Abstract
Alligators are crocodilians and among few species that endured the Cretaceous–Paleogene extinction event. With long life spans, low metabolic rates, unusual immunological characteristics, including strong antibacterial and antiviral ability, and cancer resistance, crocodilians may hold information for molecular pathways underlying such physiological traits. Peptidylarginine deiminases (PADs) are a group of calcium-activated enzymes that cause posttranslational protein deimination/citrullination in a range of target proteins contributing to protein moonlighting functions in health and disease. PADs are phylogenetically conserved and are also a key regulator of extracellular vesicle (EV) release, a critical part of cellular communication. As little is known about PAD-mediated mechanisms in reptile immunology, this study was aimed at profiling EVs and protein deimination in Alligator mississippiensis. Alligator plasma EVs were found to be polydispersed in a 50–400-nm size range. Key immune, metabolic, and gene regulatory proteins were identified to be posttranslationally deiminated in plasma and plasma EVs, with some overlapping hits, while some were unique to either plasma or plasma EVs. In whole plasma, 112 target proteins were identified to be deiminated, while 77 proteins were found as deiminated protein hits in plasma EVs, whereof 31 were specific for EVs only, including proteins specific for gene regulatory functions (e.g., histones). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed KEGG pathways specific to deiminated proteins in whole plasma related to adipocytokine signaling, while KEGG pathways of deiminated proteins specific to EVs included ribosome, biosynthesis of amino acids, and glycolysis/gluconeogenesis pathways as well as core histones. This highlights roles for EV-mediated export of deiminated protein cargo with roles in metabolism and gene regulation, also related to cancer. The identification of posttranslational deimination and EV-mediated communication in alligator plasma revealed here contributes to current understanding of protein moonlighting functions and EV-mediated communication in these ancient reptiles, providing novel insight into their unusual immune systems and physiological traits. In addition, our findings may shed light on pathways underlying cancer resistance, antibacterial and antiviral resistance, with translatable value to human pathologies.
Highlights
Alligators are crocodilians, with two living species, the American alligator (Alligator mississippiensis) and the Chinese alligator (Alligator sinensis)
Plasma extracellular vesicles (EVs) were assessed by nanoparticle tracking analysis (NTA) for particle numbers and size distribution using the NanoSight NS300 system, revealing a poly-dispersed population of EVs in the size range of mainly 50–400 nm, albeit with some individual variation in EV profiles within these size ranges and peaks at smaller (30 nm) and larger (500 nm) sizes (Figure 1A)
In plasma EVs, cross-reaction with antihuman PAD2 antibody was prominent, and cross-reaction with antihuman PAD3 was detected at low levels, while the EVs did not show positive against the anti-human PAD4 antibody (Figure 2B)
Summary
Alligators are crocodilians, with two living species, the American alligator (Alligator mississippiensis) and the Chinese alligator (Alligator sinensis). Alligators are long-lived ancient animals and, alongside crocodiles, are among the few species who endured the Cretaceous–Paleogene extinction event. Crocodilians appeared ∼240 million years ago, during the Middle Triassic. Crocodilians are similar in appearance to other reptiles, they are only distantly related to lizards and belong to the closest extant relatives of birds, occupying an important evolutionary position [1,2,3]. Alligators can endure and occupy unsanitary environments, withstand radiation of high levels, and be routinely exposed to heavy metals but are rarely reported to develop cancer [4]. With long life spans and unusual immunological characteristics, including unique antimicrobial responses [5,6,7,8], antiviral activity against enveloped viruses, including HIV [9] and low metabolic rate [10], crocodilians may hold information for molecular pathways underlying such unusual physiological traits
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