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.

Keywords: Chia; acidic chitinase; carnivores; digestive enzyme; gene loss; insectivores.

Fig. 1.

Two amino acid substitutions can activate dog Chia. (A) Schematic representation of E. coli-expressed Chia chimeric proteins. The amino acid sequences are color-coded as follows: pink, mouse sequence; blue, dog sequence. (B) Comparison of the chitinolytic activities of Chia proteins outlined in (A). (C) Schematic representation of E. coli-expressed Chia mutant proteins. (D) Comparison of the chitinolytic activities of mutant proteins outlined in panel C with mouse and dog Chia. (E) A homology model of the CatD shows that the identified amino acid substitutions (pink in mouse, left; blue in dog, right) are close to the residues interacting with the carbohydrate chain (gray).

Fig. 2.

Chia gene loss in Carnivora. (A) Phylogenetic tree of 40 carnivorans. Phylogenetic relationships and divergence times were obtained from TimeTree version 3.0 (http://www.timetree.org/). A scientific name in bold means that Chia sequences were annotated genes and otherwise assembled manually from the genome by BLAST using the dog Chia cDNA sequence. Stars indicate a pseudogenization event, but the timing is unknown. (B) The main diet in each species is shown in the illustration; pink, meats; purple, insects or crustaceans; yellow, fruits or nectar; green, plants. (C) The protein-coding region of Chia. X, the positions of loss-of-function mutations and the mutations shared in each family are indicated by boxes. (D) Whether it is a protein-encoding gene is indicated by the closed circle; pink, the protein-coding gene with 214L and 216G (active type); blue, protein-coding gene with 214F and 216A (inactive type); black, lack of full-length protein-coding region (pseudogene). The illustrations in (A) and (B) (copyright AC Works Co., Ltd.) are used with permission.

Fig. 3.

Insect-eating species encode full-length chitinase and exhibit high chitinase activity. (A) Schematic representation of E. coli-expressed dog and insectivorous Chia. C, Cys residues conserved in active Chia proteins; FA or LG, the amino acids that controlled the activity of Chia are color-coded as follows: pink, mouse sequence; blue, dog sequence. (B) Comparison of the chitinolytic activities of dog, banded mongoose, meerkat, common dwarf mongoose, striped hyena, and skunk Chia proteins using 4-MU-(GlcNAc)₂. Error bars represent the mean ± SD from a single experiment conducted in triplicate.

Fig. 4.

The timing of Chia’s inactivating substitution events in Canidae. (A) Phylogenetic tree of ten canids, including outgroup species. The scale bar indicates the divergence times (20 Mya) obtained from TimeTree version 3.0 (http://www.timetree.org/). (B) Amino acid sequences of exon 7 regions of canids and outgroup Chia. The amino acids at positions 214 and 216 that control the activity of Chia are color-coded as follows: pink, mouse sequence; blue, dog sequence. (C) Schematic representation of WT lycaon Chia (WT-lycaon) and Chia mutant protein named Cys-lycaon. (D) Comparison of the chitinolytic activities of Chia proteins outlined in (C). The relative activity when the WT lycaon Chia activity level at pH 2.0 was set to 100% is shown. Error bars represent the mean ± SD from a single experiment conducted in triplicate. The illustrations were drawn by Eri Tabata.

Fig. 5.

The evolutionary process of the cat Chia pseudogene from ancestrally active proteins. (A) Graphical display of the coding region of cat Chia and the positions of the inactivating mutations. ATG, start codon; indel, with the number above indicating the number of bases deleted; X, stop codon. Cre-cat, creation of functional Chia proteins that restored their ORFs from their genes. C (black), a Cys position conserved in mammalian chitinase, and C (blue), a Cys distinct from the conserved position among mammalian chitinases. FA, the amino acid that controls the activity of Chia and is the same as the mouse sequence. Cys-cat, creation of functional Chia proteins that restored their positions and number of Cys. (B) Comparison of the chitinolytic activities of Chia proteins outlined in (A) and (E). We showed relative activity when the meerkat Chia activity level at pH 2.0 was set to 100%. Error bars represent the mean ± SD from a single experiment conducted in triplicate. (C) Schematic representation of Chia chimeric proteins narrowing down the responsible region. The amino acid sequences are color-coded as follows: pink, meerkat sequence; blue, Cys-cat sequence. (D) Comparison of the chitinolytic activity of Chia proteins outlined (C) and their comparison with a meerkat and Cys-cat Chia. (E) Schematic representation of the Chia mutant protein named Cys-cat-5 Mut. (F) A homology model of the cat Chia mutant proteins shows Chia pseudogene evolution from an ancestrally active enzyme. The identified amino acid substitutions were color-coded red.

Fig. 6.

Model explaining the evolution of the Chia gene in Carnivora. Schematic illustration of Chia gene evolution from an insectivorous ancestor with dietary changes. Species that feed on insects retain Chia with high chitinolytic activity (Active-type Chia). In species that do not feed on insects, the structure and activity of the gene have changed. The activity of Chia in Canidae was reduced by two amino acid substitutions (Inactive-type Chia). Except for Canidae, Chia has evolved into a nonfunctional gene (Functional loss-type Chia) due to the relaxation of functional constraints and the accumulation of mutations that cause stop codons and/or loss of activity.