Mild behavioral impairment in early Alzheimer's disease and its association with APOE and BDNF risk genetic polymorphisms.

BACKGROUND: Mild behavioral impairment (MBI) has been commonly reported in early Alzheimer's disease (AD) but rarely using biomarker-defined samples. It is also unclear whether genetic polymorphisms influence MBI in such individuals. We thus aimed to examine the association between the cognitive status of participants (amnestic mild cognitive impairment (aMCI-AD) vs cognitively normal (CN) older adults) and MBI severity. Within aMCI-AD, we further examined the association between APOE and BDNF risk genetic polymorphisms and MBI severity. METHODS: We included 62 aMCI-AD participants and 50 CN older adults from the Czech Brain Aging Study. The participants underwent neurological, comprehensive neuropsychological examination, APOE and BDNF genotyping, and magnetic resonance imaging. MBI was diagnosed with the Mild Behavioral Impairment Checklist (MBI-C), and the diagnosis was based on the MBI-C total score ≥ 7. Additionally, self-report instruments for anxiety (the Beck Anxiety Inventory) and depressive symptoms (the Geriatric Depression Scale-15) were administered. The participants were stratified based on the presence of at least one risk allele in genes for APOE (i.e., e4 carriers and non-carriers) and BDNF (i.e., Met carriers and non-carriers). We used linear regressions to examine the associations. RESULTS: MBI was present in 48.4% of the aMCI-AD individuals. Compared to the CN, aMCI-AD was associated with more affective, apathy, and impulse dyscontrol but not social inappropriateness or psychotic symptoms. Furthermore, aMCI-AD was related to more depressive but not anxiety symptoms on self-report measures. Within the aMCI-AD, there were no associations between APOE e4 and BDNF Met and MBI-C severity. However, a positive association between Met carriership and self-reported anxiety appeared. CONCLUSIONS: MBI is frequent in aMCI-AD and related to more severe affective, apathy, and impulse dyscontrol symptoms. APOE and BDNF polymorphisms were not associated with MBI severity separately; however, their combined effect warrants further investigation.

Crab-Eating Monkey Acidic Chitinase (CHIA) Efficiently Degrades Chitin and Chitosan under Acidic and High-Temperature Conditions.

Chitooligosaccharides, the degradation products of chitin and chitosan, possess anti-bacterial, anti-tumor, and anti-inflammatory activities. The enzymatic production of chitooligosaccharides may increase the interest in their potential biomedical or agricultural usability in terms of the safety and simplicity of the manufacturing process. Crab-eating monkey acidic chitinase (CHIA) is an enzyme with robust activity in various environments. Here, we report the efficient degradation of chitin and chitosan by monkey CHIA under acidic and high-temperature conditions. Monkey CHIA hydrolyzed α-chitin at 50 °C, producing N-acetyl-d-glucosamine (GlcNAc) dimers more efficiently than at 37 °C. Moreover, the degradation rate increased with a longer incubation time (up to 72 h) without the inactivation of the enzyme. Five substrates (α-chitin, colloidal chitin, P-chitin, block-type, and random-type chitosan substrates) were exposed to monkey CHIS at pH 2.0 or pH 5.0 at 50 °C. P-chitin and random-type chitosan appeared to be the best sources of GlcNAc dimers and broad-scale chitooligosaccharides, respectively. In addition, the pattern of the products from the block-type chitosan was different between pH conditions (pH 2.0 and pH 5.0). Thus, monkey CHIA can degrade chitin and chitosan efficiently without inactivation under high-temperature or low pH conditions. Our results show that certain chitooligosaccharides are enriched by using different substrates under different conditions. Therefore, the reaction conditions can be adjusted to obtain desired oligomers. Crab-eating monkey CHIA can potentially become an efficient tool in producing chitooligosaccharide sets for agricultural and biomedical purposes.

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

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.

Mouse Acidic Chitinase Effectively Degrades Random-Type Chitosan to Chitooligosaccharides of Variable Lengths under Stomach and Lung Tissue pH Conditions.

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.

Robust chitinolytic activity of crab-eating monkey (Macaca fascicularis) acidic chitinase under a broad pH and temperature range.

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 â„ƒ) 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.

The Combined Effect of APOE and BDNF Val66Met Polymorphisms on Spatial Navigation in Older Adults.

BACKGROUND: The apolipoprotein E (APOE) ɛ4 allele is associated with episodic memory and spatial navigation deficits. The brain-derived neurotrophic factor (BDNF) Met allele may further worsen memory impairment in APOEɛ4 carriers but its role in APOEɛ4-related spatial navigation deficits has not been established. OBJECTIVE: We examined influence of APOE and BDNF Val66Met polymorphism combination on spatial navigation and volumes of selected navigation-related brain regions in cognitively unimpaired (CU) older adults and those with amnestic mild cognitive impairment (aMCI). METHODS: 187 participants (aMCI [n = 116] and CU [n = 71]) from the Czech Brain Aging Study were stratified based on APOE and BDNF Val66Met polymorphisms into four groups: ɛ4-/BDNFVal/Val, ɛ4-/BDNFMet, ɛ4+/BDNFVal/Val, and ɛ4+/BDNFMet. The participants underwent comprehensive neuropsychological examination, brain MRI, and spatial navigation testing of egocentric, allocentric, and allocentric delayed navigation in a real-space human analogue of the Morris water maze. RESULTS: Among the aMCI participants, the ɛ4+/BDNFMet group had the least accurate egocentric navigation performance (p < 0.05) and lower verbal memory performance than the ɛ4-/BDNFVal/Val group (p = 0.007). The ɛ4+/BDNFMet group had smaller hippocampal and entorhinal cortical volumes than the ɛ4-/BDNFVal/Val (p≤0.019) and ɛ4-/BDNFMet (p≤0.020) groups. Among the CU participants, the ɛ4+/BDNFMet group had less accurate allocentric and allocentric delayed navigation performance than the ɛ4-/BDNFVal/Val group (p < 0.05). CONCLUSION: The combination of APOEɛ4 and BDNF Met polymorphisms is associated with more pronounced egocentric navigation impairment and atrophy of the medial temporal lobe regions in individuals with aMCI and less accurate allocentric navigation in CU older adults.

Comparative functional analysis between human and mouse chitotriosidase: Substitution at amino acid 218 modulates the chitinolytic and transglycosylation activity.

Chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) have been attracting research interest due to their involvement in various pathological conditions such as Gaucher's disease and asthma, respectively. Both enzymes are highly expressed in mice, while the level of AMCase mRNA was low in human tissues. In addition, the chitinolytic activity of the recombinant human AMCase was significantly lower than that of the mouse counterpart. Here, we revealed a substantially higher chitinolytic and transglycosylation activity of human Chit1 against artificial and natural chitin substrates as compared to the mouse enzyme. We found that the substitution of leucine (L) by tryptophan (W) at position 218 markedly reduced both activities in human Chit1. Conversely, the L218W substitution in mouse Chit1 increased the activity of the enzyme. These results suggest that Chit1 may compensate for the low of AMCase activity in humans, while in mice, highly active AMCase may supplements low Chit1 activity.

Quantification of chitooligosaccharides by FACE method: Determination of combinatory effects of mouse chitinases.

Fluorophore-assisted carbohydrate electrophoresis (FACE) enables detection and quantification of degradation products from artificial and natural chitin substrates such as 4-NP-(GlcNAc)2, (GlcNAc)4 and colloidal chitin. The FACE method has been improved by our group for analysis of chitooligosaccharides in the presence of several buffer systems commonly used in the biochemical evaluation of chitinolytic activities of enzymes at pH 2.0-8.0. FACE is a very sensitive technique detecting picomolar amounts of molecules. We optimized the detection conditions as follows: exposure type, precision; sensitivity, high resolution; exposure time, 5 s. We evaluated the (GlcNAc)2 levels using a standard curve that allows chitooligosaccharides quantification at up to 10 nmol amounts. Using the method presented here, the chitinolytic properties of different chitinases can be compared directly. Serratia chitinase A (ChiA) and chitinase B (ChiB), two well-studied bacterial chitinases, have been shown by HPLC to have a synergistic effect on the chitin degradation rate. Using the FACE method, we determined the combinatory effects of mouse chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) in natural chitin substrates processing.•FACE is a simple and quantitative method.•Our improved procedure enables the quantification of chitooligosaccharides produced by chitinases at pH 2.0-8.0.•FACE is able to quantify chitooligosaccharides at up to 10 nmol amounts.

Pharmacogenetic algorithm for predicting daily dose of warfarin in Caucasian patients of Czech origin.

OBJECTIVES: Warfarin use is limited by a low therapeutic index and significant interindividual variability of the daily dose. The most important factor predicting daily warfarin dose is individual genotype, polymorphisms of genes CYP2C9 (warfarin metabolism) and VKORC1 (sensitivity for warfarin). Algorithms using clinical and genetic variables could predict the daily dose before the initiation of therapy. The aim of this study was to develop and validate an algorithm for the prediction of warfarin daily dose in Czech patients. METHODS: Detailed clinical data of patients with known and stable warfarin daily dose were collected. All patients were genotyped for polymorphisms in genes CYP2C9 and VKORC1. RESULTS: Included patients were divided into derivation (n=175) and validation (n=223) cohorts. The final algorithm includes the following variables: Age, height, weight, treatment with amiodarone and presence of variant alleles of genes CYP2C9 and VKORC1. The adjusted coefficient of determination is 72.4% in the derivation and 62.3% in the validation cohort (p<0.001). CONCLUSIONS: Our validated algorithm for warfarin daily dose prediction in our Czech cohort had higher precision than other currently published algorithms. Pharmacogenetics of warfarin has the potential in the clinical practice in specialized centers.

Impact of APOE and BDNF Val66Met Gene Polymorphisms on Cognitive Functions in Patients with Amnestic Mild Cognitive Impairment.

Apolipoprotein (APOE) ɛ4 is a well-known risk factor for late-onset Alzheimer's disease (AD), but other AD-related gene polymorphisms might also be important, such as the polymorphism within the brain-derived neurotrophic factor (BDNF) gene. Carriage of BDNF Val66Met has been associated with faster cognitive decline and greater hippocampal atrophy in cognitively normal elderly. Thus, we examined the effects of the concurrent presence of APOE and BDNF polymorphisms on cognitive functions and brain morphometry in amnestic mild cognitive impairment (aMCI) patients. 107 aMCI patients (mean age = 72.2) were recruited from the Czech Brain Aging Study and, based on APOE and BDNF genes polymorphisms, were divided into four groups: ɛ4-BDNFVal/Val (n = 37), ɛ4-BDNFMet (n = 19), ɛ4+BDNFVal/Val (n = 35), and ɛ4+BDNFMet (n = 16). All patients underwent clinical examination, magnetic resonance imaging, and complex neuropsychological battery. The combination of APOEɛ4+ and BDNF Met was associated with significantly worse memory performance in immediate and delayed recall compared to other polymorphism groups. We did not observe increased atrophy in areas related to memory function in the ɛ4+BDNFMet group. Our findings suggest that carriage of ɛ4+BDNFMet is associated with more pronounced memory dysfunction, a typical feature of early AD, but not with structural brain changes in aMCI patients. These findings suggest that in APOEɛ4/BDNF Met carriers, synaptic dysfunction affecting memory may precede pronounced structural changes.

Residues of acidic chitinase cause chitinolytic activity degrading chitosan in porcine pepsin preparations.

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.

Neurons Induced From Fibroblasts of c9ALS/FTD Patients Reproduce the Pathology Seen in the Central Nervous System.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are incurable neurodegenerative conditions. A non-coding hexanucleotide (GGGGCC) repeat expansion in the c9orf72 gene is the most common genetic cause of ALS/FTD. We present a cellular model of c9ALS/FTD where induced neurons (iNeurons) are generated within 2 weeks by direct conversion of patients' dermal fibroblasts through down-regulation of polypyrimidine-tract-binding protein 1 (PTB1). While sense (S) and anti-sense (AS) intranuclear RNA foci were observed in both fibroblasts and iNeurons, the accumulation of (S) and (AS) repeat-associated non-ATG translation (RANT) products were detected only in iNeurons. Importantly, anti-sense oligonucleotides (ASOs) against the (S) repeat transcript lead to decreased (S) RNA foci staining and a reduction of the corresponding RANT products without affecting its (AS) counterparts. ASOs treatment also rescued the cell viability upon stressful stimulus. The results indicate that iNeurons is an advantageous model that not only recapitulates c9ALS/FTD hallmark features but can also help uncover promising therapeutics.

Direct comparison of chitinolytic properties and determination of combinatory effects of mouse chitotriosidase and acidic mammalian chitinase.

Chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) have been implicated in food processing and various pathophysiological conditions such as chronic inflammatory diseases. By combination of the colorimetric analysis and fluorophore-assisted carbohydrate electrophoresis (FACE) method, we directly compared the chitinolytic properties of mouse Chit1 and AMCase and determined their combinatory effects in artificial and natural chitin substrates processing. Chit1 and AMCase display different dynamics of chitinolytic properties through acidic to neutral conditions. At pH2.0, the activity of AMCase was higher than that of Chit1 and stronger or comparable with that of Serratia marcescens chitinase B, a well-characterized bacterium chitinase. Changes of degradation products using different substrates indicate that AMCase and Chit1 have diverse properties under various pH conditions. Exposure of the chitin substrates to both Chit1 and AMCase did not indicate any mutual interference of these enzymes and showed no synergistic effect, in contrast to observations regarding some bacterial chitinases. Our results suggest that Chit1 and AMCase have no synergistic effect under physiological conditions.

Warfarin loading dose guided by pharmacogenetics is effective and safe in cardioembolic stroke patients - a randomized, prospective study.

Warfarin treatment is commonly started with a fixed loading dose that might be associated with an increased risk of bleeding. An individual maintenance dose can then be estimated based on a pharmacogenetic algorithm. Starting treatment with the estimated dose implies a longer time to reach the therapeutic range. Our goal was to compare the safety and efficacy of initiating warfarin treatment with a loading dose guided by pharmacogenetics versus a maintenance dose. The primary endpoint was time in the therapeutic range (TTR) in the first 10 days of treatment. Secondary endpoints were time to the first international normalized ratio (INR) in therapeutic range (2.0-3.0) and occurrence of serious adverse events. Consenting cardioembolic stroke patients were genotyped for CYP2C9 (cytochrome P450 2C9 gene) and VKORC1 (vitamin K epoxide reductase complex, subunit 1 gene) polymorphisms and a maintenance warfarin dose was estimated. Patients were randomized into two groups. The loading dose group (LDG) patients received twice the estimated dose in the first 2 days of treatment. The maintenance dose group (MDG) patients received the estimated dose directly from day one. The TTR in the first 10 days was significantly higher in the LDG than in the MDG (50.5% vs. 38.3%, p = 0.003). The time to the first INR in this range was significantly shorter in the LDG (5.24 vs. 7.3 days). There were no significant differences in the INR above this range or serious adverse events. Warfarin loading dose guided by pharmacogenetics after recent cardioembolic stroke improved the efficacy of warfarin initiation without increasing the risk of adverse events.

High expression of acidic chitinase and chitin digestibility in the stomach of common marmoset (Callithrix jacchus), an insectivorous nonhuman primate.

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.

Chitinase mRNA Levels Determined by QPCR in Crab-Eating Monkey (Macaca fascicularis) Tissues: Species-Specific Expression of Acidic Mammalian Chitinase and Chitotriosidase.

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.

Chitin digestibility is dependent on feeding behaviors, which determine acidic chitinase mRNA levels in mammalian and poultry stomachs.

Chitin, a polymer of N-acetyl-D-glucosamine (GlcNAc), functions as a major structural component in chitin-containing organism including crustaceans, insects and fungi. Recently, we reported that acidic chitinase (Chia) is highly expressed in mouse, chicken and pig stomach tissues and that it can digest chitin in the respective gastrointestinal tracts (GIT). In this study, we focus on major livestock and domestic animals and show that the levels of Chia mRNA in their stomach tissues are governed by the feeding behavior. Chia mRNA levels were significantly lower in the bovine (herbivores) and dog (carnivores) stomach than those in mouse, pig and chicken (omnivores). Consistent with the mRNA levels, Chia protein was very low in bovine stomach. In addition, the chitinolytic activity of E. coli-expressed bovine and dog Chia enzymes were moderately but significantly lower compared with those of the omnivorous Chia enzymes. Recombinant bovine and dog Chia enzymes can degrade chitin substrates under the artificial GIT conditions. Furthermore, genomes of some herbivorous animals such as rabbit and guinea pig do not contain functional Chia genes. These results indicate that feeding behavior affects Chia expression levels as well as chitinolytic activity of the enzyme, and determines chitin digestibility in the particular animals.

Acidic Chitinase-Chitin Complex Is Dissociated in a Competitive Manner by Acetic Acid: Purification of Natural Enzyme for Supplementation Purposes.

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.

Protease resistance of porcine acidic mammalian chitinase under gastrointestinal conditions implies that chitin-containing organisms can be sustainable dietary resources.

Chitin, a polymer of N-acetyl-D-glucosamine (GlcNAc), is a major structural component in chitin-containing organism including crustaceans, insects and fungi. Mammals express two chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Here, we report that pig AMCase is stable in the presence of other digestive proteases and functions as chitinolytic enzyme under the gastrointestinal conditions. Quantification of chitinases expression in pig tissues using quantitative real-time PCR showed that Chit1 mRNA was highly expressed in eyes, whereas the AMCase mRNA was predominantly expressed in stomach at even higher levels than the housekeeping genes. AMCase purified from pig stomach has highest activity at pH of around 2-4 and remains active at up to pH 7.0. It was resistant to robust proteolytic activities of pepsin at pH 2.0 and trypsin and chymotrypsin at pH 7.6. AMCase degraded polymeric chitin substrates including mealworm shells to GlcNAc dimers. Furthermore, we visualized chitin digestion of fly wings by endogenous AMCase and pepsin in stomach extract. Thus, pig AMCase can function as a protease resistant chitin digestive enzyme at broad pH range present in stomach as well as in the intestine. These results indicate that chitin-containing organisms may be a sustainable feed ingredient in pig diet.

Mouse acidic mammalian chitinase exhibits transglycosylation activity at somatic tissue pH.

Mouse acidic mammalian chitinase (AMCase) degrades chitin with highest efficiency at pH 2.0 and is active up to pH 8.0. Here, we report that mouse AMCase also exhibits transglycosylation activity under neutral conditions. We incubated natural and artificial chitin substrates with mouse AMCase at pH 2.0 or 7.0 and analyzed the resulting oligomers using an improved method of fluorescence-assisted carbohydrate electrophoresis. Mouse AMCase produces primarily dimers of N-acetyl-d-glucosamine [(GlcNAc)2 ] under both pH conditions while generating transglycosylated (GlcNAc)3 primarily at pH 7.0 and at lower levels at pH 2.0. These results indicate that mouse AMCase catalyzes hydrolysis as well as transglycosylation and suggest that this enzyme can play a novel role under physiological conditions in peripheral tissues, such as the lungs.