Uncovering Generation Mechanisms of IgNAR High Diversity in Chiloscyllium Plagiosum via Transcriptome Analysis

Abstract

Cartilaginous fishes are the phylogenetically oldest extant jawed vertebrates possessing an immunoglobulin (Ig)-based adaptive immune system. Sharks and other cartilaginous fish are ideal models for studying the evolution of adaptive immunity, due to their phylogenetic significance and production of a special high diversity heavy-chain only antibody called IgNAR. However, the lack of established transcriptome and IgNAR sequences has impeded progress in understanding the high diversity or other unique characteristics of IgNAR and even early immune evolution. Here, we systematically study the mRNA and IgNAR immunoglobulins V region (vNARs) of Chiloscyllium plagiosum for the first time, to reveal mechanisms and key genes involved in IgNAR diversity generation.

In this study, 104 samples from 20 tissues of 3 adult female, 1 junior female and 1 junior male sharks are sequenced and 15227 mRNAs are identified and analyzed. Cross-species conservation analysis demonstrates that mRNAs that exclusively appear in bamboo shark species (setA) are immune-related. This is further confirmed by weighted correlation network analysis (WGCNA) analysis as the largest number of bamboo shark less-conserved mRNAs fall into immune-related modules. We next investigate the tissue expression of genes in the intersection between immune-related modules and setA, and find spleen and gill play a major role in bamboo shark adaptive immune system. Potential determinant genes are also identified, including AID, responsible for Ig somatic hypermutation and its regulatory genes, suggesting a distinct Ig diversity generation mechanism in bamboo sharks.

To further investigate the roles of identified genes in IgNAR diversity generation, immune repertoire analysis is conducted. High level of IgNAR diversity is discovered and is mainly concentrated in CDR3 region. The previously reported AID hotspots WRC/RGYW/AGCT also exist in Chiloscyllium plagiosum CDR regions (CDR3 by analyzing D gene, CDR1, HV2, HV4), but a new hotspot TGBDWK shows particularly high frequency in the CDR3 region, indicating the earliest-diverged AID exhibit unique substrate specificities. We construct AID structures of selected 5 species by AlphaFold and verify that Chiloscyllium plagiosum AID is indeed very different in structure and catalytic pocket.

Altogether, our findings prove that the unique AID and mechanisms of Chiloscyllium plagiosum lead to the high IgNAR diversity, presenting immunological characteristics that are distinct from those of recently evolved species. This lays a foundation for understanding the generation and evolution mechanisms of Ig diversity, providing novel insights to the early adaptive immune evolution.

Date of Award	8 Jul 2024
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Xin DENG (Supervisor) & Jiahai SHI (Co-supervisor)