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

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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.

No MeSH data available.


AMCase is not degraded by pepsin.Soluble proteins obtained from mouse stomach were incubated at 37 °C for 0, 5, 10, 20, 40 and 60 min at pH 7.6 or 2.0. (a) Total protein analysis by Coomassie Brilliant Blue (CBB) staining, (b) Total protein levels quantification, (c) Western blot and (d) chitinolytic activity assay measured at pH 2.0 as described in Methods. Values in (b) and (d) represent mean ± SD from a single experiment conducted in triplicate. *p < 0.05. P-values were determined using Student’s t-test.
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f1: AMCase is not degraded by pepsin.Soluble proteins obtained from mouse stomach were incubated at 37 °C for 0, 5, 10, 20, 40 and 60 min at pH 7.6 or 2.0. (a) Total protein analysis by Coomassie Brilliant Blue (CBB) staining, (b) Total protein levels quantification, (c) Western blot and (d) chitinolytic activity assay measured at pH 2.0 as described in Methods. Values in (b) and (d) represent mean ± SD from a single experiment conducted in triplicate. *p < 0.05. P-values were determined using Student’s t-test.

Mentions: At pH 7.6, no changes in the band pattern and intensities were noticeable during the 60 min incubation (Fig. 1a and b, left panels). In contrast to the pH 7.6 condition, we observed a 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 unmodified within 60 min of incubation (Fig. 1a and b, right panels). Western blot analysis indicated that the conversion from pepsinogen C to pepsin C occurred within first 5 min of incubation at pH 2.0 (Fig. 1c, upper right panel and Supplementary Fig. S1). Beta-actin, a soluble protein, was stably present during the 60 min of incubation at pH 7.6 (Fig. 1c and Supplementary Fig. S1), whereas it was only slightly detectable at pH 2.0 at time 0 and degraded completely within first 5 min of incubation (Fig. 1c).


Acidic mammalian chitinase is a proteases-resistant glycosidase in mouse digestive system
AMCase is not degraded by pepsin.Soluble proteins obtained from mouse stomach were incubated at 37 °C for 0, 5, 10, 20, 40 and 60 min at pH 7.6 or 2.0. (a) Total protein analysis by Coomassie Brilliant Blue (CBB) staining, (b) Total protein levels quantification, (c) Western blot and (d) chitinolytic activity assay measured at pH 2.0 as described in Methods. Values in (b) and (d) represent mean ± SD from a single experiment conducted in triplicate. *p < 0.05. P-values were determined using Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: AMCase is not degraded by pepsin.Soluble proteins obtained from mouse stomach were incubated at 37 °C for 0, 5, 10, 20, 40 and 60 min at pH 7.6 or 2.0. (a) Total protein analysis by Coomassie Brilliant Blue (CBB) staining, (b) Total protein levels quantification, (c) Western blot and (d) chitinolytic activity assay measured at pH 2.0 as described in Methods. Values in (b) and (d) represent mean ± SD from a single experiment conducted in triplicate. *p < 0.05. P-values were determined using Student’s t-test.
Mentions: At pH 7.6, no changes in the band pattern and intensities were noticeable during the 60 min incubation (Fig. 1a and b, left panels). In contrast to the pH 7.6 condition, we observed a 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 unmodified within 60 min of incubation (Fig. 1a and b, right panels). Western blot analysis indicated that the conversion from pepsinogen C to pepsin C occurred within first 5 min of incubation at pH 2.0 (Fig. 1c, upper right panel and Supplementary Fig. S1). Beta-actin, a soluble protein, was stably present during the 60 min of incubation at pH 7.6 (Fig. 1c and Supplementary Fig. S1), whereas it was only slightly detectable at pH 2.0 at time 0 and degraded completely within first 5 min of incubation (Fig. 1c).

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.