Shark class II invariant chain reveals ancient conserved relationships with cathepsins and MHC class II

Abstract

The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates.

Figure 1. Nurse shark cDNAs with homology to Ii chain

Nucleotide and putative amino acid sequence of nurse shark cDNA clones 104D3 (top) and D2 (bottom). Gaps introduced for alignment are shown as dashes. Single base point mutations and insertion/deletions are highlighted. Amino acids of the endosomal targeting motif are highlighted in pink, the transmembrane domain in green, CLIP yellow, trimerization domain blue and Tg domain red. NCBI has given these sequences accession numbers JF507710 and JF507711.

Figure 2. Amino acid alignment identifies nurse shark and other chondrichthian Ii when compared to other vertebrate sequences

Yellow highlighting (spanning two residues) indicates phase zero, green highlighting indicates phase one and blue highlighting indicates phase two intron positioning. Genbank accession numbers are shown in Supplemental Table 2. Endosomal targeting motifs (L-L/I/V) are underlined, as are the six conserved cysteines of Tg-like domains and (in the human sequences) the three alpha helices of the trimerization domain. Asparagine-linked glycosylation motifs (N-X-S/T, X≠P) are in italics. Also in italics are polar residues corresponding to the hydrophilic patch of the transmembrane domain implicated in Ii trimerization.

Figure 3. Invariant chain domain conservation in cartilaginous fish and tetrapods

Representative predicted protein sequences were chosen to compare the entire longest splice forms of invariant chain homologs found. Xenopus laevis was used for frog. Human is shown with earlier alternative start site that gives 16 additional amino acids to the cytoplasmic tail, shown in red. Length is measured in amino acids.

Figure 4. Exon-intron organization and relative size similar from shark to man

Exons encoding the transmembrane domain are shown in green, the exons approximately encoding the three alpha-helices of the trimerization domain are shown in blue, and the Tg domain in red. Question marks denote missing data from elephant shark scaffolds, exon content in each set of parentheses is on one unmapped scaffold. Drawn to scale, distance in base pairs is shown at bottom.

Figure 5. Tissue expression of shark Ii mRNA

Northern blot hybridization of nurse shark tissues (Br, brain; Ep, epigonal; Gi, gills; He, SV, spiral valve; Ki, kidney; Li, liver; Pa, pancreas; PBL, peripheral blood leukocytes; Sp, spleen; Te, testis; and Th, thymus) with the Gici-Ii probe demonstrates that three transcripts for Ii are expressed at similar levels relative to MHCIIA. Size in bases of the transcripts is shown on the left.

Figure 6. CIITA of cartilaginous fish

Amino acid alignment of partial putative elephant shark (eShark) ortholog of CIITA. Yellow highlighting indicates identity with other vertebrate CIITA proteins. Predicted domains of protein shown under alignment. Other sequences included in the alignment: finch XP002195062, opossum XM001376433, zebra danio XP001343072, and human NP000237.

Figure 7. Predicted cathepsin L inhibition by Tg domain in cartilaginous fish

A. Bead amino acid schematic of Tg-like domain of nurse shark. Amino acids of Ii predicted from mammalian crystal structures to be important contacts with cathepsin L and conserved in shark are shown as larger circles. Conserved cathepsin amino acids that are predicted to interact with Ii are shown as green boxes, dotted lines show hydrogen bonds and electrostatic interactions, dashed lines show hydrophobic interactions. Disulfide bonded cysteines are shown joined by a line. B. Five cathepsins from elasmobranchs having similarities to cathepsin L aligned with cathepsin L and S from human. Residues predicted to form key conserved interactions are in green. C. Amino acid alignment of Tg – like domain from elephant shark, nurse shark, Pacific electric ray, frog (X. laevis), anole lizard, chicken and human. Residues conserved in at least 5 of the 7 aligned sequences are shown in red in panel C and red beads in panel A. Nurse shark Val255 and orthologous residues are highlighted in orange as this position was usually maintained as an aliphatic, hydrophobic residue. Underlined amino acids make key contacts between Ii Tg-like domain and cathepsin L in human. Structure interactions adapted from Guncar and Turk (Guncar et al., 1999b).

Figure 8. Cartilaginous fish Ii groups with other Ii in phylogenetic analysis

Neighbor joining tree of Ii amino acid sequences aligned and analyzed in MEGA. Alignment (with sequences not included in this tree) is found in Supplemental Figure 1, sequence accession numbers and names given in Supplemental Table 2. Bootstrap values at nodes inferred from 1000 replicates. Evolutionary distance is shown with the scale bar in the units of amino acid substitutions per site. Boxes to the center-right highlight different vertebrate phylogenetic clusters of Ii and far right mark loss of Tg and trimerization domains in some teleost Ii forms, compared to the complete Ii.