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.


Results
Gene expression analysis of Chit1 and AMCase mRNAs in pig tissues. To study the in vivo regulation of pig Chit1 and AMCase genes expression, total RNA from various normal pig tissues was analyzed using a qPCR assay with a specifically designed standard DNA ( Supplementary Fig. S1) as described in Methods.
Clear tissue-specific pattern was observed in both chitinases mRNAs expression ( Fig. 1a and b, upper panels). High levels of Chit1 mRNA were detected in the eye (Fig. 1a, upper panel), followed by liver, salivary gland, intestine and lung (Fig. 1a, lower panel). AMCase mRNA was predominantly detected in the stomach (Fig. 1b,  upper panel), followed by intestine, liver, salivary gland and lung (Fig. 1b, lower panel). The levels of AMCase in Figure 1. Expression of Chit1 and AMCase mRNAs in pig tissues. Quantification of (a) Chit1 and (b) AMCase mRNAs in ten major pig tissues. Both chitinases were quantified by qPCR using the standard DNA. All values obtained were expressed as molecules per 10 ng of total RNA. (c) Ratios of AMCase to Chit1. All mRNA copy numbers were derived based on the same standard dilutions. The upper panels indicate the actual number, whereas the lower panel shows each value on logarithmic scale. Each reaction was performed in triplicate. **p < 0.01. P-values were determined using Student's t-test.
AMCase mRNA levels in pig stomach. Next, we evaluated the AMCase mRNA levels in pig stomach in detail. The mRNAs of pepsinogen A and C, H + /K + -ATPase and AMCase were expressed at much higher levels than those of the housekeeping genes (Fig. 2). The expression of pepsinogen A and C mRNAs were ~300 times and ~15 times higher than that of AMCase, respectively (Fig. 2, upper panel). AMCase mRNA level was comparable to H + /K + -ATPase and 26 times higher than that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5 times higher than β-actin, and hundreds-to-thousands of times higher than other four tested gastric mucosa genes (Fig. 2, lower panel). These results indicate that AMCase is one of the major transcript in the pig stomach.
Endogenous pepsins degrade soluble proteins in the stomach extract. Next, we investigated the protease activity of endogenous pepsins in artificially created pig stomach environment at pH 2.0 and 37 °C. Soluble protein fraction was prepared from the pig stomach in the absence of protease inhibitor and incubated at pH 7.6 or pH 2.0 for up to 60 min. The protein fractions were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), followed by Coomassie Brilliant Blue (CBB) staining ( Fig. 3a). At pH 7.6, no changes in the band pattern and intensities were noticed within 60 min incubation (Fig. 3a). In contrast, we observed time-dependent decrease of the soluble proteins with a marked reduction after as early as 5 min of incubation at pH 2.0, although several bands remained unchanged after 60 min of incubation (Fig. 3a). Western blot analysis using anti-pepsin antibody showed a shift of the respective bands within 5 min of incubation at pH 2.0, indicating pepsinogen-to-pepsin conversion (Fig. 3b).
Western blot analysis using anti-mouse AMCase showed that this enzyme was stable during the 60-min incubation at both pH 7.6 and 2.0 (Fig. 3c). Moreover, the chitinolytic activity measured using 4-nitrophenyl N,N′ -diacetyl-β-D-chitobioside (4-NP-chitobioside) also remained virtually unchanged (Fig. 3d). These results indicated that AMCase has chitinolytic activity even in the presence of large amount of endogenous pepsins in the pig stomach extract.
Purification and characterization of pig AMCase from stomach tissue. For further characterization, we purified AMCase by application of the stomach extract onto the chitin beads column. Bound chitinase was eluted from the column with 8 M urea, subsequently removed from the sample as described in Methods. SDS-PAGE analysis of the protein fractions showed one major band at 54 kDa (Fig. 4a). Thus, we obtained purified AMCase usable for in vitro enzymatic assays. Purification of the enzyme is summarized in Table 1.
The pH optima were determined by monitoring enzyme activity at different pH in 0.1 M Gly-HCl (pH 1.0-3.0) or McIlvaine's (pH 2.0-8.0) buffers using 4-NP-chitobioside as a substrate for 30 min at 37 °C. Highest activity was detected at pH 2.0 in 0.1 M Gly-HCl buffer. Using McIlvaine's buffer, higher enzymatic activity was observed at pH 2.0-5.0 with peaks at pH 2.0 and pH 4.0 with gradual decrease in less acidic environments (pH 6.0-8.0) (Fig. 4b). Thus, the chitinolytic activity of AMCase has a slightly different pH-related pattern depending on used buffer. The effect of temperature on enzyme activity was determined in 0.1 M Gly-HCl buffer at pH 2.0 at temperatures ranging from 30 to 64 °C using same substrate for 30 min. As shown in Fig. 3c, the rate of the AMCase-catalyzed reaction was gradually enhanced with increasing temperature and reached the maximum level at 60 °C, then abruptly declined.
We next determined the pH stability of the pig AMCase. The enzyme was pre-incubated on ice for 60 min at various pH in Gly-HCl or McIlvaine's buffers. The enzyme activity was then analyzed at 37 °C and pH 2.0. As shown in Fig. 4d, the pig AMCase has a remarkable acid stability as the pre-incubation at pH 2.0 caused no measurable decrease in chitinase activity.
Pig AMCase degrades polymeric chitin substrates under the stomach condition. We incubated the purified protein with equal amount of pepsin (0.4 µg) at pH 2.0 for 1 hour and we confirmed the above-observed stability of AMCase at pH 2.0 ( Fig. 5a) as well as the maintenance of its chitinolytic activity (Fig. 5b).
Next, we tested whether AMCase can degrade chitin substrates under the pig stomach condition. We incubated colloidal or crystalline chitin with purified AMCase and analyzed the products by an improved fluorophore-assisted carbohydrate electrophoresis (FACE) 37,38 . Purified enzyme degraded both substrates at pH 2.0 as early as after 1 hour incubation and produced primarily (GlcNAc) 2 fragments in the presence of pepsin ( Fig. 5c and d).
Pig AMCase is resistant to trypsin and chymotrypsin and degrades chitin substrates under intestinal condition. Next, we investigated whether pig AMCase is also stable under intestinal condition.
We incubated the purified protein with equal amount of trypsin/chymotrypsin (0.4 µg) at pH 7.6 and found that AMCase remained stable and active throughout the incubation ( Fig. 6a and b).
We incubated crystalline and colloidal chitin with purified AMCase and equal amount of trypsin/chymotrypsin (0.4 µg) at pH 7.6 for 1 hour and the degradation products were analyzed as described above ( Fig. 6c and d). Similarly to stomach condition, (GlcNAc) 2 was produced under the intestine-like condition by AMCase activity. Then, we mimicked the GIT physiology regarding the movement of stomach contents to intestine by pre-incubation of the stomach extract at pH 2.0 for 1 hour, followed by neutralization to pH 7.6 and addition of trypsin/chymotrypsin and further 1-hour incubation ( Fig. 6e and f). We observed more (GlcNAc) 2 degradation Chitin in mealworm shells and fruit fly wings is degraded by AMCase and pepsin in the stomach extract. Next, we tested whether AMCase and pepsin can degrade chitin-protein substrates present in chitin-containing organism under pig stomach condition. We incubated mealworm (Tenebrio molitor) larvae shells with stomach extract at pH 2.0 followed by FACE analysis as described above. AMCase in the stomach extract degraded mealworm shells at pH 2.0 after 16 hours incubation and produced different GlcNAc fragments with the dimer being most abundant (Fig. 7a).
To gain further insights into the digestibility of chitin-containing organisms, we next tested fruit fly (Drosophila melanogaster Oregon-R) wings. We first homogenized wings and incubated with the stomach extract and analyzed the products by FACE. We detected (GlcNAc) 2 fragments (Fig. 7b).
We also visualized the fruit fly wings surface using stereo microscope ( Fig. 7c and d) and scanning electron microscope (SEM) (Fig. 7e-h) after 16 hours treatment with (Fig. 7d,f and h) or without (Fig. 7c,e and g) stomach extract in 0.1 M Gly-HCl (pH 2.0). The extract-treated fruit fly wings and the solution became hazy containing some particles (Fig. 7d) and we observed partially damaged regions ( Fig. 7f and h). We detected no such damage in wings incubated only with the buffer (Fig. 7c,e and g). These results indicate that AMCase in the stomach extract can deteriorate chitinous fruit fly wing integrity.   Table 1. Purification of AMCase from pig stomach. The purified protein was prepared from 1 G of stomach tissue as described in the Methods.

Discussion
Mammalian chitinases have extensively been studied mainly in mice and humans, however relatively little is known about the enzymes in other mammals. In this report, we showed that AMCase mRNA was predominantly expressed in pig stomach tissue and it was much higher than Chit1, housekeeping genes and gastric mucosal genes. We purified AMCase from pig stomach and determined its optimal activity at pH 2.0-4.0 and 60 °C. This enzyme degraded polymeric chitin substrates including mealworm shells and fruit fly wings in the environment mimicking pig GIT conditions. These results support our hypothesis on AMCase functioning as a protease-resistant glycoside hydrolase in the pig digestive system. AMCase mRNA level was comparable with of H + /K + -ATPase (role in maintaining the stomach acidity) 39 , and it was substantially higher than mRNA of two housekeeping genes and other gastric mucosa proteins in the pig stomach. This is in agreement with our previous report on high AMCase expression in mouse stomach 32,35 . These data suggest that AMCase in these animals might be able to digest chitin in those animal bodies.
It has been reported that AMCase expression is elevated under several pathological conditions including dry eye syndrome 20,21 . Our qPCR analysis showed that AMCase mRNA expression was ten times lower than that of Chit1 in healthy pig eye. These data imply that AMCase may be a key mediator of innate immune responses in certain ocular pathologies. Chit1 mRNA, on the other hand, is constitutively expressed in healthy mouse eye 32 , human lacrimal gland 40 and pig eye. Lysozyme is thought to have anti-bacterial effects, whereas Chit1 protects against fungi. Thus, Chit1 probably protects mammalian eyes from chitin-containing pathogens such as fungi, whose cell wall contains high levels of chitin.
Pig pepsin A and C have been purified, crystallized and extensively studied, in the past [41][42][43][44][45] . However, to our knowledge, multiple comparisons of pepsin with other mucosal genes and reference genes mRNA levels have not been performed. Here, we show that pepsinogen A mRNA level was substantially higher than housekeeping genes and gastric mucosa genes in the stomach while exceeding 20 times that the level of pepsinogen C. Although the functional difference between pepsin A and C is still unclear, our results suggest that pepsin A is a major protease and pepsin C acts as a co-protease in the pig stomach.
Pig AMCase was more active in Gly-HCl buffer than in McIlvaine at pH 2.0 (Fig. 4b). Thus, the chitinolytic activity of AMCase has a slightly different pH-related pattern depending on used buffer. Similar results were also obtained in mouse AMCase 35 and chicken Chia 36 , although with less significant difference in chicken Chia between the two buffers. The reason is not well understood, but some lessons could be learned from human pancreatic α-amylase, whose activation has been shown to be catalyzed by chloride ion 46 . Some members of the amylase protein family require chloride for maximal activity [47][48][49] . Hydrochloric acid is secreted in the stomach, creating acidic conditions (pH ~2), which may induce similar activation of AMCase. This assumption warrants further scrutiny. Murine AMCase has been well studied and its optimal activity has been reported at pH 2.0 with a decrease at less acidic conditions (pH 3.0-7.0) 15,35,38 . Also, chicken Chia (AMCase homologue) was most active at pH 2.0 and it retained at less acidic condition 36 . We analyzed effect of pH on the pig AMCase chitinolytic activity and showed that the activity was highest in pH 2.0-4.0 and remained active at up to pH 7.0. Although the pig AMCase shares 81 to 89% primary sequence homology with mouse and chicken counterparts, those specificities to pH and buffer are different. These species particularities can be attributed to differences during the molecular evolution based on changes in feeding habitat.
In our study, we showed that pig AMCase degraded chitin substrates including shells of mealworm larvae and fruit fly wings as well as crystalline and colloidal chitins in the presence of digestive proteases and produced (GlcNAc) 2 . Accordingly, mouse and pig AMCase as well as chicken Chia mainly produced (GlcNAc) 2 31,35,36 . In addition, we found that mealworm shells digestion by pig AMCase also resulted in (GlcNAc) 3 and (GlcNAc) 4 fragments. The product patterns were slightly different from those resulting from colloidal and crystalline chitin degradation. Similarly, we confirmed formation of longer chitooligosaccharides from mealworm larvae shells by chicken Chia 36 . Importantly, we have recently shown that mouse AMCase catalyzes transglycosylation as well as hydrolysis 50 . Thus, it is feasible to assume that distinct chitooligosaccharides can be produced from partially deacetylated chitin in mealworm shells or transglycosylation by pig AMCase.
Pig is one of the major protein resource for humans worldwide. Increasing demand of meat protein requires more feed resources for the livestock. There have been published several studies reporting on application of insects as a sustainable high protein feed ingredient for pig. For example, Jin et al. 51 showed that supplementation of dried mealworm in weaning pigs' diet improves their growth performance and nutrient digestibility without any detrimental effect on immune responses 51 . Furthermore, chitin derivatives can enhance the immune response and act as an antibiotic/probiotic in pregnant pig 52 . Various biological activities, and especially anti-cancer and anti-inflammatory action of chitin oligosaccharide and chitosan oligosaccharide, have been well studied 9,53-55 . Distinct chitooligosaccharides from chitin-containing organisms may improve immune systems or act as probiotics providing benefits for animal health. Therefore, further evaluation of nutrient value, digestibility and potential side-effects of chitin-containing organisms used as feed ingredient on pig growth performance is needed.
In this report, we showed that AMCase mRNA was highly expressed in pig stomach, having a remarkable protease resistance and degraded chitin or chitin-containing organisms into (GlcNAc) 2 and several chitooligosaccharides under the GIT condition. We previously reported that similar properties of AMCase and Chia were found in mouse 35 and chicken 36 , respectively. These animals primarily feed on chitin-containing organisms such as insects and fungi. It is plausible to suggest that their food habitat leads to high expression levels of these enzymes in the stomach and their intense chitinolytic activity in the GIT. According to recent knowledge, chitin-containing organisms can be used as good energy source in pig, chicken and mouse organisms because proteolytic enzyme accessibility is improved by degrading chitinous cuticles of insects and crustaceans by respective AMCase enzymes. From practical point of view, we need further research in other animal species including those with limited ingestion of chitin-containing organisms to reveal whether chitinous diets could be implemented in such species including cattle, sheep, horse, dog, etc.

Methods
Pig stomach tissues. Six months-old male pig stomach tissues (Landrace F1) were purchased from Funakoshi Co., Ltd (Tokyo, Japan), which were dissected from the animals, quickly frozen on dry ice and kept at −80 °C.
RNA and cDNA preparation. Pig Total RNA Panel (Zyagen, San Diego, CA, USA) was used to examine the distribution of transcripts in various tissues. In addition, total RNA was isolated from the pig stomach tissues using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) per manufacturer's instructions and reverse transcribed into cDNA essentially as described previously 32,35,36 . Selection of primer pairs for qPCR. Primers for qPCR were designed using PrimerQuest Input (Integrated DNA Technologies, Coralville, IA, USA) and their suitability was evaluated based on a single product generation, as reflected by a single melting temperature as describe previously 32,33,36 . The primers' sequences are listed in Supplementary Table S1. Construction of the DNA standard and qPCR. Construction of the 10 genes standard DNA coding sequences of AMCase, pepsinogen A, pepsinogen C, H + /K + -ATPase, gastrin, gastric intrinsic factor and mucin were commercially synthesized and inserted into pTAKN-2 vector (Eurofins Genomics, Tokyo, Japan). The standard DNA (956 bases; see Supplementary Fig. S1) was prepared by PCR reamplification from the plasmid DNA using the forward primer 5′-TTGCCGTCCGTGCATATT-3′ and the reverse primer 5′-CAAGGTCAAGGCCATCAAA-3′ and was thereafter used as the standard DNA for qPCR. Each reaction was performed in triplicate.
Pig stomach extract preparation. Soluble fraction was prepared from pig stomach tissues (0.2 g) by homogenization followed by centrifugation at 15,000 g for 10 min at 4 °C 35,36 . The supernatants were used as the stomach extract and pre-incubated at 37 °C for 0, 10, 20, 40 or 60 min at pH 7.6 or pH 2.0. After incubation at pH 2.0 and 37 °C, the solutions were neutralized. Chitinase enzymatic assays. Chitinolytic activity was determined using a synthetic chromogenic substrate, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside (4-NP-chitobioside, Sigma-Aldrich) essentially as described previously 31 . All enzymatic reactions for optimum pH and temperature determination of were conducted in a volume of 50 μL as described previously 31,56 .

SDS
Purification of pig AMCase. AMCase was purified from pig stomach tissue (1 g) using chitin beads column and eluted with 8 M urea as performed previously 36 . Protein concentrations were determined by the Bradford Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA) using the BioPhotometer Plus UV/Vis photometer (Eppendorf, Hamburg, Germany), with bovine serum albumin as a standard. AMCase unit definition was also reported previously 31  Mealworms. Jumbo mealworm (Tenebrio molitor) larvae were purchased from local commercial supplier (Lumberjack Co., Ltd., Tokyo, Japan). We used the shells containing connective tissue as chitin-protein polymer substrates as described previously 36 . Degradation of colloidal and crystalline chitin substrates and mealworm larvae shell. Colloidal and crystalline chitin were incubated in a volume of 50 µL containing purified AMCase (4 mU) or soluble protein (4 mU) from pig stomach as described previously 35,36 . Mealworm larvae shells were also incubated with soluble protein fraction (20 mU) in an analogous way. Generated chitin fragments were analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) as originally described by Jackson 37 and recently improved by us 38 . Chitin degradation of fruit fly wings by stomach extract. Fruit flies (D. melanogaster Oregon-R) were bred at the facility in Okayama University. Flies were immersed once in ethanol. Fifty wings were homogenized using TaKaRa BioMasher Standard (TaKaRa Bio, Shiga, Japan) and then treated with stomach extract as described in mealworm. After incubation at 37 °C for 16 hours, degradation products were analyzed by FACE as described above. For morphological examination, wing was treated with stomach extract containing 9 mU AMCase activity in 0.1 M Gly-HCl (pH 2.0) at final volume of 10 µL using a glass slide printed with water-repellent mark (TF1205, Matsunami Glass Ind., Ltd., Osaka, Japan). The morphological changes of the wings were assessed using a stereo microscope (M205 C, Leica Microsystems, Wetzlar, Germany). Observations of fly wings by scanning electron microscope (SEM, JCM-6000, acceleration voltage: 15 kV, JEOL Ltd., Tokyo, Japan) were achieved by ionic liquids coating method without accumulation of electron charges, indicating that the liquid behaves as an electronically conducting material 57 . Observed sample was immersed into mixture of ionic liquid, 10% 1-hexyl-3-methylimidazolium bis (fluorosulfonyl) imide (Mitsubishi Materials Electronic Chemicals Co., Ltd., Akita, Japan) in ethanol. Immersed fly wing samples were dried at room temperature over 1 hour prior to SEM observations for removing ethanol. Thin and uniform ionic liquid coating layer enabled clear SEM observation without charge up of surface of measurement samples.