Exploration of the Nurse Shark (Ginglymostoma cirratum) Plasma Immunoproteome Using High-Resolution LC-MS/MS

Abstract

Many animals of scientific importance lack species-specific reagents (e.g., monoclonal antibodies) for in-depth studies of immune proteins. Mass spectrometry (MS)-based proteomics has emerged as a useful method for monitoring changes in protein abundance and modifications in non-model species. It can be used to quantify hundreds of candidate immune molecules simultaneously without the generation of new reagents. Here, we used MS-based proteomics to identify and quantify candidate immune proteins in the plasma of the nurse shark (Ginglymostoma cirratum), a cartilaginous fish and representative of the most basal extant vertebrate lineage with an immunoglobulin-based immune system. Mass spectrometry-based LC-MS/MS was performed on the blood plasma of nurse sharks immunized with human serum albumin (n=4) or sham immunized (n=1), and sampled at days 0 (baseline control), 1, 2, 3, 5, 7, 14, 21, 28, 25, 42 and 49. An antigen-specific antibody response was experimentally confirmed post-immunization. To provide a high-quality reference to identify proteins, we assembled and annotated a multi-tissue de novo transcriptome integrating long- and short-read sequence data. This comprised 62,682 contigs containing open reading frames (ORFs) with a length >80 amino acids. Using this transcriptome, we reliably identified 626 plasma proteins which were broadly categorized into coagulation, immune, and metabolic functional groups. To assess the feasibility of performing LC-MS/MS proteomics in nurse shark in the absence of species-specific protein annotations, we compared the results to an alternative strategy, mapping peptides to proteins predicted in the genome assembly of a related species, the whale shark (Rhincodon typus). This approach reliably identified 297 proteins, indicating that useful data on the plasma proteome may be obtained in many instances despite the absence of a species-specific reference protein database. Among the plasma proteins defined against the nurse shark transcriptome, fifteen showed consistent changes in abundance across the immunized shark individuals, indicating a role in the immune response. These included alpha-2-macroglobulin (A2M) and a novel protein yet to be characterized in diverse vertebrate lineages. Overall, this study enhances genetic and protein-level resources for nurse shark research and vastly improves our understanding of the elasmobranch plasma proteome, including its remodelling following immune stimulation.

Secreted novel AID/APOBEC-like deaminase 1 (SNAD1) – a new important player in fish immunology

“…This could be facilitated by the delivery of SNAD1 via extracellular vesicles (EVs), which would reconcile the secretory nature of these proteins with their potential involvement in diverse intracellular processes. Moreover, another possibility is that SNADs are delivered from the plasma into virally infected cells, perhaps via endocytosis, where they exert antiviral activity by mutating viral genomes, similar to the observed activity of mammalian APOBEC3 enzymes against HIV ( 46 ).…”

“…( 4 ) and SNAD1 and SNAD2 from fish previously examined by Bakke et al. ( 46 ). The sequences were analysed for their phylogenetic relationships to other SNAD sequences found in GenBank.…”

“…SNAD1 has been shown to be upregulated in response to cold exposure in fish, suggesting that it may play a role in boosting the innate immune response under cold conditions ( 7 ). This finding is crucial due to the possibility, that an expanded innate immunity, in addition to lower pathogenic pressures in a cold environment reduced the pressure to maintain robust secondary antibody diversification ( 5 , 46 ).…”

Decoding the Transcriptome of Sharks, Rays, and Chimaeras: Insights into Their Physiology, Morphology, Evolution, and Biomedical Applications

“…These discoveries can help increase of efficiency of the production of these antibodies, depending on the interest of each antibody. Moreover, the usage of multi-tissue transcriptomics aided with the identification of more than 626 nurse shark plasma proteins [188], which helped identifying the proteins involved on the response of immune stressors on Ginglymostoma cirratum. This can be important for understanding how chondrichthyes respond to immune stimulants and help with the production of antibodies.…”

Comparative Proteome Analysis of Four Stages of Spermatogenesis in the Small-Spotted Catshark (Scyliorhinus canicula), Using High-Resolution NanoLC-ESI-MS/MS

“…milii to a 4600 Mb length for Carcharodon carcharias and feature large introns and a large content of transposable elements rather than massive gene duplication. − Analysis of origins and losses of vertebrate gene families showed that a significant number of gene families were present at the root of the Gnathostomata branch, establishing the gene families present in bony vertebrates and cartilaginous fishes. , So, the jawed vertebrate ancestor was found to possess the gene repertories regulating homeostasis and reproduction and Chondrichthyes are the most basal extant jawed vertebrates with glycoprotein hormones differentiated into gonadotropins (FSH and LH), thyrotropins (TSH1 and TSH2) and thyrostimulin (GPA2GPB5) . However, several gains and losses of genes have been described between Elasmobranchs and Osteichthyes ,,− but also between Elasmobranchs and the Holocephalans in the Chondrichthyes, two groups which diverged early after the estimated divergence time of Chondrichthyes and Osteichthyes. ,, In addition to the importance of identifying chondrichthyan genomes for the understanding of genome evolution, these data are also fundamental for large-scale-omic analyses and several transcriptomic and proteomic data elements are now available for Elasmobranchs, − providing important molecular tools for physiological studies in Chondrichthyans. In the present study, we explored the proteome of the testis using a high-resolution nanoLC-ESI-MS/MS approach to study the evolution of the regulation of spermatogenesis and to enrich the proteomic data for Elasmobranchs.…”