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Acidic mammalian chitinase is a proteases-resistant glycosidase in mouse digestive system

View Article: PubMed Central - PubMed

ABSTRACT

Chitinases are enzymes that hydrolyze chitin, a polymer of β-1, 4-linked N-acetyl-D-glucosamine (GlcNAc). Chitin has long been considered as a source of dietary fiber that is not digested in the mammalian digestive system. Here, we provide evidence that acidic mammalian chitinase (AMCase) can function as a major digestive enzyme that constitutively degrades chitin substrates and produces (GlcNAc)2 fragments in the mouse gastrointestinal environment. AMCase was resistant to endogenous pepsin C digestion and remained active in the mouse stomach extract at pH 2.0. The AMCase mRNA levels were much higher than those of four major gastric proteins and two housekeeping genes and comparable to the level of pepsinogen C in the mouse stomach tissues. Furthermore, AMCase was expressed in the gastric pepsinogen-synthesizing chief cells. The enzyme was also stable and active in the presence of trypsin and chymotrypsin at pH 7.6, where pepsin C was completely degraded. Mouse AMCase degraded polymeric colloidal and crystalline chitin substrates in the gastrointestinal environments in presence of the proteolytic enzymes. Thus, AMCase can function as a protease-resistant major glycosidase under the conditions of stomach and intestine and degrade chitin substrates to produce (GlcNAc)2, a source of carbon, nitrogen and energy.

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Related in: MedlinePlus

AMCase is resistant to digestion by trypsin and chymotrypsin.Soluble proteins fraction from mouse stomach was incubated under stomach-like condition, followed by incubation in intestine-like environment containing trypsin and chymotrypsin at equal or 50-fold excess to the amount of the soluble proteins. The samples were analyzed by (a) CBB staining, (b,c) Western blot and (d) measurement of chitinolytic activity. All incubations were performed at 37 °C. Values represent mean ± SD. **p < 0.01 P-values were determined using Student’s t-test.
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f3: AMCase is resistant to digestion by trypsin and chymotrypsin.Soluble proteins fraction from mouse stomach was incubated under stomach-like condition, followed by incubation in intestine-like environment containing trypsin and chymotrypsin at equal or 50-fold excess to the amount of the soluble proteins. The samples were analyzed by (a) CBB staining, (b,c) Western blot and (d) measurement of chitinolytic activity. All incubations were performed at 37 °C. Values represent mean ± SD. **p < 0.01 P-values were determined using Student’s t-test.

Mentions: Stomach empties its content that includes pepsin C and AMCase into the intestinal lumen where the acid is neutralized by sodium bicarbonate and where trypsin and chymotrypsin are secreted. Here, the predigested proteins are further degraded into smaller peptides. We artificially generated intestinal environment and investigated the stability of AMCase in the presence of trypsin and chymotrypsin. We incubated the soluble protein fraction from mouse stomach at pH 2.0 and 37 °C for 1 hour, followed by incubation under intestine-like neutral condition (pH 7.6) in the presence of equal or 50-fold higher levels of trypsin and chymotrypsin at 37 °C for 3 hours. In agreement to the previous experiment (Fig. 1), the total protein quantity drastically decreased during the incubation at pH 2.0. Subsequent incubation at pH 7.6 in the presence of trypsin and chymotrypsin resulted in further degradation of total protein (Fig. 3a). Pepsin C was completely digested, whereas AMCase levels were not affected in the presence of equal amounts of trypsin and chymotrypsin (Fig. 3b and Supplementary Fig. S5). Anti C-terminal AMCase antibody recognized the 50 kDa band, which was decreased during the incubation at stomach pH conditions. However, AMCase level was not reduced by the equal or 50-fold excess of both enzymes (Fig. 3b and Supplementary Fig. S5). Using the anti-N-terminal AMCase antibody, we also detected the 41 kDa band in addition to that of 50 kDa, which were not altered significantly by the trypsin and chymotrypsin (Fig. 3c and Supplementary Fig. S5). Moreover, the chitinolytic activity was not significantly affected by any of the incubation conditions (Fig. 3d). These data indicate that AMCase and its cleaved CatD are stable even in the presence of trypsin and chymotrypsin at pH 7.6.


Acidic mammalian chitinase is a proteases-resistant glycosidase in mouse digestive system
AMCase is resistant to digestion by trypsin and chymotrypsin.Soluble proteins fraction from mouse stomach was incubated under stomach-like condition, followed by incubation in intestine-like environment containing trypsin and chymotrypsin at equal or 50-fold excess to the amount of the soluble proteins. The samples were analyzed by (a) CBB staining, (b,c) Western blot and (d) measurement of chitinolytic activity. All incubations were performed at 37 °C. Values represent mean ± SD. **p < 0.01 P-values were determined using Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121897&req=5

f3: AMCase is resistant to digestion by trypsin and chymotrypsin.Soluble proteins fraction from mouse stomach was incubated under stomach-like condition, followed by incubation in intestine-like environment containing trypsin and chymotrypsin at equal or 50-fold excess to the amount of the soluble proteins. The samples were analyzed by (a) CBB staining, (b,c) Western blot and (d) measurement of chitinolytic activity. All incubations were performed at 37 °C. Values represent mean ± SD. **p < 0.01 P-values were determined using Student’s t-test.
Mentions: Stomach empties its content that includes pepsin C and AMCase into the intestinal lumen where the acid is neutralized by sodium bicarbonate and where trypsin and chymotrypsin are secreted. Here, the predigested proteins are further degraded into smaller peptides. We artificially generated intestinal environment and investigated the stability of AMCase in the presence of trypsin and chymotrypsin. We incubated the soluble protein fraction from mouse stomach at pH 2.0 and 37 °C for 1 hour, followed by incubation under intestine-like neutral condition (pH 7.6) in the presence of equal or 50-fold higher levels of trypsin and chymotrypsin at 37 °C for 3 hours. In agreement to the previous experiment (Fig. 1), the total protein quantity drastically decreased during the incubation at pH 2.0. Subsequent incubation at pH 7.6 in the presence of trypsin and chymotrypsin resulted in further degradation of total protein (Fig. 3a). Pepsin C was completely digested, whereas AMCase levels were not affected in the presence of equal amounts of trypsin and chymotrypsin (Fig. 3b and Supplementary Fig. S5). Anti C-terminal AMCase antibody recognized the 50 kDa band, which was decreased during the incubation at stomach pH conditions. However, AMCase level was not reduced by the equal or 50-fold excess of both enzymes (Fig. 3b and Supplementary Fig. S5). Using the anti-N-terminal AMCase antibody, we also detected the 41 kDa band in addition to that of 50 kDa, which were not altered significantly by the trypsin and chymotrypsin (Fig. 3c and Supplementary Fig. S5). Moreover, the chitinolytic activity was not significantly affected by any of the incubation conditions (Fig. 3d). These data indicate that AMCase and its cleaved CatD are stable even in the presence of trypsin and chymotrypsin at pH 7.6.

View Article: PubMed Central - PubMed

ABSTRACT

Chitinases are enzymes that hydrolyze chitin, a polymer of &beta;-1, 4-linked N-acetyl-D-glucosamine (GlcNAc). Chitin has long been considered as a source of dietary fiber that is not digested in the mammalian digestive system. Here, we provide evidence that acidic mammalian chitinase (AMCase) can function as a major digestive enzyme that constitutively degrades chitin substrates and produces (GlcNAc)2 fragments in the mouse gastrointestinal environment. AMCase was resistant to endogenous pepsin C digestion and remained active in the mouse stomach extract at pH 2.0. The AMCase mRNA levels were much higher than those of four major gastric proteins and two housekeeping genes and comparable to the level of pepsinogen C in the mouse stomach tissues. Furthermore, AMCase was expressed in the gastric pepsinogen-synthesizing chief cells. The enzyme was also stable and active in the presence of trypsin and chymotrypsin at pH 7.6, where pepsin C was completely degraded. Mouse AMCase degraded polymeric colloidal and crystalline chitin substrates in the gastrointestinal environments in presence of the proteolytic enzymes. Thus, AMCase can function as a protease-resistant major glycosidase under the conditions of stomach and intestine and degrade chitin substrates to produce (GlcNAc)2, a source of carbon, nitrogen and energy.

No MeSH data available.


Related in: MedlinePlus