Chitinase mRNA levels by quantitative PCR using the single standard DNA: acidic mammalian chitinase is a major transcript in the mouse stomach.

Chitinases hydrolyze the β-1-4 glycosidic bonds of chitin, a major structural component of fungi, crustaceans and insects. Although mammals do not produce chitin or its synthase, they express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). These mammalian chitinases have attracted considerable attention due to their increased expression in individuals with a number of pathological conditions, including Gaucher disease, Alzheimer's disease and asthma. However, the contribution of these enzymes to the pathophysiology of these diseases remains to be determined. The quantification of the Chit1 and AMCase mRNA levels and the comparison of those levels with the levels of well-known reference genes can generate useful and biomedically relevant information. In the beginning, we established a quantitative real-time PCR system that uses standard DNA produced by ligating the cDNA fragments of the target genes. This system enabled us to quantify and compare the expression levels of the chitinases and the reference genes on the same scale. We found that AMCase mRNA is synthesized at extraordinarily high levels in the mouse stomach. The level of this mRNA in the mouse stomach was 7- to 10-fold higher than the levels of the housekeeping genes and was comparable to that the level of the mRNA for pepsinogen C (progastricsin), a major component of the gastric mucosa. Thus, AMCase mRNA is a major transcript in mouse stomach, suggesting that AMCase functions as a digestive enzyme that breaks down polymeric chitin and as part of the host defense against chitin-containing pathogens in the gastric contents. Our methodology is applicable to the quantification of mRNAs for multiple genes across multiple specimens using the same scale

Quantitative Real-Time PCR Analysis of YKL-40 and Its Comparison with Mammalian Chitinase mRNAs in Normal Human Tissues Using a Single Standard DNA

YKL-40 (YKL for the first three N-terminal residues of a 40 kDa protein) belongs to a group of human chitinase-like proteins (CLPs), which are similar to chitinases but lack chitinolytic activity. YKL-40 mRNA and its protein levels have been reported elevated in multiple disorders including asthma, cystic fibrosis, rheumatoid arthritis and malignant tumors. Here, we quantified the YKL-40 mRNA levels and compared them with chitinases and housekeeping genes in normal human tissues. To establish the quantitative real-time PCR (qPCR) system for evaluation of relative YKL-40 mRNA levels, we constructed a human standard DNA molecule by ligating cDNAs of YKL-40, two mammalian chitinases and two housekeeping genes in a one-to-one ratio. We generated cDNAs from various normal human tissues and analyzed the YKL-40 mRNA expression levels using a qPCR system with the standard DNA. We found that YKL-40 mRNA is present widely in human tissues while its expression patterns exhibit clear tissue specificity. Highest YKL-40 mRNA levels were detected in the liver, followed by kidney, trachea and lung. The levels of YKL-40 mRNA in the kidney and liver were more than 100-times higher than those of chitotriosidase mRNA. Our study provides for the first time a comprehensive analysis of the relative expression levels of YKL-40 mRNA versus mammalian chitinases in normal human tissues

Expression of Chit1 and AMCase mRNAs in mouse tissues.

<p>Quantification of Chit1 (A) and AMCase (B) mRNAs in mouse tissues. Both chitinases were quantified by real-time PCR using the standard template 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 panel indicates the actual number, whereas the lower panel shows the logarithm of the values.</p

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

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

Establishment and validation of a quantitative real-time PCR system using the standard template DNA.

<p>The checked DNAs are as follows: A, Chit1; B, AMCase; C, GAPDH; D, β-actin; E, pepsinogen C. (Left) Real-time PCR quantification of each 10-fold serial dilution of the standard template DNA using primer pairs targeting each gene. (Right) The standard curves by using the standard template DNA containing the five cDNA fragments (brown closed circles). In addition, the quantification of full coding cDNAs was carried out using primer pairs for each gene. The target cDNA was amplified from a dilution of the full coding cDNA with a known concentration (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050381#pone.0050381.s002" target="_blank">Figure S2</a></b>) and subsequently analyzed as an unknown sample (blue closed rhombuses). Equal quantities were observed for each tested dilution of the standard curve and full coding cDNA.</p

Strategy for comparing the gene expression levels of five genes.

<p>The expression levels of the Chit1 and AMCase genes were compared. To evaluate the chitinase levels, we used two housekeeping genes, GAPDH and β-actin, which are constitutively expressed at high levels in most tissues and cells. In addition, we chose pepsinogen C, which is a major component of the gastric mucosa, as a reference gene for the stomach tissues. Using these three reference genes, we evaluated the gene expression levels of Chit1 and AMCase in mouse tissues.</p

Analysis of Chit1 and AMCase mRNAs and reference gene mRNAs in lung and stomach tissues.

<p>The expression levels of the five genes determined using the cDNAs prepared from lung (A) or stomach (B) tissues from 3-month-old mice (n = 5) were quantified by real-time PCR. The upper panel indicates the actual value, whereas the lower panel shows the logarithm of each value.</p

Construction of standard template DNA.

<p>A. Schematic representation of the standard template DNA used for real-time PCR. The target fragments of the AMCase, pepsinogen C, Chit1, GAPDH and β-actin cDNAs with flanking sequences and restriction sites were amplified and ligated in a one-to-one ratio into a DNA fragment, which was then cloned into a cloning vector. The linearized standard template DNA was amplified from the plasmid DNA and used as the standard DNA. B. Nucleotide sequence of the standard template DNA. The template DNA, 913 nucleotides long, contained five cDNA fragments (shown in different colors) covering the PCR-target region (shown in bold underline) plus 9–120 bases of the flanking regions and containing restriction sites for <i>Bgl</i>II, <i>Xho</i>I, <i>Pst</i>I, or <i>Not</i>I (bold italic).</p

Evaluation of primer suitability for real-time PCR.

<p>PCR primers were selected based on whether they gave one melting temperature (A–E) and a single PCR product on a 10% polyacrylamide gel (F). To verify the specificity of the primers, the dissociation curves of the PCR products for five genes generated using mouse tissue cDNA mixture were examined. The PCR products were analyzed on a 10% polyacrylamide gel, followed by ethidium bromide staining. The nucleotide sequences of the PCR products were confirmed.</p

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 &alpha;-chitin at 50 &deg;C, producing N-acetyl-d-glucosamine (GlcNAc) dimers more efficiently than at 37 &deg;C. Moreover, the degradation rate increased with a longer incubation time (up to 72 h) without the inactivation of the enzyme. Five substrates (&alpha;-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 &deg;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