Accumulation of environmental chitin in the lungs can lead to pulmonary fibrosis, characterized by inflammatory infiltration and fibrosis in acidic chitinase (Chia)-deficient mice. Transgenic expression of Chia in these mice ameliorated the symptoms, indicating the potential of enzyme supplementation as a promising therapeutic strategy for related lung diseases. This study focuses on utilizing hyperactivated human Chia, which exhibits low activity. We achieved significant activation of human Chia by incorporating nine amino acids derived from the crab-eating monkey (Macaca fascicularis) Chia, known for its robust chitin-degrading activity. The modified human Chia retained high activity across a broad pH spectrum and exhibited enhanced thermal stability. The amino acid substitutions associated with hyperactivation of human Chia activity occurred species specifically in monkey Chia. This discovery highlights the potential of hyperactivated Chia in treating pulmonary diseases resulting from chitin accumulation in human lungs.

• Keywords
• acidic chitinase (Chia), amino acid substitutions, chitin, enzyme engineering, evolution, exon swapping, hyperactivation, primate lineage, treating pulmonary diseases,
• MeSH
• Chitin metabolism   MeSH
• Chitinases * genetics   metabolism   chemistry   MeSH
• Hydrogen-Ion Concentration MeSH
• Humans MeSH
• Mice MeSH
• Enzyme Stability MeSH
• Amino Acid Substitution MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CHIA protein, human MeSH Browser
• Chitin MeSH
• Chitinases * MeSH

Placental mammals' ancestors were insectivores, suggesting that modern mammals may have inherited the ability to digest insects. Acidic chitinase (Chia) is a crucial enzyme hydrolyzing significant component of insects' exoskeleton in many species. On the other hand, herbivorous animal groups, such as cattle, have extremely low chitinase activity compared to omnivorous species, e.g., mice. The low activity of cattle Chia has been attributed to R128H mutation. The presence of either of these amino acids correlates with the feeding behavior of different bovid species with R and H determining the high and low enzymatic activity, respectively. Evolutionary analysis indicated that selective constraints were relaxed in 67 herbivorous Chia in Cetartiodactyla. Despite searching for another Chia paralog that could compensate for the reduced chitinase activity, no active paralogs were found in this order. Herbivorous animals' Chia underwent genetic alterations and evolved into a molecule with low activity due to the chitin-free diet.

• Keywords
• Evolutionary biology, Molecular biology, Zoology,
• Publication type
• Journal Article MeSH

Ym1 (chitinase-like 3, Chil3) expressed in mice is a nonenzymatic chitinase-like protein, which shows 67% identity with mouse acidic chitinase (Chia). Similar to Chia, Ym1 is overexpressed in asthma and parasitic infections in mouse lungs. Due to the lack of chitin-degrading activity, the biomedical role of Ym1 under these pathophysiological conditions remains to be determined. In this study, we investigated what region and amino acid changes in Ym1 resulted in the loss of enzymatic activity. Replacing two amino acids at the catalytic motif to obtain a Chia-like sequence (N136D and Q140E; MT-Ym1) did not activate the protein. We conducted a comparative study of Ym1 and Chia. We found that three protein segments-(i) the catalytic motif residues, (ii) exons 6 and 7, and (iii) exon 10-are responsible for chitinase activity loss in Ym1. We show that replacing each of these three segments in Chia that are also involved in substrate recognition and binding by the Ym1 sequence can fully abolish the enzymatic activity. In addition, we show that there have been extensive gene duplication events at the Ym1 locus specific to the rodent lineages. Consistent with this result, Ym1 orthologs from the rodent genome were under positive selection when analyzed through the CODEML program. These data suggest that numerous amino acid substitutions in the regions involved in the chitin recognition, binding, and degradation ability of the ancestor Ym1 molecule lead to the irreversible inactivation of the protein.

• Keywords
• chitin, chitinase, chitinase-like protein Ym1, enzyme inactivation, mouse,
• MeSH
• Biological Evolution MeSH
• Chitin chemistry   MeSH
• Chitinases * chemistry   MeSH
• Mice MeSH
• Amino Acid Substitution MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chil3 protein, mouse MeSH Browser
• Chitin MeSH
• Chitinases * MeSH

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,
• MeSH
• Carnivora * metabolism   MeSH
• Chitin chemistry   metabolism   MeSH
• Chitinases * genetics   metabolism   MeSH
• Diet MeSH
• Mice MeSH
• Dogs MeSH
• Amino Acid Sequence MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Dogs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chitin MeSH
• Chitinases * MeSH

Chitooligosaccharides exhibit several biomedical activities, such as inflammation and tumorigenesis reduction in mammals. The mechanism of the chitooligosaccharides' formation in vivo has been, however, poorly understood. Here we report that mouse acidic chitinase (Chia), which is widely expressed in mouse tissues, can produce chitooligosaccharides from deacetylated chitin (chitosan) at pH levels corresponding to stomach and lung tissues. Chia degraded chitin to produce N-acetyl-d-glucosamine (GlcNAc) dimers. The block-type chitosan (heterogenous deacetylation) is soluble at pH 2.0 (optimal condition for mouse Chia) and was degraded into chitooligosaccharides with various sizes ranging from di- to nonamers. The random-type chitosan (homogenous deacetylation) is soluble in water that enables us to examine its degradation at pH 2.0, 5.0, and 7.0. Incubation of these substrates with Chia resulted in the more efficient production of chitooligosaccharides with more variable sizes was from random-type chitosan than from the block-type form of the molecule. The data presented here indicate that Chia digests chitosan acquired by homogenous deacetylation of chitin in vitro and in vivo. The degradation products may then influence different physiological or pathological processes. Our results also suggest that bioactive chitooligosaccharides can be obtained conveniently using homogenously deacetylated chitosan and Chia for various biomedical applications.

• Keywords
• FACE method, acidic chitinase, block-type chitosan, chitin, chitooligosaccharides, random-type chitosan,
• MeSH
• Chitinases chemistry   metabolism   MeSH
• Chitosan chemistry   metabolism   MeSH
• X-Ray Diffraction MeSH
• Hydrolysis MeSH
• Hydrogen-Ion Concentration * MeSH
• Mice MeSH
• Oligosaccharides chemistry   metabolism   MeSH
• Organ Specificity MeSH
• Lung metabolism   MeSH
• Substrate Specificity MeSH
• Stomach metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chitinases MeSH
• Chitosan MeSH
• oligochitosan MeSH Browser
• Oligosaccharides MeSH

Diet of the crab-eating monkey (Macaca fascicularis) consists of both plants and animals, including chitin-containing organisms such as crabs and insects. This omnivorous monkey has a high expression of acidic chitinase (CHIA) in the stomach and here, we report on its enzymatic properties under different conditions. When we compared with Mus musculus CHIA (Mm-CHIA), Macaca fascicularis CHIA (Mf-CHIA) exhibits higher chitinolytic activity at broad pH (1.0-7.0) and temperature (30-70 ℃) range. Interestingly, at its optimum pH (5.0), Mf-CHIA showed the highest activity at 65 °C while maintaining it at robust levels between 50 and 70 °C. The degradation efficiency of Mf-CHIA was superior to Mm-CHIA toward both polymeric chitin as well as an artificial chromogenic substrate. Our results show that unique features of Mf-CHIA including its thermostability warrant the nomination of this enzyme for potential agricultural and biomedical applications.

• MeSH
• Chitin chemistry   MeSH
• Chitinases chemistry   MeSH
• Escherichia coli MeSH
• Hydrogen-Ion Concentration MeSH
• Macaca fascicularis MeSH
• Mice MeSH
• Oligosaccharides chemistry   MeSH
• Polymers chemistry   MeSH
• Polysaccharides chemistry   MeSH
• Recombinant Proteins chemistry   MeSH
• Carbohydrates chemistry   MeSH
• Temperature MeSH
• Stomach metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chitin MeSH
• Chitinases MeSH
• Oligosaccharides MeSH
• Polymers MeSH
• Polysaccharides MeSH
• Recombinant Proteins MeSH
• Carbohydrates MeSH

Commercially available porcine pepsin preparations have been used for the production of chitooligosaccharides with various biomedical activities. However, the origin of this activity is not well understood. Here we show that the chitosan-degrading activity is conferred by residues with chitinolytic activity of truncated forms of acidic chitinase (Chia) persisting in the pepsin preparation. Chia is an acid-stable and pepsin-resistant enzyme that degrades chitin to produce N-acetyl-D-glucosamine dimer. We found that Chia can be truncated by pepsin under stomach-like conditions while maintaining its enzymatic activity. Similarly to the full-length protein, truncated Chia as well as the pepsin preparations digested chitosan with different degrees of deacetylation (DD: 69-84%) with comparable degradation products. The efficiency was DD-dependent with a marked decrease with higher DD, indicating that the chitosan-degrading activity in the pepsin preparation is due to the chitinolytic activity rather than chitosanolytic activity. We suggest that natural or recombinant porcine Chia are suitable for producing chitooligosaccharides for biomedical purposes.

• MeSH
• Chitinases metabolism   MeSH
• Chitosan metabolism   MeSH
• Hydrolysis MeSH
• Hydrogen-Ion Concentration MeSH
• Pepsin A metabolism   MeSH
• Swine MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chitinases MeSH
• Chitosan MeSH
• Pepsin A MeSH

Chitin is a polymer of N-acetyl-D-glucosamine (GlcNAc) and a main constituent of insects' exoskeleton. Insects are rich in protein with high energy conversion efficiency. Recently, we have reported that acidic chitinases (Chia) act as digestive enzymes in mouse, pig and chicken (omnivorous) but not in dog (carnivorous) and bovine (herbivorous), indicating that feeding behavior affects Chia expression levels, and determines chitin digestibility in the particular animals. Common marmoset (Callithrix jacchus) belongs to New World monkey family and provides a potential bridge between mouse models and human diseases. Common marmoset is an insectivorous nonhuman primate with unknown expression levels and enzymatic functions of the Chia homologue, CHIA. Here, we report that common marmoset highly expresses pepsin-, trypsin- and chymotrypsin-resistant CHIA in the stomach. We show that CHIA is most active at pH 2.0 and degrades chitin and mealworm shells into GlcNAc dimers under gastrointestinal conditions. Although common marmoset and crab-eating monkey (Old World monkey) have two CHIA genes in their genomes, they primarily express one gene in the stomach. Thus, this study is the first to investigate expression levels and enzymatic functions of CHIA in a New World primate, contributing to the understanding of dietary adaptation and digestion in this taxon.

• MeSH
• Callithrix metabolism   MeSH
• Chitin metabolism   MeSH
• Chitinases * chemistry   metabolism   MeSH
• Diet MeSH
• Feeding Behavior psychology   MeSH
• Stomach enzymology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Chitin MeSH
• Chitinases * MeSH

Mice and humans express two active chitinases: acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1). Both chitinases are thought to play important roles in specific pathophysiological conditions. The crab-eating monkey (Macaca fascicularis) is one of the most frequently used nonhuman primate models in basic and applied biomedical research. Here, we performed gene expression analysis of two chitinases in normal crab-eating monkey tissues by way of quantitative real-time polymerase chain reaction (qPCR) using a single standard DNA molecule. Levels of AMCase and CHIT1 messenger RNAs (mRNAs) were highest in the stomach and the lung, respectively, when compared to other tissues. Comparative gene expression analysis of mouse, monkey, and human using monkey⁻mouse⁻human hybrid standard DNA showed that the AMCase mRNA levels were exceptionally high in mouse and monkey stomachs while very low in the human stomach. As for the CHIT1 mRNA, we detected higher levels in the monkey lung when compared with those of mouse and human. The differences of mRNA expression between the species in the stomach tissues were basically reflecting the levels of the chitinolytic activities. These results indicate that gene expression of AMCase and CHIT1 differs between mammalian species and requiring special attention in handling data in chitinase-related studies in particular organisms.

• Keywords
• acidic mammalian chitinase, asthma, chitinolytic activity, chitotriosidase, crab-eating monkey, human, mouse, nonhuman primate model, qPCR, species-specific gene expression,
• Publication type
• Journal Article MeSH

Acidic chitinase (Chia) has been implicated in asthma, allergic inflammations, and food processing. We have purified Chia enzymes with striking acid stability and protease resistance from chicken and pig stomach tissues using a chitin column and 8 M urea (urea-Chia). Here, we report that acetic acid is a suitable agent for native Chia purification from the stomach tissues using a chitin column (acetic acid-Chia). Chia protein can be eluted from a chitin column using 0.1 M acetic acid (pH 2.8), but not by using Gly-HCl (pH 2.5) or sodium acetate (pH 4.0 or 5.5). The melting temperatures of Chia are not affected substantially in the elution buffers, as assessed by differential scanning fluorimetry. Interestingly, acetic acid appears to be more effective for Chia-chitin dissociation than do other organic acids with similar structures. We propose a novel concept of this dissociation based on competitive interaction between chitin and acetic acid rather than on acid denaturation. Acetic acid-Chia also showed similar chitinolytic activity to urea-Chia, indicating that Chia is extremely stable against acid, proteases, and denaturing agents. Both acetic acid- and urea-Chia seem to have good potential for supplementation or compensatory purposes in agriculture or even biomedicine.

• Keywords
• GlcNAc, acetic acid, acidic chitinase, chitin, chitin column, competitive manner, natural enzyme, supplementation purposes, therapeutic agents, urea,
• MeSH
• Chitin chemistry   metabolism   MeSH
• Chitinases chemistry   metabolism   MeSH
• Chickens MeSH
• Acetic Acid chemistry   MeSH
• Swine MeSH
• Protein Binding MeSH
• Stomach enzymology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chitin MeSH
• Chitinases MeSH
• Acetic Acid MeSH