Noninsect-Based Diet Leads to Structural and Functional Changes of Acidic Chitinase in Carnivora

Abstract

Acidic chitinase (Chia) digests the chitin of insects in the omnivorous stomach and the chitinase activity in carnivorous Chia is significantly lower than that of the omnivorous enzyme. However, mechanistic and evolutionary insights into the functional changes in Chia remain unclear. Here we show that a noninsect-based diet has caused structural and functional changes in Chia during the course of evolution in Carnivora. By creating mouse-dog chimeric Chia proteins and modifying the amino acid sequences, we revealed that F214L and A216G substitutions led to the dog enzyme activation. In 31 Carnivora, Chia was present as a pseudogene with stop codons in the open reading frame (ORF) region. Importantly, the Chia proteins of skunk, meerkat, mongoose, and hyena, which are insect-eating species, showed high chitinolytic activity. The cat Chia pseudogene product was still inactive even after ORF restoration. However, the enzyme was activated by matching the number and position of Cys residues to an active form and by introducing five meerkat Chia residues. Mutations affecting the Chia conformation and activity after pseudogenization have accumulated in the common ancestor of Felidae due to functional constraints. Evolutionary analysis indicates that Chia genes are under relaxed selective constraint in species with noninsect-based diets except for Canidae. These results suggest that there are two types of inactivating processes in Carnivora and that dietary changes affect the structure and activity of Chia.

Comparative transcriptomics reveals divergent paths of chitinase evolution underlying dietary convergence in ant-eating mammals

“…The presence of frameshift mutations and stop codons was inspected in all nine chitinase genes in the southern aardwolf genome (Allio et al 2021). As expected, CHIA1, CHIA2, CHIA3, CHIA4 were indeed found to be non functional, and CHI3L2 seems to be absent from the genome of the southern aardwolf as in most members of Carnivora (Emerling et al 2018;Tabata et al 2022). While no inactivating mutations could be detected in the coding sequences of CHIA5, CHI3L1, CHIT1 or OVGP1, we cannot rule out the possibility that some specific mutations in regulatory elements inactivating the expression of these genes could have appeared in P. cristatus.…”

“…Our study did not find this gene expressed in giant anteater salivary glands. On the other hand, in insectivorous carnivores (Carnivora) and pangolins (Pholidota), CHIA5 is functional whereas CHIA1-4 are pseudogenized (Emerling et al 2018; Tabata et al 2022). Similar inactivating mutations were observed in the CHIA1 gene in carnivores and pangolins and were dated to at least 67 Mya, well before the origin of carnivores (46.2 Ma) and pangolins (26.5 Ma) (Emerling et al 2018).…”

“…Chitinase gene family evolution seems to have been strongly influenced by historical contingency events related to gene loss following adaptation to a specific diet (Emerling et al 2018; Janiak et al 2018; Chen and Zhao 2019; Tabata et al 2022). For instance, fossil evidence showing that stem penguins primarily relied on large prey items like fish and squid has been invoked to explain the loss of all functional CHIA genes in all extant penguin species despite the recent specialization of some species towards a chitin-rich crustacean diet (Cole et al 2022).…”

“…CHIA5b cannot bind to chitin unlike CHIA5c and CHIA5d, suggesting different roles for these three paralogous proteins. The evolution of the different CHIA1-5 genes involved changes in their catalytic sites, which have consequences on the secondary structure of enzymes and potentially affect their optimal pH or function as it has recently been shown for CHIA5 in Carnivora (Tabata et al 2022). Experimentally testing the chitin degradation activity on different substrates and at different pH of enzymes produced from the ancestral sequences reconstructed for each of the five CHIA paralogs would allow a better understanding of their enzymatic activity.…”

“…The presence of the sole CHIA5 in pangolins was interpreted as the consequence of historical contingency with the probable loss of CHIA1-4 functionality in the last common ancestor of Pholidota and Carnivora (Emerling et al 2018). In Carnivora, it has recently been confirmed that a non insect-based diet has caused structural and functional changes in the CHIA gene repertoire resulting in multiple losses of function with only few species including insects in their diet retaining a fully functional CHIA5 gene (Tabata et al 2022). The fact that CHIA5 was found to be highly expressed in the main digestive organs of the Malayan pangolin (Ma et al 2017; Ma et al 2019; Cheng et al 2022) suggests that pangolins might compensate for their reduced chitinase repertoire by an increased ubiquitous expression of their only remaining functional paralog in multiple organs.…”