Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues

Misa Ohno,Yuto Togashi,Kazuaki Okawa,Kyoko Tsuda,Yasusato Sugahara,Fumitaka Oyama,Masayoshi Sakaguchi,Minori Kamaya,Emiko Mizoguchi

doi:10.1371/journal.pone.0067399

Abstract

Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific.

Highlights

Chitin, which is a polymer of N-acetyl-D-glucosamine, is the second most abundant polysaccharide in nature [1]
As in mouse tissues [17], we used the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and b-actin as reference genes because they are constitutively expressed at high levels in most cells [18]
We chose pepsinogen C as a reference gene in the stomach because the level of acidic mammalian chitinase (AMCase) mRNA in the mouse stomach was comparable to the mRNA level of pepsinogen C, which constitutes a major component of the gastric mucosa [19]

Summary

Introduction

Chitin, which is a polymer of N-acetyl-D-glucosamine, is the second most abundant polysaccharide in nature [1]. It is an integral component of the exoskeletons of crustaceans and insects, the microfilarial sheaths of parasitic nematodes and fungal cell walls [1,2]. Chitinases are enzymes that digest the chitin polymer. Mammals do not produce chitin, humans and mice have two genes that encode active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) [2,3]. AMCase, which is the second most active chitinase in mammals, was identified as a compensatory enzyme for Chit and named for its optimal activity in acidic conditions [6]

Methods

Results

Conclusion