species used were one chimaera (spotted ratfish Hydrolagus colliei); 3 rays (cownose ray Rhinoptera bonasus, little skate Leucoraja erinacea, thornback ray Raja clavata) and 8 sharks (nurse shark Ginglymostoma cirratum, bamboo shark Chiloscyllium punctatum, horn shark Heterodontus francisci, sand-tiger shark Carcharias taurus, catshark Scyliorhinus canicula, lemon shark Negaprion brevirostris, spiny dogfish Squalus acanthias, blue shark Prionace glauca). The marker size (kb) is shown on the left. Bands in the gel are marked with arrows.

Figure 6.

UDA is monomorphic in wild nurse shark individuals. A) Alignment of nucleotide sequences obtained from cloning of PCR products of α1 and α2 UAA and UDA from four wild nurse shark individuals (1, 2, 3 and 4). B) Trace files of nucleotide sequences obtained by direct sequencing of PCR products of α1 UAA (one individual) and of α1 and α2 UDA for the same four wild nurse shark individuals (1, 2, 3 and 4). Dots indicate nucleotides identical to nurse shark #1 (highlight).

Figure 7.

UDA is linked to the nurse shark MHC. The Southern blot was performed with genomic DNA from 39 siblings in a nurse shark family (animal number shown above the blot) and their mother using BamHI restriction fragments hybridized with nurse shark α3 domain of UDA probe. The previously identified 13 MHC groups a-m identified with a UAA probe are indicated below (Ohta et al., 2000, Ohta et al., 2002). Marker size (kb) is shown on the left.